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Abstracts from the 9th Biennial Scientific Meeting of the Asia Pacific Paediatric Endocrine Society (APPES) and the 50th Annual Meeting of the Japanese Society for Pediatric Endocrinology (JSPE)

Tokyo, Japan. 17-20 November 2016
International Journal of Pediatric Endocrinology20172017 (Suppl 1) :15

  • Published:

PS1 Fat fate and disease - from science to global policy

Peter Gluckman

Office of Chief Science Advsor to the Prime Minister

Attempts to deal with the obesity epidemic based solely on adult behavioural change have been rather disappointing. Indeed the evidence that biological, developmental and contextual factors are operating from the earliest stages in development and indeed across generations is compelling. The marked individual differences in the sensitivity to the obesogenic environment need to be understood at both the individual and population level. There is considerable evidence showing that the fetus and neonate are affected by the environment created by its mother and these have longer-term metabolic consequences including obesity and the implications for non-communicable disease (NCD). But while this evidence emerged over 30 years ago, it is only in the past 5 years it has gained traction in the policy domain. A number of barriers, both scientific and societal, existed to the incorporation of this knowledge into public policy. These barriers included an over-simplistic understanding of the phenomenon, a failure to appreciate its normative nature and multiple mechanisms that are best understood in a evolutionary medicine context, a lack of compelling biological mechanistic explanations and estimates of the size of the developmental component. These issues have largely been addressed to the extent needed for policy recommendations although there are areas of research that need further promotion – in particular those related to the development of eating behaviours and satiety control. However in part because of the Millennium Development Goals that brought an emphasis to maternal and girls’ health and because of scientific progress as reflected in the “ first 1000 days” movement, the first recognition of the developmental and epigenetic component to obesity was noted in the political declaration of the UN General Assembly in 2011. This led the WHO and an increasing number of governments to start to formulate developmental strategies to confront obesity. The World Health Assembly in May 2016 adopted the report of the Commission on Ending Childhood Obesity and this has major implications for national and global health policy. I will discuss these developments from both a scientific and public policy perspective.

PS2 Bone Fragility in Children - Genes and Treatments

Frank Rauch

Shriners Hospitals for Children

Heritable forms of primary bone fragility in children typically lead to a clinical diagnosis of either osteogenesis imperfecta (OI) or juvenile osteoporosis (JO). OI is usually caused by dominant mutations affecting one of the two genes that code for two collagen type I, but a recessive form of OI is present in 5-10% of individuals with a clinical diagnosis of OI. Most of the involved genes code for proteins that play a role in the processing of collagen type I protein (BMP1, CREB3L1, CRTAP, LEPRE1, P4HB, PPIB, FKBP10, PLOD2, SERPINF1, SERPINH1, SEC24D, SPARC,

TMEM38B), or interfere with osteoblast function (SP7, WNT1). Specific phenotypes are caused by mutations in SERPINF1 (recessive OI type VI), P4HB (Cole-Carpenter syndrome) and SEC24D (‘Cole-Carpenter like’). Patients with heritable bone fragility who do not have extraskeletal manifestations of OI are often diagnosed with JO. The JO phenotype can be caused by mutations in LRP5 and PLS3, but also by mutations in some of the genes that cause OI. For most of these gene defects the mechanisms linking mutation to phenotype remain to be elucidated. Regarding specific medical therapy of bone fragility in children, bisphosphonates are currently the main treatment option. Recent data indicate that children with moderate to severe OI have the ability to reshape most compressed vertebra, if treatment is started early enough and is continued throughout the growing period. However, bisphosphonate therapy does not have a major effect on the development of scoliosis and the incidence of long- bone fractures remains elevated. Even though children with moderate OI (OI type IV) typically achieve independent ambulation, this is rarely the case for children with severe OI (OI type III). Newer medications are being evaluated in an attempt to improve on the therapeutic efficacy of bisphosphonates but the available information about their action in OI is very limited at present.

PS3 Unique Newly Discovered Muse Cells May Lead to the Paradigm Shift of Stem Cell Therapy

Mari Dezawa

Department of Stem Cell Biology and History & Department of Anatomy and Anthropology, Tohoku University Graduate School of Medicine

Multilineage-differentiating stress enduring (Muse) cells are naturally existing unique stem cells that are non- tumorigenic and are pluripotent-like because they can generate cells representative of all three germ layers from a single cell, express pluripotency markers, and are able to self-renew and to spontaneously differentiate into cells compatible to the tissue they homed in vivo after engraftment. Whenever they are injected intravenously, they can escape from being trapped in the lung and spleen, unlike mesenchymal stem cells (MSCs), and efficiently home into damaged tissue, suggesting that robust repair can be delivered by intravenous injection of naïve Muse cells. Such unique functions of Muse cells were demonstrated in animal models of stroke, partial hepatectomy, skin ulcer of diabetes mellitus and muscle degeneration. They do not have to be “induced,” or genetically manipulated, to be pluripotent or be purposive cells before transplantation as required with some other cell varieties - they already display inherent pluripotent-like properties after isolation and, with their acquired properties of purposive cells, Muse cells spontaneously repair damaged sites based on their unique mechanisms.

They can be collected as cells positive for SSEA-3, a surface marker for pluripotent stem cells, from readily accessible sources such as the bone marrow (~0.03% of the total mononucleated cell population), and from cultured fibroblasts (several %), as well as from the dermis and adipose tissue. Thus, they are expected to be practical cells for clinical application. Recently, Muse cells are shown to circulate in peripheral blood in healthy donors, and the number increases in stroke patients in an acute phase, suggesting that endogenous Muse cells are mobilized into peripheral blood to repair tissues while their number is not sufficient to recover, and that supply of exogenous Muse cells is expected to deliver clinically relevant functional recovery. Overall, results suggest that Muse cells are a feasible and promising source for cell-based approaches.

PS4 RASopathy - from molecular mechanism to clinical practice

Yoichi Matsubara

National Center for Child Health and Development

Studies in the past decade revealed that a group of genetic disorders results from dysregulation of the Ras/ MAPK (mitogen-activated protein kinase) signaling pathway. These syndromes are comprehensively termed “Ras/ MAPK pathway syndromes” or “RASopathies”. These disorders include Noonan syndrome, Noonan syndrome with multiple lentigines (formerly called LEOPARD syndrome), Costello syndrome, cardiofaciocutaneous (CFC) syndrome, Noonan-like syndrome, neurofibromatosis type I, NF-llike syndrome, hereditary gingival fibromatosis, Legius syndrome and capillary malformation-arteriovenous malformation. Patients with these syndromes share many clinical features such as distinct facial appearances, developmental delays, cardiac defects, growth delays, and neurological disturbances. Molecular analyses identified mutations in genes related to the Ras/MAPK signaling pathway, namely PTPN11, SOS1, RAF1, KRAS, BRAF, NRAS, HRAS, MAP2K1/2, SHOC2, CBL, NF1, SPRED1 and RASA1.

More recently, novel gene variants, including RIT1, RRAS, RASA2, A2ML1, SOS2 and LZTR1, have been shown to be associated with RASopathies, further expanding the disease entity. Molecular characterization of these syndromes help to better elucidate an understanding of the pathogenesis of these disorders and aid in the development of potential therapeutic approaches. Model organisms, such as zebrafish and mice, harboring disease-causing mutations have been produced and they exhibit phenotypes resembling clinical pictures of patients. A series of compounds acting on the Ras/MAPK pathway seem to ameliorate various symptoms, holding promise for treating patients with these syndromes.

SY1-1 Growth: New era of growth disorders

How to approach growth disorders: genetics for better clinical care

Andrew Dauber

Cincinnati Center for Growth Disorders, Division of Endocrinology, Cincinnati Children fs Hospital Medical Center

Human growth is a complex process governed by a multitude of biological process. Traditionally, genetic analysis has played a limited role in the pediatric endocrinologist’s evaluation of patients with growth disorders. Focused candidate gene testing is employed in cases of distinct syndromes (i.e. Leri-Weill Dyschondrosteosis, Noonan Syndrome, Russell Silver Syndrome) or clear defects in the growth hormone/IGF-I axis. We will first explore the role of candidate gene testing in the evaluation of short stature and growth hormone deficiency. We will then turn to the use of genomic technologies including whole exome sequencing and chromosomal microarrays for the identification of rare genetic sequence and copy number variants underlying individual patient’s short stature. We will review a proposed algorithm for the diagnosis of growth disorders.

SY1-2 Growth: New era of growth disorders

IGF2 mutation in Silver-Russell syndrome

Thomas Eggermann

Institute of Human Genetics, University Hospital, Aachen Technical

With the identification of 11p15.5 disturbances in Silver-Russell syndrome (SRS) ten years ago, the central role of at least the telomeric imprinted region ICR1 in 11p15.5 for the etiology of the disease became obvious. Two of the genes regulated by the ICR1 are IGF2 and H19. Due to its physiological function and its imprinting status, IGF2 had been suggested as the causative gene for SRS, but the final clue was still missing. We recently identified a paternally inherited IGF2 mutation in a family with growth retardation and SRS, we could confirm the hypothesis that IGF2 is the key factor for the growth retardation and other features in SRS. However, molecular studies in further SRS families as well as in sporadic patients did not identify further cases, thus IGF2 mutations do not significantly contribute to the mutation spectrum in this disorder. However, in the last decade it has emerged that IGF2 and other imprinted genes belong to common functional networks, either because of their physiological function or because they belong to the imprinted genes network (IGN), and molecular alterations of genes of IGF2 interaction partners also contribute to growth retardation. Comprehensive studies in our patient cohort (n>500) referred for SRS diagnostic testing helped us to identify further molecular alterations in the 11p15.5 region itself, but also in other genes and chromosomes (e.g. GRB10 in 7p13, MEST in 7q31, IGF1R in 15q26). Our data and those from animal and cellular models indicate that IGF2 and the coregulated H19 gene as well as further imprinted and/or functionally related factors belong to the IGN and the IGFII-IGF1R signaling cascade by cis and trans acting mechanisms. These findings contribute to a better understanding of human growth and its disturbances. The translational use of this knowledge has significantly improved molecular diagnostics of SRS and will have an impact on personalized patient management.

SY1-3 Growth: New era of growth disorders

Treatment Strategies for Short Stature in Achondroplasia

Hiroshi Kitoh

Department of Orthopaedic Surgery, Nagoya University Graduate School of Medicine

Achodnroplasia (ACH) is one of the most common short-limbed skeletal dysplasias characterized by rhizomelic shortening of the extremities, frontal bossing with midface hypoplasia, and trident hands. ACH is caused by gain- of-function mutations in the fibroblast growth factor receptor 3 (FGFR3) gene, which is a negative regulator of longitudinal bone growth. No effective treatments for short stature in ACH are currently available. The limb lengthening is a therapeutic option to gain bone length, but the treatment period for bone maturation is very long which results in higher rates of complications.

We have established a novel cell therapy using culture-expanded bone marrow cells (BMC) and platelet rich plasma (PRP) during the limb lengthening to accelerate callus formation. We have performed this cell therapy in more than 130 bones since 2002. The clinical outcome of the limb lengthening in ACH was significantly improved by BMC and PRP transplantation. The effectiveness of this cell therapy, however, was limited for skeletally mature patients due to their limited osteoblastic differentiation of BMC.

For further improvement of our cell therapy, we tried to search clinically applicable drugs that promote the activity of Runx2, which plays a pivotal role in the differentiation of mesenchymal cells to the osteoblastic lineages. By drug repositioning (DR) strategy, we found that a proton pump inhibitor, lansoprazole, enhanced nuclear accumulation of Runx2, promoted expressions of various osteoblastic genes, and induced osteoblastogenesis of human BMC. Systemic administration of lansoprazole to a rat femoral fracture model increased osteoblastogenesis. Lansoprazole could be applicable for ex vivo culture to obtain better quality of BMC.

We next tried to search drugs that suppress abnormally activated FGFR3 signaling in ACH. We found that meclozine, an antihistamine drug that has long been used for motion sickness, facilitated chondrocyte proliferation and mitigated loss of extracellular matrix in ACH model cells. Meclozine also suppressed abnormally activated FGFR3 signaling in various chondrocytic cells that were infected with human mutants. In bone explant culture, meclozine increased the longitudinal growth of embryonic tibia from ACH model mice. After 3 weeks administration of meclozine, the body and tail length, and the individual bone length was significantly longer in meclozine-treated ACH mice than in untreated ACH mice. The plasma concentration of meclozine during treatment was within the range that has been used in clinical settings for motion sickness. These results indicated potential clinical feasibility of meclozine for the improvement of short stature in ACH.

SY2-1 Puberty: Mechanisms and disorders of puberty

Neuroendocrine mechanism regulating puberty onset

Hiroko Tsukamura

Nagoya University

Puberty, which is associated with a maturation of the gonadal axis, is timed by an increase in tonic gonadotropin release from the anterior pituitary, where synthesis and release of gonadotropins are stimulated by gonadotropin- releasing hormone (GnRH) released from the hypothalamus. Kisspeptin, encoded by Kiss1, is a potent stimulator of gonadotropin release via inducing GnRH release, and plays a critical role in puberty onset, because inactivating mutation of Kiss1 or GPR54, a kisspeptin receptor gene, causes pubertal failure in rodents and humans. Accumulating evidence suggests that KNDy neurons, which express kisspeptin, neurokinin B and dynorphin A, in the hypothalamic arcuate nucleus (ARC) are a key regulator of tonic GnRH/gonadotropin release, which governs gonadal activity, i.e., gametogenesis and steroidogenesis in both sexes. Most probably, pubertal maturation of gonadal axis is triggered by an increase in Kiss1/kisspeptin expression in the ARC KNDy neurons in rodents. It is likely that estrogen derived from the immature ovary plays a critical role in prepubertal restraint of ARC Kiss1/ kisspeptin expression, and that a decrease in sensitivity to the estrogen negative feedback during peripubertal period is a key event for pubertal maturation of the hypothalamic mechanism regulating GnRH and then gonadal activity in rodents.

SY2-2 Puberty: Mechanisms and disorders of puberty

Central precocious puberty caused by mutations in the imprinted gene MKRN3

Ana Paula Abreu

Universidade de Sao Paulo/ Brigham and Women's Hospital

Puberty and reproduction are controlled by the hypothalamic-pituitary-gonadal axis. This axis is active during the embryonic and neonatal stages of human life but then suppressed during childhood, then ultimately re-activated, first detected as an increase in amplitude and frequency of gonadotropin-releasing hormone (GnRH) pulses, to initiate puberty. The precise mechanisms that regulate GnRH secretion to constrain the HPG axis during infancy and childhood and subsequently trigger the initiation of puberty remain elusive. Complex interactions among genetic, nutritional, and environmental factors influence pubertal initiation. Pubertal timing is associated with risks of subsequent disease. Early activation of the hypothalamic–pituitary–gonadal axis results in gonadotropin- dependent or central precocious puberty, which is clinically evidenced by pubertal initiation before 8 years old in girls and 9 years old in boys. The genetic determinants of the timing of human pubertal development and in particular CPP are largely unknown. In 2013, we identified loss-of-function mutations in a single gene, MKRN3, encoding makorin ring finger protein 3, in five of 15 families with central precocious puberty, using an exome sequencing approach. MKRN3 is located on chromosome 15q, in the Prader-Willi Syndrome critical region, and is maternally imprinted, expressed only from the paternally inherited allele. Following this inheritance pattern, all MKRN3 mutations identified in patients with central precocious puberty were inherited from their fathers. To date, MKRN3 is the most common genetic defect associated with central precocious puberty in families from several different ethnic groups. MKRN3 belongs to the Makorin family, a family of E3 ubiquitin ligases. Its protein structure has a ubiquitin ligase domain. The mutations identified in patients with central precocious puberty are expected to disrupt protein function. MKRN3 expression in the arcuate nucleus of mice is high prepubertally and decreases before puberty initiation, reaching very low levels in adult life. Taken together, these findings suggest that the loss of function of MKRN3 results in the release of a restraint mechanism on the GnRH pulse generator, which in turn releases the brake on puberty. MKRN3 is the first gene with an inhibitory role on GnRH secretion with mutations identified in humans.

SY2-3 Puberty: Mechanisms and disorders of puberty

Mechanisms of pubertal growth spurt and growth plate closure

Kye Shik Shim

Kyung Hee University Hospital at Gangdong

Growth of height is increased in early or mid-puberty and is known as pubertal growth spurt, but decreased and ceased after growth plate closure in late puberty. Therefore, the pubertal growth spurt has a growth-promoting effect and the growth plate closure reveals a limiting effect on bone elongation. However, the mechanisms behind these contrary phenomena during puberty are still not exactly known.

The elongation of long bones results from chondrocyte differentiation, proliferation and hypertrophy, and extracellular matrix secretion in proliferative and hypertrophic zone of epiphyseal plates.

The growth plate closure is thought as the result of epiphyseal senescence due to the loss of differentiation potential of chondrocytes in resting zone of epiphyseal plates.

The complex networks of genetic, nutritional, cellular, paracrine, and endocrine factors are considered closely related with pubertal growth spurt and growth plate closure.

The genetic factors include the genes linked with intracellular, extracellular, paracrine and endocrine factors. The intracellular factors contain chondrocyte transcription factors, Ras-MAPK signaling, and centrosomal proteins, etc. The extracellular factors are the cartilage extracellular matrixes that contain specific collagen, non-collagenous proteins, and proteoglycans, etc.

The important paracrine factors that regulate the growth plate chondrocytes are parathyroid hormone-related protein/Indian hedgehog pathway, fibroblast growth factor and C-type natriuretic peptide signaling.

The major endocrine systems that control pubertal growth are GH-IGF-I and estrogen signaling.

Therefore, a lot of factors include the cellular factors involved with chondrocyte differentiation, proliferation and hypertrophy, and multiple molecular pathways related with chondrocyte differentiation, vascularization, and ossification in the growth plate are considered influencing each other. However, the complex interactions of these factors are still unclear. Elucidation of the detailed mechanisms of the pubertal growth spurt and growth plate closure will allow a better understanding of the molecular mechanisms responsible for idiopathic short stature, precocious puberty, and skeletal disorders. It will also contribute to the development of new therapeutic modalities for those disorders with better efficacy and. fewer side effects.

SY3 Iodine deficiency disorders

Somchit Jaruratanasirikul

Songkla University

Iodine is a trace element that is essential for thyroid hormone synthesis, and the adequate production of thyroid hormone is essential for brain development, physical growth and control of metabolic processes in the body throughout the lifespan. In pregnant women, thyroid hormone is essential for neurological development of the fetus. During the infancy period, iodine is also essential for neurological development, being involved in neuronal cell migration, myelination and various other functions.

The major source of iodine is from exogenous or dietary intake. However, dietary sources provide only a small amount of iodine which is usually insufficient to meet the daily requirement. Long duration of iodine insufficiency leads to iodine deficiency disorder which is a significant public health problem in many countries. Iodine deficiency- related problems such as goiter, hypothyroidism, growth stunting, and cognitive impairment are relatively common. Various national policies have been enacted to eliminate conditions related to iodine insufficiency. Iodine supplementation through salt iodization is an effective and sustainable strategy for preventing iodine deficiency. Biomarkers used to monitor iodine status at the population level include urinary iodine excretion (UIE), goiter rates, and serum thyroid stimulating hormone (TSH) levels. In recent decades the more extreme manifestations of iodine deficiency such as cretinism or multinodular goiter have significantly decreased, however the various less severe manifestations noted above are still prevalent. Hence, national surveillance strategies towards establishing the iodine status in a country’s population should be regularly carried out. In common clinical practice, spot urinary iodine levels for measuring the median iodine excretion are generally used to assess iodine status in population studies.

The most susceptible group for iodine deficiency disorder is women of reproductive age, whose offspring, if iodine deficient in utero, are at high risk of irreversible mental impairment. The other susceptible group is women providing breast milk to their children, as this may be the only source of iodine during the first 6 months of life. Low concentrations of thyroid hormone during the fetal stage and early infancy are associated with irreversible brain damage, including mental retardation and neurological abnormalities. The main factors influencing the extent and magnitude of neurological complications arising from iodine deficiency are the timing and severity of the deficiency and consequent thyroid hormone deficits. Subtle impairment of cognitive function is likely to occur even in offspring of pregnant women with mild or asymptomatic hypothyroidism.

SY3-2 Thyroid: Management of thyroid disorders

The long term outcome of subclinical congenital hypothyroidism

Sarah Mathai

Department of Pediatrics, Christian Medical College

The biochemical diagnosis of congenital primary hypothyroidism(CH) is made when serum TSH level is elevated and Free T4(FT4) level is low/low normal. Newborn babies have a physiological TSH surge soon after birth (70- 100 mIU/ml) which declines by 72 hours of age. TSH level remains rather stable (< 6 mIU/ml) between one month of age and adult life. Elevated TSH level (>6mIU/ml) with normal FT4 levels beyond the neonatal period may be defined as subclinical congenital hypothyroidism(SCCH).

Hypothyroidism adversely affects growth & cause irreversible developmental delay especially in young children, therefore urgent initiation of levothyroxine(LT4) supplements is recommended in CH. However overtreatment may cause hyperactivity, bone age advancement and craniosynostosis. In children with SCCH, decision to treat or not during early infancy is very difficult. The American Academy of Pediatrics recommend treatment if TSH is persistently > 10 mIU/ml beyond 2 weeks of age irrespective of T4 levels. There are no definite guidelines regarding initiation of treatment in infants with TSH levels 6-10 mIU/ml.

Among the factors implicated in subclinical hypothyroidism, the most important is maternal iodine deficiency. In fact maternal iodine status rather than the maternal thyroid status has been reported to adversely affect the cognitive development in children according to a recent study and this group has demonstrated improvement in neurodevelopmental outcome with maternal iodine supplementation.

There is limited data on the long term outcome of SCCH in children. The outcomes reported are ongoing subclinical hypothyroidism even up to late adolescence, adverse neurodevelopmental outcome especially verbal skills and development of overt hypothyroidism. Children who had persistent TSH elevation have been reported to have abnormal thyroid scintiscans. While better cognitive outcome has been reported with initiation of low dose LT4 supplements during infancy, some studies have also reported bone age advancement. In our institution we have screened 150,000 newborns for CH over the last 15 years. In our practice we often do not commence LT4 supplements for children with SCCH but periodic monitoring is done. We have assessed their growth and development and the results are awaited.

In conclusion, CH should be diagnosed based on S.TSH and T4/FT4 levels to prevent overdiagnosis and overtreatment. Demonstration of abnormal thyroid scintiscan in children with SCCH may predict persistence of TSH elevation. All children with SCCH whether treated or not need long term monitoring. Optimising maternal iodine and thyroid status may be simple and effective in reducing SCCH prevalence and its adverse long term impact on children.

SY3-3 Thyroid: Management of thyroid disorders

Management of autoimmune thyroiditis

Andrew Bauer

The Children's Hospital of Philadelphia; Perelman School of Medicine, The University of Pennsylvania

Autoimmune thyroid disease (AITD) is comprised of two distinct but related disorders, Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). In both disorders T-cells infiltrate the thyroid resulting in tissue disruption as well as activating B-cells to produce anti-thyroid antibodies. GD is associated with the production of a TSH-receptor antibody that stimulates dysregulated overproduction of thyroid hormone. In contrast, HT is associated with thyrocyte apoptosis and production of anti-thyroglobulin (TgAb) and anti-thyroid peroxidase (TPOAb), with TPOAb further reducing thyroid hormone production via decreased organification. Iodine excess may increase the risk for developing AITD, in particular HT. Early identification and treatment of autoimmune thyroid disease in children and adolescents is critical to optimize growth and development. In addition, there is evidence that both GD and HT may increase the risk for developing thyroid nodules and thyroid cancer. We will review the evaluation and management of these two entities with a focus on subclinical hypothyroidism and the definitive treatment of GD.

SY4-1 New Strategy

Next-generation sequencing and array-based comparative genomic hybridization for patients with pediatric endocrine disorders

Maki Fukami

National Research Institute for Child Health and Development

Recent advances in molecular technology including next-generation sequencing (NGS) and array-based comparative genomic hybridization (aCGH) enabled researchers to perform high-throughput mutation screening and genome-wide copy number analysis for multiple samples. To date, we have performed NGS and aCGH analyses for more than 500 patients with pediatric endocrine disorders. Representative results include the flowing. [1] Molecular diagnosis of patients with hypogonadism or hypospadias: We performed NGS-based mutation screening of all known causative genes and aCGH-based genome-wide copy-number analysis. We analyzed 58 patients with hypogonadotropic hypogonadism and identified pathogenic molecular defects in 14 patients. Oligogenicity was not evident in our patient group. As rare abnormalities, we identified a submicroscopic deletion involving FGFR1, an SOX3 polyalanine deletion, and a WDR11 splice site mutation. Then, we also analyzed 62 patients with non-syndromic hypospadias. We found that monogenic and digenic mutations in known causative genes and cryptic copy-number alterations account for more than 10% of cases. [2] Identification of genomic rearrangements underlying aromatase excess syndrome: We performed aCGH for male patients with hereditary gynecomastia. The results suggest that microdeletions, microduplications, and complex rearrangements around CYP19A1 encoding aromatase lead to estrogen overproduction and resultant gynecomastia. These data provides novel models of human disorders caused by cryptic genomic rearrangements. [3] Whole exome sequencing of patients with disorders of sex development (DSD): We performed whole exome sequencing for individuals with various types of disorders of sex development. As a result, we detected intragenic mutations and upstream deletion of SOX9 in 46,XY DSD patients without campomelic dysplasia. These data expand the phenotypic spectrum of known monogenic disorders. Furthermore, we identified novel gene mutations that may be associated with DSD.

This session aims to introduce the usefulness of NGS and aCGH in the field of pediatric endocrinology.

SY4-2 New Strategy

Generating Functional Miniature Organs (Organoids) in a Dish

Hidenori Akutsu

Department of Reproductive Medicine, National Research Institute for Child Health and Development

Pluripotent stem cells can generate virtually any cell type and, as such, can be used to model development and disease and even hold the promise of providing cell-replacement therapies. Structures resembling whole organs, termed organoids, have been generated from stem cells through the development of three dimensional culture systems. Organoids are derived from pluripotent stem cells that differentiate to form an organlike tissue exhibiting multiple cell types. However, no one reported that organoids in vitro possessed similar functions of “real” organs. Intestines are generated through a highly orchestrated, serial developmental process and are composed of cell types from all three primary germ layers. Directed differentiation of human pluripotent stem cells (hPSCs) can yield gut-specific cell types; however, these structures do not replicate some of the critical functional interactions between cell types of different germ layers. Recently, we have developed a simple protocol based on a tissue self- organization concept for generation of mature functional intestinal organoids from hPSCs under xenogeneic- free conditions. The organoids showed the gut tube-like architecture consisted of mucosa and submucosa by histological, immunofluorescence and electron micrograph examinations. Stem cell-derived miniature guts (mini-guts) were derived from all three germ layers and contained distinct types of intestinal cells, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. They demonstrated intestinal functions including peptide absorption and innervated bowel movements responsive in response to stimulation with histamine and anticholinergic drugs. Here, I present our recent achievements of mini-guts and discuss about future applications with 3D organoids.

SY4-3 New Strategy

Clinical steroid mass spectrometry and metabolomics

Cedric HL Shackleton

Institute of Metabolism and systems research (IMSR), University of Birmingham / UCSF Benioff Children fs Hospital Oakland Research Institute

This talk will summarise the 80 year history of steroid metabolomics in diagnosis, ending with latest developments in tandem MS and machine interpretation of GC/MS metabolomic data.

The characterization and measurement of steroids in health and disease has been fundamental to endocrinology since the 1930s. The first example of a diagnostic steroid for an endocrine disorder was the discovery of pregnanetriol in the urine of patients with the adrenogenital syndrome (CAH) by Guy Marrian in 1937. For decades, paper and TLC dominated metabolomic (urinary steroid) approaches to steroid analysis, while from the 1960s RIA of circulating hormones and precursors was pre-eminent and remains useful.

Mass spectrometry now dominates the steroid analytical field but it has taken a long time to reach this position. The breakthrough came in the mid-1960s, when a Swedish company introduced the LKB 9000 GC/MS instrument combining the separation powers of gas chromatography with the structural information of mass spectrometry. In 1967 this presenter had the privilege to use the first British LKB in Professor Charles Brooks’ lab in Glasgow to document the structures of steroids excreted by the human newborn.

In the 50 years of steroid mass spectrometry there have been continuous advances, notably for GC/MS the introduction of capillary columns, chemical derivatisation methods and advanced data systems.

A most important advance was combining HPLC and MS and the introduction of MS/MS to provides high specificity and sensitivity. Automated HPLC MS/MS systems now allow quick separations and accurate measurement of vanishingly small amounts of hormonal steroids. The high throughput has rendered HPLC/tandem MS the desired method for routine clinical serum steroid measurements. In contrast to serum steroids, HPLC/MS of urinary steroids was challenging because of the metabolite structures involved but in the last two years quality comprehensive urinary steroid profiles have been produced and the method will be central to metabolomics.

In spite of being labour intensive GC/MS still holds its own for metabolomic studies, although an ongoing drawback of complex metabolomic data production is interpretation by the operator and explaining diagnostic findings to the interested clinician. To that end my colleagues in Birmingham (Prof Wiebke Arlt. Elizabeth Baranowski, Angela Taylor, Tulay Gulan, Michael Biehl, Kerstin Bunte, Peter Tino and others) have developed a novel interpretable machine learning technique, Angle Learning Vector Quantisation , designed to distinguish multiple biosynthetic error conditions in adults and children using the urinary steroid metabolome. Predictions with close to 100% specificity and sensitivity. Interestingly, pregnanetriol, the mother of steroid metabolites, remains a central analyte with our metabolic profile studies almost 80 years after Marrian’s painstaking identification.

SY5-1 Obesity: Increasing risk of morbidity in children and adolescents

The role of MC4R in pediatric obesity: above and beyond the central nervous system

Feihong Luo

Deparment of Pediatric Endocrinology and Inborn Metabolic Diseases, Shanghai, China

Obesity is a worldwide health problem. Approximately 21-24% children and adolescents are overweight, and 16-18% of individuals have abdominal obesity. Obese 10- to 13-year-old children are 6 to 7 times more likely to become obese adults, when compared with their nonobese peers 50% to 65% of adults with severe obesity (>180% of ideal weight) were obese as children. Obesity is a complex, multifactorial condition with high heritability. Increasing evidence that environmental and nutritional influences during infant and early childhood’s development can have lasting effects on adult’s predisposition to obesity and metabolic syndrome. We analyzed the successive anthropometric data from birth to 24 months and found parental pre-pregnant BMI played the most important role in their offspring’s body size after 6 months age; the total gestational weight gaining during pregnancy of the mother steadily explains nearly a quarter (25 ± 5%) of the relative importance of children’s weight; the weight of the parents is the major factor influencing the children’s weight after 6 months of age, this complex feature of human early growth highlights the underlying association with epigenetic and genetic regulation. While, among the known obese-causing single gene defects which result in human obesity, MC4R deficiency is probably the most frequent. The prevalence of loss-of-function MC4R mutations ranges from 0.5% to 5.8% in childhood onset obesity. A generational effect was observed, with a penetrance of 40% in MC4R-deficient adults aged>52 years, 60% in 18-52 year-old adults and 79% in children. MC4R is expressed by distinct neurons in the CNS that synapse with neurons that express the pomc gene, and produce a α -MSH-related end-product. The role of MC4R as a regulator of feeding behavior in mice was established not long after the characterization of the mc4r gene. The binding of α -MSH to neurons in the hypothalamus that express mc4r will decrease feeding behavior. Conversely, point mutations of the mc4r gene that render the receptor nonfunctional result in the onset of obesity. The patients with MC4R mutations were obese from an early age, but with increase in both fat and lean masses, and were excessively hungry from 6–8 months of age with hyperphagia and persistent food-seeking behavior. Besides of the well- established central MC4R pathways, recent research revealed that MC4Rs expressed on intestinal enteroendocrine L cells regulate PYY and GLP-1 secretion, two gut hormones implicated in the regulation of energy and glucose homeostasis. Increasing evidences support the notion that MC4R may have a variety of physiological roles in the pathogenesis of pediatric early onset obesity.

SY5-2 Obesity: Increasing risk of morbidity in children and adolescents

Management of hypothalamic obesity

Mitchell Eugene Geffner

Keck School of Medicine of USC

Hypothalamic obesity is typically an intractable form of obesity that was initially described in patients with craniopharyngiomas usually following surgical treatment and unintentional damage to hypothalamic nuclei, which results in disruption of energy regulation. However, this definition has now expanded to include obesity developing after a variety of hypothalamic insults, both acquired and congenital (such as other hypothalamic tumors, pituitary macroadenomas with suprasellar extension, gliomas, meningiomas, teratomas, and germ cell tumors; radiotherapy, Prader-Willi syndrome, and mutations in the leptin, leptin receptor, POMC , MC4R , and CART genes). The pathogenic mechanisms underlying hypothalamic obesity are complex and multifactorial, including damage to the ventromedial hypothalamus, which leads to hyperphagia; a low-resting metabolic rate; autonomic imbalance; hypomobility; and insomnia. Additional mechanisms by which the brain regulates adipose tissue and β -cells of the pancreas include the sympathetic nervous system, vagally-mediated hyperinsulinemia, and the endocrine system, namely the growth hormone, thyroid, and hypothalamo-pituitary-adrenal axes. Medical therapy to date (methamphetamine derivatives, somatostatin analogs, GLP-1 and its analogs, oxytocin, and MetAP2 inhibitors have been tried with varying degrees of success), with bariatric surgery now coming under study. Understanding the central role of the hypothalamus in the regulation of feeding and energy metabolism may also be critical to help gain insight into the pathogenesis and management of common obesity.

SY5-3 Obesity: Increasing risk of morbidity in children and adolescents

Determinants, consequences and prevention of childhood overweight and obesity

Muhammad Yazid Jalaludin

Department of Paediatrics, Faculty of Medicine, University Malaya

Over the recent years, the prevalence of childhood obesity has increased rapidly worldwide, likely as a result of complex interactions between genes, dietary intake, physical activity, and the environment. The determinants of childhood obesity is multifactorial and involves a complex relationship among various non-modifiable and modifiable factors. Apart from genetic factors, maternal obesity, gestational diabetes and adiposity rebound are some of the important non-modifiable factors. Evaluating modifiable factors contributing to obesity (weight gain during infancy, physical activity, diet, sleep, parental feeding practices, screentime, parental obesity and families from lower socioeconomic status) is an important step towards effective intervention.

Childhood obesity is associated with significant short and long-term health and social consequences. Childhood obesity affects nearly every organ of the body. Short term health impacts such as hypertension, dyslipidaemia and insulin resistance (metabolic syndrome) which were earlier considered markers of adult cardiovascular disease, are now common among obese children. Long term health impacts of childhood obesity although less commonly seen by paediatrician, include premature cardiovascular diseases, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), obstructive sleep apnoea syndrome (OSAS), infertility and orthopaedic pain.

Prevention is more effective than treatment in dealing with the rising prevalence of childhood obesity. Roots of obesity can be traced to very early in life and appropriate prenatal and postnatal nutrition can help to reduce the risk of overweight/obesity in adulthood. Establishing healthy dietary and lifestyle habits after birth and throughout the childhood and adolescent period can help reduce the risk of childhood obesity. Child health clinics, school health screening programmes, as well as general paediatricians/family physicians are at the first line of defense even before the BMI crosses the acceptable range and hence play a crucial role in prevention and treatment of childhood obesity. Routine anthropometric measurement are essential during clinic visits to detect overweight/ obesity at an early stage, thus permitting necessary actions to be taken.

Once the child’s BMI crosses the overweight percentile, individualise interventions are critical for successful weight management. The Staged Approach recommended by the American Academy of Paediatrics (AAP) involves multi- disciplinary team as early as Stage 2. The need for multi-disciplinary team involvement increases as the problem escalate.

SY6-1 Which is most dangerous for future cognition in young children with diabetes: hypo- or hyperglycemia?

Diabetes dysglycaemia, cognition and the developing brain

Fergus Cameron

Royal Children fs Hospital

Glucose is the preferred metabolite of the brain with 25% of circulating blood glucose in adults destined for cerebral metabolism. It is intuitive then that type 1 diabetes mellitus (T1DM), a disorder characterised by perturbations in blood glucose (“dysglycaemia”), should cause acute and chronic brain dysfunction. These cognitive and affective impacts appear to be greatest in the developing brain of children and adolescents with T1DM. Aspects of diabetic dysglycaemia that appear to be most significant are hypoglycaemia, hyperglycaemia and diabetic ketoacidosis (DKA). Some early in vitro work suggest that glycaemic variability may also play a significant role in neural cell injury. Prospective observational data from the point of diagnosis to neuromaturation in the Royal Children’s Hospital Diabetes Cohort Study revealed a rather lamentable “rule of thirds”- one third of subjects developed a DSM IV threshold mental health disorder, one third did not complete secondary schooling and one third did not continue in adult care after transition. This was coincident with a 0.3 SD loss of full scale IQ and changes in regional brain volumes. More recent functional imaging studies have provided insight into some of the mechanistic aspects of dysglycaemia-induced brain injury. DKA at the point of diagnosis is associated with acute grey and white matter volume and spectroscopic changes that are associated with neurocognitive outcome in the medium term. Clamp studies combined with MRI have shown that hypo- and hyperglycaemia result in distinct regional changes in brain perfusion and metabolic activity. Potential synergies of chronic and additive dysglycaemic insults are difficult to quantitate largely due to an inability to fully record all aspects of glycaemic perturbation over a life course. In addition to this, pre-conditioning and programming may also play significant mediating roles. However, developmental age at the time of diabetes onset appears to have a critical influence upon outcome. A nascent understanding of mechanism of neural injury is providing some insights as to potential non-glycaemic interventions that might be used to protect the developing brain.

SY6-2 Which is most dangerous for future cognition in young children with diabetes: hypo- or hyperglycemia?

Improving metabolic control without increasing severe hypoglycemia

Karin Åkesson

National Swedish Padiatric Diabetes Registry

Over the last years the metabolic control in children with type 1 diabetes in Sweden has improved and the mean HbA1c in year 2015 is 57.3 mmol/mol, IFCC (7.4 %, DCCT). Data on almost all (>97 %) children, 0-18 years of age, with type 1 diabetes in Sweden is registered in Swediabkids, the Swedish Pediatric Diabetes Quality Registry. Swediabkids includes data on HbA1c, BMI, insulin dose and frequency of insulin pumps as well as on acute and chronic complications. This allows us to continuously follow changes in the frequency of severe hypoglycemia when HbA1c is decreasing. The new technology, with Continuous Glucose Monitoring and advanced insulin pumps, has made it possible to have a tight control of the children’s glucose level. The children are taught to correct their glucose level if it is below 4 or above 8 mmol/mol and to always give dextrose to normalize a low glucose level. These recommendations make the children and their families highly involved in their own treatment. The families as well as the personnel in schools and day care centers are taught to count carbohydrates and to give insulin correction doses during the days. Since July 2009 a new law is implemented in Sweden to secure the rights of children with diabetes. The pediatric diabetologist or nurse has to make a detailed agreement with the parents or other caregivers to what extent the child needs help with the diabetes self-care during school day. This agreement should be written down in a national uniform action plan form. By giving the child and its close environment the possibility to take command over the treatment and by extra support from the pediatric diabetes team when this is needed, it is possible to improve metabolic control without increasing severe hypoglycemia.

SY6-3 Which is most dangerous for future cognition in young children with diabetes: hypo- or hyperglycemia?

Closed loop systems to avoid extreme hypo- and hyperglycemia

Martin de Bock

Princess Margaret Hospital
Closed loop insulin delivery systems aim to closely regulate blood glucose levels by using algorithms that interpret real-time sensor glucose values and adjust insulin delivery in real time, without increasing the risk of hypoglycaemia. These systems have been extensively tested in closely regulated scenarios including hotels and diabetes camps – all of which have shown favourable glycaemic outcomes compared to standard pump therapy. The first long term, unsupervised outpatient trials have now been conducted, confirming that these results can be translated into real life settings. Closed loop insulin delivery systems are expected to be commercially available within the next 2 years, heralding a new era for patients with type 1 diabetes, where tight glycaemic control can be achieved, without the burden of hypoglycaemia or requirement for frequent patient initiated management strategies.
Fig. 1 (abstract SY6-3).
Fig. 1 (abstract SY6-3).

See text for description

SY7-1 DSD: Variations in Sex Development and in Gender Identity

Initial approach to Disorders of Sexual Differentiation

Kah Yin Loke

National University of Singapore

The disorders of sexual differentiation (DSD) comprise many conditions of diverse pathophysiology which present in the newborn with atypical external genitalia or in the adolescent with atypical sexual development during the pubertal years. These clinical situations are difficult to manage and an initial comprehensive evaluation is necessary to (1) assign the appropriate gender, (2) develop a logical, pragmatic plan for investigation and (3) organize the necessary medical, surgical and psychological intervention.

The initial diagnostic clinical evaluation requires a thorough prenatal and family history. Dysmorphic features and midline facial defects may provide clues to the underlying cause. External genitalia should be examined for the presence and symmetry of gonads in the labioscrotal folds, fusion of the folds, phallic size and site of the urinary meatus on the phallus, from which the external masculinization score can be derived.

For infants with ambiguous genitalia, investigations can be ordered based on:

Bilateral non-palpable gonads: first line tests include sex chromosome karyotyping, serum electrolytes, serum 17-hydroxyprogesterone [17OHP], testosterone, LH, FSH, and a pelvic ultrasound for female internal organs.

The normal 46 XX chromosomal female with elevated 17OHP levels and the presence of a uterus suggests congenital adrenal hyperplasia (CAH). (ii) For infants with chromosomal karyotype other than 46 XX, the second line tests aimed to determine the presence of a testes and the adequacy of androgen production include measuring the anti-Mullerian hormone, HCG stimulation test, with further imaging and laparoscopy. Confirmation of a specific diagnosis may require genetic analysis.

Palpable gonads (likely to be testis / ovotestis): sex chromosomal karyotyping should be performed, followed by second line tests indicated above.

Adolescents with DSD may present as:

Girls with primary amenorrhoea, which may be caused by DSD with 46 XY sex reversal (gonadal dysgenesis, testosterone biosynthetic defects, androgen insensitivity syndrome [AIS], LH receptor mutations). Initial relevant investigations will include a chromosomal karyotype, LH, FSH, androgen levels and pelvic ultrasound, followed by an HCG stimulation test.

Girls who virilise at puberty, which can be caused by DSDs with undetectable Mullerian structures (17 β

-hydroxysteroid dehydrogenase deficiency, 5 α -reductase deficiency, partial AIS), and DSDs with detectable Mullerian structures (partial gonadal dysgenesis, ovotesticular DSD, CAH, androgen secreting tumours). Relevant investigations include a pelvic ultrasound, LH, FSH, dehydroepiandrosterone, serum androgen with their precursor levels and 17OHP.

SY7-2 DSD: Variations in Sex Development and in Gender Identity

Long-term outcome of early surgical intervention for hypospadias; What happens at Puberty?

Kimihiko Moriya

Department of Renal and Genitourinary Surgery, Hokkaido University

Hypospadias (HS) is one of the most common congenital anomalies in male children. The goal of HS surgery is reconstructing the urethra to the tip of the glans and straightening the penis. Successful HS surgery ensures a cosmetic penile appearance, voiding in the standing position and unhampered sexual function in adulthood. Despite great surgical interest in short-term results, there are only a few studies regarding the long-term outcome. In this lecture, I will present a long term outcome of HS surgery, focusing on the cosmetic and endocrinological outcome at puberty from our long-term outcome study.

As a first study, to clarify the cosmetic and sexual outcome, a questionnaire was mailed to HS patients and control subjects at 18 years old or older. This study demonstrated that although their sexual behavior was not different from that in control subjects, HS patients had a higher rate of dissatisfaction with smaller penile size. Following the outcome of the questionnaire study, actual penile length of HS patients was evaluated. As a result, while the severity of HS and endocrinological abnormality at post-pubertal evaluation were factors affecting penile size, penile length in patients with severe HS was shorter even in cases without endocrinological abnormality. These results suggest that severe HS is not only a disorder of urethral development, but also a disorder of penile development.

A multifactorial etiology is considered to be involved in the genesis of HS. Among them, androgen actions have an important role for urethral development during fetal period. Several studies have shown a prepubertal hormonal abnormality in patients with HS from the endocrinological viewpoint. However, reports of hormonal status in HS patients at puberty are scarce. Therefore, we retrospectively reviewed endocrinological status at puberty. Endocrinological abnormality was identified in patients with mild as well as severe HS. While severity of HS did not affected the incidence of high serum FSH, history of the undescended testis was a risk factor for high serum FSH at post-pubertal evaluation.

In conclusion, penile size, which is most dissatisfied issue from the patients’ view point at puberty, is affected by severity of HS. In addition, history of undescended testis is an important issue from the endocrinological view point, especially on reproductive function. Since both testicular descent and urethral formation are influenced by androgen action, coexistence of these pathology would be a severe form in the spectrum of male genital anomaly form the reproductive view point.

SY7-3 DSD: Variations in Sex Development and in Gender Identity

Transgender Youth: Current Concepts, Management, and Priorities for Research

Stephen Rosenthal

Division of Pediatric Endocrinology, Child and Adolescent Gender Center, University of California, San Francisco

An increasing number of preadolescents and adolescents identifying as “gender nonconforming”, “gender expansive”, or “transgender”, are seeking medical services to enable the development of physical characteristics consistent with their experienced gender. The onset of puberty in transgender youth is often accompanied by increased gender dysphoria, the persistent distress associated with incongruence between assigned sex at birth and experienced gender identity. While recent reports from the Netherlands have shown that gender dysphoria may be ameliorated by a gender-affirming model of care, including puberty suppression, cross sex hormones, and gender confirming surgery, there are relatively limited outcomes data, and many questions remain unanswered. This presentation will highlight current concepts of the biology of gender identity development, management of gender dysphoria, gaps in knowledge, and priorities for research. It is hoped that such research will inform optimal care of transgender youth and promote further understanding and acceptance of these individuals.

SY8-1 Workshop on Developing countries

A report on the first year of the APPES-CLAN Equity Working Group. Where to from here? What can you do to help?

Kate Armstrong

President & Founder, CLAN (Caring & Living As Neighbours)

The APPES-CLAN Equity (ACE) Working Group was established in 2015 as a formal collaboration between the Asia Pacific Pediatric Endocrinology Society (APPES) and CLAN (Caring & Living As Neighbours) to drive long-term, sustainable change that redresses inequity and improves quality of life for children living with endocrine conditions in the Asia Pacific region.

