CHARGE is a phenotypically heterogeneous autosomal dominant disorder that has been recognized as a non-random cohesive syndrome since the identification of CHD7 mutations as an underlying etiology [1, 2, 7]. CHD7, at 8q12.1, encodes a protein of the chromodomain family [17, 18]. The exact function of CHD7 has not been elucidated, however, in situ hybridization analysis during human development has demonstrated expression of this gene in the central nervous system, semicircular canals and the neural crest of the pharyngeal arches; thereby implicating the embryologic role of CHD7 in the development of the respective organs [19, 20]. Furthermore, CHD7 mRNA expression was documented in the hypothalamus, pituitary and olfactory bulb in the rat and also demonstrated in both migratory and post-migratory GnRH neuronal cell lines . Our subject carried a heterozygous G744S, not seen in controls; this missense change has been reported in one previous series and designated a polymorphism, though no functional studies were conducted and controls were not tested . In this instance, the G744S change was seen in a family with three children with clinical CHARGE born to two different women and one man. A larger CHD7 rearrangement was additionally seen in all affected children, and found in mosaic form in the father's sperm cell DNA but not in lymphocytes. The G744S change, however, was found in two of the CHARGE children (the third was not tested) and in the completely unaffected father. Since the father was heterozygous and not mosaic for G744S, the authors considered G744S to be a non-pathogenic variant (Kohlhase J, unpublished data). CHD7 mutations have clinically variable expression, and clear genotype-phenotype correlations are not observed, even among patients with identical CHD7 mutations [1–3]. Asymptomatic carriers are reported as well, particularly in inherited forms . We can speculate that the disparate putative effect of this mutation is subject to yet undefined secondary genetic, epigenetic or environmental influences; this has been demonstrated in other genetic disease models, such as idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome [23–25]. Our bioinformatics based structural analysis, protein alignment, and DNA sequencing in normal controls, provide supportive evidence that the G744S is a true deleterious mutation involving a highly conserved amino acid, likely disrupting a crucial protein interaction site.
In this age of sophisticated and readily available genetic analysis, the diagnosis of CHARGE appears to be transforming from the rigid fulfillment of conglomerates of specific major and minor criteria, towards an approach more inclusive of patients with atypical or attenuated phenotypes, as was demonstrated by Kim et al . CHARGE and Kallmann syndromes (KS, KAL5, MIM 612370), though distinct developmental disorders, were noted to share features of impaired olfaction and hypogonadism; thus CHD7 was hypothesized to be involved in the pathogenesis of KS even in the absence of the CHARGE phenotype. Among 197 patients, they identified seven heterozygous mutations (two splice and five missense, absent in ≥ 180 controls) in three sporadic KS and four sporadic normosmic IHH patients. Thus, sporadic CHD7 mutations occurred in 6% of IHH/KS patients studied, allowing them to conclude that IHH/KS can represent a milder allelic variant of CHARGE syndrome. Furthermore, Jongmans et al also identified de novo CHD7 mutations in 3 of 56 mixed KS and nIHH subjects. Interestingly, in retrospect, their IHH patients with CHD7 mutations had some CHARGE features, including colobomas, deafness, ear anomalies, cleft palate and short stature; however, not to the degree that would fulfill traditional criteria . Supported by this rational, we can concluded that our subject, with an atypical eyelid coloboma, hearing loss, severe developmental delay, ventricular septal defect, short stature, and abnormal facies, in addition to other more recently described features, such as a limb anomaly, primary hypoparathyroidism and interrupted pubertal development, may be included in this designation.
