Phenotypic spectrum of CHARGE syndrome in fetuses with CHD7 truncating mutations correlates with expression during human development.

BACKGROUND: The acronym CHARGE refers to a non-random cluster of malformations including coloboma, heart malformation, choanal atresia, retardation of growth and/or development, genital anomalies, and ear anomalies. This set of multiple congenital anomalies is frequent, despite rare patients with normal intelligence, and prognosis remains poor. Recently, CHD7 gene mutations have been identified in CHARGE patients; however, the function of CHD7 during development remains unknown. METHODS: We studied a series of 10 antenatal cases in whom the diagnosis of CHARGE syndrome was suspected, considering that a careful pathological description would shed light on the CHD7 function during development. CHD7 sequence analysis and in situ hybridisation were employed. RESULTS: The diagnosis of CHARGE syndrome was confirmed in all 10 fetuses by the identification of a CHD7 heterozygous truncating mutation. Interestingly, arhinencephaly and semi-circular canal agenesis were two constant features which are not included in formal diagnostic criteria so far. In situ hybridisation analysis of the CHD7 gene during early human development emphasised the role of CHD7 in the development of the central nervous system, internal ear, and neural crest of pharyngeal arches, and more generally showed a good correlation between specific CHD7 expression pattern and the developmental anomalies observed in CHARGE syndrome. CONCLUSIONS: These results allowed us to further refine the phenotypic spectrum of developmental anomalies resulting from CHD7 dysfunction.

PAX2 mutations in renal-coloboma syndrome: mutational hotspot and germline mosaicism.

The renal-coloboma syndrome (RCS, MIM 120330) is an autosomal dominant disorder caused by PAX2 gene mutations. We screened the entire coding sequence of the PAX2 gene for mutations in nine patients with RCS. We found five heterozygous PAX2 gene mutations: a dinucleotide insertion (2G) at position 619 in one sporadic RCS case, a single nucleotide insertion (619 + G) in three unrelated cases, and a single nucleotide deletion in a familial case. In this familial case, three affected sibs showed a striking ocular phenotypic variability. Each of the sibs carried a 619insG mutation, whilst unaffected parents did not, suggesting the presence of germline mosaicism. Interestingly, the 619insG mutation has been previously reported in several patients and is also responsible for the Pax21Neu mouse mutant, an animal model of human RCS. This study confirms the critical role of the PAX2 gene in human renal and ocular development. In addition, it emphasises the high variability of ocular defects associated with PAX2 mutations ranging from subtle optic disc anomalies to microphthalmia. Finally, the presence of PAX2 germline mosaicism highlights the difficulties associated with genetic counselling for PAX2 mutations.

Expression of the PAX2 gene in human embryos and exclusion in the CHARGE syndrome.

The CHARGE syndrome comprises ocular coloboma, heart malformation, choanal atresia, retarded growth and development, central nervous system malformations, genital hypoplasia, ear abnormalities, or deafness. The cause of the CHARGE syndrome remains unknown. In the present study, we analyzed the distribution pattern of the PAX2 gene in human embryos and found that PAX2 gene expression occurs in the primordia affected in the CHARGE syndrome. These data prompted us to consider the PAX2 gene a candidate gene in the CHARGE "association." We analyzed the PAX2 gene in 34 patients fulfilling the diagnostic criteria of the CHARGE syndrome for deletion and nucleotidic variations of the coding sequence and identified only polymorphisms. Our data suggest that mutation of the PAX2 gene is not a cause of the CHARGE association. However, the pattern of expression of PAX2 suggests that genes encoding downstream targets effectors could be candidate genes for the CHARGE syndrome.

Familial CHARGE syndrome because of CHD7 mutation: clinical intra- and interfamilial variability.