The ACE Working Group agreed to adopt a rights-based, community development approach to achieving equity for children living with endocrine conditions, and committed to the principles of: participation; accountability; non- discrimination and equality; empowerment; and legal, policy and ethical frameworks. CLAN’s strategic framework for action and five pillars further informed the ACE Working Group partnership as early priorities were considered:
  • Affordable access to medicines and equipment

  • Education, research and advocacy

  • Optimal medical management

  • Encouragement of family support groups and

  • Reducing financial burdens on families.

Deliverables for the first six months of the ACE Working Group included: a mapping exercise to determine the most vulnerable paediatric endocrine communities in the Asia Pacific region; development of equity markers to track achievements and improvements in health outcomes for children affected by endocrine conditions; -collaborative action with CAH communities and APPES members to address barriers to accessing essential medicines and equipment identification of existing educational resources for paediatric endocrine communities, with information made publicly available online; development of a clear strategic plan to inform action improving access to hydrocortisone and fludrocortisone for the CAH Community in the region; support of fellows training, informed by community priorities; utilisation of communication platforms to promote activities of the partnership; advocacy and action relating to universal access to newborn screening for congenital hypothyroidism in the region; strengthening networks between paediatric endocrine communities and associated professional societies clarification of barriers to financial independence for paediatric endocrine communities, with particular focus on school attendance and health insurance.

This presentation will report on the achievements of the ACE Working Group in its first year, including practical examples of collaborative action already transforming the lives of children in the Asia Pacific region. The session will be an opportunity for APPES members and multisectoral partners to engage with ACE Working Group members and collaboratively explore opportunities for involvement in future activities.

SY8-2 Workshop on Developing countries

Challenges for Pediatric Endocrinology in a resource constrained environment

Syed Jamal Raza

National Institute of Child Health

Development of Pediatric subspecialties is a relatively new frontier for certain countries where overall healthcare scenario is still far behind the developed world. Lack of adequate finances is a major but not the only constraint that obstructs growth of specialties.

There are many other barriers and challenges in developing, establishing and flourishing of the subspecialty. Access to health care, which is a general problem, also affects disorders of endocrinology especially in situations where you need urgent care like Diabetic Ketoacidosis, salt wasting adrenal crises etc.

However, the most pressing and difficult problem is the lack of trained personal. In Pakistan, a country of population over 180 million have only a handful of trained Pediatric Endocrinologist. Lack of trained personal not only makes it difficult to establish the discipline but also results in a dearth of training opportunities for local doctors. This situation can be helped greatly by other countries by providing funded training opportunities for doctors from resource-constrained countries. Once enough trained personal become available, local training programs can be developed, subsequently reducing the need for people to go abroad from training.

Lack of availability of affordable endocrine investigations have also been and will remain a barrier in developing services. With more sophisticated (and hence expensive) tests becoming available which on one hand improve the diagnostic acumen but remain difficult to perform even in large towns and centers and even more in peripheral or small towns with limited health facility. Molecular work in diagnosis has now become a norm, and most of the conditions can only be diagnosed with certainty with molecular testing (especially DSD). This not only has prognostic implications for patients but also imperative for prenatal testing. Facilities for molecular testing are not only expensive, but also require technical manpower which is difficult to obtain and retain in public sectors which is only place the majority can access.

Drug availability has also unfortunately been a problem in many such countries and simple, essential life saving drugs like hydrocortisone and fludrocortisone are not registered and regularly available in country like Pakistan. This brings a disastrous situation for the patients when despite the diagnosis they cannot be adequately treated.

So, in conclusion, there are many barriers and challenges still withholding development in resource poor countries. Most can be overcome with advocacy, planning and some help from the developed world.

SY8-3 Workshop on Developing countries

Building Newborn Screening Systems

Dianne Webster

Newborn Metabolic Screening Programme / Antenatal Screening for Down Syndrome & Other Conditions

Newborn baby screening for congenital hypothyroidism (CH) is a proven, well accepted public health initiative but implementation generally is problematic in developing economies. The first consideration is that of prioritisation of public health initiatives within the jurisdiction and newborn screening may be perceived as of lower benefit and priority than eg immunisation or elimination of disease carrying vectors or provision of clean water. Following a decision to screen items for consideration include who (start with private payers? With hospitals?); what condition/s? what cutoff/s? who will collect samples, test, organise followup and treatment? Payment for treatment? Audit and monitoring? Of high importance is definition of the goals of screening in terms of health outcomes eg for CH growth and intellectual development optimal for the individual. Achievement of health outcomes requires definition of a newborn screening system incorporating not only the sampling and laboratory testing (relatively easy to establish) but also the maternity services and facilities, diagnostic and treatment pathways including paediatrics, pharmacy, followup monitoring testing, family information and support that are essential to ensure that an infant with a high TSH level achieves their full potential. Collection and transport of samples is generally difficult especially outside urban areas and consideration should be given in the initial stages of a programme of testing for conditions where the baby will benefit from detection of the condition even if this is delayed beyond what many programmes would consider optimal (receipt in laboratory by 4-5 days of age) such as CH; and to starting in an urban setting, with families able to pay if this is the only way to begin screening. Followup of positive tests has been an issue in some programmes and an argument can be made for setting the screening cutoffs at values which give high positive predictive values until the followup system is established. Conditions such as CAH where the testing and notification is time critical can be added, the population screened broadened and cutoffs lowered when the infrastructure is in place. The gradual approach to implementation enables building of the multiple relationships and processes which ensure the best outcomes for affected infants.

SY9-1 Hot topics

Epigenetics as the mediator of gene:environment interactions and programming of non-communicable disease

Richard Saffery

Murdoch Childrens Research Insitute and Department of Pediatrics, University of Melbourne

The Developmental Origins of Health and Disease (DOHaD) hypothesis is based largely on observational studies in humans and direct experimentation in animal models. Several mechanisms have been proposed to account for the latency between an “exposure” in utero and ‘programming’ of later disease risk, including epigenetic variation induced early in development. Environmental sensitvity is a hallmark of epigenetic variation and exogenous exposures have the potential to influence epigenetic profile, modifying the developmental trajectory and risk of disease.

Animal studies have demonstrated conclusively that the in utero environment can modify the epigenetic profile in progeny in association with long term (even intergenerational) phenotypic consequences, though replication in such models remains fragmented. Equivalent data in humans are also beginning to emerge. However, to establish epigenetics as the mediator of ‘programming’ in humans several criteria must be met. First, interindividual differences in epigenetic profile must be present in early life. Second, environmental exposures implicated in later life phenotypic outcomes, must be linked to specific epigenetic variants. Third, such epigenetic variants should be reproducibly associated with later life phenotypic outcomes. Fourth, specific epigenetic variants must have demonstrated functional consequences on gene activity or cellular function.

It is now clear that genetic variation ‘shapes’ the human epigenetic profile throughout life and may play a role in mediating the effects of environmental exposures on epigenetic variation. Specific in utero exposures (such as maternal smoking) induce defined epigenetic changes in newborns, some of which persists into adulthood.

Mounting evidence also links early epigenetic variation to later onset phenotypes and non-communicable diseases. The field is advancing at a rapid pace but remains in its infancy. The recent adoption of standardised platforms for epigentic analysis, the advent of several very large prospective birth cohorts, and the formation of large consortia to build sample sizes with sufficient power to detect small magnitude effects, represent major advances. Ultimately, a complementary approach encompassing (i), well controlled and reproducible animal studies, (ii) ongoing human longitudinal observational cohorts, and (iii) well-informed interventional studies, represents the best way forward to firmly establish epigentic variation as a mediator of DOHaD-related health outcomes in humans.

Author details: Professor Richard Saffery is National Health & Medical Research Council (Australia) Senior Research Fellow with 20 years experience in Molecular and Cellular Biology, including 15 in the field of Epigenetics. He heads a team investigating the determinants of the human epigenetics profile in early life and its role in later health outcomes. This involves state-of-the-art multidisciplinary approaches, encompassing genetic, environmental and epigenetic analysis. Central to our work has been the establishment of several longitudinal birth cohorts with detailed environmental, anthropometric, clinical and other data, with associated biorepository.

Prof Saffery has published over 170 papers, including key discoveries demonstrating the interaction of genetic, environmental, temporal and tissue-specific factors in regulating the early life epigenetic profile in humans. His team has received over $15M in funding and has developed an international reputation in early life human epigenetics research. Prof Saffery currently leads the Epigenetics Work Package for a large multinational European Commission (FP7) funded project examining the early life determinants of metabolic health in children.

SY9-2 Hot topics

Obesity and Brain Insulin Resistance: Cause of Consequence

Hans-Ulrich Häring

Department of Internal Medicine IV, University Hospital Tübingen

The Tübingen family study collects people with increased risk to develop type 2 diabetes and comprises by now approx. 3.000 deeply phenotyped individuals. We used wholebody MRI imaging to assess bodyfat distribution patterns. We could identify subphenotypes of bodyfat distribution, a metabolically healthy phenotype of obesity (MHO) versus a metabolically unhealthy phenotype (MUHO) as well as distinct patterns of perivascular adiopose tissue as well as neckfat.

In a subgroup of approx. 250 individuals we studied brain insulin effects by MEG and fMRI and observed brain insulin resistance in obese people. We used nasal insulin as a tool to study brain effects in the fMRI.

Stimulation of brain insulin signalling by nasal insulin caused brain responses related to homeostatic regulation, eating behaviour but also caused changes of peripheral insulin sensitivity as determined by glucose clamp. Based on stable isotope techniques this brain derived signals affect both glucose appearance from the liver as well as glucose disappearance. Low hypothalamic insulin sensitivity correlated with increased visceral fat and decreased subcutaneous fat. We hypothesize that the brain insulin resistance causes unfavourable fat distribution patterns as a consequence of impaired signalling from the brain to the periphery favouring accumulation of visceral fat. This mechanism might also favour the develoment of NAFLD.

SY9-3 Hot topics

MIRAGE Syndrome: A New Adrenal hypoplasia Syndrome Caused by Heterozygous SAMD9 Mutations

Naoko Amano

Department of Pediatrics, Keio University School of Medicine Department of Pediatrics, Saiseikai Central Hospital

Most patients with primary adrenal insufficiency (PAI), such as 21-hydroxylase deficiency, can be diagnosed biochemically by measuring urine or serum steroid metabolites, while a minor subset of PAI patients lacks diagnostic biochemical features. We analyzed known causative genes in biochemically uncharacterized PAI patients and diagnosed genetically about 70% of them. Nonetheless, etiologies of the remaining 30% were not elucidated. By exome sequencing and follow-up genetic analyses in 24 early-onset PAI patients with biochemically and genetically unknown etiologies, we identified heterozygous SAMD9 mutations in 11 patients. According to the common clinical features among these 11 patients, we named this disorder ‘MIRAGE’ syndrome. 1) Myelodysplasia; All patients showed transient thrombocytopenia and/or anemia in early infancy. Two patients subsequently developed myelodysplastic syndrome and died in early childhood. 2) Infection; All patients experienced severe invasive infections, and six patients died before age two years. 3) Restriction of growth; All patients were born preterm (gestational age, 25 to 35 weeks), small-for-gestational age (birth weight, -4.0 to -2.2 SD), and also had severe growth restriction after birth. 4) Adrenal hypoplasia; All seven evaluated patients showed adrenal hypoplasia by ultrasonography. Postmortem analysis of two female patients revealed that their adrenal glands were extremely small and highly disorganized. The adrenal medulla was only partially surrounded by the adrenal cortex, and the cytoplasm of dysgenetic adrenocortical cells were foamy in appearance. 5) Genital phenotypes; All seven patients with 46,XY karyotype had underdeveloped external genitalia. Postmortem analysis of the two female patients revealed that their ovaries were extremely small and had very few primordial cells. 6) Enteropathy; All nine evaluated patients had chronic diarrhea with colonic dilation.

In this symposium, I will talk about identification of a new causative gene for PAI, SAMD9 , clinical features of MIRAGE syndrome and some functional studies of the identified SAMD9 mutations.

MTE1 Bone

Pediatric Osteoporosis

Craig Munns

The Children's Hospital at Westmead

Up to 50% of children fracture before age 18 years, with a peak incidence during early adolescents. Of these who fracture, approximately 20% will sustain a second fracture. Although childhood fracture is usually associated with trauma there are increasing data to indicate that fracture incidence in children is related to reduced bone mineral density and relative skeletal fragility.

It is impractical and unnecessary for the bone health to be assessed in every child who presents with fracture. The ISCD 2013 Pediatric Official Position on Fracture Prediction and the Definition of Osteoporosis in Children and Adolescents (1) has recommended:

Evaluation of bone health should identify children and adolescents who may benefit from intervention to decrease their elevated risk of a clinically significant fracture

A clinically significant fracture history is one or more of the following:
  • Two or more long bone fractures by age 10 yr

  • Three or more long bone fractures at any age up to 16 yr.

Vertebral compression fracture (loss of 20% height at any point).

One or more vertebral compression fractures are indicative of osteoporosis. In such children and adolescents, measuring BMD adds to the overall assessment of bone health. Children who fulfil the above criteria should undergo a bone health evaluation.

So, what investigations should be performed in a child with recurrent fracture? This can be guided by the clinical features of the child but there are ‘routine screening’ investigations that may be considered in the majority of children. It is important to assess mineral homeostasis and vitamin D levels. Coeliac and thyroid disease should be screened. Dependent on the age of the child, gonadal status may be evaluated. All children with recurrent fracture should have a lateral spine xray to assess for vertebral compression fracture. This is especially the case in children with low trauma fracture, osteogenesis imperfecta or if there is a history of glucocorticoid therapy. Duel energy xray absorptiometry (DXA) remains the gold standard for assessment of reduced bone density and bone mineral content for age. The caveats of DXA in children will be discussed.

Management of recurrent fracture in children centres around reduction of risk factors and improvement in muscle and bone mass. Optimising vitamin D, calcium and pubertal progression are also important as is the judicious use of bisphosphonates and other bone specific therapy.


Bishop et al. 2014 Journal of Clinical Densitometry: Assessment & Management of Musculoskeletal Health Volume 17.

MTE2 Endocrine Imaging

The state-of-the-art applications of PET in endocrinology

Tohru Yorifuji

Endocrinology and Metabolism, Children's Medical Center, Osaka City General Hospital

MTE3 Growth & Genetics

Genetic Approaches to make a Diagnosis in Short Stature

Peter Clayton

Faculty of Biology, Medicine & Health, University of Manchester

Over the last 30 years many monogenic causes of growth disorders, including hormonal, primordial, skeletal and syndrome-associated, have been identified by examining candidate genes or hunting for the causative gene in regions of homozygosity or gene rearrangement. For instance mutations in most of the components of the GH-IGF axis have been identified, as have the causes of major skeletal dysplasias.

In more recent years, genome wide association studies (GWAS) have found variants in a number of these genes contribute to adult stature, and revealed that many other genes, as yet not implicated in growth disorders, also contribute. In addition array studies have shown that copy number variants (CNVs) are more commonly found in short stature. Alongside these observations, advances in genetic technologies have led to the increasing use of targeted gene panels (e.g. for rasopathies) and whole exome (or genome) sequencing (WES) in growth disorders. This is leading to diagnoses of a particular condition that had not been anticipated based on phenotype.

We therefore have powerful tools to achieve a diagnosis, but how should they be used in the approach to a child presenting with significant short stature (height SDS <-2). Attention to defining auxology (including limb to axial skeleton proportions) and growth pattern in relation to stature and development in the family, obtaining a comprehensive history of potential risk factors and/or occult system disease, and carefully documenting overall phenotype remains the cornerstone of initial assessment. This clinical evaluation is likely to trigger a request for investigations that may include a screen for system disease, a skeletal survey, hormonal evaluation and an array study. In some cases, the clinical features may immediately point to a specific genetic condition, and if so, then a traditional search for a mutation in a single gene could be undertaken. If there are suggestive features, a targeted gene panel would be appropriate. In those whose short stature appears to be of undefined aetiology (so called idiopathic short stature [ISS]), then WES can provide an opportunity to make a diagnosis.

These testing strategies have led to the recognition that genes known to cause short stature syndromes with well-defined clinical features may be mutated in ISS children. This extends the phenotype, and can lead to the recognition of more subtle features associated with that condition. These strategies may also identify genes implicated in growth physiology but not previously recognized to cause human disease. The CNV and whole exome/genome approaches may lead to genes never previously recognized as related to growth being implicated as causes of short stature. In this situation it is important that defects in these genes are found in multiple families to confirm causality.

The use of genetic technologies will vary considerably dependent on the health-care resource setting. Studies evaluating large cohorts of unselected ISS children are required to evaluate the cost-effectiveness of using next generation sequencing to improve molecular diagnosis rates.

MTE4 Growth

New data on long-term safety of GH treatment in SGA


Erasmus University Medical Center

Background: Children born SGA have a higher risk to develop metabolic diseases in adulthood. GH treatment increases adult height but also causes insulin resistance, which have raised concerns about the long-term consequences of GH treatment in children born SGA. Published data of the SAGhE project suggested an increased cardiovascular mortality in adults born SGA who were treated with GH during childhood. The main limitation of the SAGhE project is that data of ex-GH-treated adult patients are compared with national reference values and not with an age-matched untreated adult patients. To study the long-term effects of GH treatment on the risk of diabetes mellitus type 2 and cardiovascular diseases, it is important to compare data of previously GH-treated adults born SGA with those of untreated adults born SGA.

Outline: This session will give an overview of published GH safety data and will present recently published data of the largest follow-up study in 199 previously GH-treated SGA young adults who were longitudinally studied for five years after attainment of adult height, at GH-cessation, six months, two and five years thereafter. Body composition, insulin sensitivity and β -cell function were determined and data at five years after GH-cessation were compared with untreated age-matched controls: 51 untreated short SGA adults, 92 SGA adults with spontaneous catch-up growth, and 142 adults born appropriate for gestational age.

Main findings: Data show a steady increase in fat mass during five years after GH treatment, indicating the loss of pharmacologic effects of GH. Reassuringly, the GH-induced reduction in insulin sensitivity was fully reversed within six months after cessation of GH treatment and remained similar thereafter, despite the increase in fat mass. At 5 years after GH-cessation, fat mass, insulin sensitivity and β -cell function of previously GH-treated SGA adults were similar to untreated short SGA adults, indicating that long-term GH treatment in children born SGA has no unfavourable effect on metabolic health in early adulthood.

MTE5 Water and electrolytes

Management of water and electrolytes imbalance

Joseph Majzoub

Boston Children's Hospital, Harvard Medical School

Maintenance of the tonicity of extracellular fluids within a very narrow range is crucial for proper cell function. Extracellular osmolality regulates cell shape as well as intracellular concentrations of ions and other osmolytes. Furthermore, proper extracellular ionic concentrations are necessary for the correct function of ion channels, action potentials, and other modes of intercellular communication. Extracellular fluid tonicity is regulated almost exclusively by the amount of water intake and excretion, whereas extracellular volume is regulated by the level of sodium chloride intake and excretion. Normal blood tonicity is maintained over a 10-fold variation in water intake by a coordinated interaction among thirst, arginine vasopressin (AVP)/anti-diuretic hormone (ADH), and renal systems. Dysfunction in any of these systems can result in abnormal regulation of blood osmolality, which if not properly recognized and treated may cause dysfunction in neuronal and other cellular activities. When the relationships among water intake, vasopressin status, and renal responsiveness are not understood, life-threatening hyponatremia or hypernatremia may occur, whereas knowledge of these systems almost always allows for proper management of sodium and water balance without these complications.

Learning objectives: As a result of participating in this session, learners should be able:
  • To understand how to treat a patient with diabetes insipidus or SIADH with combinations of fluid and AVP or DDAVP, without causing hyper- or hypo-natremia. When AVP/DDAVP is high (anti-diuretic state), a patient can only tolerate 1/10th as much fluid as when vasopressin/DDAVP is low (diuretic state).

  • To diagnose cause of polyuria: central diabetes insipidus versus primary increased fluid intake

  • To use measurement of blood copeptin instead of AVP

  • To diagnose cause of hyponatremia: dehydration/reduced intravascular volume versus SIAD

  • To treat diabetes insipidus in neonates without causing hyponatremia

MTE6 Adrenals

Diagnosis and Management of CAH

Toshihiro Tajima

Jichi Children's Medical Center Tochigi

Congenital adrenal hyperplasia (CAH) refers to a range of autosomal recessive diseases resulting from deficiency of cortisol secretion. The incidence is 1/10,000-20,000 globally and 21-OHD is the most frequent disease among CAH cases. Many patients with 21-OHD have pigmentation, virilization of the external genitalia in females, failure to thrive and poor weight gain, but others have 21-OHD with only very mild clinical symptoms. Elevated blood 17-hydroxyprogesterone is the best biochemical diagnostic marker fro diagnosis. Furthermore, tests for diagnosis and understanding of the pathological condition include plasma ACTH, serum electrolytes, plasma glucose, plasma aldosterone, plasma renin activity or concentration, and blood gas. If 21-OHD is suspected due to clinical symptoms and abnormal test results, and a manifestation of adrenal insufficiency is found, treatment must be the priority.

The principles of treatment of 21-OHD are to supplement insufficient glucocorticoid and mineralocorticoid levels, inhibit enhanced adrenal androgen production, and maintain growth and maturation similar to those of healthy children. Treatment continues for life. Insufficient treatment causes adrenal crisis (acute and severe adrenal insufficiency) due to decreased tolerance to physical stress and short stature due to bone age advancement. Excessive treatment causes iatrogenic Cushing's syndrome, including short stature, obesity and hypertension.

In the neonatal stage, since adrenal androgen production is markedly enhanced, high dose of hydrocortisone (HC) (25-100 mg/m2) is needed to suppress adrenal androgen. HC is also used as maintenance therapy and the dose of glucocorticoids during maintenance therapy must be carefully determined to prevent overdose and underdose. Salt wasting 21-OHD is not sufficiently treated with HC alone and requires fludrocortisone (FC). Sodium intake from breast milk and bottle formula is insufficient for treatment in the infant stage, and sodium chloride replacement is also necessary. A dose of FC in maintenance therapy is 0.025 to 0.2 mg/day. The FC dose should be adjusted based on the plasma renin activity or concentration, electrolyte levels; and body weight gain. Adverse reactions including increased blood pressure and edema should be monitored.

It is also very important to avoid adrenal insufficiency. If a patient has febrile illness, gastroenteritis with dehydration, surgery or trauma, it is necessary to increase the dose of glucocorticoids transiently. A child at an early age has a risk for hypoglycemia and electrolyte imbalance; therefore, long-term fasting conditions should be avoided, and intravenous administration of glucose and sodium should be performed as required.

MTE7 Thyroid

Medical and Surgical Management of Pediatric Thyroid Nodules and Cancer

Andrew Bauer1,2, Ken Kazahaya3,4

1The Children's Hospital of Philadelphia; 2Perelman School of Medicine, The University of Pennsylvania; 3Division of Pediatric Otolaryngology, The Children's Hospital of Philadelphia; 4Department of Otorhinolaryngology, Head and Neck Surgery, University of Pennsylvania

Evaluation and management of nodular thyroid disease in the pediatric population is different from the adult population. Thyroid nodules and cancer in the pediatric population have distinct biology, pathophysiology, clinical findings, and prognosis. For example, thyroid nodules are less frequent in childhood than in adulthood, but are more often malignant. Previous guidelines for the management of thyroid nodules and cancers were geared toward adults. Diagnostic work up or treatment that may be recommended for an adult may not be appropriate for a child. In 2015, the American Thyroid Association (ATA) formulated and published guidelines for evaluation and management of children and adolescents with thyroid tumors. The intent of the guideline was to provide recommendations to ensure stratification of care based on complete and accurate pre-operative assessment with thyroid/neck ultrasound and fine needle aspiration. Pediatric specific post-operative risk levels were also created to identify patients at risk of persistent disease that would benefit from radioiodine therapy. The evaluation and management of patients with persistent and/or recurrent disease were also reviewed with recommendations directed at reducing surgical complications as well potential complications associated with repeat radioiodine therapy.

We will address the initial work up, operative management and postop care in the pediatric population. In addition to review of the key points outlined in the ATA pediatric guidelines, we will also discuss additional strategies from our practice to reduce complications of therapy as well as more recent data from the literature.
Table 1 (abstract MTE7).

See text for description

ATA Pediatric Risk Level


Initial Post-operative Staging


Disease confined to the thyroid with minimal level VI lymph node metastasis

TSH suppressed Tg at 3 month intervals with neck ultrasound at 6 months


Extrathyroidal extension with extensive level VI or lateral neck lymph node metastasis

TSH-stimulated Tg and diagnostic 123I whole body scan


Regionally extensive disease with or without distant metastasis

TSH-stimulated Tg and diagnostic 123I

whole body scan

LS9 New Genetic Causes of Short Stature: A Primer for Endocrinologists

Andrew Dauber

Cincinnati Center for Growth Disorders, Division of Endocrinology, Cincinnati Children's Hospital Medical Center

In this talk, we will review recent advances in the field of growth genetics. These defects include mutations in genes within the growth hormone/IGF-I axis, including the recently discovered mutations in the PAPPA2 and IGF2 genes, as well as mutations in genes outside this axis. We will review a few genes with direct effects at the growth plates (ACAN and NPR2). We will also briefly discuss new findings in primordial dwarfism including a network of DNA damage repair genes in which mutations cause severe short stature. Finally, there will be a brief overview of the lessons learned from genomewide association studies about the genetics of height in the general population.

LS10 Current Status of the long-term Safety of GH Treatment

Peter Clayton

Faculty of Biology, Medicine & Health, University of Manchester

Pituitary-derived growth hormone (GH) was first used in the 1950s and withdrawn in the mid-1980s because of its link with Creutzfeld-Jacob disease, with recombinant human GH (r-hGH) now being in use for >30 years. Over that time its safety and efficacy in children and adults has been subject to considerable scrutiny. R-hGH is used under licence in a wide range of disorders, including growth failure associated with a number of diverse aetiologies in children, GH deficiency in adults with onset in childhood or adulthood, and HIV/AIDS-wasting in adults. It has also been used in clinical trials to assess its impact in skeletal dysplasias, steroid-induced growth failure, poor growth associated with inflammatory disorders and fracture healing, as well as off-label use.

There are therefore significant challenges in evaluating safety in all these contexts. These include: lack of an untreated comparator population, relatively small sample sizes, inadequate long-term data on GH exposure, in particular dosages, inconsistent definitions of diagnoses and outcomes, and reporting bias of adverse events. Nevertheless there is a large literature primarily from observational studies while on r-hGH that indicates an overall good safety record, with no increase in de-novo cancer or tumour recurrence in children. There are a number of well recognised but infrequent side-effects (intracranial hypertension, musculoskeletal symptoms, obstructive sleep apnoea, bone problems [usually related to the underlying condition e.g. scoliosis], and alterations in thyroid and cortisol metabolism).

Despite r-hGH’s safety record, there have been underlying concerns related to the potential impact of the physiological actions of r-hGH on cell growth and metabolism, that could associate with cancer risk and cardiometabolic effects, and hence an overall effect on mortality.

To address these concerns, large cohorts of patients with well-characterised diagnoses and treatment exposures need to be assembled and followed long-term, both while on r-hGH and in the years after treatment is completed. The ‘Safety and Appropriateness of GH treatments in Europe’ study was established to assemble a large group of patients, who were at least 18 years of age and had been treated in childhood only with r-hGH. This cohort was first assembled in France, followed by 7 other European countries. This has resulted in a metacohort of ~25,000 individuals, most commonly treated for growth failure due to isolated GH deficiency, idiopathic short stature or short stature in children born small for gestational age (53%), Turner syndrome (13%) and growth hormone deficiency linked to neoplasia (12%), with >400,000 person years of follow-up and an average duration of 17 years per patient.

To date results on cancer incidence, mortality and vascular complications have been reported from the French cohort (n=6298), and mortality has been reported from a combined Swedish, Belgium and Dutch (S/B/D) cohort (n=2543). In the French cohort, there was an increased standardised mortality ratio (SMR) for ‘ill-defined conditions’ (SMR 3.35), bone neoplasms (SMR 5) and cerebrovascular disease (SMR 5.29), while in S/B/D cohort there were no deaths from cancer or vascular disease. In the French study, high GH doses (>50μg/kg/day) were associated with an increased SMR (3.41). These different results illustrate the importance of assembling very large datasets, and this will be addressed in the full SAGhE cohort.

A recent position statement, generated by an international panel of experts, which had evaluated published safety data and current pharma databases, concluded that ‘GH continues to have a good safety record when used for approved indications and at recommended doses’. Importantly the group recommended that long-term pharmacovigilance of all exposed to r-hGH should occur over the years after treatment.

LS11 How GH treatment changes the phenotype of children with Prader Willi Syndrome

Anita Hokken-Koelega

Erasmus University Medical Center

The most important reason for treating children with Prader-Willi Syndrome (PWS) with growth hormone (GH) is to optimize their body composition.

Setting Longitudinal study to evaluate efficacy and safety data during 8 years of GH treatment, in a Dutch cohort of 60 prepubertal children with genetically proven Prader Willi syndrome, treated with 1 mg GH/m2/day (0.035 mg GH/kg/day). Annual measurements of lean body mass (LBM) and fat mass percentage (%) by the same dual-energy x-ray absorptiometry (DXA) machine in all children.

Results: After a significant increase during the first year of GH treatment (P < 0.0001), LBM remained stable for 7 years at a level above baseline (P < 0.0001). After a significant decrease in the first year, fat mass % SDS and body mass index (BMI) SDS remained stable at a level not significantly higher than at baseline (P = 0.06, P = 0.14, resp.). BMI SDS based on PWS references was, however, significantly lower after 8 years of GH treatment than at baseline (P < 0.0001). After 8 years of treatment, height SDS and head circumference SDS had completely normalized. Mean IGF-I SDS increased to + 2.36 SDS during the first year of treatment (P < 0.0001) and remained stable since then. GH treatment did not adversely affect glucose homeostasis, serum lipids, blood pressure and bone maturation.

Conclusion: This 8-year study demonstrates that long-term GH treatment is able to change the Prader Willi phenotype, by counteracting the natural course of increasing obesity and abnormal body composition in PWS.

LS12 Safety and Efficacy of PEG-rhGH in Growth Hormone Deficiency Children: Results of Multicenter Clinical Studies in China

Pinchas Cohen1, Xiaoping Luo2

1SC Leonard Davis School of Gerontology; 2Chinese Society of Pediatric Endocrinology and Metabolism / Department of Pediatrics, Tongji Hospital, Tongji Medical College of Huazhong University of Sciense & Technology

Introduction: Jintrolong® (PEG-rhGH) is a long-acting rhGH prepared by conjugating a branched polyethylene glycol molecule to rhGH. A phase-I pharmacokinetics study showed it to be suitable for weekly dosing regimen in children with GHD. A phase-II study established the efficacious dose of weekly PEG-rhGH to be 0.2-mg/kg/wk. Phase III and IV studies were designed to evaluate short-term and long-term efficacy and safety of PEG-rhGH in large scale of children with GHD.

Methods: A phase-III, multicenter, randomized, open-label, controlled trial enrolled treatment-naïve, prepubertal GHD children >3 years. 108 patients were randomized in a 2:1 ratio to s.c. injection weekly PEG-rhGH at a dose of 0.2-mg/ kg/wk or to daily rhGH at a dose of 0.25-mg/kg/wk for 25-weeks. The primary endpoint was height velocity (HV) increase at 25 weeks. The phase IV study included 4 sub-studies, that enrolled prepubertal GHD children ≥3 years from 80 hospitals, that were treated for 26-weeks. The study design is showed in figure-1. The primary endpoint was height standard deviation score increase (ΔHtSDS) at 26 weeks. All patients were enrolled to extension studies after 26-weeks with optimal PEG-rhGH dosage until final height.

Results: Significantly greater HV increase was associated with PEG-rhGH treatment vs. daily rhGH (P<0.05) at 25 weeks in the phase-III study. Adverse events (AEs) were comparable between two groups. In the phase-IV study, 944 patients from 4 sub-studies completed the 26-week treatment. TheΔHtSDS were comparable between the different dosage PEG-rhGH groups in sub-study-1, and sub-study-2. Although the ΔHtSDS were comparable within bi-weekly PEG- rhGH, weekly PEG-rhGH and daily rhGH in sub-study-3, a borderline significance trend was observed after 39-weeks follow-up between the bi-weekly and weekly PEG-rhGH. In the observational, open-label sub-study-4, the most frequent treatment dosage was weekly PEG-rhGH at 0.15-mg/kg/wk. AEs were comparable between PEG-rhGH and daily rhGH in all 4 sub-studies.

Conclusions: PEG-rhGH showed excellent safety and efficacy in GHD children treated with doses from 0.1 to 0.2-mg/kg/wk. Preliminary results from phase IV studies need to be confirmed with additional patients and long-term follow-up.
Fig. 1 (abstract LS12).
Fig. 1 (abstract LS12).

Phase IV study design

LS13 Moving Towards an International Consensus on the Future of GH therapy

Pinchas Cohen

USC Leonard Davis School of Gerontology

As growth hormone (GH) therapy completes its sixth decade, it has become a staple of endocrine therapy for both children and adults. Thirty years ago, recombinant daily GH replaced pituitary GH therapy that was associated with risk of Jakob-Creutzfeldt Disease. However, as daily rhGH treatment continued to be refined, concerns about its safety (primarily related to cancer risk) lingered, even as large observational phase-IV studies did not demonstrate specific risks. Over the last several years, preliminary results of a retrospective analysis of a large cohort with long term outcomes of GH recipients in Europe (SAGhE) raised serious concerns about the possibility of increased mortality in a French sub-cohort that were related mainly to cardiovascular causes. Analysis of other sub-cohorts including Scandinavian data did not confirm this finding. The international pediatric endocrine community responded to this concern with serious reconsideration of the issues that culminated in a consensus workshop co-sponsored by all of the major relevant societies that recognized some inherent concerns related to GH use, particularly as they relate to second malignancies among cancer survivors, but did not find evidence for elevated cancer risk in other populations and concluded that overall mortality did not appear to be increased when the entire SAGhE population was taken into account (1). These important lessons will be critical in the clinical integration of a new generation of long-acting growth hormone products, with over six different distinct preparations currently being tested in phase-II and –III studies. Another consensus workshop recently held and published, examined the various issues involved in developing this new product class (2). While challenges in optimizing safety and efficacy of these new drugs clearly exist, early data, and a rationale clinical development strategy, make it very likely that several long-acting products will soon be available for the use of the endocrine community, which will enhance physician-patient choices, improve convenience, and potentially overcome adherence and compliance issues, without compromising the excellent safety record of GH.


1. Allen DB, Backeljauw P, Bidlingmaier M, Biller B, Boguszewski M, Burman P, Butler G, Chihara K, Christiansen JS, Cianfarani S, Clayton PE, Clemmons D, Cohen P, Darendeliler F, Deal C, Dunger DP, Erfurth EM, Fuqua J, Grimberg A, Haymond M, Higham C, Ho KK, Hoffman AR, Hokken-Koelega AC, Johannsson G, Juul A, Kopchick JJ, Lee P, Pollak M, Radovick S, Robison L, Rosenfeld R, Ross RJ, Savendahl L, Saenger P, Toft Sorensen H, Stochholm K, Strasburger CJ, Swerdlow A, Thorner MO. Growth Hormone Safety Workshop Position Paper: a critical appraisal of recombinant human growth hormone therapy in children and adults. Eur J Endocrinol. 2015; 174:1–9.

2. Christiansen JS, Backeljauw P, Bidlingmaier M, Biller B, Boguszewski M, Casanueva FF, Chanson P, Chatelain P, Choong CS, Clemmons DR, Cohen L, Cohen P, Frystyk J, Grimberg A, Hasegawa Y, Haymond M, Ho K, Hoffman AR, Holly JM, Horikawa R, Hoybye C, Jorgensen JO, Johannsson G, Juul A, Katznelson L, Kopchick JJ, Lee KO, Lee KW, Luo XP, Melmed S, Miller B, Misra M, Popović V, Rosenfeld RG, Ross J, Ross RJ, Saenger P, Strasburger CJ, Thorner MO, Werner H, Yuen KC. Growth Hormone Research Society Perspective on the Development of Long- Acting Growth Hormone Preparations. Eur J Endocrinol. 2016; 174:1–9.

LS14 When one gene does it all: monogenic forms of autoimmune diabetes

Elisa De Franco

Naomi Berrie Fellow in Diabetes Research, Molecular Genetics, University of Exeter Medical School

Neonatal diabetes is diagnosed before 6 months of age and is generally caused by a mutation in a single gene. Traditionally it has been considered as a separate entity from the more common type 1 diabetes, which is generally diagnosed after 6 months and is caused by a combination of genetic predisposition and environmental triggers. Last generation genetic approaches have allowed great advances in the knowledge of the genetic basis of both type 1 and neonatal diabetes, challenging the conventional view of neonatal diabetes and type 1 diabetes as two completely distinct diseases.

Mutations in 23 genes have been identified to date to cause neonatal diabetes, providing a genetic diagnosis for 82% of patients diagnosed before 6 months of age. The majority of these genes are involved in insulin sensing/ secretion and beta cell function/development. A notable exception are mutations in three genes (FOXP3, STAT3, and IL2RA) which cause neonatal diabetes with additional autoimmune features. These three genes are not directly involved in beta cell development or function, in fact they encode for proteins that are important for regulation of the immune system and prevention of autoimmunity. Patients with mutations in these genes have diabetes soon after birth as a result of early autoimmune destruction of their beta cells. Furthermore, these patients generally have low birth weight, suggesting that the autoimmune reaction starts in utero. These patients generally develop additional autoimmune features early in infancy and allogenic stem cell transplantion is currently the only therapeutic option. The Exeter team has been specifically looking for the genetic causes of early onset diabetes and autoimmunity and has currently identified 2 further genes causing neonatal diabetes through immune dysregulation.

But how accurate is the cut-off of 6 months for a differential diagnosis of neonatal diabetes versus type 1 diabetes? We have assessed this using a T1D genetic risk score generated from type 1 diabetes-associated common genetic variants in patients diagnosed before 6 months of age in whom a genetic cause could not be identified. This approach has identified patients who are likely to have early onset type 1 diabetes who were indeed diagnosed before the age of 6 months.

Uncovering the genetic basis of neonatal and type 1 diabetes broadens our understanding of the mechanisms involved in the the pathogenesis of both diseases. Emerging evidence points towards a continuum model for different diabetes subtypes: from monogenic to more common forms of diabetes.

LS15 African Efe Pygmies: From Anthropology to Endocrinology

Mitchell E. Geffner

Keck School of Medicine of USC, Center for Endocrinology, Diabetes & Metabolism, Children's Hospital Los Angeles

Pygmy populations occupy a vast territory extending west-to-east along the central equatorial African belt from the Congo Basin to Lake Victoria, but are also found in other parts of the world, e.g. , Papua, New Guinea. Their short stature is thought to be adaptive (as opposed to a defect) in terms of ease of locomotion through dense forest understory, minimization of food requirements during cyclical periods of undernutrition, and selection for survival in hot, humid forest environment where greater surface area:body mass ratio is necessary for sufficient thermoregulation. The evolutionary history of the human pygmy phenotype (small body size), a characteristic of African rainforest hunter-gatherers, is largely unknown. Certain genomic regions are significantly associated with the pygmy phenotype in some rainforest hunter-gatherer populations from east central Africa and have multiple attributes that provide supporting evidence of genuine association with the pygmy phenotype, including enrichments for SNPs previously associated with stature variation in Europeans and for genes with growth hormone receptor and regulatory functions consistent with early suggestions that the short stature of pygmies results from GH resistance and an absent adolescent growth spurt. Efe pygmies of northeast Zaire have the shortest mean adult stature of any population on earth. We previously described GH and IGF-I unresponsiveness in T-lymphoblast cell lines derived from Efe pygmies. Using this model, we also found markedly decreased cell surface expression of type 1 IGF receptors (IGF-I receptors) with normal ligand binding affinity. The pygmy IGF-I receptors were not autophosphorylated and did not transmit a signal in response to physiological concentrations of IGF-I. There was a substantially decreased level of IGF-I receptor mRNA in the pygmy cells with a normal mRNA half-life. No consistent mutation was found in the exons encoding the IGF-I receptor. These results indicate decreased IGF-I receptor gene transcription and IGF-I receptor signaling as the primary variation in the pygmy cell lines, with GH resistance a secondary back-up phenomenon. Our findings point to the IGF-I receptor as the locus governing short stature in the African Efe pygmy and suggest that human stature may be genetically controlled by expression of the IGF-I receptor.

LS16 Skeletal, Growth, and Functional Improvements in Children with Hypophosphatasia Treated with Asfotase Alfa for 5 years

Jill H Simmons

Vanderbilt University School of Medicine

Hypophosphatasia (HPP) is an ultra-rare orphan disease caused by mutation(s) in the ALPL gene resulting in deficiency of tissue-nonspecific alkaline phosphatase (ALP). Disease presentation is variable, ranging from frequently fatal in the perinatal or infantile forms to primarily causing musculoskeletal abnormalities and pain in juveniles and adults. Infantile HPP features poor bone mineralization leading to rickets and is sometimes complicated by seizures, respiratory compromise, poor growth, and delayed motor function. Juvenile HPP features include delayed/ impaired motor function, generalized muscle pain, poor bone mineralization, abnormal gait, and early tooth loss. Historically, HPP patients were treated with surgical management, dental hygiene, respiratory support, and dietary modifications when necessary, but until recently, there was no disease-specific therapy available. Recombinant bone-targeted ALP is available for patients with HPP, and studies have demonstrated benefits upon the perinatal, infantile, and juvenile forms. Asfotase Alfa (AA) is given subcutaneously at a dose of 2 mg/ kg/ day 3 days per week or 1 mg/ kg/ day 6 days per week in Japan.

Five- year survival in perinatal/ infantile HPP patients treated with AA was 84%(n=37) , which was higher than that observed in a historical control group (27%). Changes in HPP-related skeletal disease have been demonstrated, with the Radiographic Global Impression of Change (RGI-C) scale (-3=severe worsening; +3=near/complete healing) improving to +2.7 (+1.3, +3.0) by year 1 (n=9) and continuing at +2.0 (+2.0, +3.0) at year 5 (n=9) Infants treated with AA also have significant improvement in respiratory status, growth and motor skills development. Thirteen 6-12 year-old childhood-onset HPP patients treated with AA for 5 years were compared with 16 untreated pediatric historical controls with HPP. Improvements were seen in RGI-C scale of treated patients by 6 weeks [+1 (0.0, +2.0), p=0.001] and persisted through 5 years [+2.2 (+1.7, +2.7), p=0.0005]; no change was seen in the RGI-C scale of historical controls. In the treated group, improvements occurred in weight and height z-scores and developmental, pain, and disability assessments.

In all patient populations studied, serious adverse events have been rare but injection site reactions (ISR) are common and include erythema, warmth, and lipohypertrophy at injection sites. ISRs and AES have not led to medication discontinuation.

In conclusion, AA enzyme replacement therapy has been demonstrated to improve survival, skeletal mineralization, growth, and motor skills in children with HPP. Side effects have been minimal and have not led to drug discontinuation. Follow-up for long-term effects needs to continue.

ES1-1 Total management of Rickets: causes, prevention and treatment

Global Consensus Recommendations on Prevention and Management of Nutritional Rickets: Definition and Diagnosis

Lorna Ramos-Abad

University of the Philippines College of Medicine, Philippine General Hospital Metro Manila

Nutritional Rickets (NR), a disorder of defective chondrocyte differentia- tion and mineralization of the growth plate and defective osteoid mineralization, is caused by vitamin D deficiency and/or low calcium intake in children. Osteo- malacia is abnormal matrix mineralization in established bone, and although present in children with rickets, it is used to describe bone mineralization defects after com- pletion of growth.

Diagnosis of NR is made on the basis of history, physical examination, and biochemical testing and is confirmed by radiographs. Osseous signs and symptoms include: swelling wrists and ankles Delayed fontanelle closure (normally closed by the age of 2 years) Delayed tooth eruption (no incisors by the age of 10 months, no molars by 18 months), Leg deformity (genu varum, genu valgum, windswept deformity) Rachitic rosary (enlarged costochondral joints – felt anteriorly, lateral to the nipple line) Frontal bossing Craniotabes (softening of skull bones, usually evident on palpation of cranial sutures in the first 3 months) Bone pain, restlessness, and irritability. Radiographic features are: splaying, fraying, cupping, and coarse trabecular pattern of metaphyses, widening of the growth plate, Osteopenia. There could be nonosseous features such as Hypocalcemic seizure and tetany, Hypocalcemic dilated cardiomyopathy (heart failure, arrhythmia, cardiac arrest, death) Failure to thrive and poor linear growth, delayed gross motor development with muscle weakness, raised intracranial pressure.

Biochemical testing alone is not sufficient to diagnose NR and may not differentiate whether the primary cause of NR is vitamin D or dietary calcium deficiency because combined deficiencies are common. Vitamin D status is assessed by measuring blood levels of total 25OHD. Classification of vitamin D status is based on serum 25-hydroxyvitamin as: sufficiency, >50 nmol/l , Insufficiency, 30–50 nmol/l Deficiency, <30 nmol/l .

ES1-2 Total management of Rickets: causes, prevention and treatment

Global Consensus Recommendations on Prevention and Management of Nutritional Rickets

Navoda Atapattu

Consultant Paediatric Endocrinologist

Nutritional rickets (NR) has been increasingly reported in high and low income countries. Resurgence of NR has been observed in the high income countries due to increase in immigrants and refugee groups. There is a considerable variation in the definition, diagnosis and management of NR. European Society for Pediatric Endocrinology decided to formulate evidence-based recommendations together with the PES, JSPE, SLEP, APEG, ISPAE, ASPAE, CSPEM, ESPGHAN. The consensus paper includes evidence up to the end of 2014.

Nutritional rickets is defined as defective chondrocyte differentiation and mineralization of growth plate due to vitamin D deficiency and or low calcium intake. Defective mineralization results in growth plate widening, metaphyseal cupping and fraying seen on the radiography. Vitamin D status is assessed by measuring blood levels of total 25 OH vitamin D. Dietary calcium deficiency is diagnosed by obtaining a calcium intake history with the help of dietary calcium intake questionnaire specific to their country/region. Vitamin D level of >50 nmol/ l is considered sufficient, 30-50 nmol/l is considered as insufficient and < 30 nmol/L is deficient and >250nmol/ l with hypercalciuria is considered as vitamin D toxicity. It is recommended to provide 400IU of vitamin D daily for all infants from birth irrespective of mode of feeding and 600IU is recommended for children and adults through diet or supplementation. In countries where food fortification is not a routine practice, vitamin D supplementation is needed for children with a history of vitamin D deficiency, children and adult at risk of vitamin D deficiency and pregnant women. Minimal dose of vitamin D to treat rickets is 2000IU daily for 3 months. Oral route of vitamin D is recommended. Calcium 500mg as dietary intake or supplement is routinely needed together with vitamin D in addition to vitamin D supplement. Complementary feeding rich in calcium should be started no later than 26 weeks to prevent rickets. Population based studies should determine the burden of rickets and screening should be done based on clinical features. NR can have a major impact on the health of infants, children, and adolescents. It is important to recognize NR as a preventable global public health problem and implement rickets preventing programs by adhering recommended vitamin D and calcium intakes, monitoring for adherence and promoting staple food fortification programs.