CHARGE syndrome was initially not considered to involve the limb. Several subsequent reports have shown associated limb anomalies and one series reported at least one limb anomaly in over one third of 172 CHARGE patients; furthermore, there did not appear to be a common limb anomaly in their cohort and minor abnormalities were included . Prasad et al in 1997 were among the first to report severe limb abnormalities, including camptodactyly, tibial hemimelia and severe club-foot, in a patient with clinical CHARGE Syndrome . It is notable that "club-foot", or congenital talipes equinovarus, is typically not due to osseous malformation. In 2007, Van de Laar et al reported 3 patients with heterozygous CHD7 truncating mutations in distinct exons, who displayed several limb malformations, including tibial aplasia, monodactyly and bifid femora . Other limb defects, including triphalangeal thumb, polydactyly of the foot, ectrodactyly, and radial aplasia, have been reported as well [1, 19, 29, 31]. Sanlaville et al in 2006, studied expression of CHD7 during human embryonic development detecting a weak signal in limb bud mesenchym at C14 . Our patient, added to those previously described, highlighted by our demonstration of strong Chd7 expression in murine limb buds, further supports the suggestion that limb abnormalities should be a more recognized feature within the phenotypic spectrum of CHARGE syndrome. Moreover, homology modeling revealed a similarity to a bacterial flagellae protein , which in turn has high homology with the human TCN gene, encoding the human cytoskeletal protein titin; in concert with actin, titin plays a dominant role in human cytoskeletal development. Interestingly, direct comparison of titin and 1UCU sequences revealed a 19 amino acid gap of highly charge amino acids (spanning aa 741-759, EDPG VQKRRSSRQVKRKRY), which are most likely involved in functional differences between the two proteins and hence confer particular vulnerability to functional changes upon mutation.
In CHARGE Syndrome, anomalies of the urinary tract are reported in 10-40%, and include neurogenic bladder, duplex kidneys, renal ectopia or agenesis, horseshoe kidneys, and ureteral anomalies. [1, 32, 33]. To our knowledge, our patient is the first CHARGE patient to be reported with the severe phenotype of MCDK, requiring renal replacement therapy with dialysis followed by transplant. Cystic renal dysplasia is an anomaly of differentiation of the fetal kidney, whereby the kidney contains primitive ducts and non-renal tissues such as cartilage, fat, hematopoietic tissue, and often cysts. The most severe form of cystic renal dysplasia is MCDK, and most cases are unilateral. In subjects with CHARGE, only few reports of simple renal cysts exist [1, 2, 30]. Interestingly, one CHARGE subject with a unilateral right-sided dysplastic kidney also had significant limb anomalies, including right tibia aplasia, left tibia hypoplasia, and bilateral club feet . The molecular events involved in multicystic dysplastic kidneys, in general, remain to be elucidated, though studies have suggested involvement of WNT-1 , FGFR3 , and PAX2 . The PAX2 gene is associated with the renal coloboma syndrome (MIM 120330), a syndrome characterized by renal hypoplasia and insufficiency, vesicoureteric reflux, and optic disc coloboma. Interestingly, multicystic dysplastic kidney has been reported in one family with Renal Coloboma Syndrome . In a study of the distribution pattern of the PAX2 gene in human embryos, Tellier et al demonstrated that PAX2 gene expression occurs in the primordia affected with CHARGE syndrome. Therefore, PAX2 was further analyzed in 34 patients fulfilling the diagnostic criteria of the CHARGE syndrome, though no deletions or nucleotide variations of the coding sequence were detected, suggesting that mutations of the PAX2 gene was not a cause of the CHARGE . Considering the embryonic expression of PAX2 reported, and the common clinical features of Renal Coloboma Syndrome with CHARGE, one can hypothesize that CHD7 may have a role in regulating PAX2 gene and therefore this overlapping pathway might be explored in CHARGE etiology, and perhaps contributes to the variable expression observed.
The significant clinical overlap and inherently variable features of CHARGE and DiGeorge Syndromes can make differentiating these initial diagnoses particularly challenging. Hypocalcemia has been reported in CHARGE, though hypoparathyroidism, specifically, has been implicated in only few cases [38, 39]. A study comparing 25 CHARGE subjects with CHD7 mutations to a large cohort of subjects with 22q11.2 deletion syndrome, noted that features found more commonly in CHARGE syndrome included coloboma, choanal atresia, facial nerve palsy, tracheoesophageal fistula, and genital hypoplasia in boys. Interestingly, a high incidence of marked hypocalcemia was observed in their CHARGE study group (72%), and a pronounced spectrum of cell-mediated immunodeficiency ranging from lymphopenia (60%) to severe combined immunodeficiency (8%), was seen as well. Defects in humoral immunity were documented in 4 CHARGE patients and included severe hypogammaglobulinemia with decreased T-cell numbers, transient hypogammaglobulinemia during infancy, and immunoglobulin A deficiency . An accurate distinction between these two entities can, therefore, be challenging but will influence genetic counseling; CHD7 mutations more typically occur sporadically, whereas 22q11.2 deletions are familial in 10% of cases [2, 41].