CHARGE syndrome (OMIM #214800) is a multiple malformation syndrome with distinctive diagnostic criteria, usually because of CHD7 (chromodomain helicase DNA binding 7) haploinsufficiency. Familial occurrence of CHARGE syndrome is rare. We report six patients from two Caucasian families (both with one parent and two children) affected by mild to severe CHARGE syndrome. Direct sequencing of the CHD7 gene was performed in these two unrelated families. A mutation in exon 8 (c.2501C>T - p.S834F) in first chromodomain was found in family A and a nonsense mutation in exon 2 (c.469C>T - p.R157X) in family B. Both mutations are de novo in the parents. In family A, the elder son had bilateral cleft lip and palate, esophageal atresia with fistula, complex heart defect and vertebral abnormalities, while the younger had a posterior coloboma. Their mother had asymptomatic vestibular dysfunction and retinal coloboma, identified after the molecular diagnosis of her children. In family B, both affected children had severe expression of CHARGE syndrome. The father carrying the mutation only had asymmetric anomaly of the pinnae. These familial reports describe the intrafamilial variability of CHARGE syndrome, and underline the presence of CHD7 mutations in patients who do not fit the 'classical clinical criteria' for CHARGE syndrome.

JAGGED1 gene expression during human embryogenesis elucidates the wide phenotypic spectrum of Alagille syndrome.

Mutations of the JAGGED1 gene, encoding a NOTCH receptor ligand, cause Alagille syndrome (AGS), a complex malformative disorder affecting mainly the liver, heart, vertebrae, eye, and face. Minor and occasional features involving kidney, pharynx, systemic arteries, skeleton, and ear are in some cases associated with the syndrome. To describe the expression of JAGGED1 during human embryogenesis and to study its relationship with all the features of AGS, we performed in situ hybridization studies on human embryos and fetal tissue sections. JAGGED1 was mainly expressed in the cardiovascular system. In the liver, JAGGED1 transcripts were only detected in blood vessels. JAGGED1 was also expressed in other structures of mesenchymal origin (distal mesenchyme of limb buds; mesonephric and metanephric tubules of the kidney) and in epithelial structures including the ciliary margin of the retina and the posterior part of the lens, the ventral epithelium of the otic vesicle, the neurosensory epithelium of the ear vestibule, the epithelium of pharyngeal arches, and the developing central nervous system. The strong JAGGED1 expression during human embryo- and feto-genesis both in the vascular system and in other mesenchymal and epithelial tissues implicates abnormal angiogenesis in the pathogenesis of Alagille syndrome and particularly the paucity of interlobular bile ducts. However, it is probably not the only mechanism of the disease. Except for the central nervous system, there is a strong correlation between JAGGED1 expression and all the features of AGS. This implies that the features occasionally associated with the syndrome are not coincidental.

Expression of the sonic hedgehog gene in human embryos with neural tube defects.

BACKGROUND: To estimate the rate of malformations observed during early human development, a series of 38,913 first-trimester abortions were studied. Neural tube defects (NTD) were found in 57 cases. METHODS: A histological study of serial sections performed in 25 embryos revealed a spectrum of axial structure abnormalities. Expression of the SHH gene was studied by in situ hybridization in one case of CRS and in two cases of SB. RESULTS: A cervical notochord duplication was always found in craniorachischisis (CRS, n = 8), but not in spina bifida (SB, n = 10) or diplomyelia (split cord malformation, n = 3). In the embryo with CRS, expression of SHH was found in both domains, corresponding to the duplicated part of the notochord, whereas a single signal was observed in the nonduplicated part. This expression was associated at the cervical level of the open neural tube with a broad SHH expression domain and with two or even three domains in its lumbar region, suggesting multiple functional floor plates. Similarly, in two embryos with SB, two domains of SHH expression were found in the ventral neural tube. CONCLUSIONS: Our findings suggest that notochord splitting in the cervical region might be involved in the pathogenesis of CRS. Interestingly, similar notochord abnormality and altered expression of the shh gene are observed in Lp mice with NTD. This suggests that the Lp gene could be a candidate gene for human CRS. Further studies are needed to establish the primary event responsible for the notochord splitting and for the abnormal expression of the SHH gene in the floor plate in embryos with CRS and SB.