ES1-3 Total management of Rickets: causes, prevention and treatment

Hypophosphatemic Rickets: Current Status and Future Perspectives

Toshimi Michigami

Department of Bone and Mineral Metabolism, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho, Izumi, Osaka Japan

Phosphate is a component of hydroxyapatite and is indispensable for skeletal mineralization. Prolonged deficiency of phosphate can be caused by impaired intestinal absorption and/or renal phosphate wasting, resulting in rickets in children and osteomalacia in adults. Although phosphate is abundant in food, patients with malnutrition or malabsorption may have phosphate deficiency, leading to hypophosphatemic rickets. Renal phosphate wasting can be observed in the conditions with impaired function of renal proximal tubules, such as Fanconi syndrome and some forms of renal tubular acidosis. Renal reabsorption of phosphate is mostly mediated by type 2a and 2c sodium-phosphate co-transporters (NPT2a and NPT2c) in the proximal tubules. Loss-of-function mutations in SLC34A3 encoding NPT2c cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH).

Several kinds of hypophosphatemic rickets and osteomalacia are caused by excessive actions of fibroblast growth factor 23 (FGF23). FGF23 is produced mainly by osteocytes in the bone and exerts its effects on the distant organs. In the kidney, FGF23 increases renal phosphate excretion by suppressing the expression of NPT2a and NPT2c. In addition, FGF23 reduces the level of 1,25-dihydroxyvitamin D. FGF23-related hypophosphatemic rickets/ osteomalacia is characterized by renal phosphate wasting, hypophosphatemia, and inappropriately low level of serum 1,25(OH)2D. Mutations in FGF23 that make the protein resistant to proteolytic cleavage cause autosomal dominant hypophosphatemic rickets (ADHR). X-linked hypophosphatemic rickets (XLH), the most common form of FGF23-related hereditary hypophosphatemic rickets, is caused by inactivating mutations in the phosphate- regulating gene homologous to endopeptidase on X chromosome (PHEX). Genetic evidence has revealed that loss- of-function mutations in dentin matrix protein 1 (DMP1), ectonucleotide pyrophosphatase phosphodiesterase-1 (ENPP1) , and family with sequence similarity 20, member C (FAM20C) lead to FGF23-related hypophosphatemic rickets/osteomalacia of autosomal recessive inheritance. Other conditions such as McCune-Albright syndrome, linear nevus sebaceous syndrome, and tumor-induced osteomalacia, are also associated with FGF23-related hypophosphatemia. Measurement of serum FGF23 levels is useful to distinguish FGF23-related hypophosphatemia from rickets or osteomalacia of other causes, because they are rather low in vitamin D deficiency and intrinsic defects in renal proximal tubules. Patients with XLH are currently treated with active vitamin D and phosphorus. However, the administration of active vitamin D and phosphorus further increases the level of FGF23, which might worsen the disease. A neutralizing antibody against FGF23 has been developed recently as a new therapy for XLH, and clinical trials are currently undergoing.

ES2 Adrenarche


Joseph Majzoub

Royal Children's Hospital

Adrenarche is the initiation of dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) secretion from the zona reticularis of the adrenal gland during childhood. Adrenarche is a gradual process beginning during the first five years of life, whereas pubarche, the appearance of pubic hair and an end result of adrenarche, normally occurs when children are eight or more years of age. Premature adrenarche is among the most common pediatric endocrine disorders, usually considered benign but occasionally the first sign of an adrenal tumor or enzyme deficiency. In some children, premature adrenarche may be a forerunner of the polycystic ovary syndrome or the metabolic syndrome. The trigger for adrenarche remains unknown, and initiating factors leading to production of adrenal androgens are poorly defined.

Learning objectives: As a result of participating in this session, learners should be able:
  • To understand the changes that occur in 17, 20 lyase and 3 β HSD2 enzyme activities during adrenarche

  • To understand changes in the zona reticularis during adrenarche

  • To understand how to identify true, genetic 3 β HSD2 deficiency

  • To understand whether mild, nonclassical 3 β HSD2 exists

  • To discuss the possible role of intracrine, intra-adrenal cortisol in the onset of adrenarche

  • To speculate on why benign premature adrenarche is associated with obesity

Kaichi-Kida 1 Thymic deletion of ICA69 induces autoimmune diabetes and other endocrine diseases

Asako Tajima1,2, Ichiro Miyata1, Hiroyuki Ida1, Massimo Trucco2, Yong Fan2

1Department of Pediatrics, Jikei University School of Medicine; 2Allegheny Health Network, Institute of Cellular Therapeutics

Aim: Islet autoantigen 69 (ICA69), encoded by the Ica1 gene, is one of the known autoantigens in type 1 diabetes (T1D). In addition to pancreatic beta cells, ICA69 is also present in extrapancreatic endocrine and exocrine tissues, such as the thyroid and salivary gland. Notably, ICA69 is ectopically expressed in the thymus, and its level of expression is significantly decreased in T1D prone NOD mice, compared to that of T1D resistant C57BL/6 mice. Immunization with ICA69 peptides in NOD mice not only accelerates diabetes progression, but also induces autoimmune response in the salivary glands. These results highlight the antigenic role of ICA69 in the progression of these diseases and suggest that thymic ICA69 expression is essential to establish immune tolerance of ICA69-expressing organs. In this study we attempted to elucidate the underlying mechanism of ICA69 and its role in the induction of autoimmunity in the pancreas and other endocrine tissues.

Materials and Methods: C57BL/6 mice with systemic Ica1 gene deficiency (B6.ICAdel/wt for heterozygous and B6.ICAdel/del for homozygous ICA69 deletion) were first generated to observe the spontaneous effect of ICA69 in the endocrine tissues. Next, ICA69 was eliminated specifically in the thymus using the Cre-lox system (Aire-ICA69). Endocrine functions were assessed by intraperitoneal glucose tolerance tests and saliva flow rate. Flow cytometry, Enzyme-Linked ImmunoSpot assay, and immunohistochemistry were performed to investigate the potential link between the level of thymic ICA69 expression and peripheral anti-ICA69 autoimmunity.

Results: Significant decrease of thymic ICA69 expression was observed in B6.ICAdel/wt mice. When immunized with ICA69 peptides, B6.ICAdel/wt mice develop both insulitis and thyroiditis. Consistently, Aire-ICA69 mice displayed an impaired glucose tolerance as early as 12 weeks of age. In addition to insulitis, immunohistochemical analyses revealed the spontaneous development of autoimmunity in multiple extrapancreatic organs, including sialadenitis, thyroiditis, and gastritis.

Conclusion: Our findings establish a direct link between compromised thymic ICA69 expression and autoimmunity against multiple ICA69- expressing organs, and identify a potential mechanism for the development of T1D-associated autoimmune disorders, as well as other multi- organ diseases, such as autoimmune polyglandular syndromes 2 and 3.

Kaichi-Kida 2 DNA methylation defects in short children born small for gestational age

Akie Nakamura1, Takanobu Inoue1, Keiko Matsubara1, Shinichiro Sano2, Yasuhiro Naiki3, Shuichi Yatsuga4, Junko Nishioka4, Keisuke Nagasaki5, Koji Muroya6, Sachiko Kitanaka7, Toshihiro Tajima8, Reiko Horikawa3, Tsutomu Ogata2, Maki Fukami1, Masayo Kagami1

1Department of Moledular Endocrinology, National Research Institute of Child Health and Development; 2Department of Pediatrics, Hamamatsu University School of Medicine; 3Division of Endocrinology and Metabolism, National Medical Center for Children and Mothers; 4Department of Pediatrics and Child Health, Kurume University School of Medicine; 5Department of Homeostatic Regulation and Development, Niigata University Graduate School of Medical and Dental Sciences; 6Division of Pediatrics; 7Department of Endocrinology and Metabolism, Kanagawa Children's Medical Center; 8Department of Pediatrics, The University of Tokyo; 9Department of Pediatrics, Jichi Children's Medical Center Tochigi

Background: DNA methylation defects at differentially methylated regions (DMRs) of imprinted loci are one of the cause of intrauterine growth restriction (IUGR) and postnatal growth failure. Among eight known imprinting disorders, Silver-Russell syndrome (SRS), Temple syndrome and Prader-Willi syndrome patients show pre- and postnatal growth retardation. Furthermore, maternal uniparental disomy of chromosome 6 (UPD(6)mat) and 20 (UPD(20)mat) have also been associated with growth failure. However, there is no single report of systematic methylation analysis of a large cohort of patients with IUGR and postnatal growth failure. In this study, we aimed to clarify the frequency of methylation defects in short children born small for gestational age (SGA).

Participants: A total of 183 short children (height < -2.0 SD after the age of 2 years or before growth hormone treatment) born SGA were recruited in this study. Inclusion criteria of SGA were defined as birth weight and birth length SDS less than 10th percentile for gestational age. Patients with apparent chromosomal or monogenic abnormalities which induce IUGR were excluded. 28 patients were classified as ‘Likely-SRS’ according to the Nechine-Haribison clinical scoring system.

Methods: We performed quantitative DNA methylation analysis at DMRs of 9 imprinted loci (PLAGL1 , PEG1 , PEG10 , H19 , Kv, DLK1-MEG3 intergenic (IG), MEG3 , SNRPN and GNAS A/B) by pyrosequencing using genomic DNA isolated from peripheral blood leukocytes. When we detected methylation defects, we carried out further studies, such as comparative genomic hybridization, multiplex-ligation dependent probe amplification and microsatellite analysis, to determine the genetic causes of methylation abnormalities (microscopic chromosomal rearrangement, UPD or epimutation).

Results: We identified loss of methylation (LOM) at H19 -DMR in 19 cases, UPD(7)mat in 6 cases, LOM at IG-DMR and MEG3 -DMR in one case, UPD(14)mat in 5 cases, UPD(15)mat in one case, UPD(6)mat in 3 cases and UPD(20)mat in 2 cases. In addition, we detected one case with partial LOM at IG-DMR and MEG3 -DMR due to trisomy 14 mosaicism with UPD(14)mat, and another case with partial LOM at A/B DMR due to 20pter microdeletion and 20qter microduplication.

Conclusions: Methylation defects were detected in 39 of 183 (21%) short children born SGA. Our results highlight the clinical importance of methylation defects as genetic causes of short stature born SGA.

Kaichi-Kida 3 The gut microbiome and insulin resistance in children born very preterm

Valentina Chiavaroli1,2, Thilini N Jayasinghe1, Sachin Jayan1, Cameron Ekblad1, Jose’ Derraik1, Paul Hofman1,2, Elizabeth McKenzie1, Justin O'Sullivan1,2, Wayne Cutfield1,2

1University of Auckland, Liggins Institute; 2Gravida: National Centre for Growth and Development

Background: In recent years the gut microbiome has been shown to influence the development of obesity and type 2 diabetes mellitus. We hypothesize that adverse early life events in preterm children may lead to alterations in the gut microbiome, which contribute to later metabolic disease. Children born preterm are at increased risk for insulin resistance, obesity, and cardiovascular disease.

Aim: To correlate the metabolic phenotype with the gut microbiome composition and functional capacity in children born very preterm compared to those born at term.

Results: Participants were healthy prepubertal children aged 5 to 11 years born very preterm (-4 · min-1(mU/l); p=0.0007). Stool metatranscriptomics identified Coriobacteriaceae and Collinsella species as being associated with children who were born preterm (highly significant with LDA score >4.0, LefSe). There were also functional changes in the activity of the microbiome in children born preterm, including glutamate and arginine metabolism, known to be involved in glucose metabolism. Changes to the metabolites within the preterm fecal and plasma correlated with the observed phenotypes.

Conclusion: Children born very preterm are insulin resistant with differences in gut microbiome species and activity. In preterm children we speculate that (i) these changes in the gut microbiome were established in early infancy, and (ii) the altered gut microbiome contributes to insulin resistance.

Kaichi-Kida 4 Genetic etiology study of 90 complete growth hormone deficiency patients by targeted next-generation sequencing

Jia-Tong Hou1, Shi-Yao Wang1, Han-Ze Du1, Hui-Juan Zhu1, Ci-Ren A-San2, Hong-Bo Yang1, Lin-Jie Wang1, Feng-Yng Gong1, Hui Pan1

1Department of Endocrinology, Key Laboratory of Endocrinology of Ministry of Health, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College; 2Binhai Genomics Institute, BGI-Tianjin, Tianjin Enterprise Key Laboratory of Clinical Molecular Diagnostic

Objectives: This study aimed to discover the genetic mutations and identify the etiologies in a cohort of complete growth hormone deficiency patients, and to establish a new genetic testing method—targeted next-generation sequencing (NGS) in genetic evaluation of complete growth hormone deficiency and verify its potential utility.

Methods: A total of 90 complete growth hormone deficiency patients were included in the study. Twenty-three genes related to growth hormone deficiency were sequenced parallel by the combination of NGS and targeted genomic enrichments. The clinical significances of variants detected in patients were evaluated on the basis of the extant clinical guideline.

Results: These patients were divided into two categories based on clinical diagnosis, including 56 with idiopathic growth hormone deficiency (IGHD) and 34 with multiple pituitary hormone deficiencies (MPHD). We identified 20 gene variants as the possible genetic causes for 18 (20%) of the 90 growth hormone deficiency patients, which included 12(66.7%) of IGHD patients and 8 (33.3%) of MPHD patients. Of the variants detected, 6 were previously reported and the others were not reported. Of the 12 IGHD patients with possible genetic causes, 4 were identified with CHD7 mutation, 2 with GHSR mutation, 2 with VPS13B mutation, 1 with ALMS1 mutation, 1 with SEMA3E mutation, 1 with a novel homozygous deletion in chromosome 17: 61994532- 61996228 of the whole GH1 gene and 1 with a novel heterozygous duplication in chr3 F57231913-57234310 of the whole HESX1 gene. Among the 6 MPHD patients with possible genetic causes, 2 were identified with GLI2 mutation, 3 with SOX3 mutation, 1 with LHX4 mutation and 1 with a novel homozygous deletion in chromosome 17: 61994532- 61996228 of the whole GH1 gene.

Conclusions: The method of combination of NGS and targeted genomic enrichments is able to explain 20% of the molecular etiologies of the GHD patients. This study confirmed the high efficiency and precise of such a comprehensive genetic evaluation in clinics. It provides a good way to a large-scale genetic study of GHD. Part of patients with pathogenic gene mutations may not show the corresponding phenotypes. Different patients with the same genetic mutation can lead to variable phenotypes.

Key words: growth hormone deficiency, multiple pituitary hormone deficiency, genetic etiology, next-generation sequencing Ctargeted genomic enrichments.v

Kaichi-Kida 5 Diagnostic Application of Targeted Exome Sequencing for Skeletal Dysplasia

Sung Yoon Cho1, Seok Bae3, Nayoung K. D. Kim3, Ok-Hwa Kim4, Tae Joon Cho5, Sung Won Park6, Young Bae Sohn7, Woong-Yang Park3, Dong-Kyu Jin1

1Department of Pediatics, Samsung Medical Center; 2Department of Pediatics, Myongji Hospital; 3Samsung Genome Institute, Samsung Medical Center; 4Department of Radiology, Woorisoa Children's Hospital; 5Department of Pediatric Orthopaedics, Seoul National University Childrens Hospital; 6Department of Pediatics, Cheil General Hospital & Womans’ Health Care Center; 7Department of Medical Genetics, Ajou University Hospital

Identification of causative genes for skeletal dysplasia (SD) is important to provide an exact diagnosis and decide treatment modalities and to counsel the patients. Due to the genetic heterogeneity in SD, the high throughput method can be adapted for the efficient diagnosis. To this end, a new diagnostic pipeline were designed to screen 260 reported candidate genes for SD. A total of 22 patients diagnosed with skeletal dysplasia, or suspected to have it were recruited over one year. Their clinical diagnosis were hypochondroplasia, osteogenesis imperfect type I, Sticker syndrome, pseudohypoparathyroidism, osteopetrosis. pycnodysostosis, spondyloepiphyseal dysplasia strudwick type, and ischiospinal dysostosis. We applied targeted exome sequencing (TES) on 260 genes in the 22 probands with SD. Potential causative variants determined using TES were confirmed by filtering steps. These variants were then filtered out with clinical features, inheritance pattern and a co-segregation study in the family and allele frequency in normal 197 control subjects. Finally, we detected 18 causative variants, and these variants were in the FGFR3, GANS, COL1A1, COL1A2, COL2A1, LRP5, CLCN7, CTSK, BMPER, and TNFSF11 genes bringing the total detection rate in all the patients to be 68.2% (15/22). These variants were validated using Sanger sequencing. Despite the advent of whole genome and whole exome sequencing, we propose TES as a screening and diagnostic tool at least for SD to find mutations based upon its efficacy and cost-effectiveness.

O1-1 Diabetes Mellitus is Associated with Intrauterine Hyperglycemia regardless of Maternal Obesity

Won Im Cho1, Hye Rim Chung1, Hak C. Jang2, Joon-Seok Hong3, Jung Sub Lim4, Min Jae Kang5, Young Ah Lee6, Choong Ho Shin6, Sei Won Yang6

1Division of Endocrinology and Metabolism / Department of Pediatrics, Seoul National University Bundang Hospital; 2Division of Endocrinology / Department of Internal Medicine, Seoul National University Bundang Hospital; 3Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital; 4Department of Pediatrics, Korea Cancer Center; 5Department of Pediatrics, Hallym University Sacred Heart Hospital; 6Department of Pediatrics, Seoul National University Children's Hospital

Background: It has not been conclusively established whether intrauterine hyperglycemia affects prepubertal offspring obesity regardless of maternal obesity. This study evaluates the effect of maternal obesity and hyperglycemia on the fat mass (FM) of prepubertal offspring of mothers with gestational diabetes mellitus (OGDM).

Methods: FM was determined by dual-energy X-ray absorptiometry in 22 OGDM aged 5-6 years, and was compared with that of 49 age- matched offspring of normoglycemic mothers. The relationship between FM and the results of a 100 g oral glucose tolerance test (OGTT) during the second trimester was analyzed in OGDM.

Results: FM was higher (3,548 g vs. 2,245 g) and lean mass was lower (15,609 g vs. 16,941 g) in OGDM compared with control subjects, respectively, whereas body mass index (BMI) did not significantly differ between the two groups. FM and truncal FM positively correlated with glucose levels, especially 0 hour glucose level (r = 0.580 and P = 0.005, r = 0.655 and P = 0.001, respectively). After adjusting for maternal prepregnancy BMI, total FM (r = 0.535, P = 0.012) and truncal FM (r = 0.535, P = 0.003) of OGDM correlated with maternal 0 h glucose levels of the 100 g OGTT.

Conclusions: Increased FM of OGDM aged 5-6 years is associated with intrauterine hyperglycemia regardless of maternal obesity.

O1-2 Relationship of lower heart rate variability with risk for metabolic syndrome in patients with childhood-onset craniopharyngioma

Hae Woon Jung2, Young Ah Lee1, Hwa Young Kim3, Gyung Min Lee4, Ji Young Kim1, So Youn Kim1, Kyung A Jeong1, Keun Hee Choi1, Jung-Eun Cheon5, In-One Kim5, Choong Ho Shin1, Sei Won Yang1

1Department of Pediatrics, Seoul National University College of Medicine; 2Department of Pediatrics, Kyung Hee University Medical Center, Department of Pediatrics; 3Kangwon National University Hospital; 4Department of Radiology, Konyang University Hospital, Department of Pediatrics; 5Seoul National University College of Medicine

Objective: Autonomic nervous system (ANS) dysfunction with hypothalamic involvement (HI) is implicated in the development of obesity and metabolic complications. In patients treated for childhood onset craniopharyngioma, we investigated changes in ANS activity according to the extent of HI and presence of obesity, using measures of heart rate variability (HRV). Risks for being metabolically unhealthy were analyzed in relation to HRV changes.

Methods: From March 2014 to January 2016, HRV indices of overall variability [standard deviation NN interval (SDNN) and total power (TP)], parasympathetic modulation [root mean square of successive RR interval differences (RMSSD) and high frequency (HF)], and sympathetic or sympathovagal modulation [low frequency (LF) and LF/HF ratio] were measured in 48 patients (28 males) aged 10-30 years with HI after craniopharyngioma treatment at Seoul National University Children’s Hospital. The extent of HI was graded on magnetic resonance imaging. Anthropometric measurements, fasting glucose, insulin, lipid panel, and blood pressure were obtained.

Results: Patients with extensive HI showed increased BMI z-scores (P = 0.008), waist circumference (P = 0.037) and insulin resistance (homeostasis model assessment of insulin resistance, HOMA-IR, P = 0.043). HRV indices were decreased with extensive HI, but showed no differences between obese and non-obese. SDNN, TP, RMSSD, and LF were all decreased with extensive HI. Obese patients with concomitantly reduced overall variability (by SDNN or TP) showed increased HOMA-IR (P < 0.05, for both), triglycerides (P < 0.05, for both), blood pressure (P < 0.05, for both), and decreased HDL cholesterol (P < 0.05, for both). Risk of being metabolically unhealthy was increased in patients with both obesity and reduced overall variability (P < 0.05, for both).

Conclusion: Extensive HI is associated with obesity as well as decreases in overall variability and parasympathetic modulation. Obese patients with concomitant reduced HRV had higher risk of being metabolically unhealthy.

O1-3 Relationship between Asymmetric dimethylarginine in Umbilical Cord Plasma and Birth Weight Follows a U-shaped Curve

Junji Takaya1,2, Yuko Tanabe2, Yuichi Kuroyanagi2, Kazunari Kaneko2

1Department of Pediatrics, Kawachi General Hospital, Department of Pediatrics; 2Kansai Medical University

Objective: Placental transport of nutrients is dependent on vascular development, which determines blood flow to the placenta. The ability of the uterine artery to dilate during pregnancy may be specifically related to upregulation of multiple pathways for production of nitric oxide (NO). Asymmetric dimethylarginine (ADMA), an L-arginine analog and a nonselective NO synthase inhibitor is associated with cardiovascular and metabolic disorders. In addition, epidemiological studies confirm that the relationships between human birth weight and adult obesity, hypertension, or insulin resistance follow U-shaped curves. We hypothesized that increased ADMA and decreased NO might underlie the initial pathophysiologic events leading to insulin resistance. To test the hypothesis, the relationship between birth weight and ADMA or nitric oxide parameters was evaluated.

Methods: The study group consisted of 41 singleton subjects with gestational ages ranging from 36-41 wk, and birth weights ranging from 1,798-3,822 g. The subjects were divided into 9 infants with small for gestational age (SGA) and 32 with appropriate for gestational age (AGA). Their cord plasma ADMA, insulin, insulin-like growth factor-1(IGF-1), and adipocytokine levels were determined using enzyme-linked immunosorbent assays. The plasma NOX [nitrite (NO2-)+nitrate (NO3-)] levels were measured using the colorimetric assay.

Results: The relationship between birth weight and ADMA levels followed a U-shaped curve rather than inverse linear associations expected over a full range of birth weight distribution. ADMA positively correlated with birth weight in AGA group (p< 0.001, R=0.590), and inversely correlated with birth weight in SGA group (p< 0.05, R=-0.741). Plasma ADMA was inversely correlated with adiponectin (p< 0.05, R=-0.289) and quantitative insulin sensitivity check index (QUICKI) (p< 0.05, R=-0.294) in all subjects, while not correlated with NOx. Plasma glucose, ADMA, NOx, and resistin concentrations did not differ significantly between SGA and AGA group. However, plasma insulin, IGF-1, leptin, adiponectin and QUICKI were lower in the SGA group than in the AGA group.

Conclusions: Plasma ADMA levels in cord blood might be an early marker of fetal growth and insulin resistance. High plasma ADMA levels may represent prenatal programming of insulin resistance, and may be used as a predictor of adult diseases. An analysis of all of these reports would generate that the relationship between birth weight and these metabolic abnormalities in adult life can be described by a U-shaped curve. Our results indicate a possible role of ADMA in fetal life for future disorders characterized by insulin resistance.

O1-4 The Effect of Sfrp5, Wnt5a, Adiponectin, and Chemerin on Blood Pressure Regulation in Obese Children

Yan C Yin

The Second Affiliated Hospital of Xi'an Jiaotong University, Pediatric

The aim was to evaluate the associations of Sfrp5 and Wnt5a with blood pressure (BP), and to examine whether BP can be influenced by changes in adipocytokines, such as Sfrp5, adiponectin, chemerin, and hsCRP, in obese children after lifestyle intervention. A cross-sectional study was conducted in 263 obese children. In addition, a 6-month lifestyle intervention was performed in a subgroup of 89 obese children with hypertension. Anthropometric parameters, clinical data, adiponectin, chemerin, Sfrp5, and Wnt5a were measured at baseline and after lifestyle intervention. Sfrp5 and adiponectin levels were significantly lower in obese children with hypertension, but Wnt5a, hsCRP, and chemerin levels were elevated in obese children with hypertension. In multivariable linear regression analysis, Sfrp5, Wnt5a, adiponectin, chemerin, and hsCRP were associated with both standard deviation score-systolic blood pressure (SDS-SBP) and -diastolic blood pressure (SDS-DBP). Lifestyle intervention resulted in a significant improvement in BP and weight loss. These were accompanied by significant decreases in hsCRP and chemerin, and significant increases in Sfrp5 and adiponectin, whereas Wnt5a was not changed. Furthermore, the changes in Sfrp5, adiponectin, chemerin, and hsCRP act as partial mediators of the relationship between weight loss and BP reduction. Although Sfrp5 and Wnt5a levels correlated with BP at baseline, after lifestyle intervention, Sfrp5 is more sensitive to reduction in BMI and BP compared to Wnt5a, and the relationship between weight loss and BP reduction were partially mediated by changes in Sfrp5. So we speculate if Sfrp5 and Wnt5a each play a role in regulating BP, it must be different roles.

O1-5 The association of HLA-DR, DQ genotypes and CTLA4 polymorphisms with a presence of thyroid antibody in Japanese children with Type 1A diabetes

Tadayuki Ayabe, Misako Okuno, Tomoyuki Kawamura, Tokuo Mukai, Takahiro Mochizuki, Shouji Nakayama, Emiko Tachikawa, Yasusada Kawada, Ichiro Yokota, Shigetaka Sugihara

Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes (JSGIT)

Introduction: We have made it clear that among Japanese children with Type 1A diabetes (T1AD), the thyroid autoantibody (T-Ab) positive rate is 26.6%, higher in females, and rises depending on age in females. The aim of our study was to clear the genetic factors which associate with a high risk of T-Ab possession in Japanese pediatric T1AD.

Subjects and Methods: HLA-DRB1 and DQB1 were analyzed for the registered 909 T1AD patients (the average age at the time of the registration was 12 years and 5 months old and 217 cases had T-Ab) in the Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes, and the relation with the T-Ab (someone with more than one of the anti-thyroid peroxidase antibody and the anti- thyroglobulin antibody) was considered. CTLA4 rs231775 and rs3087243 polymorphisms were analyzed for 905 T1AD patients and compared with 455 healthy controls. Then, the influence on T-Ab possession of CTLA4 SNPs for 905 T1AD patients was considered. The contribution to the T-Ab possession was considered by using logistic regression analysis, making these genotypes (HLA-DRB1, DQB1, and CTLA4 SNPs), gender and age explanatory variables.

Results: The susceptible HLA-DRB1 and DQB1 alleles, haplotypes and genotypes to T1AD didn’t associate with the risk of T-Ab possession. In pediatric T1AD, CTLA4 rs231775 had a significantly high frequency of G allele compared with controls regardless T-Ab presence (OR: 1.56, p<0.001) and rs3087243 had a significantly high frequency of G allele compared with controls only in T-Ab positive T1AD (OR: 1.87, p<0.001). 2 SNPs of CTLA4 had a significantly high frequencies of G allele compared with T-Ab negative T1AD in T-Ab positive T1AD. The age (OR: 3.55~4.98), the sex (OR: 2.11) and CTLA4 rs3087243 (OR: 1.63) were statistically significant effects on the risk of the T-Ab possession with logistic analysis.

Conclusions: This study newly demonstrated CTLA4 rs231775 conferred susceptibility to T1AD regardless T-Ab presence, and females with allele G of CTLA4 rs3087243 were at high risk to possess T-Ab in the age dependence in Japanese pediatric T1AD.

O1-6 The Protective Effects of Adenovirus-mediated IL-10 Gene and Anti-CD20 Monoclonal Antibody on the Pancreatic β Cells of NOD Mice in the Early Stage of Natural T1D Onset

Li Tang1,2, Li Cheng2, Fan Lei2, Tian Fei2, Tang Aiping2

1Division of Endocrinology and Metabolism, QingDao Women and Children's Hospital1; 2Department of Pediatricsm QingDao University

Objective: To investigate the protective effects of Adenovirus-mediated IL-10 gene and anti-CD20 monoclonal antibody (mAbs) on the pancreatic β cells in nonobese diabetes (NOD) mice with type 1 diabetes mellitus (T1D) at early stage.

Methods: 35 female NOD mice at onset of diabetes and aged 17–20 weeks old were randomly divided into 5 groups. Mouse 1,2,3,4 and 5 groups were intravenously injected 500ug of anti-CD20 mAbs, 500ug of anti-CD20 mAbs combined with 100ul of Ad-IL-10, 100ul of Ad- IL-10, 100ul of Ad-GFP, 100ul of normal saline respectively. All mice were monitored for blood glycose everyday, and sacrificed 9 weeks after injection. Their serum levels of C-peptide were measured and the degree of insulitis were observed. Apoptosis related gene and protein were detected.

Results: (1) The blood glucose level of mice in group 2 was reduced. (2) A majority of the insulitis was grade 0-1 in group 2, and grade 2-3 in group 5. (3) The apoptosis rate of pancreatic β cells was significantly lower in group 2 than that in the group 3,4,5, p<0.05. (4) Immunohistochemistry indicated that IL-10 could be highly expressed locally in the pancreatic islets. (5) The results of qPCR and western blot showed that combined intervention exerted protective effects on pancreatic β cells through activating Bcl-2 anti-apoptosis pathway, inhibiting the expression of TNF- α and Fas, and blocking caspase-8--caspase-3 as well as caspase-9--caspase-3 apoptosis pathways.

Conclusion: Combined intervention could reduce the apoptosis of pancreatic β cells of NOD mice in the early stage of T1D, and had certain protective effects on the residual pancreatic β cell function.

O2-1 Clinical outcome and mutation spectrum of the StAR gene in patients with congenital lipoid adrenal hyperplasia

Eungu Kang1, Yoon-Myung Kim1, Jin-Ho Choi1, Gu-Hwan Kim2, Beom Hee Lee1,2, Han-Wook Yoo1,2

1Department of Pediatrics, Asan Medical Center Children's Hospital, University of Ulsan College of Medicine; 2Asan Medical Center Children's Hospital, University of Ulsan College of Medicine, Medical Genetics Center

Purpose: Congenital lipoid adrenal hyperplasia (CLAH) is caused by mutations in the StAR gene and StAR protein plays a critical role for transport of cholesterol to mitochondria. The aim of this study was to investigate StAR mutation spectrum and associated clinical and endocrinologic characteristics in patients with CLAH.

Methods: The study included 45 patients with CLAH from 42 unrelated families diagnosed by clinical features, endocrine profile, and mutation analysis. Clinical features, endocrine data, and radiologic finding were reviewed retrospectively. Seven exons and associated intronic flanking regions of the StAR gene were amplified by PCR and directly sequenced.

Results: Most patients (42/45, 93.3%) with StAR defect presented with adrenal crisis in the neonatal period, while 3 late-onset patients with skin hyperpigmentation after age 2 years. Among 45 patients, 19 were genetic males with 46,XY karyotype, 21 had a 46,XX, whereas the other 5 patients’ karyotype was not available. Patients with non-classic type CLAH presented with skin hyperpigmentation and chronic adrenal insufficiency without salt-wasting crisis after age 12 months. Two genetic females having compound heterozygous mutations showed late onset adrenal insufficiency, while one genetic male with compound heterozygous for p.Q258* and p.R272H was incompletely virilized and raised as a boy. Three pubertal-aged genetic females exhibited spontaneous breast development at age 10 to 13 years. One of them showed spontaneous menarche at age 13 years and experienced ovarian cyst torsion at age 14 years. Mental retardation was found in two patients. One patient died of adrenal crisis at the age of 2 months due to poor adherence to medication. The genotype of the StAR was clarified in all patients, identifying 9 different mutations. Of these, p.Q258* was the most common (86.9%, 73/84 alleles), suggesting founder effect.

Conclusion: CLAH has wide clinical spectrum from life-threatening adrenal insufficiency in early infancy to chronic adrenal insufficiency thatcan present much later in life. CLAH was known to be mainly caused by p.Q258* mutation in Korea by founder effect. However, this study indicate that mutations of the StAR gene have diverse spectrum with various mutations.

O2-2 Biochemical diagnosis of 17 α -hydroxylase deficiency by urinary steroid metabolites in Japanese children

Yuhei Koyama1, Keiko Homma2, Eishin Ogawa3,4, Ichiro Miyata5, azumichi Onigata6,7, Tomonobu Hasegawa8

1LSI Medience Co. Special Pharmacology Analysis Department, Controlled Substance erated Tsting Group; 2Keio University Hospital, Central Clinical Laboratories; 3Department of Pediatrics, Teikyo University School of Medicine; 4Department of Pediatrics, Tohoku niversity School of Medicine; 5Department of Pediatrics, The Jikei University School of edicine; 6Department of Pediatrics, Shimane University Faculty of Medicine; 7Department of Pediatrics, Gunma University School of Medicine, Department of Pediatrics; 8Keio niversity School of Medicine

Background: 17 α -hydroxylase deficiency (17OHD) is one of congenital adrenal hyperplasia due to CYP17A1 gene abnormality. Affected 46,XY patients usually have female external genitalia. Corticosterone (B) metabolites and cortisol (F) metabolites ratio, a theoretical biochemical marker of 17a-hydroxylase activity, was reported to be increased in 17OHD. However, no data are available to distinguish 17OHD from cytochrome P450 oxidoreductase deficiency (PORD) which is a disease theoretically showing similar urinary steroid metabolites pattern.

Objective: The objective of this study was to establish differential diagnostic approach of 17OHD from PORD using urinary steroid profile.

Methods: We recruited genetically confirmed 5 Japanese children of 46,XY 17OHD, age between 6 months - 17 years, and 254 controls (22 PORD, 25 disorders of sex development (DSD), and 207 non-disease controls. We measured urinary steroid metabolites by gas chromatography mass spectrometry (mg/g creatinine) including B metabolites (B-group; 5a-, 5b-tetrahydrocorticosterone and 5a-, 5b-tetrahydro-11-dehydrocorticosterone), F metabolites (F-group; 5a-, 5b-tetrahydrocortisol and 5a-, 5b-tetrahydrocortisone), and pregnanetriolone (Ptl). We calculated B-group/F-group ratio.

Results: Twelve patients (all five 17OHD and 7 out of 22 PORD) showed B-group/F-group ratio of more than 1 (upper figure). Among 12 patients, Ptl showed no overlap between 17OHD and PORD using cutoff value of 0.3 mg/g creatinine (lower figure).

Discussion: These data indicated that biochemical differential diagnosis of 17OHD from PORD in Japanese children was possible by urinary steroid metabolites; B-group/F-group ratio and Ptl with cutoff values of 1 and 0.3 mg/ g creatinine, respectively. Future studies are needed to assess these urinary biochemical markers can distinguish 17OHD from isolated 17,20-lyase deficiency, another phenotype of CYP17A1 gene abnormality and adrenal tumor, likely showing similar urinary steroid metabolites pattern of 17OHD.
Fig. 1 (abstract O2-2).
Fig. 1 (abstract O2-2).

See text for description

O2-3 Induction of Cyp21a1 with AAV vector ameliorates systemic steroid metabolism in a mouse model of congenital adrenal hyperplasia

Yasuhiro Naiki1, Mami Miyado2, Reiko Horikawa1, Noriyuki Katsumata2, Shuji Takada3, Maki Fukami2

1Division of Endocrinology and Metabolism, National Center for Child Health and Development; 2Department of Molecular Endocrinology, National Research Institute for Child Health and Development; 3Department of Systems BioMedicine, National Research Institute for Child Health and Development

Background: Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase (21-OH) deficiency (21-OHD) is an autosomal recessive disorder, in which CYP21A2 mutations or deletions result in underproduction of glucocorticoid and mineralocorticoid, and overproduction of androgens. Patients with CAH are treated with oral steroid supplementation, but optimal control of blood steroid levels remains difficult. Thus, new therapeutic approaches are still needed. Previously, adenovirus-mediated administration of human CYP21A2 to adrenal glands rescued the phenotype of a mouse model of 21-OHD. To date, adeno-associated virus vectors became a good candidate with its weak immunogenicity and are capability of delivering genes to various tissues to maintain stable expression. In this study, we examined whether transduction of murine Cyp21a1 in extra-adrenal tissues could rescue steroid metabolism in 21- OHD mice.

Methods: A naturally occurring mouse model of 21-OHD was obtained by mating heterozygous pairs. Heterozygous pregnant mothers received daily injections of dexamethasone from late pregnancy to the day of delivery, to prevent deaths of newborn pups. Homozygous newborn mice received daily injections of corticosterone and fludrocortisone during the first 3 weeks after birth. A serotype-2 AAV vector containing Cyp21a1 cDNA and a cytomegalovirus promoter was constructed and we injected the AAV vector to the thigh muscles of the homozygote mice aged between 3 and 10 months (n = 4 for Cyp21a1- containing vector and n = 2 for control). Serum progesterone and DOC levels were measured before and 4 weeks after injection. One AAV-injected mouse was subjected to monthly blood sampling during 8 months after injection.Progesterone and DOC in serum samples were measured by liquid chromatography tandem mass spectrometry.

Results: Serum progesterone/DOC ratios were markedly reduced in all four animals at 4 weeks after injection. The effect had been continued until 8 months after injection. (Figure)

Conclusion: These results indicate that extra-adrenal induction of Cyp21a1 ameliorates steroid metabolism in 21-OHD mice for long term. This study suggests a novel therapeutic strategy for congenital adrenal hyperplasia, which warrants further investigations.
Fig. 1 (abstract O2-3).
Fig. 1 (abstract O2-3).

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O2-4 The effect of glucocorticoid treatment on bone mineral density in children with congenital adrenal hyperplasia: systematic review and meta-analysis

Agustini Utari1,2, Siti RF Saldi3, Bambang Tridjaja1, Jose R Batubara1

1Division of Pediatric Endocrinology, Department of Pediatric, Faculty of Medicine , University of Indonesia/ Cipto Mangunkusumo Hospital; 2Division of Pediatric Endocrinology, epartment of Pediatric, Faculty of Medicine , Diponegoro University ; 3Clinical Epidemiology and Evidence-Based Medicine (CEEBM) Unit, Faculty of Medicine , University of Indonesia/ Cipto Mangunkusumo Hospital

Background: Congenital Adrenal Hyperplasia (CAH) is a disorder characterized by the impaired activity of one of the enzymes needed for cortisol synthesis. The most common cause of CAH is the 21-hydroxylase deficiency that leads to cortisol and aldosterone deficiency in addition to androgen excess. Therapy in these cases is a long-life treatment of glucocorticoid and often mineralocorticoid. Many reports consider these patients were at risk of glucocorticoid induces osteoporosis. However, bone tissue is significantly affected by androgens and glucocorticoid. There were conflicting results regarding the effect of glucocorticoid treatment on bone mineral density (BMD) in CAH patients. Objectives: The objectives of this present study were to evaluate the effect of glucocorticoid treatment on BMD in children with CAH compared to normal healthy children.

Search methods: We performed literature search of studies using Cochrane Library, MEDLINE, EBSCO, PROQUEST, and another database to identify studies of BMD and CAH through May 2015. We also searched the reference lists of articles and contacted researchers in the field. We retrieved English language articles for review, and we searched for both published and unpublished studies.

Selection criteria: Randomized controlled trial, cohort, case control and cross sectional studies was considered. The type of participants was CAH patient who has already on glucocorticoid treatment at least two years. The comparison for these patients were normal healthy children. The outcome measure in this review was Whole BMD Z-score and Lumbar Spine BMD Z-score, which were measured by dual-energy X-ray absorptiometry (DXA).

Data collection and analysis: Two authors reviewed (AU and JB) independently abstracts for inclusion and read full- text articles to extract data. The risk of bias was assessed regarding randomisation, allocation sequence concealment, blinding, incomplete outcome data, selective outcome reporting, and other biases.

Main Results: We included nine studies involving 222 patients for systematic review; however only four studies involving 84 patients could be included in the meta-analysis. There were no randomized studies that met inclusion criteria. Thus we use non-randomised study that fulfilled the inclusion criteria. The meta-analysis showed that there was no significant mean difference between Whole BMD Z-Score and Lumbar spine BMD Z-Score among children with CAH who treated with glucocorticoid compared to normal healthy child (p=0.57, 95% CI, -0.46-0.84 and p = 0,86; CI 95%, -2,3 – 1,94, respectively).

Conclusions: Whole BMD and Lumbar spine BMD Z-Score in children with CAH treated with the glucocorticoid is similar with normal children.

O2-5 Late-onset glucocorticoid responsive circulatory instability of preterm infants

Hye Rim Chung1, Won Im Cho1, Chang Won Choi2, Byeong il Kim2

1Division of Endocrinology and Metabolism / Department of Pediatrics, Seoul National University Bundang Hospital; 2Division of Neonatology / Department of Pediatrics, Seoul National University Bundang Hospital

Background: An increasing number of preterm infants were treated with glucocorticoids for late-onset circulatory instability (CI) thought to be caused adrenal insufficiency. However, the presence of adrenal insufficiency, which is presented with late-onset CI in preterm infants, is not fully verified yet. We aimed to describe the incidence and clinical features of late-onset CI which responded to glucocorticoid replacement, and to determine the frequency of low serum cortisol values that meet criteria for relative adrenal insufficiency in these preterm infants.

Methods: Among 1,840 preterm infants who were admitted to a single neonatal intensive care unit between 2010 and August 2014, 47, who showed clinical signs of CI after 7 days-old and were recovered shortly after hydrocortisone replacement, were enrolled. Clinical signs of CI included hypotension, hyponatremia, hyperkalemia and oliguria. Infants with infection, hypovolemia, and cardiac problems were excluded. Medical records including serum cortisol levels during CI were reviewed retrospectively.

Results: Median gestational age (GA) of 47 infants was 28+2 weeks (range, 24+0 - 33+6 weeks), and median birth weight was 995 g (range, 430 - 2,275 g). Median age of development of CI was 20 days (range, 8 - 49 days). Incidence of CI was 2.6% (47/1,840) in all preterm infants; 8.65% (47/543) in infants of ≤ 34 weeks of GA; 18.2% (22/110) in infants of ≤ 28 weeks of GA. Incidence of CI in infants below 1,500g birth weight was 13% (40/302). Median serum cortisol level during CI was 8.0 μ g/dL (range, 2.0 - 12.0 μ g/dL), which met the criteria of vs. 129.3 mmol/L, P <0.001), and hourly urine output (3.7 vs . 1.7 mL/kg/hour, P <0.001) were decreased, and serum potassium levels (4.64 vs .5.99 mmol/L, P <0.001) were increased when compared the levels before CI. The clinical signs of CI were improved within 24 hours after hydrocortisone replacement in all 47 infants.

Conclusions: Our findings suggest that adrenal insufficiency is presented with late-onset glucocorticoid responsive CI, and is not rare in preterm infants.

O2-6 Comprehensive Steroid Profile in Classic 21-Hydroxylase Deficiency: Clinical and Hormonal Correlations

Karn Wejaphikul1,2, Hataichanok B. Kongmanas1, Pongsathorn ittichan3, Watchara Sirisuwan3, Thiti Snabboon3, Taninee Sahakitrungruang1

1Faculty of Medicine, Department of Pediatrics, Chulalongkorn University; 2Faculty of Medicine, Department of Pediatrics, Chiang Mai University; 3Faculty of Medicine, Department of Internal Medicine, Chulalongkorn University

Introduction: Optimal treatment of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) requires monitoring of clinical parameters and steroid markers such as 17-hydroxyprogesterone (17-OHP), androstenedione, and testosterone. However, an increase of 17-OHP levels is commonly observed during pubertal period, due to gonadal co-secretion of 17-OHP. Since the accumulated 17-OHP can be converted to 21-deoxycortisol (21-DOC) via 11-hydroxylation in adrenals only. We hypothesize that 21-DOC may serve as a better adrenal steroid marker for treatment monitoring in CAH youth.

Objectives: We aim to evaluate the utility of comprehensive serum adrenal steroids by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as markers of clinical control in CAH youth, and compare two different 17-OHP and testosterone assays (LC-MS/MS vs. commercial immunoassays).

Methods: A cross-sectional study of 37 patients (24 females) with classic 21-OHD aged 1-26 years were enrolled. Single serum samples were collected at 8 AM before the morning dose of glucocorticoid to measure steroid panels by LC-MS/MS and immunoassays. Patients were classified as being in “good control” or “poor control” based on clinical criteria including signs of androgen excess, degree of hyperpigmentation, bone age advancement, and ACTH levels. Comparisons of serum steroid concentrations were performed between two groups. The receiver operating characteristic (ROC) curves were used to determine the cut-off values for diagnosing “poor control”.

Results: Serum 17-OHP, progesterone, androstenedione, testosterone, and DHEA concentrations were higher in “poor control” group, but 21-DOC and DHEAS levels were not different. The ROC curves showed that 17-OHP, androstenedione, progesterone, and testosterone concentrations were the best hormonal predictors of clinical poor control with areas under the curves (95%CI) of 0.91 (0.79-1.0), 0.92 (0.82- 1.0), 0.88 (0.76-1.0), 0.87 (0.73-0,99), P 0.001, respectively. By the Bland & Altman plot, the differences of 17-OHP and testosterone levels between LC-MS/MS and immunoassay were 9.2 ng/mL (95%CI -82 to 101), and 0.6 ng/mL (95%CI -2.1 to 3.2), respectively. The higher levels of 17-OHP or testosterone exhibited higher discrepancy between two methods.

Conclusion: Serum 17-OHP, androstenedione, and testosterone concentrations are of significance in diagnosing clinical poor control. 21-DOC is not useful for treatment monitoring in CAH. Despite decent overall correlation, absolute 17-OHP and testosterone concentrations differed substantially between two different assays. Frequent monitoring of clinical parameters and standardized methods of steroid assays are crucial to diminish the

O3-1 Analysis of endocrinopathy as late effects in Childhood cancer survivors at a single institute in Japan

Tomoko Yoshida1, Kanako Nakao1, Yuta Chiba1, Yumiko Terada1, Asuko Ogiwara1, Keisuke Yoshii1, Chikako Kiyotani1,2, Yoko Shioda2, Tomoo Osumi2, Daisuke Tomizawa2, Motohiro Kato2, Kimikazu Matsumoto2, Yasuhiro Naiki1, Reiko Horikawa1

1Division of Endocrinology and Metabolism, National Center for Child Health and Development; 2National Center for Child Health and Development, Children's Cancer Center

Background: The survival rates for children and adolescents with malignant cancers have increased these days, along with the improvement of treatments modality. Consequently, the childhood cancer survivors (CCSs) with late treatment-related complications, especially with endocrinopathy are increasing.

Objective: To assess the frequency and characteristics of endocrinopathy of CCSs in single institute in Japan.

Methods: 272 CCSs from 2002 to 2016 were involved in this study. Patients with craniopharyngioma were excluded because of no chemotherapy and radiotherapy. 71 patients with benign disorders, complicated with other syndromes, or last cancer treatment within one year were excluded from 272 CCSs. The number of original diseases are: solid tumors 83 (neuroblastoma, langerhans cell histiocytosis, retinoblastoma, rhabdomyosarcoma, hepatoblastoma, Wilms’ tumor), brain tumors 51 (medulloblastoma, germinoma, teratoma, optic glioma, ependymoma), hematological malignancies 67 (Acute lymphocytic leukemia, acute myeloid leukemia, lymphoma). The medical history and the hormonal data of 201 patients (98 males, 103 females, aged 2 ~ 19 (average 12) years) at our endocrine follow-up clinic for CCSs were obtained retrospectively.

Results: Over all, growth hormone deficiency were observed in 32 patients (20 in brain tumor (39.2% in brain tumor) and 12 in others (8.0% in other tumor)), thyroid dysfunction in 22 (12 in brain tumor (23.5%) and 10 in others (6.6%)), adrenal dysfunction in 18(8 in brain tumor (15.7%) and 10 in others (6.6%)), respectively. Among those with brain tumors, AVP replacement was performed in 9 (17.6%). In 146 patients with pubertal age (male>13 years and female>11years), sex hormone replacement were necessary in 15 (10.3%). GnRH agonist was prescribed for 21 (10.4%) in children below 12 years old, except 1 case, because of central precocious puberty or early puberty for height. Hormone replacement was more common in patients with hypothalamic-pituitary tumors. Patients with neuroblastoma (n=27) also showed higher frequency of hormone replacement therapy. Adult height was obtained from 5 patients with neuroblastoma, 3 of them showed adult height less than -4SD without GHD.

Assessment: Patients with brain tumor was at higher risk of pituitary dysfunction. Patients with neuroblastoma, when compared to those with other peripheral solid tumor, tended to have growth failure and hormone deficiency suggesting that intense chemotherapy and radiation therapy may have disrupting effect on endocrine system and resistance to growth hormone.

Conclusion: Endocrinopathy is common for CCSs. We need to continue surveillance to know more to follow them up appropriately.

O3-2 Genome-wide analysis of differential DNA methylation in Silver-Russell syndrome

Di Wu, Chunxiu Gong

Department of Endocrinology, Genetics and Metabolism, Beijing Children's Hospital, Capital Medical University

Background: Silver-Russell Syndrome (SRS) is clinically heterogeneous disorder characterized by low birth weight, postnatal growth restriction and variable dysmorphic features. The aetiology of SRS is complex and current evidence strongly implicates imprinted genes. Although approximately half of all patients exhibit DNA hypomethylation at the H19/IGF2 imprinted domain, and around 7%-10% have maternal uniparental disomy of chromosome 7(UPD (7) mat), no clear mechanism is apparent in the other patients. In this study, we aim to look for further DNA methylation defects in SRS patients.

Methods: We measured DNA methylation in 7 SRS patients and 5 controls at >485,000 CpG sites using DNA methylation microarrays. We analyzed methylation changes genome-wide as well as at known imprinted regions to identify SRS-associated epimutations. Then we used bisulfite sequencing and digital PCR to identify the differentially methylated regions (DMRs) we found.

Results: Our analysis identifies epimutations at the previously characterised domains of H19/ IGF2, providing proof of principle that our methodology can detect DNA methylation changes at imprinted loci. In addition we discovered a novel imprinted gene OSBPL5 associated with SRS and located at chromosome 11p14with the probe cg25963939 hypomethylated in 4/7 patients (P=0.023, β =-0.243). We also report DMRs in other genes including TGF β 3 AHSF1 AGAP43 ANOTCH4 AMYH14, which might be associated with SRS by GO pathway analysis.

Conclusions: We identified the probe cg25963939, located at the 5’UTR of imprinted gene OSBPL5, as a noval DMRs, which associated with SRS. This findings provide a new mechanism of SRS etiology and aid the further stratification of SRS patients by molecular phenotypes.

O3-3 Serum antimüllerian hormone and inhibin B as potential markers for progressive central precocious puberty in girls

Linqi Chen, Ting Chen, Haiying Wu, Fengyun Wang, Xiuli Chen, Ngrong Xie

Division of Endocrinology and Metabolism / Department of Pediatrics, The Children’s Hospital of Soochow University

This abstract is not included as it has already published.

O3-4 Genotype and phenotype of 101 Vietnamese Patients with Congenital Hyperinsulinism

Dung Chi Vu1, Duong Anh Dang1, Ngoc Thi Bich Can1, Khanh Ngoc Nguyen1, Thao Phuong Bui1, Hai Thanh Le1, Dat Phu Nguyen1, Dien Minh Tran1, Ellard Sian2, Flanagan E Sarah2

1Department of Endocrinology, Metabolism and Genetics, National Children's Hospital; 2University of Exeter Medical School. UK, Molecular Genetics, Royal Devon & Exeter NHS

Background: Hyperinsulinemic hypoglycemia (HH) is a consequence of unregulated insulin secretion by pancreatic β -cells. Congenital HH is caused by mutations in genes involved in regulation of insulin secretion (ABCC8 , KCNJ11 , GLUD1 , CGK , HADH , SLC16A1, HNF4A and UCP2 ). Severe forms of congenital HH are caused by inactivating mutations in ABCC8 and KCNJ11, which encode the two components of the pancreatic β -cell ATP-sensitive potassium channel.

Objective and hypotheses: Our aim is to identify mutations in the ABCC8 and KCNJ11, HNF4A and GLUD genes, and to describe genotype and phenotype correlations of Vietnamese children with congenital hyperinsulinism.

Method: A prospective study was conducted on 101 cases with congenital hyperinsulinism diagnosed and treated at Vietnam Children’s Hospital from January 2007 to June 2016. Patients were selected by using inclusion criteria of Hussain K (2008). All exons of ABCC8; KCNJ11 , HNF4A and GLUD1 were amplified from genomic DNA and directly sequenced.

Results: Mutations were identified in 52 cases (51.5%) including mutations of ABCC8 gene (46 cases; 45.5%), Among these cases 26 with homozygous/compound heterozygous of ABCC8 and 20 cases with one paternal/maternal mutation of ABCC8 gene); KCNJ11 (5 cases; 5.0%), HNF4A (1 case; 1.0%). 100% of cases with homozygous/compound heterozygous recessive mutations or one paternal dominant mutation of ABCC8 gene did not respond to diazoxide treatment and required 95% pancreatectomy or octreotide injection. Other cases without identified mutations responded to diazoxide and/or glucose infusion.

Conclusion: children with congenital hyperinsulinism should be performed mutation analysis which helps in making diagnosis and treatment decision. Families of children with congenital hyperinsulinism should be given genetic counseling. Prenatal diagnosis should be performed as well as follow - up and treatment should be given to children with congenital hyperinsulinism immediately after birth.

O3-5 Short and long-term outcome of patients with congenital hyperinsulinism

Yumiko Terada, Yusuke Fujisawa, Yuta Chiba, Yasuko Ogiwara, Tomoko Yoshida, Kanako Nakao, Keisuke Yoshii, Yasuhiro Naiki, Reiko Horikawa

Division of Endocrinology and Metabolism, National Center for Child Health and Development

Background: Congenital hyperinsulinism (CHI) occurs in 1/25000 to 1/50000 birth and frequently becomes the cause of persistent hypoglycemia in neonates. Most of persistent hypoglycemia states are caused by genetic defects. Some can be managed by medication, some can be cured by surgical resection of focal pancreatic lesion, but some are intractable and uncontrolled by any medication.

Objective: To evaluate the adequacy of treatment choice and long-term prognosis for CHI patients in one institute.

Methods: We retrospectively investigated clinical course, laboratory data, diagnosis, treatment modality, and short and long-term outcome in patients with hyperinsulinemic hypoglycemia through their medical records from 2002 to 2016.

Results: 41 patients from 3-month-old to 46-year-old (current age), diagnosed as transient or persistent hyperinsulinemic hypoglycemia were involved in this study. Observation periods are from 3 months to 14 years. Most of the patients were diagnosed in their first to second days after birth, while 9 patients were diagnosed at 3 to 9-month-old. 12 cases (31.6%) were transient, and 10 out of these 12 were born preterm or LFD (Light for date). Among 41 patients, 12 (31.6%) were born HFD (Heavy for date), 9 of which had ATP-sensitive potassium channel abnormalities. 19 patients (46%) were diagnosed as CHI. Genetic evaluations were performed in 21 patients; particular gene mutations were found in 12 patients (57%) (ABCC8 gene mutation in 9, KCNJ11 gene mutation in 1, GLUD1 gene mutation in 2 ). The long-term prognosis of 19 CHI patients, 1 case was treated only by diet control, 8 cases continue diazoxide, 1 case was controlled by octreotide, 1 case by diazoxide and octreotide combination, and 8 cases had partial pancreas resection or subtotal pancreas resection. The decision of partial resection was decided upon the result of 18F-DOPA PET-CT. Among those who had subtotal (>95%) resection, 4 patients became insulin-dependent diabetes mellitus, and 2 cases continuing the treatment for hyperinsulinemic hypoglycemia. Considerable mental retardation is observed in only one grown-up case.

Conclusions: Genetic testing and imaging study using 18F-DOPA PET-CT, in addition to clinical evaluation, are helpful for the decision of treatment choice. Long-term neurological outcome with adequate medical and surgical treatment seems to be better in recent cases when compared to old case, however, further evaluation is necessary.

O3-6 Rare frequency of mutations in pituitary transcription factor genes in patients with combined pituitary hormone deficiency or isolated growth hormone deficiency in Korea

Yoon-Myung Kim Kim1, Eungu Kang1, Jin-Ho Choi1, Chang-Woo Jung1, Sun Hee Heo2, Minji Kang2, Gu-Hwan Kim3, Beom Hee Lee1, Han-Wook Yoo1

1Department of Pediatrics, Asan Medical Center Children's Hospital, University of Ulsan College of Medicine; 2Asan Medical Center Children's Hospital, University of Ulsan College of Medicine, Asan Institute for Life Sciences; 3Asan Medical Center Children's Hospital, University of Ulsan College of Medicine, Medical Genetics Center

Purpose: Combined pituitary hormone deficiency (CPHD) is caused by mutations in pituitary transcription factors involved in the development of the hypothalamic-pituitary axis. Minor allele frequencies of genes involved in CPHD are so low and varies substantially between ethnicities. This study evaluated the frequency of mutations in the most relevant transcription factor genes (POU1F1 , PROP1 , LHX3 , LHX4 , and HESX1 ) in patients with CPHD and isolated growth hormone deficiency (IGHD).

Method: This study included 27 patients with IGHD and CPHD. The following parameters were analyzed: age at diagnosis, height, weight, birth history, combined anterior pituitary function tests, and sellar magnetic resonance image (MRI) findings. Mutation analysis of the POU1F1 , PROP1 , LHX3 , LHX4 and HESX1 genes were performed using genomic DNA from peripheral blood leukocytes.

Results: IGHD was observed in 4 patients and CPHD in 23 patients. Age at diagnosis was 8.28 ± 7.25 years (range, 0.2–16.9 years) in IGHD and 13.48 ± 10.46 years (range, 0.2–35 years) in CPHD (P = 0.37). Serum IGF-1 and peak GH levels after GH provocation tests were significantly low in patients with CPHD compared to those of IGHD (P <0.05). The sellar MRI findings found structural abnormalities in 3 patients with IGHD (75%) and 21 patients with CPHD (91.3%) (P = 0.62). Mutation analysis identified homozygous c.326G>A (p.R109Q) mutations in HESX1 in a patient with CPHD. No mutation was identified in POU1F1 , PROP1 , LHX3 , and LHX4 genes in the other patients.

Conclusion: Patients with CPHD had severe GHD than those with IGHD. Patients with GHD should be evaluated for other systemic diseases and endocrine functions by means of ophthalmologic examination, sellar MRI, and combined anterior pituitary function tests. As the mutation frequency of pituitary transcription factors is rare in patients with CPHD and IGHD, further research should be done to investigate other causative genes or environmental factors.

O4-1 A reversible albumin-binding once-weekly growth hormone (GH) treatment (somapacitan; NNC0195-0092) induces a dose- dependent IGF-I response similar to daily GH therapy in children with growth hormone deficiency (GHD)

Michael Højby Rasmussen2, Tadej Battelino1, Jean de Schepper3, Nehama Zuckerman-Levin4, Zoran Gucev5, Lars Savendahl6

1University of Ljubljana, Faculty of Medicine; 2Novo Nordisk A/S, Global Development; 3Division of Paediatric Endocrinology, Universitair Ziekenhuis Brussel; 4Rambam Medical Center, Pediatric and Obesity Clinic; 5Medical Faculty Skopje, University Paediatrics Clinic; 6Department of Women's and Children's Health, Karolinska Universitetssjukhuset

This randomised, open-label, active-controlled, dose-escalation trial (NCT01973244) investigated single-dose exposure and insulin-like growth factor (IGF)-I response to somapacitan in 32 children with GHD after a 7–10-day GH-washout period. Pre-pubertal children (aged 6–13 years) were assigned equally to four cohorts: 0.02, 0.04, 0.08 and 0.16 mg/kg somapacitan, pending safety assessments performed at each dose level increase. Within each cohort, six children were randomised to a single dose of somapacitan and two were randomised to Norditropin® SimpleXx® once daily at a fixed dose of 0.03 mg/kg/day for a week. The once-weekly dose range of somapacitan was expected to cover the clinically relevant dose range in GHD as guided by simulations of the anticipated PK and IGF-I response in children with GHD. There were dose-dependent increases in somapacitan serum concentrations and in baseline-adjusted IGF-I AUC (0–168h) (area under the IGF-I concentration–time curve from 0–168h) and maximum concentration (Cmax). Compared with once-daily GH, IGF-I responses were lower with 0.02 mg/kg somapacitan (estimate: 0.56 ng*h/mL [95%CI: 0.44; 0.71], p <0.0001), but similar with 0.04 and 0.08 mg/kg (estimated ratios, approximately 1:1) and higher with 0.16 mg/kg (1.25 ng*h/mL [95%CI: 0.98; 1.59], p =0.0672). Only an initial peak in IGF-I response to 0.08 mg/kg was outside the reference range (+2 to –2 SDS). A similar dose-dependent response was observed for IGF-binding protein-3 (IGFBP-3) with the initial peak being largest with 0.16 mg/kg somapacitan (2.3 mg/kg vs. 0.6–1.3 mg/kg for other doses) and subsequent stabilisation within reference range. No serious adverse events with either treatment and few local tolerability issues were observed (four transient injection site reactions in three children all receiving 0.16 mg/kg somapacitan). All somapacitan doses were well tolerated with no clinically relevant safety or immunogenicity issues observed.

In a model-based analysis, steady-state PK and IGF-I responses were simulated across dose levels and compared to daily GH treatment for Cmax and average concentration (Cavg). In this model, an IGF-I dose of 0.04 mg/kg is expected to provide Cmax IGF-I levels matching average daily GH treatment; 0.08 mg/kg is expected to provide Cavg IGF-I levels matching average daily GH treatment; 0.16 mg/kg is expected to provide higher IGF-I levels, with average concentrations within +2 SDS after a single dose or at steady state.

In conclusion, the single-dose results indicate that doses of 0.04, 0.08 and 0.16 mg/kg may be suitable for once-weekly administration of somapacitan and may be effective for the treatment of childhood GHD.

O4-2 Machine Learning and Transcriptomics Accurately Predict Diagnosis and Severity of Childhood Growth Hormone Deficiency

Philip Murray1, Adam Stevens1, Ekaterina B Koledova2, Pierre Hatelain3, Peter Clayton1

1University of Manchester and Royal Manchester Children's Hospital; 2Merck KGaA; 3University Claude Bernard

Background: The diagnosis of Growth Hormone Deficiency (GHD) involves the use of multiple GH stimulation tests which require day case admission, multiple blood samples and the administration of pharmacological agents. Replacing these tests with a single blood sample would be a major advance for patients being evaluated for GHD.

Aim: To assess the utility of gene expression (GE) profiling and candidate SNP analysis for the diagnosis and classification of GHD.

Method: Pre-pubertal treatment-naïve children with GHD (n=98) were enrolled from the PREDICT study and controls (n=26) acquired from online datasets. Whole blood gene expression (GE) was determined with Affymetrix HU133v2.0 microarrays. Genotyping was performed on DNA extracted from whole blood using Illumina GoldenGate for 1536 SNPs, located on 103 candidate genes. The correlation between GE and peak GH was investigated using rank regression and a Random Forest algorithm tested for prediction of the presence of GHD and in classification of GHD into severe (peak GH>4 μg/L ). For GHD severity classification, data on age, gender, baseline IGF-I and IGFBP-3 levels were added to the Random Forest model along with SNP genotype. Performance was assessed using Area under the Receiver Operating Characteristic Curve (AUC-ROC). A biological network of GE related to peak GH levels was generated and cluster hierarchy assessed.

Results: Rank regression identified 347 probesets representing 271 genes where expression correlated with peak GH concentrations: (R = + 0.28, p<0.01). These 347 probesets gave an AUC of 0.98 (sensitivity 100%, specificity 96%) for predicting GHD status (GHD versus controls). At the DNA level, 18 SNPs in 12 genes were associated with peak GH concentrations; 16/18 were intronic and none were rare (defined as Minor Allele Frequency <1%). The function of the genes associated with the SNPs included pituitary transcription factor (POU1F1 ), generation of oestrogen (CYP19A1 ), IGF binding (IGFBP1 ), apoptosis (BCL2), cell cycle (CCND3 ) and signal transduction (PTPN1 , RARA ). Random Forest analysis was also able to accurately predict GHD severity with an AUC of 0.93 using transcriptomic data but not improved with addition of demographic, biochemical or SNP genotype data.

Conclusion: GE profiling differentiates normal subjects from those with GHD and, in a cohort of GHD subjects, accurately predicts GHD severity. It may therefore be a useful tool in clinical practice to aid in the diagnosis of GHD, potentially replacing two GH stimulation tests with a single blood sample.

O4-3 Comprehensive clinical studies in 30 patients molecularly diagnosed with Temple syndrome

Masayo Kagami1, Keisuke Nagasaki2, Rika Kosaki3, Reiko Horikawa4, Yasuhiro Naiki4, Shinji Saitoh5, Toshihiro Tajima6, Akie Nakamura1, Keiko Matsubara1, Maki Fukami1, Tsutomu Ogata1,7

1Department of Molecular Endocrinology, National Research Institute for Child Health and Development; 2Department of Homeostatic Regulation and Development, Niigata University Graduate School of Medical and Dental Science; 3Department of Clinical Laboratory Medicine, National Center for Child Health and Development; 4Division of Endocrinology and Metabolism, National Center for Child Health and Development; 5Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences; 6Department of Pediatrics, Jichi Children's Medical Center Tochigi; 7Department of Pediatrics, Hamamatsu University School of Medicine

Background: Human chromosome 14q32.2 carries paternally expressed genes and maternally expressed genes together with the germline- derived DLK1-MEG3 intergenic differentially methylated region (IG-DMR) and the postfertilization-derived MEG3 -DMR. Consistent with this, maternal uniparental disomy 14 (UPD(14)mat), and epimutations and microdeletions affecting the imprinted region of paternal origin result in discernable and non-specific clinical features such as pre- and postnatal growth failure, hypotonia, early onset puberty, and small hands. Recently, this clinically recognizable disorder has been named “Temple syndrome” (TS14) (OMIM 616222) and identified in 58 patients. Clinical features of TS14 overlap with that of Prader-Willi syndrome (PWS) and Silver-Russell syndrome (SRS). Here, we performed comprehensive clinical studies in 30 patients molecularly diagnosed with TS14.

Subjects and Methods: We performed methylation analysis for the IG-DMR and the MEG3 -DMR in 186 patients with SRS-like phenotype without 11p15 loss of methylation or UPD(7)mat,157 patients with PWS-like phenotype with normal methylation pattern of the SNRPN - DMR, 202 patients with SGA-short stature (SGA-SS), together with 33 patients with TS14 phenotype. For patients with hypomethylation of these DMRs, we carried out structural analysis to detect paternal microdeletion, and microsatellite analysis to detect UPD(14)mat. To collect detailed clinical data of all patients, we used a comprehensive questionnaire from attending physicians.

Results: Thirty Japanese patients with TS14, UPD(14)mat (n=21); epimutations (n=5); microdeletions (n=3); and unknown (n=1), were identified. Their clinical diagnoses were TS14 (n=5), PWS (n=13), SRS (n=6), SGA-SS (n=2), and others (n=4). Fifteen patients had the score of 4 or more in Netchine-Harbison SRS clinical scoring. The combinatory method of PNA-NGS can detect somaticedian SD of birth weight, birth length, present height, and present weight are –2.1, –2.7, –2.3 and –1.6, respectively. Early onset puberty was identified in 11 out of 15 patients older than 4 years. Motor development was delayed in early infancy due to muscular hypotonia. Median developmental/intellectual quotient (DQ/IQ) was 83 (rage, 53–114), and two out of 15 patients who reached school age needed special education. Hyperlipidemia was identified in four out of 21 patients whose laboratory data were obtained. The median BMI was 15.1 (range, 10.9–37.3)

Discussion: We show detailed clinical features and course of 30 Japanese TS14 patients. Because of non-specific clinical features, clinical diagnosis of TS14 was difficult. Methylation analyses for the DMRs on chromosome 14 should be considered in patients with SRS- or PWS- compatible phenotype but no abnormal methylation pattern of the DMRs on chromosome 7, 11, and 15, as well as in patients with SGA-SS and hypotonia.

O4-4 Genome-wide copy-number analysis of fifty Silver-Russell syndrome patients without known etiology

TakanobuInoue1,2, Akie Nakamura1, Tomoko Fuke1, Kazuki Yamazawa1, Shinichiro Sano1, Keiko Matsubara1, Akira Oka2, Maki Fukami1, Masayo Kagami1

1Department of molecular endocrinology, National Center for Child Health and Development; 2Department of Pediatrics, The University of Tokyo Hospital

Background: Silver-Russell syndrome (SRS) is a rare disorder associated with pre- and postnatal growth failure and characteristic features. SRS is genetically heterogeneous. H19 -differentially methylated region (DMR) hypomethylation (H19 -hypo) and maternal uniparental disomy chromosome 7 (upd(7)mat) can be detected in approximately 50% of SRS patients, and the etiology of remaining patients is unknown. Recently, some cases with Temple syndrome, upd(16)mat, upd(20)mat, and submicroscopic chromosomal abnormality were recognized in SRS patients without known etiology, but sample size of these cohort was small.

Patients and Method: This study consisted of fifty SRS patients diagnosed based on Netchine-Harbison SRS clinical scoring system (NH- CSS). To clarify the exact frequency of patients with PCNV in SRS patients without known etiology, we included only patients with normal methylation pattern for nine DMRs (H19 , Kv, IG, MEG3 , PLAGL1 , PEG1 , PEG10 , SNRPN , and GNAS A/B). We extracted genomic DNA from peripheral leucocytes. Oligoarray comparative genomic hybridization was performed in all patients using 8 × 60K catalog array (Agilent technology). To determine whether copy number variations identified in the patients were pathogenic or not, we utilized the information from Database of Genomic Variants and medical publications. The frequency of clinical features between patients with pathogenic copy number variants (PCNV) and those with H19 -hypo or upd(7)mat (previously reported by us) was analyzed by Fisher’s exact probability test.

Result: PCNV were detected in five patients in our cohort. The abnormalities included 4p microdeletion, 11p15 microduplication (two patients), 17p12 microduplication, and mosaic 18 trisomy. Clinical features of patients with PCNV are shown in the Table. Of note, among the patients with PCNV four patients presented congenital heart diseases and three patients developed epilepsy. The frequency of relative macrocephaly and body asymmetry was lower in the SRS patients with PCNV than in H19 -hypo group.

Discussion: We identified five patients with PCNV in fifty SRS patients satisfying N-H CSS. Our study revealed exact frequency of patients with PCNV in SRS patients without known etiology. We suggest that copy number analysis would improve genetic diagnosis of the SRS patients without known etiology, especially those with atypical features including congenital heart defect or epilepsy.
Table 1 (abstract O4-4).

See text for description

O4-5 Efficacy and safety of recombinant human growth hormone treatment in patients with short stature related to bone and cartilage diseases

Guangling Li1, Shiyao Wang1, Huijuan Zhu1, San A2, Hongbo Yang1, Linjie Wang1, Fengying Gong1, Zimeng Jin1, Hui Pan1

1Peking Union Medical College Hospital, Chinese Academy of Medical Science, Department of Endocrinology, Key Laboratory of Endocrinology, National Health and Family Planning Commission; 2Binhai Genomics Institute, BGI-Tianjin, Tianjin Enterprise Key Laboratory of Clinical Molecular Diagnostic

Objective: To study the clinical characteristics of patients with short stature related to bone and cartilage diseases (BCD), and evaluate the efficacy and safety of the recombinant human growth hormone (rhGH) treatment in BCD patients.

Methods: Clinical data of 33 cases of BCD patients were retrospectively analyzed. Eleven of them had undergone genetic testing. Based on receiving rhGH treatment or not, all BCD patients were divided into the treated group (BCD-T) and the untreated group (BCD-C). Twenty- one age/gender matched growth hormone deficiency (GHD) patients were collected as the control (GHD-T) group. Clinical and laboratory parameters were analyzed between each follow-up and group. The safety of rhGH treatment in BCD patients was also evaluated.

Results: Thirty-three BCD patients with mean age of 7.74 years old and mean initial height standard deviation score (Ht-SDS) of -4.50 included 18 achondroplasia/hypochondroplasia (54.5%) patients, 5 spondyloepiphyseal dysplasia (15.2%) patients, 3 multiple epiphyseal dysplasia patients, and 7 others. All patients underwent genetic testing existed gene mutations. Each of FBN1, FGFR3 and COL2A1 mutations revealed in 2 patients. The other mutations included COL9A1, NPR2, TRAPPC2, RUNX2 and CENPJ . All 15 patients in the BCD-T group showed significant improvement in growth velocity (GV) and Ht-SDS after 12-month treatment (P<0.05). While 18 patients in BCD-C group had no change in GV and Ht-SDS before and after follow-ups (p>0.05). All changes of GV and Ht-SDS in the BCD-T, BCD-C, and GHD-T groups were shown in figure1 and figure2. The rhGH dosage needed in the BCD-T group was significantly higher than that in GHD-T group (p<0.05). Insulin-like growth factor-1 (IGF-1) levels were both significantly increased in the BCD-T and GHD-T groups after rhGH treatment (p<0.05). No serious adverse reaction occurred during the follow-ups.

Conclusions: Though the effect of rhGH therapy is poorer in BCD patients compared with that in GHD patients, an appropriate dose of rhGH therapy is still helpful to improve the GV and Ht-SDS. The study of the long-term safety of rhGH treatment in BCD patients is still required.

Key words: short stature; bone and cartilage diseases; recombinant human growth hormone
Fig. 1 (abstract O4-5).
Fig. 1 (abstract O4-5).

See text for description

O4-6 A hybrid Fc-fused human growth hormone, GX-H9, shows a potential for semi-monthly administration in both adult and pediatric growth hormone deficiencies

H. Michael Keyoung1, Aryaev Mykola2, Eun Jig Lee3, Jochen Schopohl4, Tae Kyung Kim1, Young-Joo Ahn5, Jung-Won Woo1, Woo Ick Jang5, Young- Chul Sung1

1Department of pathology of the newborns and premature infants, Genexine, Inc.; 2Municipal Institution; 3Department of Internal Medicine IV, Ludwig-Maximilians University Munich; 4Yonsei University College of Medicine, Endocrinology/ Internal Medicine; 5Handok, Inc

GX-H9 is a hybrid Fc-based long-acting recombinant human growth hormone (hGH). The safety, tolerability, and PK/PD were assessed in a single ascending dose study in healthy volunteers and in multiple sequential dose studies in patients with adult (AGHD) and pediatric growth hormone deficiencies (PGHD). The efficacy of GX-H9 was compared to that of a daily recombinant hGH.

A double-blind, randomized, placebo-controlled, single ascending dose Phase 1 study of GX-H9 was conducted in 4 groups of healthy subjects (n=32) with four sequential dose levels (0.2, 0.4, 0.8 or 1.6 mg/kg). Currently, a Phase 2, randomized, active-controlled, open-label, sequential dose study of GX-H9 (0.1 mg/kg/weekly, 0.2 and 0.3 mg/kg/semi-monthly) is being conducted in patients with AGHD (n=45). In addition, a Phase 2, randomized, active-controlled, open-label, multiple dose study of GX-H9 with weekly and semi-monthly administrations is being conducted in patients with PGHD (n=48).

Single doses of GX-H9 were well tolerated at all dose levels in healthy volunteers. No safety concerns were noted, including absence of any lipoatrophy or anti-drug antibodies. Geometric mean of t1/2 ranged between 69.2 and 138.0 hours. IGF-1 serum concentrations increased in a dose-dependent manner between 0.2 and 1.6 mg/kg. The interim Phase 2 results in AGHD have indicated that administration of 0.1 mg/ kg/week and 0.3mg/kg/every other week with GX-H9 for 12 weeks were safe and efficacious. The weekly treatment of 0.1mg/kg in AGHD patients demonstrated the mean increase in IGF-1 comparable with those receiving 6 μg/kg of Genotropin® daily for 12 weeks (101.3 ± 31.2 ng/mL vs 109.1 ± 45.0 ng/mL, respectively). A limited interim analysis of PK and IGF-1 level in PGHD study was performed for the first 12 subjects after completing a single dose period, demonstrating the safety of GX-H9 and dose-dependent PK profile in pediatric patients. The administration of higher doses showed a potential for semi-monthly treatment of GX-H9 in both AGHD and PGHD. The data from ongoing Phase 2 studies will be presented.

O5-1 Function study of NKX2-1 gene c.799G>T mutation, which caused congenital hypothyroidism and central nervous system disorders

Shiyao Wang, Huijuan Zhu, Hui Pan, Hongbo Yang, Linjie Wang, Zimeng Jin, Fengying Gong

Department of Endocrinology, Key Laboratory of Endocrinology, National Health and Family Planning Commission, Peking Union Medical College Hospital, Chinese Academy of Medical Science

Background: NK2 homeobox 1 (NXK2-1) is a relatively common responsible gene in congenital hypothyroidism (CH). It plays a very important role in the development of thyroid, lung and central nervous system (CNS). We identified a de novo heterozygous NKX2-1 mutation c.799G>T (p.Val235Phe, NP_001073136.1) in a pair of twin boys who had primary CH and ataxia. As the most common NKX2-1 gene mutation, the pathologic mechanisms of the c. 799G>T mutation is still undetermined.

Methods: We introduced the mutation c.799G>T in an NKX2-1 expression vector, and then analyzed the protein expression differences in HEK293 cells between wildtype and mutant vectors through transfection experiments and Western blot. After co-transfected the expression vectors and reporter vectors harboring TG/TPO /SP-B gene promoters, we performed the dual-luciferase assay to evaluate the function of NKX2-1 c.799G>T heterozygous mutation. Furthermore, we also analyzed local structure variations of mutant NKX2-1 p.V235F using bioinformatics software.

Results: Compared with wildtype NKX2-1, the expression of mutant NKX2-1 p.V235F was significantly reduced in HEK293 cells. All TG, TPO, and SP-B promoters could be activated by wildtype NXK2-1. TG promoter was activated in a dose-dependent pattern, while TPO and SP-B promoters were not. All TG, TPO or SP-B promoters were failed to be efficiently activated by mutant NKX2-1 p.V235F. And dose changes of mutant NKX2-1 p.V235F didn’t change the transcription activity of TG, TPO or SP-B promoters induced by wildtype NKX2-1. Local structure analysis of mutant Phe235 showed abnormal contacts with nearby amino acids and Arg221. Surface charge distribution also changed in this mutant model.

Conclusions: NKX2-1 haploinsufficiency in TG promoter activation and decreased TG transcriptional activity induced by mutant NKX2-1 p.V235F may cause hypothyroidism in NXK2-1 c.799G>T patients. Our study discovered part of the pathologic mechanisms of NXK2-1 c.799G>T mutation.

Keywords: NKX2-1 related disorder; Congenital hypothyroidism; Thyroglobulin gene promoter; Thyroperoxidase gene promoter; Surfactant protein-B gene promoter
Fig. 1 (abstract O5-1).
Fig. 1 (abstract O5-1).

See text for description

O5-2 A variety of clinical presentation in congenital hypothyroidism caused by mutations in the thyroglobulin gene

Aoi Kawakita1, Yukiyo Yamamoto1, Kazuyasu Kubo1, Mami Eguchi1, Reiko Saito1, Motohide Goto1, Rinko Kawagoe1, Yasusada Kawada1, Koichi Kusuhara1, Satishi Narumi2, Tomonobu Hasagawa3

1Department of Pediatrics, University of Occupational and Environmental Health; 2Department of Molecular Endocrinology, National Research Institute for Child Health and Development; 3Department of Pediatrics, Keio University School of Medicine

Background: Mutations in the thyroglobulin (TG) are important genetic causes of congenital hypothyroidism, characterized by congenital goiter and low serum TG. Importantly, these patients are at elevated risk for developing thyroid cancer. Here, we report clinical features of four cases with various TG gene mutations.

Case report: Case 1 was born with remarkable goiter and his TSH was high (80 μ U/ml) at neonatal mass screening (MS). At 10 day-old, thyroid function tests confirmed overt hypothyroidism with low serum TG (11.5ng/ml) and L-T4 replacement started. Ultrasonography showed remarkable goiter. At re-evaluation in 3 year-old, TRH-test showed elevated basal TSH and exaggerated response (13.34 → 97.02 IU/ ml). 123I scintigraphy showed high uptake (68.89%) and negative perchlorate discharge. Ultrasonography still exhibited thyroid enlargemant.

Case 2 and 3 are male siblings of 14 and 12 year-old at first visit to our hospital. Their TSH levels were high at MS (Case 2: 110.1 μ U/ml,

Case 3: 64.3 μ U/ml). Thyroid function tests confirmed overt hypothyroidism and L-T4 replacement started. Serum TG and thyroid size were not evaluated at neonatal period. At 14 and 12 year-old, they had no goiter and examinations revealed euthyroid status with undetectable serum TG.

Case 4 is 40 day-old at first visit to our hospital. His TSH level was high (13.2 μ U/ml) at MS. Thyroid function tests confirmed overt hypothyroidism and L-T4 replacement started. No data about serum TG and thyroid size at neonatal period was available. At 40 day-old, he had no goiter and examinations revealed euthyroid status with normal TG level (47.2 ng/ml). At re-evaluation in 3 year-old, TRH-test showed elevated basal TSH and exaggerated response (17.39 → 109.3 IU/ml). 123I scintigraphy showed high uptake (54.86%) and negative perchlorate discharge. His thyroid size was normal in ultrasonography.

Genetic analysis identified heterozygous mutations in the TG gene (Case 1; c.4177A>T, pK1393X, c.3790T>C, pC1264R, Case 2 and 3; c.4177A>T, p.K1393X, c.4235delT, p.L1412fs, Case 4; c.1006T>C, p.C336A, c.7006C>T, p.R2317X).

Discussion: Cases with TG mutations have exhibited variability in clinical presentation. Goiter was often not evaluated at neonatal period and serum TG did not decrease in some cases. Careful diagnosis of goiter by ultrasonography should be performed. High uptake in 123I scintigraphy and negative perchlorate discharge are important findings at re-evaluation. According to these findings, TG mutations should be considerd even in cases with no goiter and normal serum TG. Active detection of TG mutation is expected to decrease future thyroid cancer risk.

O5-3 Genetic and imaging analyses of sibling cases with congenital hypothyroidism revealed the distinct clinical courses despite the presence of the identical mutation

Mikiko Koizumi1, Shinsuke Onuma1, Mariko Nakacho1, Yasuko Syoji1, Masanobu Kawai1, Yuri Etani1, Shinobu Ida1, Chiho Sugisawa2, Kiyomi Abe2, Satoshi Narumi2, Tomonobu Hasegawa2

1Department of Gastroenterology and Endocrinology, Medical Center and Reserch Institute for Maternal and Child Health; 2Department of Pediatrics, Keio University

Introduction: Mental retardation is a severe complication of congenital hypothyroidism (CH) and preventable by early diagnosis and therapeutic intervention. More than half of the cases with thyroid dyshormonogenesis, which accounts for 15 % of CH, is caused by the mutation of a single gene. Sibling cases of CH are occasionally observed and clinical courses are sometimes different among them. In the current study we performed imaging and genetic analysis in 7 patients from three familial cases of CH to understand the association between pathogenesis of CH and their clinical courses.

Subjects and methods: Seven patients from three familial cases of CH were recruited in the study. The type and cause of hypothyroidism was determined based on radioisotope scanning with the perchlorate discharge test and ultrasonograph. Genomic DNA was extracted from peripheral blood and genetic analysis was performed using targeted next-generation sequencing. The identified mutation was confirmed using the Sanger sequencing method.

Results: Family 1: A 19-year-old man was positive for neonatal mass screening (NMS) and diagnosed as having CH. His 15-year-old brother was negative for NMS, but subsequently diagnosed as CH at 4-month old, and synthetic thyroxin was initiated. Iodine transport defect was noted and a heterozygous mutation in the NIS gene was identified in both patients.

Family 2: A 14-year-old boy was positive for NMS and diagnosed as CH. His 11-year-old brother was also positive for NMS, but synthetic thyroxin was not initiated as TSH levels declined and thyrotropin levels were maintained within normal range. Thyroid iodine organification defect was detected and homozygous mutation in DUOX2 gene was identified in the former case, whereas a heterozygous mutation was present in the latter case.

Family 3: The proband is a 17-year-old girl. She was positive for NMS and diagnosed as CH. Her 14- and 10-year old brothers were negative for NMS, but subsequently diagnosed as subclinical CH. Thyroid hypoplasia was noted and no mutations were identified in the proband.

Discussion and Conclusion: Early therapeutic intervention is critical to improve the intellectual outcomes in CH patients; however, NMS sometimes fails to detect CH especially when the hypothyroidism is trivial. Here we present a sibling case of CH (Family 1) who followed different clinical courses despite the presence of identical mutation, suggesting the importance to monitor the thyroid function in children when there exists CH sibling(s) in the family even if the NMS test was negative.

O5-4 Comprehensive analysis of seven Toll-like receptor genes with autoimmune thyroid disease in Korean children: Sexual dimorphism in polymorphisms of TLR 4 gene might influence female predominance of AITD

Won Kyoung Cho1, Jung-Pil Jang2, Moonbae Ahn1, Min Ho Jung1, Tai-Gyu Kim2,3, Byung-Kyu Suh1, Shin Hee Kim1, Kyoung Soon Cho1, So Hyun Park1

1Department of Pediatrics, College of Medicine, The Catholic University of Korea; 2Department of Microbiology, College of Medicine, The Catholic University of Korea; 3College of Medicine, The Catholic University of Korea, Catholic Hematopoietic Stem Cell Bank

Background: The Toll-like receptors (TLRs) are germline-encoded receptors that play an essential role in initiating the immune response against pathogens. In this study, we assess the association of TLR polymorphism with autoimmune thyroid disease (AITD) in Korean children.

Methods: Seven Toll-like receptor genes (TLR-1, -2, -3, -4, -5, -6, -9) including 15 single-nucleotide polymorphisms were analyzed on 104 Korean children with AITD [Hashimoto¢s disease (HD) = 40, Graves¢ disease (GD) = 60 (thyroid-associated ophthalmopathy (TAO) = 29, non- TAO = 31)] and 192 healthy individuals.

Results: The allele frequencies of 15 SNPs in AITD patients and controls are shown in Table 3. For overall AITD cases, the frequencies of these alleles had no statistical difference with controls. When categorized by disease subgroup, GD showed lower frequencies of the TLR4 rs1927911 T allele (cP <0.018) and HD showed an lower frequencies of the TLR3 (-7) rs3775296 C allele (cP <0.044) than the control group. Between GD and HD group, the frequencies of the TLR4 rs1927911 CC genotype in HD (cP < 0.048) was lower, whereas TLR4 rs1927911 T allele in HD (cP < 0.032) showed higher frequencies than in GD. When categorized GD by sex, female-GD group showed higher frequencies of the TLR4 rs1927911 CC genotype (cP < 0.026) and lower frequency of the TLR4 rs1927911 T allele (cP < 0.017) than control. Male-GD group showed lower frequency of the the TLR4 rs1927911 CT genotype (cP < 0.031) than control. Between female and male group in GD, the frequencies of TLR4 rs10759932 CC genotype (cP <0.006) and TLR4 rs1927911 TT genotype (cP < 0.024) in male were higher, whereas TLR4 rs10759932 T allele (cP < 0.004) and TLR4 rs1927911 C allele (cP < 0.016) in male were lower than female-GD.

Conclusions: Our results suggest that TLR- 3 and -4 gene polymorphisms may contribute to the pathogenesis of AITD and TAO. Sexual dimorphism in polymorphisms of TLR 4 gene might influence female predominance of AITD.

O5-5 A Malaysian family with an activating mutation (S505R) in the Thyroid-stimulating Hormone Receptor (TSHR) gene

Noor Shafina Mohd Nor1, Johari Mohd Ali2

1Faculty of Medicine, Universiti Teknologi MARA (UiTM); 2Faculty of Medicine, University of Malaya, Department of Molecular Medicine

Introduction: Childhood hyperthyroidism occurs less commonly in children than hypothyroidism, yet far more symptomatic. Hyperthyroidism in children is mostly due to autoimmunity, predominantly as a result of Graves’ disease. Non-autoimmune hyperthyroidism caused by activating mutations in the Thyroid-stimulating Hormone Receptor (TSHR) gene occurs to a much lesser extent.

Case report: We report a Malay family with three affected individuals (a mother and her two daughters). The two sisters, aged 5 years and 1 month old and 2 years and 5 months old were under General Paediatricians follow up since infancy for symptomatic hyperthyroidism. Despite high dose of anti-thyroid medication (carbimazole), their thyroid function tests continued to be deranged (high fT4, suppressed TSH). They were then referred to our Paediatric Endocrinology clinic for further management. The family history revealed that the mother was presumed as having Graves’ disease since the age of 15 and had radioactive iodine therapy at the age of 24 years due to failure of medical treatment. She subsequently became hypothyroid and currently requiring daily L-thyroxine replacement. On examination, both siblings have palpable goiters with mildly prominent eyes. Thyroid antibodies screening were all negative. Thyroid ultrasound scan showed diffuse thyroid disease with lymphadenopathies. Bone ages were also advanced. In view of the findings, TSHR mutation analysis was done for the two siblings and the parents. Direct DNA sequencing of the TSHR gene revealed a heterozygous cytosine-to-adenine transversion in exon 10 in the two siblings and the mother. This missense mutation was not found in the father. The mutation leads to serine to arginine substitution, affecting TSHR codon 505 (S505R). The S505R mutation had previously been characterized and was observed to cause the activation of TSHR protein (gain- of-function mutation).

Discussion: In conclusion, the evidence of non-autoimmune hyperthyroidism in several family generations warrants initiation of mutation analysis to detect the possible mutation in the TSHR gene leading to the clinical entity. Affected family members harbouring the exact same mutation may exhibit variable phenotypes with regards to the age of onset and severity of hyperthyroidism.

Consent for publication: The authors declare that written informed consent was obtained for publication.

O5-6 Allogeneic stem cell transplantation (HSCT) in childhood and thyroid cancer as the most frequent secondary solid tumor following HSCT with total body irradiation

Marta Snajderova1, Petr Sedlacek2, Petra Keslova2, Renata Formankova2, Petr Riha2, Jan Stary2

1Division of Endocrinology and Diabetes / Department of Pediatrics, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol; 2Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol

Backgroud: Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for a variety of malignant and non- malignant disorders. Improved outcome leads to increasing attention to late complications in long-term survivors. Secondary cancer belongs to the most serious complications.

Aims: Occurrence of secondary solid tumors following allogeneic HSCT was analyzed.

Methods: We have evaluated clinical and laboratory data (including fT4, TSH, thyroid antibodies, thyroid function and ultrasound imaging) in 499 patients (315 M, 184 F) who underwent 545 allogeneic HSCT at a median age 8.4 years since 1989 till 2014. Of them 60% (300/499) patients are still alive and disease free.

Results: We have documented secondary malignancy in 29 patients (5.8%). Of them 13 developed post-transplant lymphoproliferative disorder at a median time 0.3 (0-1.8) years after HSCT. Secondary solid tumor was diagnosed in further 16 patients (3.2%) at a median time 11.4 (range 5.4-17.8) years after HSCT (thyroid carcinoma n=8, carcinoma of oral cavity n=3, malignant schwannoma n=2, melanoma n=1, peritoneal mesothelioma n=1, breast cancer n=1). All patients with secondary solid tumor underwent surgery and/or chemo-radiotherapy and are alive. 15/16 patients (93.8%) with secondary solid tumor received total body irradiation (TBI) 12-14.4 Gy as a part of conditioning regimen. Papillary carcinoma (in all 8 cases micronodular form, T1 or T2 stage) was the most frequent secondary solid tumor (5F, 3M; 50% of secondary solid tumors) diagnosed at a median time 10.8 years (range 5.4-17.0) after HSCT. At the time of diagnosis three patients were treated with thyroxine for autoimmune thyroid disease, one for hypothyroidism and another one for nodular goiter. All but one had HSCT for malignant disease and 7/8 received TBI.

Conclusions: The early diagnosis is one of the key tasks of long-life multidisciplinary post-transplant care including regular ultrasound evaluation of thyroid gland and neck especially more than 5 years after HSCT and namely after irradiation. The incidence of complications following allogeneic HSCT in childhood namely after TBI is increasing within time. Papillary thyroid carcinoma was the most frequent secondary solid tumor detected. Supported by MHCZ for conceptual development 00064203 University Hospital Motol.

O6-1 First FGF9 mutation in a patient with a 46,XY disorder of sex development

Makoto Ono1,2, Anthony Bird2, Stefanie Eggers3, Brittany Croft2,3, Stefan Bagheri-Fam2, Janelle Ryan2, Andrew Kueh4, Peter Stanton2, Tim Thomas4, Andrew Sinclair3, Masayo Harada5, Vincent Harley2

1Department of Paediatrics, Tokyo Bay Urayasu Ichikawa Medical Centre; 2Hudson Institute of Medical Research, Clayton, Centre for Reproductive Health; 3Murdoch Childrens Research Institute, Parkville; 4Walter and Eliza Hall Institute of Medical Research; 5Department of Clinical Anatomy, Tokyo Medical and Dental University

Background: Disorders of sex development (DSDs) include 46, XY gonadal dysgenesis (GD), where a specific genetic diagnosis is made in only ~30% of patients. Improved understanding of the genetic causes of 46, XY GD is therefore required to better inform clinical diagnosis and management. Among the genes induced by upstream SRY-SOX9 signalling to promote male sex determination is fibroblast growth factor (FGF) 9. Expressed within the pre-Sertoli cell lineage, FGF9 suppresses the female program of gonadal development via its receptor FGFR2. In mouse both are critical for testis determination as FGF9/FGFR2 knockout mice show XY sex reversal. Despite this, to date no FGF9 gene mutations/deletions/insertions have been identified in human DSD patients.

Results: We identified an FGF9 variant, a maternally derived heterozygous single nucleotide substitution c.583G>A (p.D195N), in a 46,XY GD female presented with delayed puberty, primary amenorrhea, clitromegaly, Müllerian duct remnants and raised gonadotrophin levels using 1032 DSD gene targeted parallel sequencing. In silico analysis predicted the D195N variant to be deleterious for FGF9 protein function, and in vitro studies indicated the D195 residue lies at the homodimerisation interface. Purified recombinant FGF9-D195N protein showed a reduced affinity for heparin, a property necessary for stable FGF-FGFR complexes, and reduced ability to induce Sertoli cell proliferation in vitro . To model the D195N mutation in vivo , Fgf9D195N/+ knockin mice were generated via CRISPR/Cas9 gene-editing. Pilot studies showed that E15.5 Fgf9D195N/D195N embryonic XY gonads exhibit a truncated male-specific coelomic blood vessel, and immunofluorescence analysis revealed ectopic expression of the female meiotic marker γ H2AX, indicative of sex reversal. We also investigated gonadal development in Fgf9N143T/N143T mice which also carry a mutation in the FGF9 homodimerisation domain. Likewise, E15.5 Fgf9N143T/ N143T embryonic XY gonads show a truncated coelomic blood vessel and partial sex reversal.

Conclusion: These results suggest that FGF9 homodimerisation and heparin binding are required for FGF9 function in testes determination. In addition, human FGF9 mutations may be the cause of a subset of hitherto undiagnosed human DSD patients.

O6-2 The etiology and differential diagnosis of 46,XYDSD of 88 cases

Yan Song Ning, Chun gong Xiu

Beijing Children's Hospital affiliated to Capital Medical University, Endocrine Department

Aims: Retrospectively reviewed the etiology, clinical features and differential diagnosis of 88 46,XYDSD.

Methods: Collect from September 2008 to December 2015 in Beijing Children's Hospital and gene detection of 300 cases, selecting the diagnosed of 46, XYDSD of 88 cases except CHH that compared their clinical symptoms and check and laboratory examination.

Result: The social gender of the 88 case was that male: 38, female: 50 aged 4 months to 17 years old, average 3.71 years. Clinical diagnosis were androgen insensitivity syndrome (AIS) in 33 cases (37.5%), 5 alpha reductase deficiency (5 alpha RD) in 31 cases (35.2%), 17 alpha hydroxylase deficiency in 11 cases (12.5%); relatively rare in turn Nr5a1 gene mutation in 7 cases (8.0%), 17 beta hydroxysteroid dehydrogenase deficiency in 2 cases (2.3%), 3 beta hydroxysteroid dehydrogenase deficiency in 2 cases (2.3%), Fraiser syndrome (FS) and Denys Drash syndrome (DDS) in 1 case (1.1%). The main clinical manifestations of abnormal vulva: The external Prader classification of AIS, 5 alpha RD and 17 alpha hydroxylase in children showed no significant difference; and the rest of the rare case of Prader classification for 0-3. There was no significant difference in the distribution of testicular position in various diseases. In accordance with the hCG classification experiments between Leydig cell function in normal and interstitial function is not normal statistical grouping: interstitial cells of AIS and 5 alpha RD is normal, normal T/DHT, and between the two kinds of diseases were no difference; and 17 alpha hydroxylase deficiency, Nr5a1 gene mutation, 17 beta hydroxysteroid dehydrogenase deficiency, 3 beta hydroxysteroid dehydrogenase deficiency disease,'s and DDS showed interstitial cells are not normal after hCG stimulation.

Conclusion: There are many overlapping clinical manifestations, the differential diagnosis of 46, XYDSD was difficultiesThere were no differences between the Prader grading and testicular position of the body in different cause, but significant differences in the distribution of the different causes. Other rare disorders as 17 beta - hydroxy steroid dehydrogenase deficiency, 3 beta - hydroxy steroid dehydrogenase deficiency,'s and DDS require individual specific analysis. Internal genital organs in various diseases had no significant difference, pure sex hormone detection in diagnosis is unreliable .HCG stimulation test can divid into testicular Leydig cells normal and abnormal group, but specific disease is not easy to identify. T/DHT have disorders identification, for the above, when there is doubt, should take gene detection for further confirmed.

O6-3 Mutation frequency in Japanese male patients with severe form of hypogonadotropic hypogonadism

Takeshi Sato1, Satoshi Narumi1,2, Tomohiro Ishii1, Koji Muroya3, Yumi Asakura3, Masaki Takagi4, Masanori Minagawa5, Hiroaki Suzuki6, Chiho Sugisawa7, Jun Saito7, Yukihiro Hasegawa4, Masanori dachi3, Tomonobu Hasegawa1

1Department of Pediatrics, Keio University School of Medicine; 2Department of Molecular Endocrinology, National Research Institute for Child Health and Development; 3Department of Endocrinology and Metabolism, Kanagawa Children's Medical Center; 4Department of Endocrinology and Metabolism, Tokyo Metropolitan Children's Medical Center; 5Department of Endocrinology, Chiba Children's Hospital; 6Department of Internal Medicine (Endocrinology and Metabolism), University of Tsukuba, Faculty of Medicine; 7Department of Endocrinology and Metabolism, Yokohama Rosai Hospital

Background: While previous studies have shown that 20–30% of Caucasian male hypogonadotropic hypogonadism (MHH) patients harbored causative gene mutations, genetic backgrounds of Japanese MHH patients have not been fully elucidated. Moreover, we do not know mutation frequency in MHH patients applicable to clinical practice.

Aim: To estimate mutation frequency in Japanese male patients with severe form of HH.

Methods: In this study, we defined MHH as male patients meeting all of the following criteria: (i) 18 years of age or older, (ii) no spontaneous testicular enlargement (volume ≥ 4 mL), (iii) low luteinizing hormone levels regardless of history of treatment for puberty induction and acquisition of fertility. We enrolled Japanese MHH probands, excluding CHARGE syndrome, combined pituitary hormone deficiency, and chromosomal abnormalities. We sequenced 32 genes implicated in HH using the MiSeq instrument. We defined mutations as variants meeting any of the following criteria: (i) variants reported as pathogenic, (ii) nonsense, frameshift, or splice site variants not observed in dbSNP, and Human genetic variation database, (iii) missense variants not observed in databases, conserved across species, and predicted as damaging by in silico analyses with Polyphen-2 and SIFT.

Results: Twenty male patients were included, ranging in age from 18 to 37 years, with a median age of 24 years. Nine out of 20 patients (45.0%, 95% confidence interval: 23.2–66.8%) harbored gene mutations; 4 FGFR1 , 4 KAL1 , and 1 SOX2. Oligogenic mutations were not identified.

Discussion: The proportion of detecting gene mutations in Japanese MHH patients was relatively high compared to Caucasian MHH patients, possibly due to our strict inclusion criteria. FGFR1 and KAL1 mutations were the two leading causes in Japanese MHH patients, which was consistent with the previous studies on Caucasian.

Conclusion: Mutation frequency in Japanese male patients with severe form of HH is 45.0%.

O6-4 The incidence of intracranial lesions in central precocious puberty is overestimated

Jong Seo Yoon1, Cheol Hwan So1, Hae Sang Lee1, Jung Sub Lim2, Jin Soon Hwang1

1Division of Endocrinology and Metabolism / Department of Pediatrics, Ajou University School of Medicine; 2Division of Endocrinology and Metabolism / Department of Pediatrics, Korea Cancer Center Hospital

Backgroud: Intracranial pathology has been reported in 8% of girls and 40% of boys with central precocious puberty. So the current guidelines recommend that all boys with central precocious puberty and girls with central precocious puberty at under 6 years of age should have a brain MRI. And it is controversial whether all girls who develop central precocious puberty between 6 and 8 years of age require brain MRI.

Objectives: We evaluated the intracranial lesions in Korean girls and boys with central precocious puberty.

Methods: Total 369 patients (257 girls and 112 boys) diagnosed for central precocious puberty since 2003 at Ajou university Hospital underwent brain MRI. The patients were categorized into group A (normal findings) and group B (the other findings). We evaluated clinical and biochemical factors associated with precocious puberty and studied relationship between the factors and the outcome of their MRI.

Results: Group A included 238 among 257 girls (92.6%) and 105 among 112 boys (93.8%). Group B were detected in 19 among 257 girls (7.4%) and 7 among 112 boys (6.2%). Group B consisted of pituitary gland hyperplasia, Rathke’s cleft cyst , and pineal cyst ; 11 of 19 (4.3%), 5 of 19 (1.9%) and 4 of 19 (1.1%) in girls and 4 of 7 (4.3%), 2 of 7 (1.9%) and 1 of 7 (0.9%) in boys. None of group B had a pathological brain lesion associated with central precocious puberty. Girls in group B had higher mean basal LH levels (p 0.031) and stimulated LH levels (p 0.009) than girls in group A, and the statistical difference was particularly significant in pituitary gland hyperplasia. However, there was no statistically significant difference between two groups of boys.

Conclusions: Intracranial lesions were rare in girls and boys with central precocious puberty unlike the previously known incidence. So brain MRI should not be regarded as routine screening tools for evaluation of central precocious puberty. So we think that it is necessary to discuss again for the incidence of intracranial lesion in central precocious puberty.

O6-5 Detection of somatic activating GNAS mutations in girls with isolated autonomous ovarian cyst by next generation sequencing

Hironori Shibata1, Satoshi Narumi1,2, Tomohiro Ishii1, Koji Muroya3, Yumi Asakura3, Masanori Adachi3, Goro Sasaki4, Takumi Shibazaki5, Yosuke Hara5, Tomonobu Hasegawa1

1Department of Pediatrics, Keio University School of Medicine; 2Department of Molecular Endocrinology, National Research Institute for Child Health and Development; 3Department of Endocrinology and Metabolism, Kanagawa Children's Medical Center; 4Department of Pediatrics, Tokyo Dental College Ichikawa General Hospital; 5Department of Pediatrics, Shinshu University School of Medicine

Background: McCune-Albright syndrome (MAS) caused by somatic activating GNAS mutations is characterized clinically by the classic triad of fibrous dysplasia (FD), café-au-lait skin spots, and GnRH-independent precocious puberty (PP) due to autonomous ovarian cyst. In a previous report, somatic activating GNAS mutations were found in 13 (33.3%) of 39 ovarian samples from girls with isolated autonomous ovarian cyst (J Clin Enocrinol Metab 2004; 89: 2107). In the same report, a series of nested PCR and restriction enzyme digestion detected somatic activating GNAS mutations in only 3 (7.7%) in 39 peripheral blood leucocytes (PBL) samples. We reported that next generation sequencing (NGS) detected somatic activating GNAS mutations sensitively from PBL samples in MAS (PLoS One 2013; 8: e60525).

Objective: To determine if we could detect somatic activating GNAS mutations in girls with isolated autonomous ovarian cyst by NGS using PBL samples.

Method: The study included 8 girls with GnRH-independent PP due to isolated autonomous ovarian cyst. We excluded cases with fibrous dysplasia (FD) and/or café-au-lait skin spots, the other classic triad of MAS. We performed both NGS and combinatory method of peptide nucleic acids (PNA) probe with NGS (PNA-NGS) using PBL samples from all patients.

Results: We detected somatic activating GNAS mutat ions in one (12.5%) by NGS and 5 (62.5%) by PNA-NGS of PBL samples (Table.1).

Conclusion: The combinatory method of PNA-NGS can detect somatic activating GNAS mutations sensitively from PBL samples in girls with isolated autonomous ovarian cyst. Our data suggest that somatic activating GNAS mutations is the major cause of isolated autonomous ovarian cyst.
Table 1 (abstract O6-5).

Characteristics of 8 girls with isolated autonomous ovarian cyst

O6-6 Effects of cryptorchidism on testicular function in Chinese boys

Liya Xu1, Wuhen Xu2, Pin Li1

1Department of Pediatric Endocrinology, Shanghai Children's Hospital Affiliated to Shanghai Jiaotong University School of Medicine; 2Department of Labratory Medicine, Shanghai Children's Hospital Affiliated to Shanghai Jiaotong University School of Medicine

Objective: To investigate the effects of different types of cryptorchidism on testicular Leydig-cells and Sertoli-cells function in different ages of Chinese boys.

Methods: Included were 101 Chinese prepubertal cryptorchid boys and 136 controls (0.5-13 years). Those who have been operated were also included in case group(Orchiopexy is performed in 1-3yr). Each group was divided into five subgroups: 0.5-1year (yr), 1-3yr, 3-8yr, 8-11yr, 11-13yr. The patients were analyzed by age subgroups, unilateral vs bilateral and high-position vs low-position. LHRH & hCG stimulation tests, serum-Anti-Müllerian-Hormone(AMH) and inhibin-B(InhB) were examined.

Results: Effects of different ages on testicular function: Compared with other age subgroups in case group, testosterone-values after hCG stimulation tests was significantly higher in patients 0.5-1yr(p < 0.01); Compared with the control group, serum-AMH and InhB were significantly lower in the cryptorchid boys (p< 0.01). Especially, AMH-values in 1-8yr subgroups and InhB-values in 0.5-8yr subgroups were significantly lower (p < 0.01). Effects of high-position vs Low-position on testicular function: Compared with low-position group, testosterone-values after hCG stimulation tests in high-position group were significantly lower(p=0.007). Moreover, AMH-values and InhB values in high-position group were respectively significantly lower(p=0.001, p=0.012). Effects of unilateral vs bilateral on testicular function: Testosterone-values after hCG stimulation tests, AMH-values and InhB-values between unilateral and bilateral groups all have no significant difference (p>0.05).

Conclusion: 1. The Sertoli-cells function is firstly affected in cryptorchid boys before puberty, no matter orchiopexy has been performed or not, and InhB decreased earlier than AMH which occurs during 0.5-1yr, while AMH decreased more obviously after 1yr. However, the Leydig- cells function isn't severely affected in 1yr. 2.The effects of cryptorchidism on Leydig-cells and Sertoli-cells function are related to the issue of high or low-position, but not of unilateral or bilateral cryptorchidism.

O7-1 Excessive gestational weight gain is associated with adverse health outcomes in the offspring at age 7 years

Valentina Chiavaroli1, Jose’ G B Derraik1, Sarah A Hopkins1, Janene B Biggs1, Sumudu N Seneviratne1, Lesley M E McCowan2, Wayne S Cutfield1, Paul L Hofman1

1University of Auckland, Liggins Institute; 2Department of Obstetrics and Gynaecology, Faculty of Medical and Health Science, University of Auckland

Background: Excessive gestational weight gain (GWtG) has been recognized as an important early-life risk factor for childhood obesity. We aimed to examine whether excessive GWtG was associated with alterations in body composition and metabolism in the offspring of primiparous mothers who participated in a randomised controlled trial of exercise regimen during pregnancy.

Methods: Of the initial 84 women and their offspring who participated in the trial, follow-up data were available on 52 mothers with GWtG information and their children. 35 mothers (67%) were determined to have excessive GWtG as per IOM guidelines, with the remaining 17 mothers having GWtG in the normal range. Note that the proportion of participants in each group that exercised during pregnancy was similar (60% and 54%, respectively). Children underwent clinical assessments at approximately 7.6 years, including body composition by DXA and fasting blood tests. All statistical analyses were adjusted for important confounders, in particular maternal BMI at trial recruitment (approximately 20 weeks of pregnancy).

Results: Birth weight SDS was not significantly different in the offspring of mothers with excessive or normal GWtG, when adjusted for maternal BMI (0.01 vs -0.23 SDS, respectively; p=0.34). Children born to mothers with excessive GWtG had a less favourable lipid profile with lower HDL (1.57 vs 1.75 mmol/L; p=0.038), higher triglycerides (0.87 vs 0.63 mmol/L; p=0.003), and higher total cholesterol to HDL ratio (3.06 vs 2.53 mmol/L; p=0.018), as well as increased abdominal adiposity (0.64 vs 0.55 android to gynoid fat ratio; p=0.043), even though macronutrient intake was similar in both groups. Boys born to mothers with excessive GWtG (n=19) were heavier (weight SDS 0.82 vs -0.01; p=0.010), had higher BMI SDS (0.35 vs -0.41 SDS; p=0.012) and greater abdominal adiposity (0.62 vs 0.45 android to gynoid fat ratio; p=0.010) than those born to mothers with normal GWtG (n=9). There was a small number of girls with metabolic data, but girls born to mothers with excessive GWtG (n=9) had higher fasting insulin concentrations (8.0 vs 5.4 mU/L; p=0.033) and were more insulin resistant (HOMA-IR 1.72 vs 1.16; p=0.048) than those born to mothers with normal GWtG (n=5), despite adjustment for body fat.

Conclusions: Independently of maternal BMI early in pregnancy, excessive gestational weight gain is associated with adverse health outcomes in the offspring at approximately 7.6 years of age, with some indication of sex-dependent effects.

O7-2 Growth seasonality during preschool childhood in nursery school children

Tsuyoshi Isojima1,2, Noriko Kato3, Susumu Yokoya4, Toshiaki Tanaka5, Atsushi Ono6, Hiroshi Yokomichi7, Zentaro Yamagata7, Soichiro Tanaka8, Hiroko Matsubara9, Mami Ishikuro10,11, Masahiro Kikuya10,11, Shoichi Chida12, Mitsuaki Hosoya6, Shinichi Kuriyama9,10,11, Shigeo Kure8,10

1Department of Pediatrics, Teikyo University School of Medicine; 2Department of Pediatrics, Graduate School of Medicine, The University of Tokyo; 3Department of Early Childhood and Elementary Education, Jumonji University; 4Department of Medical Subspecialties, National Center for Child Health and Development; 5Department of Pediatrics, Japanese Association for Human Auxology; 6School of Medicine, Fukushima Medical University; 7Department of Health Sciences, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi; 8Department of Pediatrics, Graduate School of Medicine, Tohoku University; 9Department of Disaster Public Health, International Research Institute of Disaster Science (IRIDeS), Tohoku University; 10Tohoku Medical Megabank Organization (ToMMo), Tohoku University; 11Department of Molecular Epidemiology, Graduate School of Medicine, Tohoku University; 12Department of Pediatrics, School of Medicine, Iwate Medical University

Background: Healthy weight children tend to increase their weight during winter and decrease weight gain during summer. In contrast, accelerated summer weight gain has been observed among elementary overweight children. It is of great concern whether this seasonal variation occurs during preschool childhood. However, few studies examined growth seasonality during preschool childhood, and studies using longitudinal data are scarce.

Methods: The study population was derived from the nationwide retrospective cohort of nursery school children born from April 2, 2004, to April 1, 2005. We could gather ten consecutive longitudinal data of 15,259 children on height and weight measured in April and October from 2006 to 2010. With these data, we calculated six-month growth of these children in height, weight, and body mass index (BMI). Growth from April to October was defined as the summer period growth, while that from October to next April as the winter period growth. We analyzed longitudinal seasonal variation of nursery school children by classifying children into six groups according to the standard deviation score (SDS) of the last measurement just before they entered an elementary school (i.e. <-2SD, -2SD~ -1SD, -1SD~ 0SD, 0SD ~ +1SD, +1SD ~ +2SD, +2SD).

Results: Distinctive seasonal growth variation in weight and BMI was detected in the two groups of children with one or more than one BMI SDS (figure). They had accelerated summer weight and BMI gains and they could be detected from 2007 (p<0.0001). Notably, they had begun since less than three years of age in children with two or more than two BMI SDS. On the other hand, such growth seasonality was not observed in height.

Conclusions: Seasonal growth variation in weight and BMI was detected in relatively overweight preschool children during infancy. These findings suggested that we should be careful of summer time weight gain even in infancy for preventing the future obesity.

O7-3 Oleate protects INS1 cells from palmitate-induced apoptosis by down-regulating ER stress and GSK3βthrought Stearoyl- CoA Desaturase 1

Shan Huang1, Cai-qi Du1, Wei Wu1, Qin Ning2, Xiao-Ping Luo1

1Department of Pediatrics, Tongji Hospital. Tongji Medical College , Huazhong University of Science and Technology; 2Department of Infectious Diseases, Tongji Hospital. Tongji Medical College , Huazhong University of Science and Technology

Backgroud: The loss of functional β -cell mass, at least in part secondary to increased β -cell apoptosis, is increasingly recognized as one of the main contributing factors to the pathogenesis of type 2 diabetes. Our previous study have identified that endoplasmic reticulum (ER) stress and GSK3 β involved in palmitate-induced β -cell apoptosis (lipotoxicity). However, the effect of oleate on β -cell is still controversial.

Objective: The present study investigated whether oleate protected β -cell from palmitate-induced apoptosis and its molecular mechanisms.

Methods: INS1 cells were cultured with varying concentrations of palmitate, oleate, TO-901317 or co-treatment of palmitate with either oleate or TO-901317. Cell viability and apoptosis were measured by CCK-8 assay, Hoechst 33342 / PI, flow cytometric assay and electron microscopy; ER stress and GSK3 β activity were assessed by RT-PCR and western blot. We also estimated intracellular triglyceride accumulation through nile red staining and electron microscopy.

Results: Contrary to lipotoxicity of palmitate, 0.25~0.5 mM oleate increased INS1 cells viability (p <0.05). Interesting, we identified that oleate rather than palmitate induced a significant intracellular triglyceride accumulation (p <0.05). Furthermore, co-treatment of oleate with palmitate apparently decreased palmitate-induced apoptosis by down-regulating ER stress and GSK3 β activity (p <0.05), Besides, we also found that the mRNA level of Stearoyl-CoA Desaturase 1 (SCD1) was up-regulated (p <0.05). Finally, co-treatment of Palmitate with LXR- agonist TO-901317, which directly up-regulated SCD1, also leaded to intracellular triglyceride accumulation and the reduction of ER stress and GSK3 β activity.

Conclusion: Oleate protected INS1 cells from palmitate-induced apoptosis by down-regulating ER stress and GSK3 β activity throught promotion of appropriate triglyceride accumulation , which related to the up-regulation of SCD1.

O7-4 Increase of body mass index from age 1.5 to 3 years is useful in screening for a high risk of childhood obesity

Go Ichikawa1,2, Junko Ichikawa1, Ayako Yoshida1, Satomi Koyama1, Osamu Arisaka1

1Department of Pediatrics, Dokkyo Medical University; 2Department of Pediatrics, Nasu Red Cross Hospital

Introduction: Childhood obesity is routinely described as a major problem in developed and developing countries. Obesity in childhood is causative for many chronic diseases, including type 2 diabetes, cardiovascular disease and hypertension. Once obesity is present, it is challenging to treat because of multiple physiological, behavioral, and cultural feedback loops. Prevention is the key to success in obesity control, but there is no effective method for screening for high risk cases. Therefore, this study was performed to develop a method for detection of a high risk of childhood obesity.

Methods: A total of 298 children in a birth cohort (town F, Tochigi, Japan) were enrolled in the study. The population of this town is 18,000, with half of the people working as farmers and half commuting to nearby large cities. Children in the study were followed up in infant health checks at a health center during the preschool period, and data were stored at a regional health center. At 12 years of age, measurement of height and weight were performed at school. Children at this age with body mass index (BMI) ≥ 95th percentile and ≥ 85th percentile according to national reference data for height and weight were diagnosed as obese and overweight, respectively. Several screening methods focusing on relationships with BMI at 1.5 and 3 years old were examined by computing the odds ratio for each method in multivariate analysis. The study was approved by the ethics committee of Dokkyo Medical University.

Results: Data were available for 215 children. The odds ratios for obesity and an overweight status at 12 years old were 18.9 (95% confidence interval 5.9-60.8, predictive accuracy 0.92) and 16.9 (5.1-55.9, 0.84), respectively, for a BMI increase 0.5 from 1.5 to 3 years and BMI 16.8 at 3 years; 3.1 (1.2-8.2, 0.70) and 2.4 (1.2-4.9, 0.68), respectively, for a BMI increase from 1.5 to 3 years; and 5.8 (2.1-16.0, 0.84) and 3.6 (1.6-8.2, 0.78) for a BMI increase 0.5 from 1.5 to 3 years.

Discussion: The current study is the first to show that children with both an increase of BMI 0.5 from 1.5 to 3 years old and BMI 16.8 at 3 years old are at high risk of later childhood obesity or an overweight status. Identification of individuals at 3 years old who are at high risk for obesity later in life may provide opportunities for early interventions.

O7-5 Cardiovascular risk factorsin children with type 1 diabetes

Ying Zhang, Pin Li, Guo Sheng

Division of Endocrinology, Shanghai children's hospital of Shanghai Jiaotong University

Background: Cardiovascular disease as a result of atherosclerosis is the major complication among children with type 1 diabetes mellitus (T1DM). The aim of this prospective study was toexamining the presence of cardiovascular risk factors in children with T1DM.

Methods: We evaluated several cardiovascular risk factorsincluding atherosclerosis, artery intima-media thickness(IMT) and metabolic responsesin 122 (66 female) diabetic children (age median 12.6 ± 2.7 years, mean HbA1c of 7.9 ± 1.1%) with T1DM, and 105 non-diabetic children (age median 12.5 ± 2.8 years) were enrolled as normal controls.

Results: The diabetic children had significantly higher carotid intima-media thickness (cIMT) and aorticIMT (aIMT) than the controls, meanwhile, the diabetic patientsalso had significantly higher values than the controls for diastolic wall stress (DWS), incremental elastic modulus (IEM) and for flow mediated dilatation (FMD). Tumor necrosis factor- α (TNF- α), Interleukin -4 (IL-4), high-sensitivity C-reactive protein (hs-CRP) and leptin were all significantly greater as compared to T1DM and controls. The difference was not significant between patients and controls for othercardiovascular risk factors.In children with T1DM, cIMTandaIMTwere both correlated with several risk factors includingage, weight, body mass index (BMI), duration of diabetes,waist/hip ratio, total cholesterol, triglyceridesand apolipoprotein B, in addition to common risk factors, cIMTwas also associated with systolic blood pressure (BP). Inflammatory factor TNF- α was just dependently associated with weight, duration of diabetes, total cholesterol, triglycerides and apolipoprotein B. Other risk factors such as height, diastolic BP, LDL/HDL-cholesterol, apolipoproteinA TandS-creatinine level were all not independent risk factors ofcardiovascular diseasein T1DMchildren.

Conclusions: Our results suggested that children patients with T1DM are associated with early impairment of the common carotid and aortic artery structure and function and that diabetic state may be the main risk factor for artery wall stiffening and thickening, which are of considerable concern as possible early events in the genesis of atheroma.

O7-6 Research on the role which FTO plays on the regulation of PI3K, MAPK pathway

Peng li Bao, Ge Li Liu

Pediatrics, General Hospital of Tianjin Medical University

Objective: FTo explore if the the change of the expression of FTO regulate the PI3K and MAPK pathways, and finally destroy the endothelial function.

Methods: Using RNA interference technology to build FTO-shRNA lentivirus vector, infect HUVEC cells to silence the FTO gene and to up-regulate the expression of FTO, then use Western Blot to prove the effect. To detect the phosphorylation levels of ERK1/2, Akt CS6K1 and eNOS, NO level was detected by nitrate reductase method.

Results: Recombinant plasmid was constructed successfully. Silence of the FTO gene leads to the phosphorylation level decreasing of Akt, eNOS, ERK, S6K1 and decreased NO production. Over expression of FTO results to PI3K and MAPK pathway activated, Akt and eNOS, ERK1/2, S6K1 phosphorylation levels increased and NO synthesis more.

Conclusion: FTO expression can regulate PI3K and MAPK pathway, suggesting that PI3K-Akt-eNOS pathway and ERK/MAPK pathway play an important role in endothelial injury leadsby obesity, providing new direction for research on Mechanism of cardiovascular function destroy results by obesity.

O8-1 Clinical Trial of Asfotase Alfa in Patients with Hypophosphatasia (HPP) in Japan

Taichi Kitaoka1, Toshihiro Tajima2, Keisuke Nagasaki3, Toru Kikuchi3, Katsusuke Yamamoto4, Toshimi Michigami5, Satoshi Okada6, Ikuma Fujiwara7, Masayuki Kokaji8, Hiroshi Mochizuki9, Tsutomu Ogata10, Koji Tatebayashi11, Atsushi Watanabe12, Shuichi Yatsuga13, Takuo Kubota1

1Department of Pediatrics, Osaka University Graduate School of Medicine; 2Department of Pediatrics, Hokkaido University School of Medicine; 3Division of Pediatrics / Department of Homeostatic Regulation and Development, Niigata University Graduate School of Medical and Dental Sciences; 4Department of Pediatric Nephrology and Metabolism, Osaka Medical Center and Research Institute for Maternal and Child Health; 5Department of Bone and Mineral Research, Osaka Medical Center and Research Institute for Maternal and Child Health; 6Department of Pediatrics, Hiroshima University Graduate School of Biomedical & Health Sciences; 7Department of Pediatrics, Tohoku University School of Medicine; 8Department of Pediatrics, Showa General Hospital; 9Division of Endocrinology and Metabolism, Saitama Children's Medical Center; 10Department of Pediatrics, Hamamatsu University School of Medicine; 11Depatment of Pediatrics, Nagara Medical Center; 12Division of Clinical Genetics, Nippon Medical School Hospital; 13Department of Pediatrics and Child Health, Kurume University School of Medicine

Background: Hypophosphatasia (HPP) is a rare skeletal disease caused by mutations in the gene coding the tissue-nonspecific alkaline phosphatase (TNSALP). The most severe type of HPP is often associated with respiratory failure due to the defect of calcification. Other types of HPP also have many complications including failure-to-thrive, hypercalcemia, premature loss of teeth and bone deformity. Recently, an enzyme replacement therapy using Asfotase Alfa (AA), a bone-targeted recombinant human TNSALP, has been developed to ameliorate the complications of HPP.

Methods: In an open-label, single-arm, multi-centered clinical study, we evaluated the safety and efficacy of treatment with AA in patients with HPP. We assessed the safety of repeated subcutaneous injections of AA as primary outcome measures along with the number of adverse events (AEs) including injection reactions. Efficacy assessments as secondary outcome measures included overall survival, respiratory status, rickets severity, physical growth, and gross motor development.

Results: Thirteen patients, 9 females and 4 males, were enrolled in this study; they ranged in age at baseline from day 0 to 34 years. Six patients had perinatal form, five had infantile form, one had childhood form, and one had adult form. Total 195 AEs reported, and the most frequent were injection site reactions (45%). Serious AEs that possibly related to the treatment were convulsion and hypocalcemia observed in the patients of prenatal type. In contrast, hypercalcemia and/or hyperphosphatemia were also observed in 3 patients of infantile form, and low calcium and/or low phosphorus formula started to these patients. As the efficacy of the treatment, all the patients survived. While total 8 patients required some forms of respiratory support at the baseline, 5 of them were free from respiratory support including oxygen supply at the end of the study. Baseline radiographs in all the patients except 2 patients with closed growth plate showed undermineralized metaphyses observed as hypophosphatasia-associated skeletal lesions. Mineralization was gradually improving with all the patients. Gross motor milestone was evaluated in 10 infants. Developmental milestones were also improved after the treatment.

Conclusions: The clinical trial revealed the improvement of skeletal, respiratory, and physical symptoms in patients with HPP by AA. Careful monitoring of serum calcium and phosphate levels is necessary because hypocalcemia, hypercalcemia, and hyperphosphatemia may occur. ( number, NCT02456038; UMIN CTR number, UMIN000014816).

O8-2 BGN Mutations in X-Linked Spondyloepimetaphyseal Dysplasia

Sung Yoon Cho1, Jun-Seok Bae2, Nayoung K.D. Kim2, Francesca Forzano3, Katta Mohan Girisha4, Dongsup Kim5, Kyoung Yeul Lee5, Shiro Ikegawa6, Noriko Miyake7, Gen Nishimura8, Andrea Superti-Furga9, Jürgen Spranger10, Ok-Hwa Kim11, Woong-Yang Park2, Dong-Kyu Jin1

1Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine; 2Samsung Genome Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine; 3Division of Medical Genetics, Galliera Hospital, Via Volta 6; 4Department of Medical Genetics, Kasturba Medical College, Manipal University; 5Department of Systems Biology, Korea Advanced Institute of Science and Technology; 6Laboratory for Bone and Joint Diseases, RIKEN Center for Integrative Medical Sciences; 7Department of Human Genetics, Yokohama City University Graduate School of Medicine; 8Department of Pediatric Imaging, Tokyo Metropolitan Children's Medical Center; 9Department of Pediatrics, Centre HospitalierUniversitaire Vaudois, University of Lausanne (CHUV); 10Im Fuchsberg 14; 11Department of Radiology, Woorisoa Children's Hospital

Spondyloepimetaphyseal dysplasias (SEMDs) comprise a heterogeneous group of autosomal-dominant and autosomal-recessive disorders. An apparent X-linked recessive (XLR) form of SEMD in a single Italian family was previously reported.We have been able to restudy this family together with a second family from Korea by segregating a severe SEMD in an X-linked pattern. Exome sequencing showed missense mutations in BGN c.439A>G (p.Lys147Glu) in the Korean family and c.776G>T (p.Gly259Val) in the Italian family; the c.439A>G (p.Lys147Glu) mutation was also identified in a further simplex SEMD case from India. Biglycan is an extracellular matrix proteoglycan that can bind transforming growth factor beta (TGF-b) and thus regulate its free concentration. In 3-dimensional simulation, both altered residues localized to the concave arc of leucine-rich repeat domains of biglycan that interact with TGF-b. The observation of recurrent BGN mutations in XLR SEMD individuals from different ethnic backgrounds allows us to define “XLR SEMD, BGN type” as a nosologic entity.
Fig. 1 (abstract O8-2).
Fig. 1 (abstract O8-2).

See text for description

Table 1 (abstract O8-2).

Clinical Findings of the Affected Individuals with XLR SEMD BGN Type

O8-3 Effect of high dose monthly maternal cholecalciferol supplementation during breastfeeding on infant and maternal vitamin d status at 5 months post-partum: a randomized controlled trial

Benjamin J Wheeler1,2, Barry J Taylor1, Peter Herbison3, Jillian J Haszard1, Adel Mikhail1, Shirley Jones1, Michelle J Harper4, Lisa A Houghton4

1Department of Women's and Children's Health, Dunedin School of Medicine, University of Otago; 2Southern District Health Board, Paediatric Endocrinology; 3Dunedin School of Medicine, University of Otago, Preventive and Social Medicine; 4Department of Human Nutrition, University of Otago

Many countries recommend daily infant vitamin D supplementation during breast feeding, but compliance is often poor. A monthly, high dose maternal regimen may offer an alternative strategy for dual maternal and infant supplementation, but its efficacy is unknown.

In a New Zealand population not offered routine vitamin D supplementation, we aimed to determine the effect of two different monthly maternal doses of cholecalciferol (D3) on both maternal and infant 25-hydroxyvitamin D (25[OH]D) status during the first 5 months of breastfeeding.

Using a randomized, double blind, placebo-controlled design, breastfeeding women (n=90, mean age 32.1 years) were assigned to receive either placebo, 50,000 IU, or 100,000 IU of D3 /mo from Week 4 to Week 20 postpartum. Change in maternal and infant serum 25(OH)D from baseline to week 20 was calculated for each group and analysed using ANCOVA.

At 20 weeks, changes in maternal serum 25(OH)D were significantly greater in those assigned to D3 versus placebo (P<0.05), mean ± SD 17.3 ± 32.7, 30.2 ± 25.9, and 40 ± 29.3 nmol/L in the placebo, 50,000 IU, and 100,000 IU groups, respectively. Mean changes in infant serum 25(OH)D were 37.4 ± 51.9, 44.7 ± 44.7, and 52.8 ± 46.4 nmol/L in the placebo, 50,000 IU, and 100,000 IU groups respectively; however, these were not significantly different between groups (p>0.1). After adjustment for season of birth, vitamin D-fortified formula intake, and infant skin colour, the effect size for the 100,000 IU group was 19.1 nmol/L (95% CI 2.5 to 35.6; p=0.025) higher compared to placebo. For infants, maternal D3 supplementation at a dose of 100,000 IU /mo during the first 5 months of breastfeeding potentially benefits vitamin D status. While attractive from a compliance perspective, further studies are required to determine optimum dose and dosing frequency before this type of strategy is recommended as an alternative to daily infant supplementation.

O8-4 Establishment of a human growth plate model with iPS cell-derived cartilage

Takeshi Kimura1,2, Kie Yasuda1, Yoko Miyoshi1, Akihiro Yamashita2, Noriyuki Tsumaki2, Keiichi Ozono1

1Department of Pediatrics, Graduate School of Medicine, Osaka University; 2Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University

Background: Endochondral ossification in the growth plate cartilage (GPC) determines the future length and shape of long bones. Several skeletal dysplasias cause growth plate dysfunction and result in severe short stature. There have been many studies on mice GPC function and development, but little is known about human GPC. We have already reported that human iPS cell-derived cartilage (hiPSC-Cart), when implanted into the subcutaneous spaces of the SCID mice for one year, formed skeleton composed of epiphyseal cartilage- and diaphyseal bone-like structure.

Aim: The aim of this study is to establish a practical hiPSC-GPC model for investigation about human growth and disease modeling.

Method: We used four hiPSC lines derived from healthy control and patients with skeletal dysplasias (thanatopholic dysplasia, achondroplasia and hypochondroplasia). hiPSC-Carts were transplanted into the subcutaneous space of SCID mice at various ages. Their ossification was observed with X-ray images over time and evaluated histologically.

Result: Histological analysis showed that transplanted hiPSC-Carts have a zonal arrangement similar to GPC, and type X collagen, a specific marker of hypertrophic chondrocytes, is positive in the transition between cartilage and bone. Furthermore, the ossification of hiPSC-Carts is promoted in younger mice. For example, GPC-like structures are formed 4 weeks after transplantation into 4 weeks-old mice. Epiphyseal cartilage-like structures express human vimentin, but other skeletal components do not. These results collectively suggest that endochondral ossification of hiPSC-Carts depends on regulation inside the host. Interestingly, we observed abnormal GPC-like structures in the patient- specific iPSC-Carts. The hypertrophic zones of these disease models showed irregular structures and cell sizes that were smaller than healthy control samples. These changes reflected the severity of the original diseases.

Conclusion: We have established an in vivo hiPSC-GPC model. The model likely contributes to further investigation for human growth.

O8-5 Vitamin D Deficiency and its Relationship with Cardiac Iron and Function in Patients with Transfusion-Dependent Thalassemia at Chiang Mai University Hospital

Prapai Dejkhamron1, Karn Wejaphikul1, Tuanjit Mahatumarat1, Suchaya Silvilairat1, Pimlak Charoenkwan1, Suwit Saekho2, Kevalee Unachak1

1Department of Pediatrics, Faculty of Medicine, Chiang Mai University; 2Department of Radiological Technology, Faculty of Associated Medical Sciences, Chiang Mai University

Background: Vitamin D deficiency (VDD) is common in patients with thalassemia. There were conflicting findings on relationship between VDD and cardiac iron. Previous study reported that VDD may be associated with iron-related cardiomyopathy plausible linking L-type calcium channel activity to cardiac iron loading in these patients. Chronic heart failure was also well described in VDD. Parathyroid hormone (PTH) was positively correlated to cardiac iron in patients with thalassemia. However, a recent study was unable to identify the association between vitamin D and cardiac iron.

Objectives: We aim to determine the prevalence of VDD and to explore the impact of VDD on cardiac iron and cardiac function in patients with transfusion-dependent thalassemia (TDT).

Method: A prospective cohort study in patients with TDT (>12 transfusions/year), aged 8-25 years, who were followed at Chiang Mai University Hospital during Sep 2013-March 2015 was conducted. Patients with liver disease, renal disease, type 1 diabetes, malabsorption, hypercortisolism, malignancy, and contraindication for magnetic resonance imaging (MRI), were excluded. Calcium, phosphate, alkaline phosphatase (ALP), parathyroid hormone (PTH), 25-hydroxy vitamin D (25(OH)D) were measured. The mean hemoglobin level in the year preceding the study was calculated. Additionally, cardiac iron (cardiac T2*) was determined by MRI, and left ventricular ejection fraction (LVEF) was evaluated by echocardiography.

Results: Sixty-one (33M/28F) patients with TDT were enrolled. The prevalence of VDD was 50.8% (31/61). The mean hemoglobin in the last 12 months and the mean ferritin during the past 5 years were significantly correlated with 25(OH)D level (p=0.026 and p=0.026, respectively). Patients with severe cardiac iron deposition (T2* ≤ 20 ms) had tendency lower 25(OH)D than those with cardiac T2*>20 ms (15.5 ± 4.7 vs 20.7 ± 7.0; p=0.065). The medians (ranges) of 25(OH)D level were 15.4 (7.9-19.8) and 21.9 (20.2-48.6) ng/mL in VDD and non- VDD group, respectively. Sex, age, serum calcium, phosphate, ALP, PTH, liver iron concentration, cardiac T2*, and LVEF were not statistically different between the groups with or without VDD. Patients with VDD had significantly lower hemoglobin levels as compared to those without VDD (7.6 ± 0.9 vs 8.1 ± 0.9; p=0.043).

Conclusion: VDD is prevalent in patients with TDT and is correlated with hemoglobin level. The correlations of VDD to cardiac iron uptake and function are warranted in the large population with TDT.

O9-1 FGFR1 mutations in four patients with congenital hypogonadotropic hypogonadism and split-hand/foot malformation: implications for the FGFR1 proximal promoter region

Kohnosuke Ohtaka1, Yasuko Fujisawa1, Rie Yamaguchi1, Fumiko Kato1, Hideaki Yagasaki1, Tatsuya Miyoshi2, Yukihiro Hasegawa2, Tomonobu Hasegawa3, Hideaki Miyoshi4, Fumio Takada5, Momori Katsumi6, Maki Fukami6, Tsutomu Ogata1

1Department of Pediatrics, Hamamatsu University School of Medicine; 2Division of Endocrinology and Metabolism, Tokyo Metropolitan Children's Medical Center; 3Department of Pediatrics, Keio University school of Medicine; 4Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine; 5Department of Medical Genetics, Kitasato University Graduate School of Medical Sciences; 6Department of Molecular Endocrinology, National Research Institute for Child Health and Development

Background: Heterozygous loss-of-function mutations of FGFR1 are known to cause multiple disorders including congenital hypogonadotropic hypogonadism (CHH), CHH with anosmia/hyposmia (Kallmann syndrome, KS), and CHH with split-hand/foot malformation (CHH-SHFM), with variable expressivity and penetrance.

Objective: The objective of this study was to examine FGFR1 in four Japanese patients with CHH-SHFM.

Patients: This study consisted of four Japanese patients (cases 1–4) with CHH-SHFM. Case 1 was a 3-month-old boy with micropenis, undetectable serum LH and testosterone at mini-puberty, and right split hand. Case 2 was a 17-year-old boy with no pubertal development, undetectable serum LH and testosterone, and bilateral split hands and feet. Case 3 was a 34-year-old female with primary amenorrhea, low serum LH (0.4 mIU/mL) and undetectable serum E2, and left split hand. Case 4 was a 2-month-old boy with micropenis, undetectable serum LH and testosterone at mini-puberty, bilateral split hands and feet, and cleft lip and palate.

Results: Direct sequencing identified two heterozygous missense mutations (p.G97S in case 1 and p.R744T in case 2) and a heterozygous splice donor site mutation (IVS12+1G>T in case 3). The two missense mutations had drastically reduced luciferase activities, without a dominant negative effect. The splice donor site mutation yielded a small amount of mRNA skipping exon 12; in addition, it was predicted to produce two different types of aberrant mRNAs satifying the condition for nonsense-mediated mRNA decay by in silico analysis. In case 4, although no mutation wasfound on the coding exons 2-18, a microdeletion around non-coding exon 1 was revealed by MLPA analysis. This microdeletion was localized to a roughly 7.2-12.7 kb region by array CGH analysis, and was determined to be 8,312 bp long by direct sequencing for a PCR product obtained with primers flanking the microdeletion. Quantitative real time PCR analysis using primers for exons 8 and 9 indcated roughly halved FGFR1 expression in immortalized lymphoblastoid cells. In silico analysis indicated the presence of promoter-compatible H3K4Me3 marks and CpG sites around exon 1, and luciferase assays using various genomic fragments around exon 1 showed high transactivation function for a 470 bp segment encompassing the 5’ part and the just upstream region of exon 1.

Conclusion: The results are consistent with heterozygous loss-of-function mutations of FGFR1 being relevant to the development of CHH- SHFM, and indicates the presence of the FGFR1 promoter around non-coding exon 1.

O9-2 Low Anti-Mullerian hormone level may indicate ovarian dysfunction in pubertal female childhood cancer survivors

Kanako Tanase-Nakao, Yusuke Fujisawa, Kazuko Mizutani, Yumiko Terada, Yuta Chiba, Tomoko Yoshida, Yasuko Ogiwara, Keisuke Yoshii, Yasuhiro Naiki, Reiko Horikawa

Division of Endocrinology and Metabolism / Department of Pediatrics, National Center for Child Health and Development

Background: Along with the improvement of cancer treatments, the number of childhood cancer survivors (CCSs) is increasing. Together with this, long-term endocrine complications such as gonadal dysfunction are gathering more attention. Anti-Mullerian hormone (AMH) implies ovarian function in female. However, the clinical utility of AMH on evaluation of gonadal function in children and adolescent of CCSs is not well established.

Objective: To investigate the relationship between AMH concentration and gonadotropin (Gn) level in female CCSs and to investigate its usefulness in evaluation of gonadal function.

Subjects & Method: Thirty-nine samples of 30 female CCSs (aged 0-18) were involved in this study. We retrospectively analyzed the data of AMH and baseline Gns and/or Gn levels after LHRH stimulation test, obtained from June 2012 to June 2016 in our center. AMH was measured by ELISA and Gns were measured by CLIA.

Result: Mean age at diagnosis for primary cancer was 6.6 ± 5.2 years old and mean age at measurement of AMH was 8.9 ± 5.5 years old. All the samples were obtained after treatments for cancer were conducted, 16 samples were within 1 year and 23 samples were beyond 1 year from the treatment. Primary cancers were 3 brain tumors, 14 solid tumors other than brain and 13 hematologic cancers. Data measured under hormone replacement therapy was excluded. Mean AMH levels of 13 samples in 12 cases with age < 6 years old was 0.7 ± 2.2ng/ ml, 8 samples in 4 cases with age >6 years old and prepuberty was < 0.1ng/ml, and 18 samples in 14 cases with puberty was 1.8 ± 3.4ng/ ml. There was no statistically significant association between AMH concentration and gonadotropin levels throughout all age group. However, Gn levels in pubertal cases with undetectable AMH level (< 0.10 ng/ml) were remarkably high when compared with pubertal cases with detectable AMH ( ≥ 0.10 ng/ml). Mean baseline LH level (n=9) was 30.4 ± 34.7 mIU/ml, mean peak LH level (n=5) was 86.9 ± 61.4 mIU/ml, mean baseline'sH level (n=9) was 52.7 ± 42.5 mIU/ml and mean peak'sH level (n=5) was 401.1 ± 708.2 mIU/ml in cases with undetectable AMH. In contrast, mean baseline LH level (n=9) was 1.8 ± 1.7 mIU/ml, mean peak LH level (n=3) was 1.1 ± 1.3 mIU/ml, mean baseline'sH level (n=9) was 3.1 ± 1.7 mIU/ml and mean peak'sH level (n=3) was 6.6 ± 5.7 mIU/ml in cases with detectable AMH.

Conclusion: Undetectably low AMH in pubertal female CCSs is associated high Gn levels and may indicate primary hypogonadism.

O9-3 Genetic diagnosis of 8 lipoatrophy patients in one single center via whole exome sequencing

Jia-tong Hou, Shi-yao Wang, Lin-jie Wang, Hong-bo Yang, Hua-bing Zhang, Feng-ying Gong, Hui Pan, Hui-juan Zhu

Department of Endocrinology, Key Laboratory of Endocrinology of Ministry of Health CPeking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College

Background: Lipoatrophy is a group of rare diseases characterized by lacking subcutaneous adipose tissue. It is agreed that lipoatrophy is closely associated with various genetic variations. Despite lots of genes had been defined as causal genes in many types of genetic lipodystrophy, the molecular mechanisms of some rare lipoatrophy patients with distinctive phenotypes remain unclear.

Methods: Eight patients with adolescent-onset lipoatrophy features were recruited from Department of Endocrinology, Peking Union Medical College Hospital. Clinical and biochemical parameters of the patients are collected in details. Whole exome sequencing was performed in all 8 patients, aiming to identify the potential gene mutations linked with their lipoatrophy manifestations.

Results: All the patients had generalized fat loss and thin mottled skin from the adolescent period. Two of them had progeroid features. Clinical features of the patients were as follows: fasting insulin (43.93 ± 7.66) μIU/ml, fasting glucose (5.7 ± 2.9) mmol/l, glycosylated hemoglobin (6.1 ± 0.8)%; triglycerides (3.57 ± 1.40) mmol/l, total cholesterol (4.31 ± 0.99) mmol/l, low-density lipoprotein cholesterol (2.52 ± 0.60) mmol/l, high density lipoprotein cholesterol (0.87 ± 0.25) mmol/l, body fat rate (28.6 ± 9.0)%. With whole-exome sequencing, we identified one POLD1 mutation (c.1812_1814delCTC, p.Ser605del) in two patients, one WRN mutation (c.3020delG, p.Gly1007Alafs*16) in one patient, one CIDEC mutation(c.686C>T, p.Thr229Met) in one patient, two INSR mutations(c.4028G>A, p.Arg1343Gln and c.1534A>G, p.Ile512Val) in two patients, and two LMNA mutations(c.898G>C, p.Asp300His and c.29C>T, p.Thr10Ile) in two patients. While one patient showed no genetic variations.

Conclusions: Through whole-exome sequencing, eight gene mutations probably linked with lipoatrophy were detected. All gene mutations detected could cause different subtypes of lipodystrophy. When patients reveal lipoatrophy and progeroid features from the adolescent period, the diagnosis of lipoatrophy should be taken into consideration. Our study expanded the spectrum of clinical manifestations and genetic variations of lipodystrophy. Like all previously reported patients, these cases were diagnosed with whole exome sequencing, highlighting the clinical utility of next generation sequencing in the diagnosis of rare, complex disorders.

Key words: Lipodystrophy, whole exome sequencing, genetics, diagnosis

O9-4 Adult height and growth pattern in patients with classic congenital adrenal hyperplasia

SeokJin Kang, Mo Kyung Jung

Yonsei University College of Medicine, Severance Children's Hospital, Endocrinologic Pediatrics

Background: Congenital adrenal hyperplasia (CAH), mostly caused by 21-hydroxylase deficiency, is autosomal recessive disorder characterized by impaired cortisol synthesis. It can be presented with a combination of aldosterone and cortisol deficiency and androgen excess. Therefore, excess production of androgen and glucocorticoid replacement can result to early bone maturation and ultimately diminished adult height (AH).

Objectives: The purpose of this study was to obtain objective data on AH with classic CAH patients and analyze the affecting factors on AH. Also we evaluated growth pattern during age increase.

Study design: We retrospectively reviewed the longitudinal medical records of 40 children with classic CAH (male [n=19]: 9 salt-wasting (SW), 10 simple-virilizing (SV); female [n=21]: 8 SW, 13 SV) who reached AH at Pediatric endocrinology clinic of Severance hospital from 1977 to 2015. We also analyzed the affecting factors on AH, and assessed growth patterns with serial height standard deviation score (SDS) dividing into following stages of growth: early childhood (0-4.99 years), mid-childhood (5-9.99 years), and adolescence (10-15 years).

Results: AH (162.7 ± 9.72 cm) was significantly shorter than the midparental height (MPH) (172.5 ± 3.40 cm) in male patients (P <0.001), and similarly, AH (154.5 ± 6.45 cm) was significantly shorter than the MPH (158.7 ± 2.96) in female patients (P =0.002). Accordingly, the AH SDS was meaningfully lower than the MPH SDS in both sex (males: P <0.001, females: P =0.002). Considering subtypes, SV had tendency to attain shorter AH than SW. In addition, the affecting factors on AH were analyzed that they were not significantly associated with subtype, age at diagnosis, dose of steroid, except MPH. Height SDS for chronologic age showed gradual decrement during childhood to adolescence (males: 0.5 ± 2.51 at early childhood, 0.8 ± 2.26 at mid-childhood, 0.2 ± 1.62 at adolescence; females: -0.4 ± 1.40, -0.2 ± 2.01, -0.3 ± 1.42 at those same periods). The final AH SDS was -1.6 ± 1.98 in males and -0.81 ± 1.45 in females.

Conclusion: Reduced AH was observed in children with classic CAH compared with their given parental height, regardless of sex and subtype. Furthermore, the height SDS tended to decrease in accordance with age increase, so this finding suggests that proper intervention about growth assessment is needed in children with CAH.

O9-5 Gene Expression Profiles of Children with GH Deficiency (GHD) prior to Treatment with Recombinant Human Growth Hormone (r-hGH) are Associated with Growth Response Over Five Years of Therapy

Adam Stevens1, Philip Murray1, Ekaterina B Koledova2, Pierre Chatelain3, Peter Clayton1

1University of Manchester and Royal Manchester Children's Hospital; 2Merck KGaA; 3University Claude Bernard

Background: The relationship between long-term growth response in GHD and pre-treatment whole genome gene expression (GE) as an integrated biomarker of the consequences of being GHD has never been explored. Prediction of long-term response to r-hGH therapy would allow better decision making about start and maintenance doses and hence cost:benefit.

Objective and hypotheses: To assess the relationship of pre-treatment GE to growth response on r-hGH in GHD children over five years of therapy.

Method: Pre-pubertal children with GHD (n=50) were enrolled from the PREDICT studies (NCT00256126 and NCT00699855). Affymetrix U133 plus 2.0 microarrays, analysed using Gene Expression Barcode 3.01, were used to determine whole blood GE prior to treatment. Growth response to r-hGH treatment was determined using Height velocity (HV) over the five years of therapy. Two groups of patients were defined by assessing growth response over the five years of treatment, (G1) always above and (G2) always below the median. The effect of gender, age and distance to target height (DTH) on GE were accounted for as covariates. Network models of gene expression and random forest analysis, a machine learning analytical technique, were used to relate GE to growth response using area under the curve (AUC) of the receiver operating characteristic. The robustness of GE markers was assessed using permutation testing (n=1000).

Results: There was no difference in gender, age and DTH (p>0.05) between the HV groups (G1 and G2) at the start of treatment. Baseline GE could predict growth response consistently above and below the median over five years with an AUC of 0.86 and 0.89 respectively. These observations were confirmed by permutation analysis. Genes were identified (p<1x10-5) unique to G1 patients (n=69) or to G2 patients (n=72). Network models of GE prioritised 94 of these 141 genes. PIK3R1 gene expression (p=1.2x10-9), associated with cell proliferation and known to be activated by GH, was related to G1. DDX58 gene expression (p=2.2x10-10), associated with RNA secondary structure, was related to G2.

Conclusion: In pre-treatment GHD we have identified gene expression associated with growth response over five years of therapy. Further assessment to determine predictive value and functional significance of gene subsets is required.


1. McCall et al Nucleic Acids Research. 2014 Jan; 42:D938–D943.

O9-6 Further evidence for the involvement of the specific ESR1 haplotype in the susceptibility to estrogenic endocrine disruptors

Yasuko Fujisawa1, Rie Yoshida2, Francesco Massart3, Naoyuki Kamatani4, Maki Fukami2, Tsutomu Ogata1

1Department of Pediatrics, Hamamatsu University School of Medicine; 2Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development; 3Department of Pediatrics, University of Pisa; 4Institute of Data Analysis, StaGen Co. Ltd

We have revealed that ESR1 encoding estrogen receptor a carries a ~ 50 kb linkage disequilibrium (LD) block in its 5’ region, and that a specific “AGATA” haplotype markedly raises the susceptibility to the development of male genital abnormalities by estrogenic endocrine disruptors (EEDs) in Japanese population. Here, we report further evidence for the involvement of the specific “AGATA” haplotype in the susceptibility to EEDs.

Identification of a 2,244 bp microdeletion in an absolute linkage disequilibrium with the specific “AGATA” haplotype We sequenced the region encompassing the LD block in two homozygotes for the specific haplotype and two subjects without this haplotype, and found a 2,244 bp microdeletion within intron 6 of the ESR1 gene in the homozygotes only. Subsequent analysis in Japanese subjects including 35 homozygotes and 151 heterozygotes for the “AGATA” haplotype and 188 subjects lacking this haplotype demonstrated an “absolute LD” between this microdeletion and the “AGATA” haplotype.

Examination of the ESR1 haplotype and the microdeletion in Italian population Next, we studied Italian boys with hypospadias (n=13) and cryptorchidism (n=80), and Italian control boys (n=150). The same LD block and the same four major haplotypes as in Japanese population were identified, and the “AGATA” haplotype was significantly associated with the external genital abnormalities as well. Furthermore, a nearly complete absolute LD was identified between the microdeletion and the specific haplotype, except for two subjects. This argues that in Italian population, the same genetic susceptibility factor is also involved in the development of male external genital abnormalities.

Comparison of the frequency of the mictodeletion between affected subjects and normal controls The frequency of the microdeletion allele was significantly higher in all groups of affected subjects. Consequent genotype analysis indicated that the frequency of the miclodeletion homozygotes was significantly higher in the Japanese HS (17.94%) than in the control boys (3.26%) as well as in Italian population (HS+CO; 3.23%, control boys; 0.67%). Comparison of the frequency of the susceptibility factor between different generations We compared the frequency of the normal homozygotes or heterozygotes for the susceptibility factors between the two generations. The frequency of the homozygotes/heterozygotes of the microdeletion was significantly lower in the normal child (39/98) than in the normal adult (167/311). This implies of that male subjects carrying the microdeletion are more susceptible to have genital abnormalities under the elevated levels of the EEDs.

P1-1-1 Status and Trends in use of insulin analog for Japanese children and adolescents with type 1 diabetes mellitus and its association with glycemic control

Yukiyo Yamamoto1,2, Toru Kikuchi1,3, Tatsuhiko Urakami1,4, Kohji Tsubouchi1,5, Goro Sasaki1,6, Haruo Mizuno1,7, Yuki Abe1,8, Kazuteru Kitsuda1,9, Shigetaka Sugihara1,10

1The Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes (JSGIT); 2Department of Pediatrics, University of Occupational and Environmental Health Japan; 3Department of Pediatrics, Faculty of Medicine, Saitama Medical University; 4Department of Pediatrics, Nihon University School of Medicine, Department of Pediatrics; 5Chuno Kosei Hospital; 6Department of Pediatrics, Tokyo Dental College Ichikawa General Hospital; 7Departments of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences; 8Department of Pediatrics, Niigata City General Hospital; 9Department of Pediatrics, Kitasato University; 10Department of Pediatrics, Tokyo Women's Medical University Medical Center East

Background and Purpose: Treatment for type I diabetes mellitus (T1DM) has been greatly changing by common use of insulin analog and continuous subcutaneous insulin infusion (CSII). We investigated current status and trends in use of insulin analog for Japanese children and adolescents with T1DM and its association with glycemic control.

Subjects: The study subjects consisted of 1090 patients, 449 males and 641 females (79 patients; 0-5 years of age, 280 ; 6-10 years of age, 731 : 11-19 years of age) enrolled in 4th cohort of JSGIT (from 1st to 3rd period: March 2013 to February 2014).

Results and Discussion: Almost all patients were being treated with multiple daily injections (MDI) or CSII. The proportion of CSII increased (MDI vs CSII; 71.8% vs 23.4%, respectively at 1st period to 69.8% vs 25.8%, respectively at 3rd period), especially in 0-5 years of age. Within patients with MDI, use of frequent injections with more than 5 times a day increased. On the other hand, use of twice basal injections decreased, suggesting that use of frequent bolus injections adapted to lifestyle were increasing. The most frequently used basal insulin was Glargine (G), followed by Detemir (D) and Degludec (T). However, G and D decreased (G: 529 patients at 1st period to 424 at 3rd period, D: 217 to 163), whereas T increased (34 to 123). The most frequently used bolus insulin was Aspart (A), followed by Lispro (L) and Glulisine (AP). The most frequently used insulin in CSII was A, followed by L and AP. In terms of bolus and CSII, trends are similar during this period. The most frequently used basal-bolus combination was G-A, followed by D-A. However, both combinations decreased (G-A: 397 to 312, D-A: 201 to 150), whereas T-A increased (25 to 96). The median hemoglobin A1c (HbA1c) in patients using each insulin analog and combination were 8.0%(G), 8.0%(D), 8.2%(T), 8.0%(A), 8.0%(L), 8.2%(AP), 7.9%(CSII-A), 8.2%(CSII-L), 7.6%(CSII-AP), 8.0 %( G-A), 7.8 %( G-L), 8.1 %( G-AP), 8.0%( D-A), 7.7 %( D-L), 7.6 %( D-AP), 8.2 %( T-A), 8.6 %( T-L), 8.4 %( T-AP). Among patients using any insulin analog for basal and CSII, patients using T and CSII-L had significantly higher levels of HbA1c. On the other hand, there was no difference among any bolus, CSII, and basal- bolus combinations. These findings are the result from database of 1st year in this cohort. Continuous investigation is needed under longer- term use.

P1-1-2 Next-generation sequencing-based screening of monogenic mutations in 43 Japanese children clinically diagnosed with type 1B diabetes

Kikumi Ushijima1, Maki Fukami1, Tadayuki Ayabe1, Misako Okuno1,2, Satoshi Narumi1, Tsutomu Ogata3, Nobuyuki Kikuchi4, Tomoyuki Kawamura5, Tatsuhiko Urakami2, Ichiro Yokota6, Shin Amemiya7, Shigetaka Sugihara8

1Department of Molecular Endocrinology, National Research Institute for Child Health and Development; 2Department of Pediatrics and Child Health, Nihon University School of Medicine; 3Department of Pediatric, Hamamatsu University School of Medicine; 4Department of Pediatric, Yokohama City Minato Red Cross Hospital; 5Department of Pediatric, Osaka City University School of Medicine; 6Division of Pediatric Endocrinology and Metabolism, Shikoku Medical Center for Children and Adults, Department of Pediatric; 7Department of Pediatric, Saitama Medical University Faculty of Medicine; 8Department of Pediatric, Tokyo Women's Medical University Medical Center East

Background: Diabetes mellitus is classified into type 1 (T1D), type 2, other specific types, and gestational diabetes. T1D lacking diabetes- associated autoantibodies are termed as type 1B (T1BD). The group of “other specific types of diabetes mellitus” includes monogenic diabetes such as neonatal diabetes and maturity-onset diabetes of the young caused by genetic defects in the insulin secretion pathway. Because clinical characteristics of those monogenic forms and T1BD are partially overlapping, children with monogenic diabetes could be clinically diagnosed with T1BD, especially during childhood.

Objectives: The objectives of this study was to clarify the prevalence and clinical consequences of monogenic mutations in Japanese children clinically diagnosed with T1BD.

Methods: We studied 43 Japanese children from 42 families diagnosed with T1D at age between 0.5 and 16.0 years and had no diabetes- associated autoantibodies. The participants were recruited from 30 hospitals during the fourth cohort study of the Japanese Study Group of Insulin Therapy for Children and Adolescent Diabetes. We performed genetic analysis using the HaloPlex target enrichment system (Agilent) and a next-generation sequencer HiSeq (Illumina) to screen mutations in 30 genes known to cause monogenic diabetes.

Results: Four of the 43 participants had heterozygous missense mutations in the insulin gene (INS ). No mutations were observed in the remaining 29 genes. The INS mutations (p.G75C, p.C96F and p.V42A) were hitherto unreported. The p.C96F mutation-carrying children were siblings, whose mother was also affected by T1D. No significant differences were observed in body mass index-Z score between the INS mutation carriers and non-carriers (-0.4 vs -0.9, p = 0.26). Age at diagnosis was significantly younger in the INS mutations carriers than that of non-carriers (2.7 vs 9.4 years, p = 0.025).

Conclusions: The results indicate that small proportion of children clinically diagnosed with T1BD children with onset age >0.5 years had monogenic mutations. Mutation screening of those children is helpful not only to understand the molecular pathogenesis but also to provide individualize management, including genetic counseling.

P1-1-3 Association Between Leptin, Soluble Leptin Receptor, and Free Leptin Index with Body Mass Index in Elementary School Children in Medan, Indonesia

Siska Mayasari Lubis1, Jose RL Batubara2, Harun Alrasyid Damanik3, Miswar Fattah4

1Pediatric Endocrinology Division, Child Health Department, Medical School, University of Sumatera; 2Pediatric Endocrinology Division, Medical School, University of Indonesia; 3Department of Nutritional Sciences, Medical School, University of Sumatera; 4Department of Molecular Biology, Prodia Widyahusada Laboratory

Introduction: In recent years, obesity is becoming an epidemic health problem. Leptin is known to play an important role in the pathogenesis of obesity. Levels of soluble leptin receptor (sOB-R) can provide an indication of free leptin, are postulated to play an important role in the pathophysiology of energy homeostasis. The free leptin index (FLI), which may be a more accurate determinant of leptin function.

Objective: To evaluate the association between leptin, sOB-R, and FLI with body mass index (BMI) in elementary school children in Medan, Indonesia.

Methods: We conducted a case control study in ten elementary schools in Medan, Leptin and sOB-R were measured by enzyme link immunosorbent assay (ELISA). The ratio of serum leptin to sOB-R provides a measure of the free leptin index (FLI).

Results: A total of 202 children between 6 and 12 years old were recruited in this study. We found significantly association between body mass index with leptin, sOB-R, and FLI (p <0.05). Soluble OB-R levels were lower in children with obesity than normal weight children (28.1 ± 8.1, 37.7 ± 9.3 ng/mL, respectively), but, leptin and FLI levels were higher than control (756.9 ± 493.4, 160,7 ± 284.7 ng/mL, respectively).

Conclusions: This study showed a decrease in levels of sOB-R and increase levels of leptin and FLI in children with obesity, and there were significantly associations between Leptin, sOB-R, and FLI with BMI.

Keywords: Leptin, free leptin index, soluble leptin receptor, body mass index, obesity

P1-1-4 Erythropoietin ameliorates obesity and glucose intolerance on high fat dietary obese mice through the activation of erythropoietin receptor/STAT3 pathway and the up-regulation of FGF21 secretion on classical brown adipose tissue

Hisakazu Nakajima1, Kazuki Kodo1, Satoru Sugimoto1,2, Ikuyo Itoh1,3, Keiichi Shigehara1, Shota Fukuhara1, Hidechika Morimoto1, Jun Mori1, Taichiro Nishikawa1,4, Kitaro Kosaka1,5, Hajime Hosoi1

1Department of Pediatrics, Kyoto Prefectural University of Medicine; 2Department of Pediatrics, Ayabe Municipal Hospital; 3Department of Pediatrics, Tanabe Central Hospital; 4Department of Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine; 5Department of Pediarics, Saiseikai Kyoto Hospital

Background, Aims and Objectives: We hypothesized that classical brown adipose tissue (cBAT) could play a crucial role in the anti-obesity effects of erythropoietin (Epo). Our study highlights the mechanism in which Epo treatment could upregulate energy expenditure and improve glucose homeostasis through cBAT in obese mice fed with a high-fat diet (HFD).

Methods: C57BL/6J mice had been fed with HFD since the age of 4 weeks (HFD mice). We administered recombinant human Epo (200IU/kg) to some HFD mice intraperitoneally, 3 times per week for 4 weeks (HFD+Epo mice). Blood glucose, serum insulin and FGF21 were monitored and an intraperitoneal glucose tolerance test (IPGTT) was performed on the two groups. We analyzed the interscapular BAT (iBAT) and the liver harvested from both groups using molecular biology and physiological methods.

Results: Body weight, blood glucose and serum insulin were decreased in HFD+Epo mice compared with HFD mice. Serum level of FGF21, interscapular surface temperature and oxygen consumption were higher in HFD+Epo mice in comparison with HFD mice. The IPGTT showed that the levels of blood glucose were lower in HFD+Epo mice than in HFD mice. The weight of iBAT was larger in HFD+Epo mice than in HFD mice, whereas that of white adipose tissue was decreased in HFD+Epo mice than in HFD mice. The mRNA and protein levels of UCP1, beta3ADR, PRDM16, PPAR α and FGF21 in the iBAT were higher in HFD+Epo than in HFD mice. However, the mRNA and protein level of FGF21 in the liver was similar between the two groups. In addition, the mRNA levels of PEPCK and G6Pase in the liver were lower in HFD+Epo mice compared with HFD mice. Epo treatment significantly stimulated phosphorylation of Epo receptor (EpoR) /STAT3 in the iBAT in a HFD condition.

Conclusions: The activation of EpoR/STAT3 pathway in the cBAT by extrinsic Epo could contribute to the upregulation of energy expenditure and the improvement of glucose homeostasis by increasing the secretion of FGF21 on the cBAT in HFD mice.

P1-1-5 Basal and bolus insulin requirement in children with type 1 diabetes on sensor augmented pump in our hospital

Yusuke Mine, Satomi Tanabe, Masako Aoki, Misako Okuno, Junichi Suzuki, Tatsuhiko Urakami

Department of Pediatrics and Child Health, Nihon University School of Medicine

Introduction: Previous s tudies have demonstrated total basal insulin dose (TBD)/ total daily insulin dose (TDD) as about 40-50% and TDD as about 0.8-1.2U/kg/day in children with type 1 diabetes on CSII. Since Feb, 2015 sensor augmented pump (SAP) is available in Japan, and we expected the improvement of HbA1c level and the adjustment of the bolus basal ratio. Therefore, we investigated the changes in HbA1c level, TDD (U/kg/day) and TBD/ TDD (%) among children under 18 years old with type 1 diabetes after starting SAP therapy in our hospital.

Subjects and methods: Subjects included 11 children with type1 diabetes (M/F= T/ U, 9.0 ± 8.7 years old). They were classified into good glycemic control group (HbA1c<7.5 n=4, 9.9 ± 8.8 years old) and the poor glycemic control group (HBA1c>8.5 n=4, 10.3 ± 5.0 years old) based on HbA1c level after starting SAP therapy. TBD/TDD and TDD also investigated between the two groups.

We compared HbA1c level, TBD/TDD and TDD after and before SAP treatment in all subjects, and also compared TBD/TDD and TDD between the good and poor glycemic control groups.

Results: HbA1c level were significantly lowered by using 0.8% after using SAP (from 8.6% to 7.8%, p=0.036). In 7 out of 11 patients, HbA1c levels were lowered by 0.5%, however, TBD/TDD and TDD didn’t change significantly after using SAP (p=0.846 vs p=0.76). BD/TDD showed significant difference between the good and poor glycemic control groups (median 28% vs 43%, P=0.03). Whereas, TDD between the two groups didn’t change significantly (median 0.855U/kg/day vs 0.875U/kg/day, p=1.0).

Discussion: SAP therapy effectively lowered HbA1c level in children with type 1 diabetes. Overall, TBD/TDD and TDD didn’t change, but TBD/TDD in the good glycemic control group was significantly lower than in the poor glycemic control group. These results suggested that good glycemic control was achieved by increasing the bolus based on the sensor glucose levels.

P1-1-6 The cutoff values of indirect indices for measuring insulin resistance in Korean children and adolescents

Heon-Seok Han1, JunWoo Kim1, Kyung Hee Yi2

1Division of Endocrinology / Department of Pediatrics, Chungbuk National University College of Medicine; 2Department of Pediatrics, Wonkwang University Sanbon Medical Center

Purpose: We investigated the prevalence rate of metabolic syndrome (MetS) among Korean children and aolescents, and the distribution of indirect index of insulin resistance (IR) using homeostasis model of insulin resistance (HOMA-IR) and triglyceride and glucose (TyG) index. Also we suggested the cutoff values of HOMA-IR and TyG index to screen high risk group of MetS.

Methods: Data from 3,3313 Korean subjects (1,756 male and 1,556 female, aged 10-18 years) were included from the Korean National Health and Nutrition Examination Survey (K-NHANES) conducted during 2007-2010. We used three different criteria of MetS by de Ferranti et al., Cook et al., and International Diabets Federation (IDF). The cutoff values of HOMA-IR and TyG indx were obtained from the receiver- operation characterisitics curves.

Results: According to the criteria of MetS in the order of de Ferranti et al., and Cook et al., and IDF, the prevalence rates of MetS in male and female were 13.9% and 12.3%, 4.6% and 3.6%, and 1.4% and 1.8%, respectively. The cutoff values of HOMA-IR and TyG index were 2.94 and 8.41, 3.29 and 8.48, and 3.54 and 8.66, respectively. Each cutoff values from the three criteria approximately corresponds to 50th, 75th, and 75th to 90th percentiles of normal distribution of HOMA-IR and TyG index.

Conclusion: We presented the prevalence rate of MetS, the distribution of indirect index of IR, and the cutoff values to screen high risk group of MetS in Korean children and adolescents. The usefulness of these criteria needs to be verified by further evaluatin.

P1-1-7 Early Development of Decreased β-cell Insulin Secretion in Children and Adolescents with HbH Disease and Its Relationship with the Hematological Severity

Pairunyar Nakavachara1, Worarat Kajchamaporn1, Vip Viprakasit2

1Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Division of Pediatric Endocrinology, Mahidol University; 2Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Division of Pediatric Hematology and Oncology, Mahidol University

Background: Endocrinopathies related to iron overload are common among transfusion-dependent thalassemia (TDT) patients. Recently, studies have shown that endocrinopathies such as adrenal insufficiency, vitamin D deficiency, low bone mass and glucose intolerance are also quite common among non-transfusion dependent thalassemia (NTDT) patients.

Although HbH is the most common NTDT in Southeast Asia, little is known regarding the glucose metabolism among these patients. The purpose of our study was to evaluate the glucose tolerance among young HbH patients.

Methods: We investigated β -cell function (HOMA- β ) and insulin sensitivity (HOMA–IR) by performing OGTT among 32 children and adolescents with HbH disease, mean aged 15.5 (range; 10.7-24.9) years. Patients were divided into two groups based on the types of HbH which were deletional (--/ α -, n = 16) and non-deletional HbH (--/ αα T, n = 16). We compared demographic, hematological data, HOMA-β and HOMA–IR between deletional vs. non-deletional groups. We also compared demographic and hematological data between normal vs. abnormal HOMA- β and normal vs. abnormal HOMA–IR groups. Statistical analyses were performed using the unpaired t-test, the Mann-Whitney U-test and the chi-squared test as appropriate. Statistical significance was considered at P value of < 0.05.

Results: All patients had normal glucose tolerance. However, 40.6% and 21.9% of patients had abnormal HOMA- β (decreased β -cell insulin secretion) and abnormal HOMA–IR (insulin resistance), respectively. Non-deletional patients had significantly lower height Z-score and functional hemoglobin levels [7.4 ± 1.2 vs. 8.5 ± 1.3 g/dL, P=0.018 ] but higher serum ferritin levels [237.9 (range 60.5 to 1881.8) vs. 87.9 (range 38.2 to 414.0) ng/mL, P=0.012 ] than deletional HbH patients. Interestingly, patients with abnormal HOMA- β had significant lower weight Z-score and functional hemoglobin levels [7.6 ± 1.5 vs.8.3 ± 1.1 g/dL, P=0.039 ] than those with normal HOMA- β . Meanwhile, patients with abnormal HOMA–IR had significant higher weight Z-score and higher percentage of overweight and obesity than those with normal HOMA-IR. The types of HbH disease were not significantly different between normal vs. abnormal HOMA- β groups and normal vs. abnormal HOMA–IR groups.

Conclusion: A high prevalence of decreased β -cell insulin secretion was shown for the first time among young HbH patients in this study. The severity of anemia as determined by the levels of functional hemoglobin seemed to be related with decreased β -cell insulin secretion independent of the types of HbH disease. Prospective studies to evaluate glucose metabolism among children and adolescents with HbH disease who have impaired β -cell insulin secretion are needed.

P1-1-8 Increased prevalence of autoimmune thyroid disease in children and young adults with type 1 diabetes in Japan

Shino Odagiri, Satsuki Nishigaki, Takashi Hamazaki, Yuko Hotta, Kayako Hashimura, Masakazu Hirose, Tomoyuki Kawamura, Haruo Shintaku

Department of Pediatrics, Osaka City University Graduate School of Medicine

Background: Type 1 diabetes (T1D) is frequently associated with other autoimmune disease, including autoimmune thyroid disease (AITD). There is a paucity of understandings of AITD in children and young adults with T1D. Here we investigated the clinical features of T1D patients with AITD.

Subjects and methods: Data were analyzed from 446 patients with T1D (2.2-51.7 years old; the mean age, 20.3 ± 9.1 years; 57.7% female), who visited our department during April 2014-March 2016, by using the medical records.

Results: Among 446 patients with T1D, total 30 patients (76.7% female) were diagnosed with any of autoimmune diseases; 29 AITD, 1 SLE, 1 celiac disease, 1 ITP. There were 2 patients with three autoimmune diseases, one was T1D with Hashimoto’s thyroiditis (HT) and ITP, and the other was T1D with HT and celiac disease. Out of the 29 T1D patients with AITD, 18 patients were diagnosed with Graves’ disease (GD) (84.2% female) and the rest of 11 patients (60% female) were diagnosed with clinical HT requiring treatments. Antithyroid antibodies (thyroglobulin and thyroid peroxidase) were examined in 271 T1D patients. 77 (28.4%) out of the 271 patients were positive for any of the antibodies. Mean ages of T1D onset were not significantly different among T1D patients with or without AITD. The mean age at diagnosis of GD and HT was 18.0 and 8.2 years old, respectively. Out of 19 patients who developed GD, 15 patients developed GD after T1D was diagnosed. Mean interval was 12.8 years. While out of 10 patients who developed HT, 6 patients developed HT after T1D was diagnosed. Mean interval was 7.1 years. All of T1D patients with GD were controlled by antithyroid drug therapy, and none of them needed surgery or radiotherapy. Relapse of GD after remission occurred in 2 patients.

Conclusions: Prevalence of AITD in T1D patients was higher compared with general population. Especially female patients with T1D have higher risk of AITD than male patients with T1D. GD tends to develop several years after the onset of T1D and careful monitoring should be considered. Antithyroid antibodies’ positivity was high in T1D patients but most of the cases did not develop clinical HT. In conclusion, this study supports the recommendation for regular examination of thyroid antibodies and thyroid function in children and young adults with T1D.

P1-1-9 Factors Associated with the Presence and Severity of Diabetic Ketoacidosis at Diagnosis of Type 1 Diabetes in Korean Children and Adolescents

Hye Jin Lee1, HyeohWon Yu1, Hae Woon Jung2, Young Ah Lee1, Jae Hyun Kim3, Hye Rim Chung4, Jae Ho Yoo5, Eun Young Kim6, Jeesuk Yu7, Choong Ho Shin1, Seong Yong Lee8, Sei Won Yang1

1Division of Endocrinology and Metabolism / Department of Pediatrics, Seoul National University Children's Hospital; 2Division of Endocrinology and Metabolism / Department of Pediatrics, Kyung Hee University Medical Center; 3Division of Endocrinology and Metabolism / Department of Pediatrics, Inje University College of Medicine, Ilsan Paik Hospital; 4Division of Endocrinology and Metabolism / Department of Pediatrics, Seoul National University Bundang Hospital; 5Division of Endocrinology and Metabolism / Department of Pediatrics, Dong A Univ Coll of Med; 6Division of Endocrinology and Metabolism / Department of Pediatrics, Chosun University, College of Medicine; 7Division of Endocrinology and Metabolism / Department of Pediatrics, Dankook University Hospital; 8Division of Endocrinology and Metabolism / Department of Pediatrics, Seoul National University College of Medicine, SNU-SMG Boramae Medical Center

Diabetic ketoacidosis (DKA) is a major life-threatening complication of type 1 diabetes (T1DM). The aim of this study was to identify the risk factors for the presence and severity of DKA at onset of T1DM in Korean children and adolescents.

A retrospective chart review of children and adolescents newly diagnosed as T1DM from January 2000 to May 2015 was done in 7 secondary and tertiary centers in South Korea. Eligible subjects were < under age of 20 years and had records on the presence or absence of DKA at T1DM diagnosis. The severity of DKA was categorized as mild (venous blood pH<7.3), moderate (venous blood pH<7.2) and severe (venous blood pH<7.1). Data was collected on age, height, body weight at diagnosis, body weight after stabilization, pubertal status, family history of diabetes, delayed diagnosis of T1DM, preceding infection, health insurance status, and parental education.

A total of 361 children and adolescents diagnosed with T1DM were included. The mean age was 8.8 ± 4.0 years and 37 patients were younger than 3 years of age and 91 patients were older than 12 years of age. 53.7% (n=194) of the patients were female. Overall 48.9% (n=177) of the patients presented with DKA at T1DM diagnosis. Thirty six percent of all patients (n=137) was not diagnosed as T1DM at the first medical consultation and in 13% (n=47), a preceding infection was present. The risk for DKA at diagnosis was 2.8 times (P=0.022) higher in patients younger than 3 years of age, and was 1.8 times (P=0.007) higher in patients older than 12 compared to patients between 3 and 12 years old. Lower serum c-peptide levels (P<0001), presence of preceding infection (P=0.001), and delayed diagnosis (P=0.02) significantly increased the risk of DKA at T1DM diagnosis. Multivariate analysis revealed that age greater than 12 (OR=2.8, PDelayed diagnosis, being too young to express symptoms or being old enough to be relatively free from parental care, and preceding infection increased the risk of DKA at T1DM diagnosis. Insurance status, parental educational level and preceding infection affected the severity of DKA. These results imply that alertness of the physician and public awareness of the symptoms of diabetes are needed to decrease the incidence and the severity of DKA at diagnosis of T1DM.

P1-1-10 Decreased serum growth differentiation factor 15 level in pediatric patients with type 1 diabetes mellitus

Takako Sasaki, Shuichi Yatsuga, Miyuki Kitamura, Junko Nishioka, Yasutoshi Koga

Department of Pediatrics and Child Health, Kurume University School of Medicine

Introduction: Growth differentiation factor 15 (GDF15) is a member of the BMP family of TGF-beta superfamily proteins, has been reported to increase in a various disorders including diabetes mellitus, cardiac/renal insufficiency or cancers. Several reports described that the elevation of GDF15 is the useful biomarker for diabetic nephropathy, or diabetic cardiomyopathy in adult. However, there are no reports about serum GDF15 in relation with the diagnostic purpose of type 1 diabetes mellitus (T1DM) in children. In this study, we investigated whether GDF15 is the good biomarker for diagnostic purpose of T1DM in children.

Methods: We collected serum samples in pediatric patients with T1DM and type 2 diabetes mellitus (T2DM), who were enrolled at Kurume University Hospital from April 2012 to June 2016. We also collected serum samples of pediatric voluntary healthy controls from July 2013 to August 2013. We measured serum GDF15 level. We also checked age, body weight, and height. Anti-GAD antibody, anti-insulin antibody, and anti-IA2 antibody were measured in all T1DM and T2DM patients at diagnosis. The study protocol was approved by the institutional review boards of all centers (coordinator: Kurume University Hospital, #13099). Written informed consent was obtained from all subjects before enrollment.

Results: Thirty-two (female; 16) pediatric T1DM, 8 (female; 4) pediatric T2DM and 60 (female; 23) controls were collected. The mean (SD) of age in T1DM, T2DM and controls was 9.80 (4.38), 9.54 (4.32), and 13.4 (2.05) years of age, respectively. The median of GDF15 in T1DM, T2DM and controls was 291.0, 414.0, and 453.5 ng/mL, respectively. No patients had diabetic retinopathy nor diabetic nephropathy. GDF15 was lower in T1DM compared to controls (p<0.0001), and T2DM (p=0.0243). GDF15 in T2DM was not significantly different compared to controls (p=0.42). GDF15 in T1DM was no significant difference from autoantibodies, respectively. (Anti-GAD antibody; p=0.66, anti-insulin antibody; p=0.57, anti-IA2 antibody; p=0.51).

Discussion: GDF15 in pediatric T1DM was significantly decreased than those seen in the controls and T2DM. Our results implied that the GDF15 may be a useful biomarker to distinguish T2DM in the patient autoantibodies were negative. GDF15 has been reported to be increased in T2DM in adult, our data did not show increase in T2DM in children. Those discrepancies may remain to be solved in future.

Conclusions: GDF15 was lower in T1DM in children than those in controls and T2DM, suggested that GDF15 may be a useful biomarker for T1DM in children.

P1-1-11 Differences level plasma visfatin on obese adolescent based on insulin resistance level acording to homeostasis model assessment insulin resistance

Eka Agustia Rini2, Indra Ihsan1, Rismawati Yaswir1

1APPES, Paediatric Endrocrinology M. Djamil Hospital Padang; 2ISPAD, Paediatric Endocrinology M. Djamil Hospital Padang

Background: Adipose tissue has been proven to be not merely a site for energy storage but also the largest endocrine organ that secretes various adipocytocine. Plasma visfatin is predominantly secreted from visceral adipose tissue, has insulin-mimetic effects and the level closely linked to insulin resistance.

Objective: The aim of this study was to investigate the differences plasma visfatin level between obese and non obese adolescents and also between obese adolecent with insulin resistance and without insulin resistance.

Methods: This was a cross-sectional study and conducted in three Senior High School in Padang. Twenty eight obese adolescents and 28 control were enrolled in the study. The age of the subjects ranged from 14-18 years. Obesity criteria was measure based on body mass index (BMI). Fasting serum glucose level measured by glucose hexokinase fotometrik method and serum insulin was measured by chemiluminessence immunoassay method. Plasma visfatin was measured with enzyme-linked immunosorbent assay (ELISA). The insulin resistance index was estimated from fasting serum insulin and glucose level using the formula. Differences in the variables were tested using independent t-test and Mann-Whitney depending on the distribution of the variables.

Result: The plasma visfatin level was higher in the obese than in the control group 2,55 (SD 1,54) ng/ml vs 1,61 (SD 0,64) ng/ml, p: 0,005, and the insulin resistance group had higher plasma visfatin level than non resistance group 3,61 (SD 1,59) vs 1,96 (SD 1,18), p: 0,004

Conclusion: There were increasing level plasma visfatin toward increase of HOMA-IR in obese adolescents.

Key word: obese adolescents, insulin resistance, plasma visfatin.

P1-1-12 Survey about Carbohydrate Education for the childhood and adolescent type 1 diabetes

Tomoyuki Kawamura1, Masakazu Hirose1, Yuko Hotta1, Sachi Tsujiai5, Yuri Ota5, Hiroko Yoshioka5, Kayako Hashimura1, Takashi Higashide2, Yoneo Kashihara2, Tomomi Hashimoto2, Kayo Kimura3, Aono Mayumi4, Aono Shigeo4

1Pediatrics, Osaka City University Graduate School of Medicine; 2Hug-Hug Kids Clinic; 3Kimu Clinic; 4Terada-cho Child Clinic; 5Osaka City University Hospital, Pediatric ward

Introduction: Carbohydrate Counting (CC) has been going to be popular for type 1 diabetes (T1D) in these days in Japan. It has passed ten years since we published the first Japanese text about CC of Japanese foods in 2006, and have started to educate our patients CC first in Japan. For educating CC to the childhood T1D, their age and development should be considered. We developed the original education method of CC. It is comprised with two parts of, the estimation carbohydrate amount and the calculation of insulin dose. The “Carb” as a unit which is equal to 10 g of carbohydrate is used. For the training of the estimation, the “Carb-flashcards” are applied. For the training of calculation, the simplified calculation sheets are used. There is few reports about the CC education for the childhood and adolescent T1D so far.

Objectives: To elucidate the educative status of CC among T1D patients in our clinic.

Methods: The questionnaire was carried out to T1D patients in our clinic.

Results: Data were obtained from 108 patients or 64 parents. The age of patients was 3-45 years old (Median 17 years old), The disease duration was 0-20 years (Median 8 years). The ratio of MDI/Pump was 46%/56%. The first education of CC was provided, 27% was only for the parents, 41% was for both the patients and parents, and 26% was only for the patients. In the cases of infant and toddler (below 6 years old), the CC education was usually provided only for the parents. More than 60% of patients above 9 years old can use CC by themselves. Some patients at 7 years old can estimate the amount of carbohydrate in food and calculate the insulin dose by CC by themselves. Seventy-one percent of patients use the “Carb”. The others use “gram”. Among the patients using Pump therapy, 60 % of them use the bolus calculator function of the pump. Many patients have mastered CC with 1 years. The duration to master CC, has become shorted significantly in the recent onset patients. That was not related to the therapies of MDI or pump.

Conclusion: The CC education should be begun at 6-7 years old of patients. Our recent education method of CC has become efficient.

P1-1-13 Trends in the prevalence of extreme obesity among Korean children and adolescents from 1998 to 2014

Hyo-Kyoung Nam, Hye Ryun Kim, Young Jun Rhie, Kee-Hyoung Lee

Department of Pediatrics, College of Medicine, Korea University

Objectives: Although recent studies reported that prevalence of childhood obesity stabilized around early 2000s, national trends of extreme obesity in Korea have not been studied. We aimed to assess the prevalence of obesity and extreme obesity for Korean children and adolescents and the impact of extreme obesity on metabolic syndrome.

Subjects and Methods: Data for 21,402 children and adolescents aged 2 to 19 years (11,188 boys and 10,214 girls), were obtained from the Korean National Health and Nutrition Examination Surveys during 1998-2014. We used age- and sex-specific body mass index (BMI) percentiles and examined the trends in the prevalence of obesity and extreme obesity based on criteria suggested by the Korean national reference standard (KCDC) and an US Centers for Disease Control and Prevention (CDC). We used the definition of metabolic syndrome from the modified the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III).

Results: The prevalence of extreme obesity using the KCDC criteria was higher than using the CDC criteria. No significant increases in the prevalence of both overweight and obesity were found among boys and girls aged 2-19 between 1998 and 2014. Significant increases in the prevalence of extreme obesity were detected among adolescents aged 10-19, especially in boys. The rates of the metabolic syndrome combined with extreme obesity were 51.6% and 34.4% in teenage boys and girls, respectively. The odds ratios of extreme obesity on metabolic syndrome compared with normal BMI were 60.7 and 34.7 in teenage boys and girls, respectively.

Conclusions: Despite the prevalence of childhood overweight and obesity in South Korea stabilized since the early 2000s, the prevalence of extreme obesity is increasing especially in teenage boys. Given the greater morbidity risks for the extreme obesity, monitoring and preventing progression to extreme obesity become more important.

P1-1-14 The seasonal variations of HbA1c chronologically reduced with improvement of glycemic control

Mie Mochizuki1, Toru Kikuchi2, Tatsuhiko Urakami3, Nobuyuki Kikuchi4, Tomoyuki Kawamura5, Kisho Kobayashi6, Koji Kobayashi1, Nobuo Matsuura7, Nozomu Sasaki2, Shigetaka Sugihara8, Shin Amemiya2, The Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes (JSGIT)9

1Department of Pediatrics, University of Yamanashi, Department of Pediatrics; 2Saitama Medial University; 3Department of Pediatrics and Child Health, Nihon University School of Medicine; 4Department of Pediatrics, Yokohama City University Medical Center; 5Department of Pediatrics; 6Kobayashi childrens clinic, Osaka City University Graduate School of Medicine; 7Department of Early Childhood Education, Seitoku University; 8Department of Pediatrics, Tokyo Women's Medical University Medical C Kobayashi Clinic enter East; 9The Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes (JSGIT)

Objective: The Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes (JSGIT) showed that glycemic control in Japanese children with type 1 diabetes had improved through changes in insulin regimens and using a combination of insulin analogues since 1995. Fluctuation of HbA1c values, well-known seasonal variations, are composed of high HbA1c values in winter and low HbA1c values in summer in diabetic patients. Although there are various reports of improved glycemic controls in diabetic patients, little is known about whether the seasonal variation has changed with times. This study was driven by the question of how the seasonal variation of glycemic control in patients with type 1 diabetes had changed from 1995 to 2012, using the data of 1995, 2000 and 2008 cohorts of JSGIT.

Methods: Participants included 662 patients in the 1995 cohort, 791 patients in the 2000 cohort and 859 subjects in the 2008 cohort. We conducted longitudinal analyses on changes in seasonal variations of HbA1c. Seasonal variation per year, HbA1c, was calculated as HbA1c value in winter minus HbA1c value in summer. Statically analysis were performed by linear regression or Mann–Whitney U test.

Results: Average HbA1c values have decreased year by year (average HbA1c (%) = -0.0672*year +143.1543, P<0.0001). HbA1c values in winter were higher than those in summer, with statistically significance in following periods except 2003 and 2007 (P<0.05). HbA1c had become smaller year by year (average HbA1c (%) = - 0.0032*year + 6.7736, P=0.0440).

Conclusions: HbA1c values in Japanese children with type 1 diabetes had significantly improved with seasonal variation; almost 0.22%. The seasonal variations of HbA1c were chronologically improved, but there were obvious seasonal variations in 2012. Further study is warranted whether advanced treatment and support for type 1 diabetes may ameliorate seasonal variation of glycemic control.

P1-1-15 Differences in levels of Retinol Binding Protein 4 ( RBP4 ) on Adolescent Obesity Based on Insulin Resistance Levels According Homeostasis Model Assessment Insulin Resistance ( HOMA - IR )

Eka Agustia Rini2, Ronaldi Noor1, Eti Yerizel1

1APPES, Paediatric Endrocrinology M. Djamil Hospital Padang; 2ISPAD, Paediatric Endocrinology M. Djamil Hospital Padang

Background: Obesity has become a global problem and continues to be a major problem in many poor and developing countries and has reached the alarm. Obesity leads to insulin resistance in childhood. Retinol binding protein (RBP4), secreted primarily from the liver and adipose tissues, was recently proposed as a link between obesity and insulin resistance. The role of RBP4 in pediatric obesity and its relationship with insulin resistance are not elucidated.

Objective: The objective of the study was to determine the status of RBP4 levels in obese and lean adolescent and the relationship between RBP4 levels and insulin resistance based on Index of Homeostasis Model Assessment Insulin Resistance (HOMA - IR ).

Method: This was a cross-sectional study and conducted in three Senior High School in Padang. Samples included 14-18 years old obesity and normal weight adolescent (n=56). We measured body mass index (BMI) and serum RBP4. Insulin resistance was assessed using HOMA-IR index.

Result: Similar RBP4 levels were found in the obese vs. non- obese group group (p > 0,05). Higher RBP4 levels were found in the insulin resistance vs. non-insulin resistance group but the differences was not significant (p > 0,05)

Conclusion: There were no significant differences in mean levels of RBP4 in the group of obese adolescents compared with non- obese adolescents . So also in the group of obese adolescents with insulin resistance compared with those without insulin resistance

Keywords: Obesity, Retinol binding protein 4 (RBP4), insulin resistance

P1-1-16 Fat or Fit- Search for predictors of metabolic complications in obese Indian children?

Anurag Bajpai1, Rashmi Kapoor2, Rishi Shukla1, Manisha Gupta1

1Division of Pediatric Endocrinology, Department of Endocrinology, Regency Hospital Limited; 2Department of Pediatrics, Regency Hospital Limited

Background: Metabolic complications are the main cause of concern in obese children. Body mass index (BMI) alone is however not a reliable predictor of these complications as they are not observed in many children with very high BMI. This heterogenity in the prevalence of metabolic complications for the same level of BMI makes identification of their pointers a desirable goal.

Aim: To identify predictors of metabolic complications in Indian children with obesity.

Design: Ongoing prospective observational study (April 1 2015 onwards). 205 children (120 boys, 5.1-18 years) with obesity underwent clinical assessment, complication screening (oral glucose tolerance test, lipid profile, fasting insulin and SGPT) and dual enhanced X Ray absorpimetery (DEXA) scan for body composition (body fat percentage and android to gynoid fat ratio). Predictors for metabolic complications were assessed using a step wise linear regression model.

Results: Metabolic complications observed in study included low HDL in 103 children (50.2%), high triglyceride levels in 51 (24.9%), pre diabetes in 44 (21.4%) and type 2 DM in 12 (5.9%). Children with metabolic complications had higher waist circumference standard deviation score (SDS, p =0.01), DEXA assessed total body fat percentage (p = 0.01) and android to gynoid ratio (p = 0.02) but similar body mass index (BMI) SDS (p = 0.1) compared to those without metabolic complications. HOMA IR levels correlated significantly with body fat, android to gynoid ratio and waist circumference SDS but not with body mass index SDS.

Conclusions: Metabolic complications are common in Indian children with obesity. High waist circumference SDS, AG ratio and total body fat are predictors to these complications. Their assessment would help target preventive and therapeutic interventions in obese children.
Fig. 1 (abstract P1-1-16).
Fig. 1 (abstract P1-1-16).

Predictor of metabolic complications in obese Indian children (N = 205)

P1-1-17 Detection of common pathogenic genes in children with special type of diabetes mellitus and its clinical application

Zhuhui Zhao, Wei Lu, Ruoqian Cheng, Li Xi, Xiaojing Li, Rong Ye, Zhong Lu, Dijing Zhi, Miaoying Zhang, Zhangqian Zheng, Feihong Luo

Department of Pediatric Endocrinology and Inborn Metabolic Diseases, Children's Hospital of Fudan Universit

Objectives: To explore the clinical value of common pathogenic gene detection in the diagnosis and treatment in hyperglycemia infants and children.

Subjects and Methods: Subjects were in-patients with hyperglycemia, age of onset before 1 year-old Cor insulin antibody negative and with family history of diabetes. Gene sequencing for ABCC8 , KCNJ11 , INS and GCK were performed and potential mutations were analyzed. The patients with ABCC8 and KCNJ11 gene mutations were treated with sulfonylurea, patients with GCK mutations were given the lifestyle intervention and others with insulin.

Results: Total 21 patients were enrolled, 15 patients were found with pathogenic gene mutations, 52.4% in ABCC8 gene and KCNJ11 gene (11/21). The patients with KCNJ11 or ABCC8 gene mutation are with average age 2.01 ± 1.62 months or 2.52 ± 2.60 months, respectively. GCK gene mutations were detected in children with age of onset more than or equal to 12 months, at 58.33 ± 43.02 months of age. There existed significant statistical difference among the onset ages of the three genetic variants, P = 0.001. The onset random blood glucose levels were significantly higher in the patients with INS gene mutation (66.70 mmol/L) than those of GCK gene mutation patients (9.73 + 1.97mmol/ L, P=0.003). 11 patients with ABCC8 or KCNJ11 gene mutation were treated with sulfonylurea and 9 patients reached euglycemia.

Conclusions: Mutations in potassium channel related genes (KCNJ11 and ABCC8) were the most common cause of neonatal diabetes in Chinese. Sulfonylurea therapy was effective and euglycemia were reached in most of the patients with the mutations in KCNJ11 and ABCC8 . Patients who were diagnosed hyperglycemia before 1 year-old Cor with negative antibody testing and family history of diabetes were referred for gene testing, even by targeted next-generation sequencing of all known related genes. The target therapy based on gene diagnosis is more effective and improvement of life quality.

P1-1-18 Maturity-onset diabetes of the young (MODY) in Hong Kong Chinese pediatric population - Are we different from the West?

Lai Ka Samantha Lee1,2, Wai Chun Sammy Wong2,3, Ho Chung Yau1,2, Wing Yan Jennifer Tsang1,2, Yuet Ping Liz Yuen2,4, Gary Wong1,2

1Department of Paediatrics, The Prince of Wales Hospital; 2Department of Paediatrics, The Chinese University of Hong Kong; 3Department of Paediatrics, The Alice Ho Miu Ling Nethersole Hospital; 4Department of Chemical Pathology, The Prince of Wales Hospital

Objectives: To investigate the period prevalence, clinical presentation, and molecular genetics of patients diagnosed MODY in the Chinese pediatric population of Hong Kong.

Methods: A retrospective study of Chinese patients with genetically confirmed MODY aged from birth to 18 years under the care of the 2 major pediatric departments of the Hong Kong New Territories East Cluster (NTEC) of Hospital Authority from 1st January 2010 to 31st December 2015. Non-Chinese patient was excluded. For the calculation of the estimated period prevalence, only patients aged below15 years were included as government population statistics stratified age at below 15 years.

The Electronic Patient Record System was employed to retrieve the clinical and genetic data. Descriptive statistics employed for data analysis.

Results: The period prevalence of Chinese patients with MODY, aged below 15 years, was 58.5 cases per 1,000,000 populations from 1st January 2010 to 31st December 2015 in the NTEC catchment areas. For those aged 18 years or below, there were 9 Chinese MODY patients, of whom 2 were related siblings. Two patients diagnosed MODY 3, the rest MODY 2. The F:M was 2:1 and mean age 10-year-5-month. All patients were non-obese. The presenting HbA1c was 5.3% - 6.7% in MODY 2, 7.5-7.7% in MODY 3. Family history was positive in 8/9, and 6/8 involved 2 generations (not counting the index patient). Anti-islet antibodies were tested and negative in 4 patients. A novel frame-shift mutation, heterozygous GCK c.1239delinsAA, p.(Tyr413*), was detected in the 2 related siblings, whereas the rest had been reported in non- Chinese studies.

Conclusion: Our study reported the first period prevalence of MODY 2 and 3 in Chinese pediatric patients in Hong Kong. This showed MODY was not uncommon in Chinese, as compared to the estimated prevalence of 68-108 cases per million (aged <= 25years) from 1996 to 2009 in UK (Shields et al. Diabetologia. 2010 Dec;53(12):2504-8). The limitation of our estimation included: (1) The case identification was based on clinical suspicion rather than protocol, (2) Due to limitation of age stratification of population statistics, only prevalence for those under 15 years was calculated. The clinical features were similar to reported in Caucasian studies. A larger sample is required to delineate any founder mutations for Chinese patients with MODY.

P1-1-19 Micronutrient deficiency in overweight / obese adolescents: Dual burden of malnutrition

Vandana Jain, Babita Upadhyaya, Anuja Agarwala

Department of Pediatrics, All India Institute of Medical Sciences

Introduction: Several micronutrients deficiencies affect functional and physical performance of the children. Frequent consumption of junk food may be associated with micronutrient deficiency. This study was conducted to assess macro- and micronutrient intake in overweight/ obese adolescents aged 10-16 yrs.

Materials and Methods: 214 overweight adolescents (147 boys) aged 10 to 16 y were enrolled. Dietary assessment was done by one day 24 hr recall and quantitative food frequency questionnaire methods. The intakes were compared with the Recommended Dietary Allowance (RDA) for age and gender for each child. Data was presented as mean ± SD and as proportion (%) with intake above or below the RDA.

Results: Mean age was 11.9 ± 1.6 y and BMI SDS 2.3 ± 1.1. The mean daily energy intake was 2922 ± 838 Kcal, with the mean contribution from protein, carbohydrate and fat

being 11.3%, 59.8% and 29.3%, respectively. Mean daily fiber intake was 8.6 ± 3.8 g. Energy and fat intake exceeded the RDA in 75% and 95% subjects, respectively, while fiber intake was less than the RDA in 95%. The mean micronutrient intake of the subjects and the proportion whose intake was less than the RDA is presented in Table 1. Deficiency of Iron, Vitamin B12 and Zinc was seen in more than 60% of the subjects, while Magnesium, Folic acid and Vitamin C intakes were optimal in a majority.

Discussion: While energy and fat intake exceed the RDA in overweight adolescents, they have lower intake of fiber and essential micronutrients, s u c h a s i r o n , B 1 2 a n d z i n c . O v e r w e i g h t adolescents have a dual burden of malnutrition and should be encouraged to have more servings of fruits, vegetables and whole grains.
Table 1 (abstract P1-1-19).

Mean micronutrient intake and proportion with intake less than RDA

P1-1-20 A case of diabetic ketoacidosis associated with rhabdomyolysis and acute kidney injury

Rieko Yazawa1, Noriyuki Takubo2, Akimi Ishikawa2, Takuro Iwasaki2, Tomoaki Yokokura2, Yu Shimizu2, Rie Yamaguchi2, Kei Fukushima2, Mayuko Tsubahara2, Tetsuo Shono2, Hidenori Haruna2, Toshiaki Shimizu1

1Department of Pediatrics and Adolescent Medicine, Juntendo University Graduate School of Medicine; 2Department of Pediatrics, Juntendo University Faculty of Medicine

Background: Diabetic ketoacidosis (DKA) associated with rhabdomyolysis has previously been reported and may significantly influence primary care.

Case: A 14-year-old female patient with a one-week history of fatigue was admitted to our hospital with dyspnea and impaired consciousness in December 2015. A urine examination performed at her school when she was 8 -years -old had been positive for sugar. On admission, a biochemical examination of the patient’s urine and blood revealed the following: pH, 6.864, HCO3-, 4.7 mmol/L, serum glucose level, 716 mg/dL (39.7mmol/L), glycated hemoglobin, 15.8%, serum ketone level, 13,066 μmol/L, urinary C-peptide, 45 μg/d and positive for urine ketones and negative for pancreas–related autoantibodies. The patient was diagnosed with type 2 diabetes mellitus (T2DM) and DKA. Metabolic acidosis was attenuated by intravenous infusion with 10 mL/kg saline and 0.1 U/kg/h insulin. Within 21 h of therapy initiation, the levels of serum creatinine (Cre, 1.33 mg/dL, 117.6μmol/L), serum creatine kinase (CK, 2359 IU/L) and urinary myoglobine (20,000 ng/ mL) were increased, and the patient presented with rhabdomyolysis and acute kidney injury. The patient’s blood pressure and urine volume were maintained, and consciousness was gradually restored. As the blood glucose levels had not decreased after admission, the dosage of insulin infusion was increased to 0.2 U/kg/h. Subsequently, the blood glucose level was normalized and urinary ketones were undetectable. Serum Cre and CK levels were improved 32 h later. However, the patient presented with oliguria and systemic edema 43 h later, and was administered 0.2 g/kg albumin and 10 mg furosemide. Systemic edema and urine volume were improved from the 4th day following hospitalization, and serum Cre and CK levels were normalized on the 7th day. The patient was discharged on the 19th day.

Conclusion: In cases of severe DKA, the complication of rhabdomyolysis should be considered and the serum CK level and renal function should be carefully observed.

Consent for publication: The authors declare that written informed consent was obtained for publication.


Suresh Chandran1, Hui Yi Chng2, Lim Joyce3, Sian Ellard4, Khalid Hussain5, Victor Samuel Rajadurai6, Fabian Yap7

1Department of Neonatology, KK Women's and Children's Hospital; 2Paediatrics, Duke NUS Medical School; 3Paediatrics, NUS Yong Loo Lin School of Medicine; 4Paediatrics, Lee Kong Chian Imperial School of Medicine, Nanyang Technological University

Background: Early identification of HNF4A mutations in neonates with HH can facilitate the management of both the child and family.

Aims: To identify HNF4A mutations in neonates with prolonged hyperinsulinaemic hypoglycemia (HH) and parental history of diabetes.

Methods: Neonates delivered at our institution requiring glucose infusion rate (GIR) >10mg/kg/min after 48h of life from April 2015 to March 2016 were identified. We collected demographic data and determined if there was history of diabetes in the baby’s parents. We analyzed the HNF4A status of those with positive parental history of diabetes.

Results: There were 4 babies whose parents had pre-existing diabetes. Of these 4 neonates with parental history (2 male, all of Malay ethnicity), 1 had paternal and 3 had maternal history of pre-existing diabetes. Patient 1 (dad with diabetes from age 15y) was diagnosed with a heterozygous novel HNF4A missense mutation p.345Y, which was paternally inherited as a de novo mutation. Patient 2 (mum with diabetes from 18y) did not consent for genetic study. Patient 3 (mum with diabetes from 13y) and Patient 4 (mum with diabetes from 18y) were tested negative for HNF4A. These neonates were among twenty-five (56% male) neonates who satisfied the criteria for HH at 48h. These 25 HH neonates were out of 11923 babies born at KK Hospital over 1 year (incidence 2.1/1000). There were 10 Chinese (40%), 10 Malay (40%) and 5 babies of other races.

Conclusion: The presence of pre-existing diabetes in parents of neonates with HH may suggest a genetic etiology. Our preliminary data indicates that HNF4A gene testing should be performed in a larger number of neonates with HH and positive parental history of pre-existing diabetes before a definitive recommendation can be made.

P1-1-22 Insulin pump therapy in type 1 diabetes: The Indian experience

Manpreet Sethi, Archana Dayal Arya

Division of Pediatric Endocrinology / Department of Pediatrics, Sir Ganga Ram Hospital

Background: Insulin pumps have been used for the management of type 1 diabetes for over 15 years in the west. However similar experience is lacking in India where multi dose insulin injections still form the mainstay of management of type 1 diabetes. In a first study of its kind from India, we attempt to highlight the effectiveness, safety and superiority of insulin pump use in type 1 diabetes.

Objective and hypotheses: To determine the impact of insulin pump therapy on short and long term glycaemic control, body mass index (BMI), rate of severe hypoglycaemia and diabetic ketoacidosis (DKA) in children, adolescents and young adults.

Method: Retrospective analysis of data from case records of patients at our clinic. Out of the 64 patients on insulin pump, 52 were included in the study. Age of the patients at initiation of insulin pump ranged from 3 years to 26 years. Reckoning age-wise categorisation at 5 year intervals, patients were stratified into 5 groups (0-5, 6-10, 11-15, 16-20, 21-25 yrs). Data regarding pre-pump HbA1C (average HbA1C in the 6 months before starting insulin pump), pre-pump BMI (BMI at last visit before pump), pre-pump episodes of hypoglycaemia and DKA and post pump HbA1C (at 6months and one year after pump initiation), post pump BMI (BMI at 6 months after pump) and post-pump episodes of hypoglycaemia and DKA (in the one year after pump) in each patient was recorded and compared.

Results: Of the 52 patients included, only 27 followed up for one year or more after initiation of pump and 25 had visits only upto 6 months. There was a drop in HbA1C and increase in BMI across all age groups with the maximum difference seen in the oldest age group after starting pump therapy. Only 3 episodes of DKA were recorded after pump therapy against 10 episodes in the pre pump period. However there were 3 episodes of severe hypoglycaemia in the post pump period as compared to 2 episodes in the pre pump period.

Conclusion: This study suggests that insulin pump therapy is effective, safe and superior in children, adolescents and young adults with type 1 diabetes.

P1-2-1 Lower adiponectin, higher ALT level, lower AST/ALT ratio, and higher VAT value may suggest a risk for the onset of type 2 diabetes in obese children

Yuki Yasuda, Hisafumi Matsuoka, Shigetaka Sugihara

Department of Pediatrics, Tokyo Women's Medical University Medical Center East

Objective: An association between type 2 diabetes mellitus and metabolic syndrome (MS) has been suggested in obese children. We compared the characteristics of fat accumulation, blood chemistry, and adipokine levels among obese children with type 2 diabetes or MS without type 2 diabetes. We then analyzed the threshold value of the risk factor for type 2 diabetes.

Methods: One hundred and seven obese children visiting our outpatient clinic were enrolled in this study. The subjects were divided into 3 groups: Group A, obese children with type 2 diabetes (n = 19); Group B, obese children with metabolic syndrome but without type 2 diabetes (n = 19); and Group C, obese children without type 2 diabetes or metabolic syndrome (n = 69). Biochemical examinations and the measurement of serum adiponectin and leptin levels were performed for each patient. The visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) levels were measured using cross-sectional computed tomography (CT) images obtained at the umbilical level. An ROC analysis was performed according to the presence of MS or diabetes mellitus. The statistical analysis was performed using JMP pro 12. This study was approved by the ethics committee of Tokyo Women’s Medical University.

Results: Group A tended to have higher VAT values and VAT/SAT ratios and lower leptin and adiponectin levels, compared with Groups B and C. In Group A, the serum ALT levels were significantly higher and the AST/ALT ratio was significantly lower than those in Group C. In Group B, the VAT and SAT values were higher than those in Group C. An ROC analysis for the onset of type 2 diabetes showed that the optimal cut-off point for adiponectin was 6.4 μ g/mL (AUC = 0.859), while those for ALT, the AST/ALT ratio, and VAT were 35 IU/L (AUC = 0.821), 0.85 (AUC= 0.794), and 78 cm2 (AUC = 0.713), respectively. No significant correlation between the HbA1c and adiponectin levels was seen in obese children without diabetes. Group A had a significantly higher frequency of a family history of type 2 diabetes than Group B.

Conclusion: These results suggest that an adiponectin level of less than 6.4 μ g/mL, an ALT level of more than 35 IU/L, an AST/ALT ratio of less than 0.85, and a VAT value of more than 78 cm2 may indicate a risk for type 2 diabetes in obese Japanese children.

P1-2-2 South Indian mothers with gestational diabetes mellitus have normal BMI: Offspring have markers of neonatal adiposity

Ahila Ayyavoo1,2, Thiyagarajan Satheesh1, Satheesh Gayathri1, Palany Raghupathy1

1Department of Pediatric Endocrinology, G. Kuppuswamy Naidu Memorial Hospital; 2University of Auckland, Liggins Institute

Background: Studies from developed countries have exposed an elevated risk for obesity and type 2 diabetes mellitus in individuals exposed to gestational diabetes mellitus(GDM) during fetal life. Maternal obesity and pregnancy weight gain are risk factors for development of GDM. Risk for GDM is seen rarely in non-obese mothers. GDM affects up to 17.8% of pregnancies in urban south India, 13.8% in semi-urban areas and 9.9% in rural regions. Children born to GDM mothers have disproportionate growth and are prone to complications in the newborn period and later life.

Aim: Analyse the growth of neonates born to GDM mothers in south-India using Ponderal index and other anthropometry.

Methodology: Birth weight, gestational age, head, chest & mid-arm circumference and length of neonates born at GKNM Hospital, Coimbatore from April 2014-March 2015 were recorded 0-48 hours after birth. History of maternal diabetes, antenatal illnesses, pre-pregnancy weight, weight before delivery and mother’s height were recorded from clinical records. Ponderal index was calculated as weight(grams) x 100/ length3(cm) and body mass index(BMI) was calculated as weight(kilograms)/ length2(metres).

Results: 745 babies were analysed (GDM: non-GDM = 192:553). Ponderal index was elevated in GDM offspring (mean difference=0.06; p=0.008). Mid-arm circumference (mean difference=0.28cms;p=0.001) and chest circumference(mean difference=0.44cms;p=0.01) were higher in neonates of GDM mothers. Head circumference (p=0.25), length (p=0.06) and birth weight (p=0.17) were similar in both groups. Proportion of boys: girls (p=0.99), duration of gestation (p=0.25), socio-economic status (p=0.66) and birth order (p=0.78) were similar too. Caesarean and instrumental deliveries were high amongst GDM mothers (79% in GDM and 68% in non-GDM;p=0.01). Pre-pregnancy BMI of GDM mothers (mean=24.94): non-GDM (mean=23.56) were within normal range, though mean difference was 1.38(p=0). GDM mothers were heavier in the pre-pregnancy period (mean difference = 2.283kgs; p=0.008). But, pregnancy weight gain was higher in non-GDM (11.96kgs) than GDM mothers (10.97kgs; p=0.019). No difference was noted in Ponderal index with respect to treatment modalities for GDM (p=0.95).

Discussion: Babies born to GDM south Indian mothers have disproportionate body composition including elevated Ponderal index, mid-arm & chest circumference suggestive of neonatal adiposity even for minor variations in BMI(difference between groups=1.38) within the normal range and lower pregnancy weight gain. Normal Indian neonates have intrauterine origin of adiposity, central adiposity and hyperinsulinemia. As observed in the Pima Indians, GDM increases the risk for later obesity in the offspring irrespective of maternal obesity.

Conclusion: Risk for later diabetes, obesity and other metabolic disorders in the unique non-obese Indian GDM scenario could be elucidated only in larger longitudinal studies.

P1-2-3 A Novel heterozygous mutation of WFS1 gene leading to constitutive ER stress is the cause of Wolfram syndrome

Shuntaro Morikawa1, Toshihiro Tajima1,2, Akie Nakamura1,3, Takeshi Yamaguchi1, Katsura Ishizu1, Tadashi Ariga1

1Department of Pediatrics, Hokkaido University Graduate School of Medicine; 2Department of Pediatrics, Jichi Children's Medical Center; 3Department of Molecular Endocrinology, National Research Institute for Child Health and Development

Background: Wolfram syndrome (WS) is a disorder characterized by the association of early-onset, insulin-dependent diabetes mellitus, diabetes insipidus, deafness, and progressive optic atrophy. The disease is caused by mutations of WFS1 gene located on 4p16 encoding protein that is called WFS1. This protein composes the tetramer which is expressed especially in pancreatic islet β -cells and the resident component of the endoplasmic reticulum (ER) membrane. During the protein synthesizing process in ER, the accumulation of misfolded and unfolded protein occurs in a certain frequency (known as “ER stress”). This ER stress is attenuated by the activation of the unfolded protein response (UPR), which recovers and maintains the ER function. Since WFS1 is known as the component of UPR, the mutant WFS1 results in unresolvable ER stress conditions and cell apoptosis. This is the major cause underlying the development of symptoms in WS.

Case report: We report a Japanese female WS patient with novel heterozygous mutation in WFS1 gene. She was admitted to our hospital for poor weight gain and DM. Her growth failure was evident at 3 months old and congenital cataract was noticed at 7 months old. Her auditory brainstem response (ABR) test revealed severe bilateral hearing loss. Her WFS1 gene showed a heterozygous twelve bases deletion in exon 8, resulting in 4 amino acid in-frame deletion (p.N325_I328del). Methods: We analyzed the functional consequence of the patient WFS1 using luciferase assay, western blotting and fluoresence analysis in vitro .

Results: (1) The patient mutant WFS1 (p.N325_I328del) raises the ER stress, ATF6 reporter activity and NFAT reporter activity in the absence of thapsigargin, indicating that this mutation induces a constitutive ER stress and elevation of the cytoplasmic Ca2+ concentration. (2) p.N325_I328del monomer has a dominant negative effect over the wild type monomer. (3) Induced constitutive ER stress is canceled by the chemical chaperone (4-phenylbutyric acid). (4) p.N325_I328del reduces the mRNA expression levels of SERCA2b (sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPase 2b).

Since SERCA2b is required for ER and cytoplasmic Ca2+ homeostasis, these results raise the possibility that p.N325_I328del causes constitutive ER stress and cell apoptosis through ER Ca2+ depletion.

Conclusion: We report a severe WS patient caused by novel heterozygous mutation of WFS1 gene. The patient mutant WFS1 induces constitutive ER stress and cell apoptosis through Ca2+ efflux from ER. These results provide new insights into the roles of WFS1 in UPR mechanism.

Consent for publication: The authors declare that written informed consent was obtained for publication.

P1-2-4 Sodium pyruvate treatment improved endogenous insulin secretion in a patient with mitochondrial diabetes

Tadayuki Ayabe1, Takeshi Inoue2, Yuji Oto2, Nobuyuki Murakami2, Yasutoshi Koga3, Ryoichi Sakuta2

1Department of Molecular Endocrinology, National Research Institute for Child Health and Development; 2Department of Pediatrics, Dokkyo Medical University Koshigaya Hospital; 3Department of Pediatrics and Child Health, Kurume University Graduate School of Medicine

Background: Mitochondrial diabetes is a rare form of diabetes mellitus, and reveals progressive decline in endogenous insulin secretion. Sodium pyruvate treatment has been reported to be a potential therapeutic choice for fatigability in patients with mitochondrial diseases. However, the effect of sodium pyruvate treatment for glucose intolerance in patients with mitochondrial diabetes remains to be clarified.

Case presentation: Water-based sodium pyruvate solutions (0.5 g/kg/day) were administrated orally to a Japanese man with mitochondrial diabetes and myopathy caused by the m.14709T-C mutation. He was already diagnosed with diabetes mellitus and started insulin self- injection. He did not have any kind of islet autoantibodies. To evaluate therapeutic effects, we measured urinary C-peptide, HbA1c and total daily insulin dose (TDD) 6 months later. His urinary C-peptide level improved from 4.3 to 17.2 mcg/day after 1 day and to 30.2 mcg/day after 6 months of sodium pyruvate treatment. He experienced no adverse event such as diarrhea resulting from sodium pyruvate treatment, except episodes of mild hypoglycemia. To avoid hypoglycemia, his TDD could be reduced from 33 Units/day to 20 Units/day. Despite reduction of TDD, his HbA1c declined from 6.5% to 5.9%.

Conclusions: Sodium pyruvate treatment improved endogenous insulin secretion and resulted in reduced TDD in a patient with mitochondrial diabetes. Sodium pyruvate treatment may be a potential therapeutic choice for patients with mitochondrial diabetes.

P1-2-5 Impact of change in leptin/adiponectin and MDA-LDL in childhood obesity associated with eosinophilic inflammation of the airway and whole body

Norihiko Azuma, Tomoko Ootani, Yuuki Yasuda, Hisafumi Matsuoka, Shigetaka Sugihara

Pediatrics, Tokyo Women's Medical University Medical Center East

Background: Childhood obesity has been suggested to be a risk factor for bronchial asthma. However, the pathological association between body fat accumulation and eosinophilic inflammation in the airway is not well known.

Objective: We examined the relationship between clinical factors of obesity and eosinophilic inflammation based on fractional exhaled nitric oxide (FeNO) measurements and peripheral blood eosinophil counts.

Methods: Thirty-two obese children (age: 6-15 years, mean age: 10.5 years) attending our outpatient clinic were enrolled in this study. Cases with diabetes mellitus, any endocrine disease, or bronchial asthma were excluded. We measured the height, weight, waist circumference, blood pressure, and FeNO of each subject. Blood samples were taken to examine the blood eosinophil count, IgE, AST, ALT, γ -GTP, TG, HDL- Chol, MDA-LDL, uric acid, insulin, hs-CRP, leptin, adiponectin and PAI-1 level in the morning after an overnight fast. The correlations between these values and FeNO or the peripheral blood eosinophil counts were then analyzed. A multivariable analysis was performed using FeNO and the peripheral blood eosinophil counts as objective variables. The statistical analysis was performed using JMP pro 12. This study was approved by the ethics committee of Tokyo Women’s Medical University.

Results: The mean BMI-z score of the 32 obese children in this study was 1.8. No single correlation between either FeNO or the eosinophil counts and the BMI-z score was found. A significant single correlation between FeNO and MDA-LDL was found (r = 0.38, P = 0.034). A multiple regression analysis revealed that leptin/adiponectin , leptin, adiponectin, the eosinophil counts, and γ GTP were selected for the

FeNO model (R2: 0.70, P < 0.0001), while AST/ALT, ALT, MDA-LDL, and IgE were selected for the eosinophil count model (R2: 0.64, P < 0.0001). Discussion: FeNO reflects eosinophil inflammation of the airway. On the other hand, the peripheral eosinophil counts reflect systemic inflammation. Our data demonstrated that the increased visceral fat accumulation associated with adipokine changes, such as the leptin/ adiponectin level, and oxidative stress markers, such as MDA-LDL, were strongly correlated with eosinophilic inflammation in the airway and whole body.

Conclusion: Increased visceral fat accumulation during childhood obesity may induce eosinophilic inflammation in both the airway and the whole body through adipokine changes and oxidative stress.

P1-2-6 The Risk Factors of Cardiac Autonomic Neuropathy in Youth with Type 1 Diabetes

Hye Jin Lee1, Hwa Young Kim2, Hae Woon Jung3, Gyung Min Lee4, So Youn Kim1, Kyung A Jeong1, Keun Hee Choi1, Young Ah Lee1, Sei Won Yang1, Choong Ho Shin1

1Division of Endocrinology and Metabolism / Department of Pediatrics, Seoul National University Children's Hospital; 2Division of Endocrinology and Metabolism / Department of Pediatrics, Kangwon National University Hospital; 3Division of Endocrinology and Metabolism / Department of Pediatrics, Kyung Hee University Medical Center; 4Division of Endocrinology and Metabolism / Department of Pediatrics, Konyang University Hospital

Cardiac autonomic neuropathy (CAN) is an often overlooked chronic and serious complication of diabetes. Reduced heart rate variability (HRV) with parasympathetic loss is the earliest subclinical marker of CAN. The purpose of this study was to investigate predictors for reduced overall HRV and parasympathetic loss in youth with childhood-onset type 1 diabetes mellitus (T1DM) without microvascular complications.

A total of 113 patients with T1DM (19.7 ± 4.4 years, 55 males), who were followed up ≥ 5 years, were enrolled between January, 2014 and June, 2015 at Seoul National University Children’s Hospital. To evaluate overall HRV and parasympathetic activity, time domain [standard deviation of mean NN intervals (SDNN) and root mean squared difference of successive NN intervals (RMSSD)] and frequency domain [total power (TP), high frequency (HF)] indices were measured using 5-min ECG recording using SA-2000E (Medicore Co. Korea). Multivariate regression model was constructed using possible covariates (age, sex, diabetes duration, mean HbA1c, diastolic blood pressure [BP], non-HDL cholesterol, smoking, drinking). The mean age at T1DM diagnosis were 7.9 ± 4.0 years and diabetes duration was 11.7 ± 4.4 years (mean HbA1c 8.3 ± 1.1%). The mean HbA1c and diastolic BP were inversely related to SDNN (P= 0.013 and P= 0.035, respectively) and TP (P= 0.012, and P= 0.007, respectively) after adjusting for covariates, indicating that both poor glycemic control and high BP were significant predictors for reduced HRV in patients with T1DM. The mean HbA1c and diastolic BP were also inversely associated with RMSSD (P= 0.012 and P= 0.002, respectively), HF (P= 0.014 and P= 0.007, respectively) after adjusting for covariates, suggesting that both poor glycemic control and high BP were significant predictors for parasympathetic denervation in patients with T1DM. Both poor glycemic control and hypertension (especially diastolic) were independent predictors for reduced overall HRV and parasympathetic denervation in youth with T1DM. Optimizing glycemic and BP control are important to maintain HRV and prevent CAN.

P1-2-7 Metabolic risk factors in Korean adolescents with severe obesity: Results from the Korea National Health and Nutrition Examination Surveys (K-NHANES) 2007-2014

Cho Won Kyoung1, Han Kyungdo2, Ahn Moon Bae1, Park Yong-Moon3, Byung-Kyu Suh1, Min Ho Jung1, Yong-Gyu Park2, Shin Hee Kim1, Kyoung Soon Cho1, So Hyun Park1

1Department of Pediatrics, College of Medicine, The Catholic University of Korea; 2Department of Biostatistics, College of Medicine, The Catholic University of Korea; 3National Institute of Environmental, Health Sciences, National Institutes of Health, Epidemiology Branch

Backgrounds: Severe obesity in adolescents has become an important global public health issue. However, little information is available for analyzing the associations between abnormal metabolic risk factors and severe obesity in Korean adolescents.

Methods: This is a cross-sectional study. Among 7197 subjects aged 10–18 years who participated in the 2007-2014 K-NHANES and checked anthropometric data, 1326 adolescents (male = 744, female = 582) with age and sex specific BMI ≥ 85th percentile were included. These obese adolescents were classified by increasing levels of BMI percentile according to the following categories: overweight (85th ≤ BMI 95th percentile), obesity (95th ≤ BMI 120% of 95th BMI), severe obesity ( ≥ 120% of 95th BMI or BMI 35, whichever was lower), and extreme severe obesity ( ≥ 140% of 95th BMI or BMI 40, whichever was lower).

Results: The prevalence of overweight, obesity, severe obesity and extreme severe obesity were 5.6%, 6.2%, 5.9% and 0.9% in 10-18-year- old Korean adolescents, respectively. Among the obese adolescents, the proportions of overweight, obesity, severe obesity and extreme severe obesity were 30%, 33.4%, 32%, and 4.7%, respectively. With increasing level of obese categoty, the mean levels of total cholesterol (TC, P for trend < 0.001), triglyceride (TG, P for trend <0.001), low density lipoprotein (LDL, P for trend <0.001), HbA1C (P for trend < 0.036), systolic blood pressure (SBP, P for trend < 0.001) were increased and high density lipoprotein (HDL, P for trend < 0.001) was decreased. With increasing level of obese categoty, the incidence of TC ≥ 200 mg/dL (P < 0.007), HDL 40 mg/dL or 50 mg/dL in girls older than 16 years-old (P <0.001), LDL ≥ 130 mg/dL (P < 0.004), TG ≥ 150 mg/dL (P < 0.003), HbA1C ≥ 5.8% (P < 0.006), SBP ≥ 130 mmHg (P < 0.003) increased, respectively.

Conclusions: Adolescents with severe obesity have more metabolic risk factors than less severe obese form adolescents. Early recognition of severe obesity is important for assessing and management of current morbidity in adolescents.

P1-2-8 Associations Between Serum Vitamin D and Glucose metabolism in 3-year-old Japanese Children - NCCHD cohort study

Yuta Chiba, Yasuko Ogiwara, Tomoko Yoshida, Yumiko Terada, Kazuko Mizutani, Yusuke Fujisawa, Kanako Nakao, Keisuke Yoshii, Yasuhiro Naiki, Reiko Horikawa

Division of Endocrinology and Metabolism, National Center for Child Health and Development

Background and Objective: Vitamin D (VD) is known to modify pancreatic β cell function and affects glucose homeostasis. VD deficiency can be a risk factor for insulin resistance and diabetes. In the previous studies, the associations between VD and glucose homeostasis were reported in late childhood and puberty. On the other hand, there were a few reports in early childhood about this relationship. The objective of this study was to examine the associations among serum concentrations of VD, glucose metabolism and its association factors in early childhood.

Subjects and methods: 653 infants (male = 341, 52.2%) at 3-years of age, participating in the NCCHD birth cohort were included in this study. Random blood samples were obtained during morning hours. Serum 25-hydroxyvitamin D (25(OH)D) was measured by LC-MS/MS. Serum insulin (IRI) was measured by CLIA, and plasma glucose level was measured by commonly used enzyme method. HOMA-IR and BMI were calculated from the biochemical and physical data. Statistical analysis was performed using linear regression analysis and t-test.

Results: Serum 25(OH)D levels were 23.8 ± 6.1 ng/ml imean ± SD Arange 9.0 `42.6 ng/ml ). There were no significant difference of 25(OH) D levels between male and female (23.9 ± 6.1 ng/ml vs 23.6 ± 6.1 ng/ml) . Significant negative correlation between 25(OH)D and serum glucose (r |0.22 Ap 0.01) was observed. The number of subjects with VD deficiency ( 20 ng/ml) and VD sufficiency (>20 mg/ml) were 28.2% (n=184) and 71.8 % (n=469), respectively. VD deficient group had significantly higher glucose level (73.7 ± 15.2 mg/dl vs 69.2 ± 15.8 mg/dl, p<0.01) and higher HOMA-IR (1.22 ± 1.25 vs 1.01 ± 0.82, p<0.02) than in VD sufficient group. Although there were nosignificant difference, VD deficient group tended to have higher insulin levels (6.3 ± 5.1 vs 5.7 ± 4.0, p 0.14) and BMI (15.8 ± 1.2 vs 15.6 ± 1.1, p 0.15) compared to VD sufficient group.

Conclusion: Although there is a limitation of interpretation since this study was conducted in the non-complete fast condition, our data suggested that Vitamin D deficiency may affect glucose homeostasis from infancy.

P1-2-9 The ratio of glycated albumin to HbA1c estimates recent past glycemic control

Ikuma Musha1,2,8, Toru Kikuchi1,8, Mie Mochizuki3,8, Junya Akatsuka1,8, Akira Ohtake1,8, Kisho Kobayashi3,8, Nobuyuki Kikuchi4,8, Tomoyuki Kawamura5,8, Tatsuhiko Urakami6,8, Shigetaka Sugihara7,8, Shin Amemiya1,8

1Department of Pediatrics, Saitama Medical University, Department of Pediatrics; 2umagaya General Hospital; 3Department of Pediatrics, Yamanashi University, Department of Pediatrics; 4Yokohama City Minato Red Cross Hospital; 5Department of Pediatrics, Osaka City University Graduate School of Medicine; 6Department of Pediatrics and Child Health, Nihon University School of Medicine; 7Department of Pediatrics, Tokyo Women's Medical University Medical Center East; 8The Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes

Objectives: While HbA1c (A1C) has been used as the gold standard of glycemic control, it is difficult to estimate glycemic control within a month in case of the measurement every 3 or 4 months. We aimed to clarify whether the change in the ratio of glycated albumin (GA) to A1C could estimate the recent past glycemic control based on the difference in these half-lives.

Methods: We examined individual long-term consistency of GA/A1C ratio in 306 childhood-onset type 1 diabetes patients whose GA and A1C were measured simultaneously more than 10 times in the observational study every 4 months as a period. A concordance between a change of A1C during previous one month ( Δ A1C) and GA/A1C ratio at test was examined, where A1C value was expressed by both NGSP (National Glycohemoglobin Standardization Program) and IFCC (International Federation of Clinical Chemistry) numbers.

Results: We confirmed individual consistency of GA/A1C ratio by the significant correlation of quadric curve between the products of GA/ A1C ratio at different period over time. The concordance rates of Δ A1C with the difference between GA/A1C ratio at test and the intrinsic value as the individual mean of GA/A1C ratios were higher than those with SD score of GA/A1C ratio at test as a whole cohort. When Δ A1C improved ≤ -0.3% (NGSP) or ≤ -2.0mmol/mol (IFCC) and worsened ≥ +0.3% or ≥ +2.0mmol/mol, the concordance rate of Δ A1C using NGSP number was more accurate to estimate the deterioration of glycemic control. However, the concordance rate of improved recent past glycemic control was not significant in using either NGSP number or IFCC number. In addition, a statistically significant positive correlation was observed between GA/A1C ratio and A1C in NGSP number.

Conclusions: A change of GA/A1C ratio from the intrinsic value was useful to estimate the deterioration in the glycemic control within a month. The discrepancy of estimation was considered to depend on the difference of A1C expressed by IFCC or NGSP number, due to what is measured as A1C in each standardized system. Since A1C in NGSP number contains non-glycated hemoglobin, GA/A1C ratio may show a positive relation as A1C gets increased in the NGSP standardization. Thus estimation using NGSP number of deteriorated glycemic control was apparently higher than that using IFCC number.

P1-2-10 Studay on autophagy genes expression and its regulation mechanism in rats with intrauterine growth restriction

Min Yang, Ying Xin, Chao Yan Li, Dan Zhang

Department of Pediatric Endocrinology, Shengjing Hospital of China Medical University

Background: The mechanism of IUGR resulting in metabolic syndrome continues poorly understood and controversial. Normal development of pancreas is crucial for the normal structure and function maintenance. Some studies suggest that autophagy plays an important role in the development and maintenance of individual cell function. The aims of this work were: (1) to compare the differences in autophagy, between β -cells in normal rats and IUGR rats; (2) to study the regulation mechanism of autophagy in rats with IUGR.

Methodology/Principal Findings: Compared with normal rats, rats with IUGR had higher levels of autophagy-related proteins LC3B-II, beclin-1. Furthermore, cytotrophoblasts cultured under hypoxia (2% oxygen) in the presence or absence of nutlin-3 (a p53 activity stimulator) had higher levels of LC3B-II. Protein expression in IUGR group compared with the normal group. mTOR and P70S6K (Thr389) expression were decreased (P <0.05), P-ULK1 (Ser757) was up-regulated (P <0.05). High fat diet could activate mTOR and P70S6K (Thr389), and P-ULK1 (Ser757) expression was down regulated (P <0.05).

Conclusion: Elevated levels of autophagy were in pancreas of rats with IUGR in the middle-late develpment period, as well as that in adult rats, which might lead to poor development of pancreas. Decreased mTOR sinaling in IUGR rats could lead to the increase of autophagy. Autophagic activity of β cells of islet of IUGR rats may be normal in early adulthood.

P1-2-11 A case study of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome

Pin Li, Zhiying Zhu, Dandan Yuan, Shasha Zhou, Guoying Yao

Division of Endocrinology and Metabolism, Affiliated Children Hospital of Shanghai Communication University

Objectives: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX syndrome) is a rare recessive X-linked hereditary disease, which is characterized by different clinical manifestations including autoimmune endocrinopathies, refractory diarrhea and eczema. It is caused by FOXP3 gene mutation, which results in abnormality of regulatory T cell, leading to destruction of autoimmune stability, causing multiple symptoms, and therefore has poor prognosis. We would like to discuss the current research of the disease by studying the diagnose and treatment of a 6-year-old boy with IPEX syndrome, in order to avoid misdiagnosing.

Methods: The initial finding of the patient was insulin-dependent diabetes mellitus iIDDM ) when he was 2 years old. Repeated respiratory tract infections during the treatment suggested the immune dysregulation with a markedly increase IgE level. Diabetes nephropathy was diagnosed when he had proteinuria at the age of 4, with concomitant repeated intestinal infection, with eczema around the mouth, electrolyte disorders and malnutrition. Therefore, he was clinically diagnosed as IPEX syndrome and took a genetic analysis.

Results: We identified a missense mutation in his FOXP3 gene (c.1010G>A Cp.Arg337Gln). His mother was an asymptomatic female gene carrier (heterozygous) while his father had no mutation in this gene loci. Hence this child was diagnosed with IPEX syndrome and treated with methylprednisolone.

Conclusions: We’ve diagnosed the IPEX syndrome through the clinical features and gene analysis. Children with early-onset IDDM, refractory diarrhea and eczema should be considered the possibility of IPEX syndrome and FOXP3 gene analysis will provide a guiding suggestion in diagnosing. During the acute stage, supporting therapy including infection control, glucose regulation and parenteral nutrition should be taken to improve symptoms, while immunosuppressant agents or hematopoietic stem cell transplantation (HSCT) will play an important role in the further treatment.

P1-2-12 Development of MyDiabetes website. A web-based education programme for children & adolescent with type 1 diabetes mellitus (T1DM)

Rokiah Ismail1, Azriyanti Anuar6, Emma Foster2, Katie Haighton3, Timothy Cheetham4, Hilary Hartley5, Ashley Adamson2, Siti Zarina Yaakop6, Muhammad Yazid Jalaluddin6

1University Malaya Medical Centre Dietetic Department; 2Newcastle University, Human Nutrition Research Centre; 3Newcastle University, Institute of Health & Society; 4Department of Paediatric Endocrinology, Institute of Genetic Medicine, Newcastle University; 5Department of Paediatric Endocrinology, Royal Victoria Infirmary, Newcastle upon Tyne; 6Department of Paediatrics, University Malaya Medical Centre, Endocrine Unit

Background: Diabetes education is essential in diabetes care. Internet can be a tool to provide educational intervention in children with T1DM.

Aim: The objective is to develop a web-based education programme to assist in diabetes management and to provide support for children with T1DM in Malaysia.

Methods: Data was collected in three phases using a mix-method approach. There were 91 participants; 64 children with T1DM, 12 parents and 3 clinicians from Malaysia. An additional 12 Malaysian children who were living in Newcastle were invited to participate in the study.

In phase one, the data was collected using both qualitative and quantitative method to understand the challenges of children with T1DM (n=52). They were asked to identify regularly consumed carbohydrate-rich foods. In phase two, a semi-structured interview and an open- ended questionnaire given to healthy children in Newcastle (n=12). This is to analyse views and general usability of the website. In the final phase, children with T1DM and their families (n=12) were recruited and introduced to the website. They were guided on how to use it at home for six months. Semi-structured interviews were conducted on these children, their parents and clinicians (n=27). An additional set of questionnaires were given to the children only (n=12) to analyse participants’ views, experiences and acceptance of the website.

Results: All children reported that the website is useful to obtain information on carbohydrate content of foods and drinks. They used it to adjust insulin accordingly. They reported that they have made changes in their food choices based on the information obtained from the website. Most of them did not record their blood glucose (BG) regularly into the BG meter diary in the website. The majority n=10 (83%) felt more confident in managing their diet, insulin, and monitoring BG. Seventeen percent (n=2) reported that the website did not help to improve confidence level or manage their diabetes better. The clinicians indicated that the website was helpful in the clinic setting to teach and review children’s BG and dietary intake. The clinicians reviewed that the website had suitable application for the children to use for self-education and self-management system.

Conclusion: This study suggested that the website provides additional benefit to the children and their parents in terms of improving knowledge on carbohydrate content, diet choices, insulin adjustment and confidence level. The website is useful for the clinicians to help educate children with T1DM during clinic visits.

P1-2-13 Sleep Deprivation Increases the Risk for Obesity in Adolescents

Ah-Reum Kwon1, Mo Kyung Jung1, Hyun Wook Chae1, Duk Hee Kim2, Ho-Seong Kim1

1Department of Pediatric Endocrinology, Severance Children's Hospital, Yonsei University College of Medicine; 2Department of Pediatric Endocrinology, Sowha Children's Hospital

Study Objective: To investigate whether sleep deprivation is a risk factor for obesity in adolescents.

Design: Cross-sectional study.

Setting: Korean National Health and Nutrition Examination Survey.

Participants: Subjects were 4,126 adolescents, aged 12-18 years old, who participated in the Korean National Health and Nutrition Examination Survey IV and V, conducted between 2007 and 2012.

Measurements and Results: Subjective sleep duration was evaluated as a risk factor for obesity. Hours of sleep were self-reported. Anthropometric measurements, a questionnaire about medical history and lifestyle, and biochemical laboratory measurements were conducted. Body mass index (BMI) z-scores were decreased with longer sleep duration. Subjects were classified according to sleep duration as those who slept ≥ 9 h/night, those who slept 7 to 9 h/night, and those who slept < 7 h/night. Odds of obesity were assessed according to sleep duration. Adolescents who slept < 7 h/night had higher odds of being obese (OR = 1.39, 95% CI 1.04-1.87) than adolescents who slept ≥ 7 h/night. One additional hour of sleep duration reduced obesity risk 11% (OR = 0.89 95% CI 0.81-0.98) and decreased BMI z-scores at the rate of 0.08. These finding were more significant in boys than in girls. Physical activity, energy intake, and stress (as index scores) did not increase the risk for obesity.

Conclusions: Sleep deprivation increases the risk for obesity in adolescents, while the degree of physical activity and energy intake may be not associated with obesity.

P1-2-14 Fulminant Type 1 Diabetes Mellitus in Japanese Children and Adolescents

Kentaro Shiga1,7, Tatsuhiko Urakami2,7, Junichi Suzuki2,7, Yasuhiko Igarashi3,7, Hanako Tajima4,7, Shin Amemiya5,7, Shigetaka Sugihara6,7

1Yokohama City University Medical Center, Children's Medical Center; 2Department of Pediatrics, Nihon University Hospital; 3Department of Pediatrics, Igarashi Children's Clinic; 4Nippon Medical School; 5Department of Pediatrics, Saitama Medical University; 6Department of Pediatrics, Tokyo Women's Medical University Medical Center East; 7The Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes (JSGIT)

Objective: Fulminant type 1 diabetes mellitus (FT1DM) is a subtype of T1DM characterized by remarkably abrupt onset. Reportedly, the frequency of FT1DM is approximately 20% of adult onset-T1DM in Japan. On the contrary, few reports have described FT1DM in pediatric patients. The purpose of this study is to determine the frequency and clinical characteristics of FT1DM in Japanese children and adolescents.

Method: A 2-phase questionnaire survey was sent to the members of the Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes (JSGIT) regarding their clinical experience with FT1DM with the age at onset below 16 years. This group is consisted of pediatric diabetologists from 79 hospitals and is the largest study group for pediatric T1DM in Japan.

Results: Responses to the questionnaires were obtained from 54 of 79 hospitals (collection rate, 68.4%). Overall, 9 hospitals had treated 17 pediatric patients with FT1DM (5 patients, 4 patients and 2 patients in 1 hospital each, and 1 patient each in 6 hospitals). The distribution of patient age was biphasic, including young children aged below 5 years and children above 8 years of age. The clinical characteristics of the disease in this population, such as severity of the symptoms at onset and precursor symptoms, did not differ from those of adult-onset FT1DM.

Conclusion: Among children and adolescents, the frequency of FT1DM is rather rare compared to those of adult. Since FT1DM is a life- threatening metabolic state, early as possible and accurate diagnosis and an appropriate therapy are crucial. Pediatricians should take enough notice of the diagnosis of FT1DM.

P1-2-15 Combined therapy with pump and long acting insulin for type 1 diabetes mellitus caused by STAT1 mutation

Takahiro Tomoda1, Misako Okuno2, Akihito Sutani1, Atsumi Tsuji-Hosokawa1, Kosuke Imai1, Kenichi Kashimada1, Tatsuhiko Urakami2, Tomohiro Morio1

1Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental University; 2Nihon University School of Medicine, Pediatrics and Child Health

Background: STAT 1 gain-of-function (GOF) is the most common genetic cause of autosomal dominant chronic mucocutaneous candidiasis (AD-CMC) and underlies a variety of infectious and autoimmune features. AD-CMC is a rare condition, and type1 diabetes mellitus (T1DM) is one of the concerning complications of GOF STAT1 mutation. However, clinical details of T1DM with AD-CMC have not been clarified. Here we report a clinical course of a case with STAT 1 GOF who developed T1DM at the age of three months.

Case report: Since developing T1DM, she received insulin therapy with continuous subcutaneous insulin infusion (CSII), and the control was acceptable for her age with 8.2% of HbA1c. Since the age of 3 years, she repeated to candidiasis at scalp, genitalia and oral cavity. She was referred to our hospital for closer examination for immunodeficiency, and heterozygous missense mutation (Met202Glu) in STAT1 was identified, diagnosing her with AD-CMC. Simultaneously, the control of her blood glucose became worse with more than 10% of HbA1c, and the control was not improved by introducing sensor augmented pump (SAP) at the age of 7 years. Her blood glucose fluctuated violently due to unstable effect of insulin therapy. By close monitoring, we learned that instability of insulin effect was more prominent on the second day after changing an infusion set and reservoir, requiring changing the set every 2 days. She frequently developed a subcutaneous nodule at infusion site, and we suspected that unexpected inflammation due to AD-CMC has occurred in site, resulting in unstable effects of the insulin therapy by blocking the tube or by affecting insulin absorption in site. For the sake of stable supply of insulin, we introduced injection of long acting insulin together with SAP, and her blood glucose level was remarkably improved from 11.3% to 8.7% of HbA1c.

Discussion: STAT 1 is involved in various signaling inflammatory pathways of IFN- γ , IFN- α / β , IL-27, IL-6 and IL-21. We assume that GOF mutation of STAT1 may activate those pathways, subsequently causing unexpected inflammatory reaction to insulin or to stimulation by catheter.

Conclusion: Insulin pump therapy accompanied with long acting insulin would be a useful option for the therapy of T1DM with STAT1 mutation.

P1-2-16 Permanent neonatal diabetes mellitus due to a G32S heterozygous mutation in the insulin gene

Xiao-Qin Xu, Ke Huang, Fang Hong

Division of Endocrinology, Children's Hospital of Zhejiang University, School of Medicine

P1-2-17 Sensitivity and specificity of USG as compared to MRI for diagnosing fatty liver in overweight adolescents

Vandana Jain2, Manisha Jana1, Alec Correa2, Babita Upadhyaya2

1Department of Radiology, All India Institute of Medical Sciences; 2Department of Pediatrics, All India Institute of Medical Sciences

Background/ Objective: Non-alcoholic fatty liver disease (NAFLD) has emerged as a significant complication of childhood obesity affecting 30-50% of all obese children in various studies. Ultrasonography (USG) is typically used as the screening tool. The present study was undertaken to determine the sensitivity and specificity of USG for diagnosis of NAFLD in overweight adolescents as compared to measurement of hepatic fat fraction by MRI.

Methodology: The study was conducted at Departments of Radiology and Pediatrics, All India Institute of Medical Sciences, New Delhi after ethical approval and voluntary informed consent. Thirty-four overweight adolescents (mean BMI SDS 2.3 +/- 1.1), aged between 10 – 15 years were included in the study. They underwent grey scale sonography in a single state of the art ultrasound scanner by a single observer. NAFLD grading was done as 0 (absent), 1 (mild), 2 (moderate) and 3 (severe) on USG. Chemical shift MR imaging with dual echo Dixon method was performed. Fat fraction (FF) was calculated from the in phase and opposed phase images as FF= signal in in-phase (IP)- signal in opposed phase (OP)/ 2 x IP.

NAFLD grading in MRI was done as present (0) and absent (1); taking a cut off of 5% FF for the presence of fatty liver.

Results: Fatty liver was present in 22 (22/34; 64.7%) cases on USG, and 16 (16/34; 47.1%) on MRI. In 22 cases the MRI and USG findings were congruent. In three cases, MRI detected fatty liver with a fat fraction of 7.5, 9 and 11.9 but USG failed to detect fatty liver. In 9 cases, USG detected NAFLD but on MRI it was not designated as fatty liver as the fat fraction was < 5% (0.85- 4.9). In 8 of these 9 cases, the USG diagnosis was mild NAFLD. Considering MRI as the gold standard, the sensitivity and specificity of USG were 0.81 and 0.50, respectively.

Conclusion: USG is more susceptible to subjectivity and inter and intra-observer variability, which may miss mild NAFLD, as well as overdiagnose it. The sensitivity is acceptable but specificity is low. Hence USG can be used as a screening test for NAFLD, but the diagnosis should ideally be confirmed by estimation of hepatic fat fraction by MRI.

P1-2-18 Think beyond type 1 and 2 diabetes

Antony Fu

Princess Margaret Hospital, Paediatrics and Adolescent Medicine

A teenage girl initially presented to our department for short stature. She was a product of non-consanguinous parents with Chinese background. She has also been followed up by opthalmology and surgery for retinopathy and sensorineural hearing loss respectively.

Almost a year after presentation, she started to developed nocturia, polyuria and polydipsia. Investigations showed hyperglycemia, ketosis without acidosis, HbA1c 14.1 %, borderline low C-peptide and negative anti-islet cell antibody. She has been put on multi-dose insulin for presumably type 1 diabetes since then.

Another year after insulin treatment, she complained of anorexia and frequent abdominal pain. Subsequent workup revealed primary hypoparathyroidism. Therefore she was put on calcium supplement and caltriol with satisfactory response.

Putting diabetes, primary hypoparathyroidism, hearing loss and retinopathy together, we kickstarted the genetic study which confirmed the diagnosis of Kearns-Sayre syndrome. Her lactate and creatine kinase levels were normal, recent echocardiogram and electrocardiogram were both unremarkable. She was treated with Riboflavin and Coenzyme Q10 thereafter.

This case outlines the importance of thinking outside the box - especially when we encounter co-morbidities beyond the scope of traditional type 1 and 2 diabetes. Earlier recognition and treatment could make a difference.

Consent for publication: The author declares that informed consent was obtained for publication.

P1-2-19 The Prevalence of Mental Health Issues among Thai Children and Adolescents with Type 1 Diabetes

Somsongla Pirompuk1, Sunsanee Ruangson2, Jeerunda Santiprabhob1, Supawadee Likitmaskul1

1Division of Endocrinology and Metabolism / Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University; 2Division of Psychology / Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University

Introduction: Type 1 diabetes mellitus (T1D) is a non-curable disease which requires continuous patient self-management for maintaining blood glucose control hence preventing related complications. Many studies have found associated incidences of psychiatric disorders in children and adolescents with T1D for example depression, anxiety or eating problems. Furthermore the pediatric patient’s parents have also suffered from conflicts over patient management of T1D and, in general, a lower quality of life resulting from the burden of taking care their children and taking time off work.

Objectives: Our study aims to determine the prevalence of mental health issues and to analyze several factors affecting patient’s mental health including gender, parental marital status, primary care givers, family income, duration of diabetes, insulin regimen and frequency of self-monitoring of blood glucose (SMBG) and the association between mental health issues and glycemic control.

Methods: A cross-sectional descriptive study had been done with T1D patients and parents who attended the pediatric diabetes clinic during 1 August 2015 to 31 January 2016 using standardized screening questionnaires. For the children, both the Children’s Depression Inventory (CDI) Thai version and the Eating Attitudes Test-26 (EAT-26) Thai version were used. For the parents, the Thai Youth Checklist (TYC) for analyzing behavioral and emotional problems was used.

Results: Seventy-nine T1D (50 females, 29 males) patients and sixty-eight care-givers were enrolled. Mean patients age was 12.3 ± 2.8 years, mean diabetes duration was 4.9 ± 2.7 years. The screening results found that T1D patients had positive behavioral/emotional problems, depression, and eating problems at 16.2% (11/68), 22.7% (17/75) and 33.3% (7/21), respectively. No positive screening results were found in patients with good glycemic control as noted by HbA1c of less than 7.5%. In addition, there were no associated factors between all mental health issues and glycemic control among patients with HbA1c <7.5%, 7.5–9% and >9% (p >0.05). The same could be said for the gender, primary care giver, family income, duration of diabetes, insulin regimen and number of SMBG per day factors. Nevertheless a divorced family status was significant indicator related to a patients’ eating problems (p = 0.024).

Conclusions: Childhood and adolescents with T1D have an increased risk of developing eating disorders, depression and behavioral/emotional problems, leading to deterioration of glycemic control, thus increased awareness as well as early detection in diabetic children and adolescents is required.

P1-2-20 Adiponectin, Interleukin-6 and high-sensitivity CRP levels in overweight/ obese Indian children

Vandana Jain1, Ajay Kumar1, Jaivinder Yadav1, Anuja Agarwala1, Lakshmy Ramakrishnan2, Naval Vikram3

1Division of Endocrinology / Department of Pediatrics, All India Institute of Medical Sciences; 2All India Institute of Medical Sciences, Cardiac Biochemistry; 3All India Institute of Medical Sciences, Medicine

Background/ objectives: Interleukin (IL)- 6 and high-sensitivity CRP (hsCRP) are markers of inflammation associated with risk of cardiovascular disease and diabetes. Low Adiponectin levels are associated with insulin resistance. The aim of this study was to assess serum IL-6, hsCRP and Adiponectin and their correlation with conventional risk factors for cardiovascular disease and diabetes in overweight/obese Indian children.

Methodology: Children between 7-15 years, with BMI > 85th centile were enrolled from Pediatric outpatient at AIIMS, New Delhi. Weight, height, waist circumference (WC) and BP were measured. Fasting blood sample was drawn for glucose (BG), total cholesterol (TC), HDL cholesterol (HDL), triglycerides ( TG), adiponectin, IL-6 and hsCRP. The following cut-offs were taken to define abnormality: BG > 100mg/dl, TC ≥ 200mg/dl, LDL ≥ 130mg/dl, HDL 150mg/dl, Adiponectin 3 mg/L and IL-6 > 10 pg/ ml. Data was expressed as mean/ median and proportion with abnormal values. Correlations were checked between adiponectin, IL-6 and hsCRP and BMI, WC and BG.

Results: Eighty-four subjects (48 boys) with mean age 10.2 ± 1.9 yrs, and mean BMI z-score 2.7 ± 0.8 were enrolled. Two-thirds were prepubertal and 67.3% had abdominal obesity. Mean systolic and diastolic BP was 113 ± 10 and 74 ± 10 mm Hg, with hypertension in 15.5%. The biochemical parameters are summarized in Table 1. Low HDL was noted in 35.1%, impaired fasting glucose in 10.7% and low Adiponectin in 16.5%. High inflammatory mediators, IL-6 in 54.4% and hsCRP in 49.4% constituted the commonest abnormality. Adiponectin was inversely correlated with WC (r= -0.28, p=0.047). IL-6 was positively correlated with BMI (r=0.23, p= 0.09), and BG (r=0.24, p=0.08). IL-6 was higher in children with impaired BG as compared to those with normal BG (107 (IQR 22.5 – 197.5) vs. 8.5 (5-116) pg/ml, p=0.06).

Conclusions: Inflammatory mediators hsCRP and IL-6 were elevated in half of the subjects. Inverse correlation between Adiponectin and waist circumference and positive correlation between IL-6 with BMI and fasting glucose indicated the usefulness of these markers in pediatric population.
Table 1 (Abstract 1-2-20)

See text for description

P1-2-21 Assessment of Oral Hygiene Status in Obese with Type 1 Diabetes Mellitus and Normal Weight Adolescents

Zerrin Orbak2, Recep Orbak1, Yerda Ozkan1

1Department of Periodontology, Ataturk University Faculty of Dentistry; 2Department of Pediatric Endocrinology, Ataturk University Faculty of Medicine

Objective: The oral hygiene level in obes with Type 1 Diabetes Mellitus needs to be elucidated. The purpose of this study was to evaluate the oral hygiene status in obese with Type 1 Diabetes mellitus and normal weight adolescents.

Methods: A cross-sectional survey of 13-15-year-old adolescents was conducted on a sample of totaly 36 who were admitted to pediatric endocrinology clinic in 2014 and 2015. The sample was divided into equal two groups: ODM (obese with Type-1 Diabetes Mellitus; n = 18; 11 boys and 7 girls, mean age 13.21) and N (normal weight; n = 18; 9 boys and 9 girls, mean age 14.01). The subjects were asked to complete a questionnaire concerning medical history and dietary/oral hygiene habits. For anthropometric evaluation, the body mass index (BMI)-for-age was used. In oral examinations, the study used Oral Hygiene Index (OHI) and Gingival Bleeding Index (GBI) for the evaluation the oral hygiene status. Statistical differences were evaluated Chi-square, odds ratio (OR), Wilcoxon and Pearson correlation tests were used (P < 0.05).

Results: In both groups, the participants displayed similar oral hygiene habits According to the total sample, 2/18 ODM (11.1 %) and 8/18N (44.4 %) had good OHI, while 7/18 ODM (38.9 %) and 2/18 N (5.5%) were classified in a low level of OHI, with a significance between the groups (p < 0.001), even after sorting by age. According to the classification of GBI, 18/18 ODM (100.0%) and 13/18 N (72.2%) had GBI 1 (bleeding gingiva), and 0/18 ODM and 5/18 N (27.7%) were classified as GBI 0 (healthy gingiva), with a significance between the groups (p <0.001), even after sorting by age.

Conclusions: This study indicated that oral hygiene level were significantly lower in the ODM group.

P1-2-22 Construction of remote monitoring system of the elementary school upper grades health checkup data

Takanori Motoki1, Maki Kariyazaki2, Satoko Tsuru2, Ichiro Miyata1

1Department of Pediatrics, Jikei University School of Medicine; 2The University of Tokyo, Health Social System Engineering Laboratory

Background: A school nurse and a school physician do screening of short stature and overweight by height and weight measured in the school health examination. Furthermore, in the municipality, practitioners are doing medical examination to preventing for children’s unhealthy lifestyle to applicants. However the pediatric endocrinologists of the ward sometimes examine the first visit patients with short stature, highly obese or type 2 diabete patients who have passed a few years from the onset.

Objective: To reveal how many children who have the extent physique problems from height and weight data obtained from school health check of the ward there are. To build a regional cooperation system not to miss the patients who hospital consultation from the onset becomes too late. This study shows the change of the frequency the elementary school upper grades child with problems in physique of 2 years.<br />

Methods: We analyzed the height and weight data which had been input to the academic affairs system and which were the total of 14 times in the school medical examination from April 2011 to September 2015. Sharing the results with the Medical.

Result: We were able to obtain the data of 10,022/10031 children (in fiscal 2014/2015) to sixth graders from fourth graders who belonged to the public elementary school of the ward. 254/320 students with tall stature (Total 2.67%/3.19%: 4th Grade 2.55%/3.05%, 5th 2.79%/3.08%, 6th 2.36%/3.44%), 147/108 students with short stature (Total 1.55%/1.08%: 1.27%/1.01%, 1.58%/0.92%, 1.59%/1.31%), 740/1054 obese students (Total 7.52%/10.51%: 7.50%/11.18%, 7.58%/11.22% 7.54%/9.14%), 560/825 slimming students (Total 5.69%/8.22%: 4.47%/10.73%, 5.88%/7.79%, 7.07%/6.23%).

Conclusion: Unlike propotion of the tall stature, that of short stature was about 1.0-1.6% not follow Gauss distribution. According to the school year it goes up, because the students that have problems in physique poor weight gain, obesity, short stature was observed tends to increase, there is a need for early intervention.

P1-3-1 Monogenic mutations in patients with non-obstructive azoospermia and oligozoospermia

Shigeru Nakamura1,2, Mami Miyado1, Kazuki Saito1,3, Momori Katsumi1,4, Yoshitomo Kobori5, Yoko Tanaka6, Hiromichi Ishikawa7, Atsumi Yoshida8, Hiroshi Okada9, Hideo Nakai2, Tsutomu Ogata1,9, Maki Fukami1

1Department of Molecular Endocrinology, National Research Institute for Child Health and Development; 2Department of Pediatric Urology, Jichi Medical University, Children's Medical Center Tochigi; 3Department of Comprehensive Reproductive Medicine, Graduate School, Tokyo Medical and Dental University; 4Department of NCCHD Child Health and Development, Graduate School, Tokyo Medical and Dental University; 5Department of Urology, Dokkyo Medical University Koshigaya Hospital; 6Department of Pediatrics, Tokyo Dental College Ichikawa General Hospital; 7Tokyo Dental College Ichikawa General Hospital, Reproduction Center; 8Department of Pediatrics, Kiba Park Clinic, Reproduction Center; 9Hamamatsu University School of Medicine

Background: Azoospermia and oligozoospermia are multifactorial conditions that affect more than 1% of adult men. To date, 26 genes have been implicated in the development of non-obstructive azo/oligozoospermia. However, there is no single report of systematic mutation screening of these genes in patient cohorts. Thus, the significance of monogenic mutations in the etiology of azo/oligozoospermia remains unknown.

Objective: To clarify the frequency of mutations in known probable pathogenic genes in Japanese patients with non-obstructive azo/ oligozoospermia.

Materials and Methods: The study group consisted of 48 Japanese patients with non-obstructive azo/oligozoospermia. Next generation sequencing was performed for coding regions of ART3 , BPY2 , DBY, DNMT3L , FKBP6, FKBPL , KLHL10, MEI1 , MSH4 , PARP2 , PLK4, PRDM9 , PYGO2 , RBMY , SEPTIN12 , SOHLH1 , SPATA17 , STRA8 , SYCP3 , TAF4B , TAF7L , TEX11 , UBR2, USP9Y, ZMYND15 and ZNF230. In this study,

we focused on nucleotide alterations that affect protein sequences or splice sites. Nucleotide changes whose frequency in the general population is more than 1.0% were excluded as common polymorphisms. Functional consequences of missence variants were predicted by two in silico assays.

Results: A total of 19 nucleotide alterations were identified in 16 of 48 patients. These variants consisted of 4 hitherto unreported substitutions and 15 rare polymorphisms. Of the 19 nucleotide alterations, 9 were assessed as “possibly/probably damaging” or “deleterious” by in silico assays. In addition, 3 substitutions affected splice sites. Of these, c.346-1G>A in SOHLH1 and c.A511G in TEX11 have been reported as the cause of azoospermia.

Conclusions: This study indicates that nucleotide alterations in known probable pathogenic genes account for a certain fraction of cases with non-obstructive azo/oligozoospermia. These results need to be validated in future studies.

P1-3-2 Recurrent pancreatitis in early infancy due to severe hypertriglyceridemia from a novel mutation of lipoprotein lipase treated with glucose insulin infusion

Ahila Ayyavoo1,2, Paul Hofman2,4, Nikhil Phadke3, Emma Glamuzina4, Palany Raghupathy1

1Department of Pediatric Endocrinology, G. Kuppuswamy Naidu Memorial Hospital; 2University of Auckland, Liggins Institute; 3Phadke Hospital, GenepathDx; 4Auckland City and Starship Children's Hospital, Adult and Paediatric National Metabolic Service

Background: Familial lipoprotein lipase(LPL) deficiency is an extremely rare autosomal recessive disorder of lipoprotein metabolism occurring in 1 per million population. LPL is responsible for the clearance of chylomicrons(large lipoprotein molecules) from circulation. LPL is produced by myocytes and adipocytes, transported to the vascular endothelium for lipolysis of triacylglycerols in chylomicrons. LPL deficiency could happen due to mutations of LPLgenes/apoCII/apoA5/GPIHBP1/LMF1.

Clinical features associated with LPL deficiency include recurrent abdominal pain, poor growth, xanthomatosis, hepatosplenomegaly, lipemic plasma. Severe complications such as recurrent pancreatitis and lipid encephalopathy could happen. Treatment options include dietary fat restriction, use of medium chain triglycerides, fibrates, plasmapheresis and alipogene tiparvovec gene therapy. Acquired hypertriglyceridemia(HTGL) observed in patients with diabetes mellitus improve with insulin, dietary fat restriction, fibrates or niacin. Acquired LPL deficiency is observed during insulin deficiency and could be improved with insulin therapy. Hence, we tried the option of glucose- insulin infusion in a 2 month old boy with recurrent pancreatitis.

Case Scenario: Third born 2 month old boy of 3rd degree consanguineous parents had developed fever and vomiting at 10 days of life. Laboratory investigations revealed lipemic serum. He had suffered four episodes of acute pancreatitis by 2 months. Lipid profile is available in Table 1. A homozygous novel mutation was detected in exon 6 of the LPL gene c.904T>C/ p.Cys302Arg which created a missense mutation in the coding region. Other investigations were normal.

Treatment: Baby was treated with pancreatitis protocol. Extremely low dose of insulin was infused simultaneously with glucose along-with hourly glucose monitoring. Baby did not have any episodes of pancreatitis after the glucose-insulin intravenous infusion. He is currently on fat-restricted diet with medium chain triglycerides(MCT) and growing normally. Another schedule of IV glucose-insulin infusion helped reduce the triglycerides back to near normalcy at 9 months of age while oral fibrates and sub-cutaneous insulin were ineffective.

Future: Subcutaneous insulin needs to be tried on a few more occasions with regular glucose monitoring to enable effective management at home. Glucose-insulin therapy should be replicated in more patients and could be a cheaper option for familial LPL deficiency.
Table 1 (abstract P1-3-2).

See text for description

P1-3-3 Glycemic Levels in Normal Newborn-Preliminary Results

Raphael DR Liberatore Junior, Nathalia Azevedo, Daniela Daltoso, Jose C Simon

Division of Pediatric Endocrinology-Pediatric Department, Ribeirao Preto Medical School

The definition of glycemic normal variation in the newborn period is one of the most difficult patterns in neonatology. Different values are proposed based mainly in data from blood glucose samples obtained in the 60’s from the last century.

We proposed to check glycemic interstitial levels in the first 24 hs in a group of normal newborns using a glycemic sensor (Medtronic IproII) implanted subcutaneously in the very first hour after born. All babies were born with more than 38 weeks of gestational age, with no medical problem, and Apgar Score of more than 8 in the first minute. The mothers were not in use of any medication and have no know medical condition. All babies received only breast milk from their own mother and nothing else, including water. The study was approved by ethical committee and the sensor was installed only after write permission from the mother and the father. The results were achieved and showed minimum, maximum, median and standard deviation.

30 normal babies with gestational age between 38 and 41 weeks were studied. The weight ate the born range between 2610 and 3825 grams. The minimum value ranged between 40 and 67 mg/dl, median 47,5 mg/dl. The maximum value ranged between 53 and 99 mg/dl, median 59,5 mg/dl. Normal curve for minute after born will be showed.

The results show that interstitial glycemic levels in normal babies are very close to normal levels in older children. Those results would be very important to define normality of glycaemia.

P1-3-4 Novel homozygous SCNN1B mutation responsible for fatal systemic Pseudohypoaldosteronism Type 1

Ahila Ayyavoo1, Sowmya Ravikumar2, Meenal Agarwal3

1Department of Pediatric Endocrinology, G. Kuppuswamy Naidu Memorial Hospital; 2Department of Neonatology, G. Kuppuswamy Naidu Memorial Hospital; 3Phadke Hospital, GenepathDx

Background: Generalised pseudohypoaldosteronism type1(PHA1) is an extremely rare salt wasting disorder with hyponatremia, hyperkalemia and metabolic acidosis with elevated levels of aldosterone. Homozygous or compound heterozygous inactivating mutations of the alpha, beta or gamma sub-units of amiloride-sensitive epithelial sodium channel(ENaC) could result in PHA1(OMIM #264350). While severe salt wasting happens from kidneys, colon, sweat, salivary glands in autosomal recessive generalised PHA1(arPHA1), the autosomal dominant PHA1 is a mild disease. The child reported here is the first SCNN1B mutation arPHA1 from India with a novel mutation too.

Case Scenario: Term boy born by caesarean section to non-consanguineous parents developed purulent conjunctivitis and acute otitis media on Day 3 of life which was unresponsive to standard management. Baby was admitted on Day7 with normal activity, 16% weight loss and dehydration.

Investigations revealed severe hyperkalemia (10.2mEq/L), hyponatremia (127mEq/L) and acidosis (pH=7.2). Serum creatinine, calcium, phosphorus and creatine kinase were normal. Child was treated with anti-hyperkalemic measures (parenteral calcium gluconate, glucose- insulin infusion, salbutamol nebulisation, bicarbonate, per-rectal potassium binders), parenteral antibiotics, oral hydrocortisone and fludrocortisone with a provisional diagnosis of congenital adrenal hyperplasia. Synacthen stimulation test revealed normal 17OH progesterone, DHEAS and cortisol peak. Serum aldosterone (>150ng/dL; normal=1-16) and plasma renin activity (12.44ng/ml/hour;normal=0.15-2.33) were elevated with persistently elevated serum potassium (K+) and reduced urinary potassium. PHA1 was diagnosed and oral salt was added after stopping hydrocortisone. Electrocardiogram revealed severe hyperkalemia. Since K+ continued to be high, peritoneal dialysis was undertaken after a difficult catheter insertion. K+ dropped to normal levels (4.5mEq/L) and baby was discharged home on oral salt and per-rectal potassium binders. Child returned within a week with severe hyperkalemia and succumbed inspite of all treatment measures.

Genetics: Modified NGS panel with custom bioinformatic pipeline run revealed homozygous novel variant in exon 8 of the SCNN1B gene c.1212C>A /p.Tyr404Ter (Y404X) which resulted in premature termination of the amino acid chain. Not previously reported in databases, but pathogenic as per all prediction algorithms.

Conclusion: Clinical suspicion, prompt diagnosis and management of electrolyte abnormalities would probably help children with less severe phenotype. Initiation of routine mutation analysis in suspected PHA1 Indian children would help in research into successful treatment measures.

Consent for publication: The authors declare that written informed consent was obtained for publication.
Fig. 1 (abstract P1-3-4).
Fig. 1 (abstract P1-3-4).

See text for description

P1-3-5 Effects of tolvaptan, an oral vasopressin antagonist, in the treatment of hyponatremia in a pediatric patient with chronic SIADH

Sunao Sasaki, Hidetoshi Sato, Yohei Ogawa, Keisuke Nagasaki, Akihiko Saitoh

Division of Pediatrics, Niigata University Medical and Dental Hospital

Background: Tolvaptan is a vasopressin V2 receptor antagonist for the treatment of euvolemic and hypervolemic hyponatremia. Tolvaptan has demonstrated eficacy in adult patients with syndrome of inappropriate antidiuretic hormone secretion (SIADH); however, there are only a few reports of SIADH in children. Moreover, use of tolvaptan for the treatment of SIADH is not covered by the medical insurance in Japan. SIADH is usually transient and self-limited and can be controlled by short-term water restriction and sodium supplementation. However, the management of chronic SIADH is more difficult, especially in children.

Patient report: The patient was a 4-year-old Japanese boy. A delayed language development was noted at a medical check-up for children, at 3 years of age. A brain magnetic resonance imaging (MRI) revealed midbrain tumor and ventricular enlargement. He had undergone an endoscopic third ventriculostomy and tumor biopsy at 3 years and 4 months of age; finally, he was diagnosed as having pilocytic astrocytoma. Since then, his serum sodium concentration fluctuated in the range of 120~130 mEq/L denoting asymptomatic hyponatremia. He had generalized tonic-clonic seizure with fever at the age of 4. We diagnosed chronic SIADH based on his clinical data. In spite of water restriction (800 mL/day) and oral sodium supplementation, his hyponatremia did not improve. Then, on anticonvulsant treatment, he developed afebrile convulsions several times. He was administered oral tolvaptan at 15 mg daily (0.5 mg/kg/day) together with mild water restriction (1000 mL/day). A serum sodium concentration of 130 -135 mEq/L was achieved, and the drug was well tolerated without any side effects. Therefore, the dose of tolvaptan was increased to 22.5 mg daily (0.8 mg/kg/day). After tolvaptan treatment, no seizures were reported.

Conclusion: In this patient with chronic SIADH after the surgical treatment for pilocytic astrocytoma, tolvaptan was effective and safe in maintaining the serum sodium concentrations at 130 mEq/L or above. Further clinical studies are necessary for understanding the effect and safety of the long-term use of tolvaptan in children.

Consent for publication: The authors declare that written informed consent was obtained for publication.

P1-3-6 The effect of letrozole on the reproductive function and linear growth in the early pubertal rat

Juan Lin, Mei Hua Ma

Department of Pediatrics, The Third Affiliated Hospital, Sun Yat-Sen University

Objective: To provide for the basis of pathological change for the effect of Letrozole on the reproductive function and linear growth in the early puberty rats.

Method: At 3 weeks (20-22 days) of age, 14 of 20 SD rats were randomly divided into experimental group (male and female half ), treated with letrozole solution 1 mg/kg/d, 6 of control group (male and female half ), treated with 0.1 ml distilled water, both groups take for 30 days. Every 3 to 4 days weighing, measuring length (nose long anal), the left tibia weight, after 30 days, anesthesia to death and take bilateral ovaries rats (female), testis (male) and left tibial tissue section, using HE staining to observe each group rats ovary (female), testis (males) and the left tibia pathological changes of organizations.

Results: 1.The testicular tissue of sperm cells development, mature sperm counts, sertoli cell development and interstitial cells of experimental group had no significant change compared with control group. 2.The experimental group had ovarian cystic structure and atresia follicles significantly increased than control group, appeared the polycystic ovary (PCOS) change, and significantly reduce the granular cell layer. 3.Letrozole had a tendency to increase on the growth plate in the early puberty rats (male, female), which is more obvious in the male rat.

Conclusion: 1.30 days use of letrozole in the early puberty male rats had no obvious influence on testicular tissue, its sperm cells development, mature sperm counts, sertoli cell development and interstitial cells had no significant change compared with control group. 2.30 days use of letrozole can cause polycystic ovary change in the early pubertal female rat. 3.30 days ues of letrozole had a tendency to increase on the growth plate in the early puberty rats (male, female), which is more obvious in the male rat.

P1-3-7 Endocrine dysfunctions in Mandibular Hypoplasia, Deafness, Progeroid Features (MDP) Syndrome: A Case Report and Review of the Literature

Ting Chen, Linqi Chen, Haiying Wu, Fengyun Wang, Xiuli Chen, Rongrong Xie

Children's Hospital of Soochow University, Endocrinology and Metabolism

Objective: Mandibular hypoplasia, deafness, and progeroid features and lipodystrophy (MDPL) syndrome is an autosomal dominant disorder characterized mainly by mandibular hypoplasia, sensorineural deafness, progressive lipodystrophy, and hypogonadism in male. This syndrome is caused by heterozygous mutations in POLD1 gene. Short stature was described in half of the reported cases.

Subjects and methods: We report here the clinical description of a 10-year-old boy first presented with short stature and hypogonadism. After GH treatment, his fat loss gradually became obvious. We clarified his genetic bases through whole-exome sequencing analysis, and verified the results via sanger sequencing. We also carefully examined the GH/IGF-1 axis, hypothalamic-pituitary-testicular axis, and islet function in the MDPL patient.

Results: This article reports the 13th MDPL patient with de novo p. S605del mutation in POLD1 , and the first MDPL patient in Eastern Asia. We found that GH levels after GH stimulation tests were normal, while baseline IGF-1 level was extremely low. The rhGH therapy was effective in accelerating growth velocity, but it would accelerate fat loss and should be contradicted. We also found that the patient had extremely low baseline AMH and testosterone after HCG stimulation. Glucose and insulin levels were normal during OGTT in the patient.

Conclusions: Our findings suggested that MDPL syndrome was a possible diagnosis in boys with both short stature and hypogonadism, and rhGH therapy should be started only when this syndrome was excluded. Our study provided better understandings of the endocrine disorders in MDPL syndrome.

P1-3-8 Mutation analysis of BSCL2 gene in two patients with type 2 congenital generalized lipodystrophy

Ruimin Chen, Xin Yuan, Ying Zhang

Division of Endocrinology and Metabolism, Fuzhou Children's Hospital of Fujian, Teaching Hospital of Fujian Medical University

Context: Type 2 congenital generalized lipodystrophy (CGL2, OMIM 269700) is a rare autosomal recessive disease, characterized by the generalized absence of adipose tissue at birth or in early infancy. Mutations in BSCL2 gene have been reported to be responsible for CGL2. Objective To analyze the clinical characteristics of two patients with CGL2, and to investigate the BSCL2 gene in two pedigrees.

Methods: Medical history, clinical manifestations, physical examination, laboratory data, and ultrasonography findings were analyzed from two patients with CGL2. Blood samples from both families were obtained for genome DNA extraction. And then the 2742 genes of inherited diseases were amplified by PCR and sequenced.

Results: Two patients mainly showed generalized lack of body fat with extreme muscularity from birth, hirsutism, skin pigmentation especially necks and armpits; abnormal faces with empty cheeks, growth acceleration, advanced bone age; hepatomegaly, one of the patients presented with macropenis, renal hypertrophy, and mental retardation; laboratory data showed hypertriglyceridemia, hypercholesterolemia, and low high-density lipoprotein cholesterol. During the follow-up, they appeared bad temper, irritability, with aggressive behavior, abdominal ultrasound of patient 1 prompted fatty liver. They were treated with low-fat, high-carbohydrate diet, blood lipids were controlled to some degree. BSCL2 gene of patient 1 showed a homozygous mutation of c.728dupG, p.Ile262Hisfs * 12, whose parents were carriers of the

heterozygous mutation; BSCL2 gene of patient 2 showed a compound heterozygous mutation: missense mutation c. 713G> A, p.Gly238Asp from paternal; nucleotide repeat c.728dupG, p.Ile262Hisfs * 12 from maternal.

Conclusion: We describe two patients with classic clinical manifestations of CGL2 confirmed by genetic sequence analysis. Of them, the c.713G>A, p.Gly238Asp is a novel mutation in BSCL2 gene, previously unreported.

P1-3-9 X chromosomal deletion due to microhomology-mediated break-induced replication in a boy with Xp22.3 contiguous gene deletion syndrome: Implications for novel genomic defects leading to Kallmann Syndrome

Koki Nagai1,2, Hirohito Shima1,3, Miki Kamimura3, Junko Kanno3, Ikuma Fujiwara4, Erina Suzuki1, Satoshi Narumi1, Akira Ishiguro2, Maki Fukami1

1Department of Molecular Endocrinology, National Research Institute for Child Health and Development; 2Department of Postgraduate Education and Training, National Center for Child Health and Development; 3Department of Pediatrics, Tohoku University School of Medicine; 4Department of Pediatric Endocrinology and Environmental Medicine, Tohoku University Graduate School of Medicine

Background: KAL1 is one of the causative genes for Kallmann syndrome (KS), a rare genetic disorder characterized by hypogonadotropic hypogonadism and anosmia. KS is frequently associated with various complications including renal aplasia. Submicroscopic X chromosomal rearrangements involving KAL1 underlie KS either as an isolated anomaly or as a component of contiguous gene deletion syndromes. The genomic basis of these rearrangements remains largely unknown, although non-allelic homologous recombination and non-homologous end- joining have been implicated in a few cases.

Objective: To report molecular findings of a patient with KS due to a contiguous gene deletion syndrome.

Case report: A 6 month-old boy presented with bilateral cryptorchidism, micropenis, and ichthyosis at his trunk and limbs. His height was 67.4 cm (-0.17 SD), stretched penile length was 2.0 cm (-3.6 SD) and testicular diameters were 8-9 mm bilaterally. His mental development was age-appropriate. GnRH stimulation test at 6 months of age showed normal LH response (< 0.07 to 2.51 mIU/mL) and increased's H response (0.68 to 24.71 mIU/mL). Brain magnetic resonance imaging detected olfactory sulcus and bulbs but no olfactory tracts nor tubercles. Abdominal ultrasonograms and renal scintigrams showed left renal aplasia.

Molecular analysis: Array-based comparative genomic hybridization detected a 2.7-Mb deletion at Xp22.3. Breakpoint characterization revealed that the deletion was 2,735,696 bp in physical length and contained NLGN4X, VCX3A, HDHD1, STS, VCX, PNPLA4, VCX2, and VCX3B , in addition to exons 8-14 of KAL1 . The breakpoints shared no long homology but had microhomology of 2-bp. Both breakpoints resided at the margin of Alu repeats. Nucleotide stretches were absent at the fusion junction.

Discussion: We characterized a microdeletion in a boy with KS. KS and ichthyosis of the boy can be ascribed to the deletion of KAL1 and STS , respectively. Since the patient lacked NLGN4X and VCX genes involved in the brain function, his mental development should be re-evaluated in future studies. Breakpoint structure indicates that the deletion arose from a break-induced replication mediated by 2-bp microhomology. Notably, the two breakpoints resided at the margin of Alu repeats. Since a high concentration of repetitive elements is known to predispose replication-based errors, the deletion in our patient could have been facilitated by Alu repeats.

Conclusion: The results indicate that microhomology-mediated break-induced replication can underlie Xp22.3 contiguous gene deletion syndromes including KS. Alu repeats may be involved in the development of replication errors involving KAL1.

Consent for publication: The authors declare that written informed consent was obtained for publication.

P1-3-10 Androgen measurement in women with polycystic ovary syndrome: Comparison between immunoassays and liquid chromatography-tandem mass spectrometry

Kazuki Saito1,2, Toshiya Matsuzaki3, Mami Miyado1, Momori Katsumi1, Shigeru Nakamura1,4, Minoru Irahara3, Tsutomu Ogata5, Maki Fukami1

1Department of Molecular Endocrinology, National Center for Child Health and Development; 2Department of Comprehensive Reproductive Medicine, Graduate school, Tokyo Medical and Dental University; 3Department of Obstetrics and Gynecology, Graduate School of Biomedical Scientces, Tokushima University; 4Department of Pediatrics, Jichi Medical University; 5Department of Pediatrics, Hamamatsu University School of Medicine

Context: Patients with polycystic ovary syndrome (PCOS) frequently develop hyperandrogenemia. So far, blood androgens in PCOS patients have been measured primarily by immunoassays. However, accumulating evidence suggests that liquid chromatography-tandem mass spectrometry (LC-MS/MS) is more accurate in steroid qualification than immunoassays.

Objective: To compare the results of immunoassays and LC-MS/MS in androgen measurement in PCOS patients and eumenorrheic women. Materials and Methods: Blood samples were obtained from 31 PCOS patients and 28 eumenorrheic women. Serum levels of testosterone and androstenedione were analyzed by chemiluminescent enzyme immunoassay (CLEIA) and radioimmunoassay (RIA) respectively, and by LC- MS/MS. The results of the patients were compared to normal ranges (the mean ± 2.0 standard deviation of the control individuals).

Results: Testosterone values of each individual were closely matched between CLEIA and LC-MS/MS (correlation coefficient in patients, 0.852; in controls, 0.898). The correlation coefficients were comparable between the patient and control groups (p = 0.470). Androstenedione values in each individual determined by LC-MS/MS and RIA were closely matched in controls (correlation coefficient, 0.908), but less correlated in patients (correlation coefficient, 0.717; p = 0.026). Testosterone measurement by CLEIA yielded false positive and negative results in one and three cases respectively, and androstenedione measurement by RIA provided false positive and negative results in six and four cases respectively.

Discussion: The results are consistent with the previously proposed notion that the results of immunoassays are affected by cross-reacting steroids. The effects of cross- reaction seem to be more prominent in androstenedione measurement than in testosterone measurement, and in PCOS patients than in eumenorrheic women. Notably, androstenedione measurement by RIA yielded false positive (indicated by gray boxes in the figure)/negative (indicated by black boxes in the figure) results in 10 of 31 PCOS patients. Since increased blood levels of testosterone and androstenedione is associated with the risk of metabolic diseases in PCOS patients, precise androgen measurement using LC-MS/MS would serve to improve the long-term prognosis of these patients.

Conclusions: In PCOS pat ients, bloo d levels of androgens, particularly those of androstenedione, should be measured by LC-MS/MS.
Fig. 1 (abstract P1-3-10).
Fig. 1 (abstract P1-3-10).

See text for description