Waardenburg-Shah syndrome (WS4, MIM_277580) is a congenital developmental disorder characterized by pigmentary abnormalities of the skin, eyes and hair, sensorineural deafness and intestinal aganglionosis (HSCR; Hirschsprung disease). Mutations in the coding regions of EDN3, EDNRB, or SOX10 account for 65-85% of the WS4 patients. These mutations are not fully penetrant, contributing to the phenotypic variability of WS4. We screened these genes in a three-generation family (14 individuals; three members affected with HSCR only and one affected with “partial’ WS4 –iris heterochromia and HSCR-). A novel heterozygous missense mutation was identified in EDNRB. EDNRB encodes the EDNRB receptor, which is essential for the differentiation of the neural crest cells into melanocytes, enteric ganglia and Schwann cells. The mutation was present in four affected and three unaffected family members. In the EDNRB isoforms 1 and 2, the mutation results in the replacement of the translation initiation codon methionine (Met) with a valine (Val) and such replacement (M1V) would theoretically abolish the use of the translation initiation codon. However, in EDNRB isoform 3, the replacement is at Met91 (M91V) and is predicted benign. Since different EDNRB transcripts are expressed concomitantly in the still developing newborn's gut, we theorized that the intra-familial variability of the phenotype could be related to the expression ratio between benign and damaging isoforms. We examined the consequences of M1V or M91V in their respective isoforms. Constructs containing either wild-type cDNA of isoform 1 and 3 or their mutated counterpart were transiently transfected into Human Embryonic Kidney 293 cell (HEK293). Confocal and immunoblot experiments showed that EDNRB M1V generated a shortened protein (starting from Met46); the wild-type-EDNRB isoform 3 or its mutant (p.M91V) were only found in the cytosol. Although EDNRB M1V was able to generate a shorter protein, the later failed to translocate onto the cell membrane, theoretically, affecting signal-transduction. Isoform 3 did not seem to play a role as cellular receptor. We also identified a c.-248G/A rare change at the 5’-untranslated region (5’UTR) of EDN3 (EDNRB ligand) which was predicted to affect translation efficiency. The presence of this variant in affected individuals but not in healthy carriers of the EDNRB mutation, suggests that both variants are necessary for the disease manifestation. Variations within the disease phenotype may be due to each individual’s genetic background. To identify other susceptibility loci, we carried out whole-genome linkage scan in this family using a high density SNP assay. Merlin software was used for parametric and non-parametric linkage. A susceptibility locus on chromosome 4q13.3-q24 was identified by both nonparametric and parametric linkage analyses, with LOD scores of 1.204 and 1.7109 respectively. Haplotype analysis refined the region to a 27.76 cM interval, in which genes involved in neuron development reside. To conclude, the novel EDNRB M1V mutation in this family may lead to HSCR and/or WS4 when in conjunction with other genetic lesions, such as the EDN3 5’UTR rare variation and/or a not yet identified susceptibility locus on chromosome 4q13.3-q24. / published_or_final_version / Surgery / Master / Master of Philosophy

Sox10 is a high mobility group (HMG) domain transcription factor which is an important regulator for neural crest development. SOX10 mutations have been identified in Waardenburg-Shah syndrome type 4 (WS4) patients who suffer from sensorineural deafness. However, the mechanisms underlying the hearing defect of SOX10-mediated WS4 are unclear. The aim of this study is to elucidate the function of Sox10 during mouse inner ear development using a mutant mouse model, in order to reveal the underlying basis for SOX10 mutation associated sensorineural deafness in WS4 patients. The mammalian inner ear originates from the otic placode epithelium as well as neural crest cells (NCCs). To understand the role of Sox10 in inner development, I investigated the contribution of cranial NCCs to the cochleovestibular ganglion (CVG) by lineage tracing analysis, using Wnt1-cre;ZEG mice in which all NCCs were marked by GFP. Co-expression of GFP-positive cells with the glial marker BFABP suggested that glial cells in the CVG were derived from NCCs. Furthermore, Sox10-expressing NCCs were found to invade the CVG at 30-somite stage. These results suggest a role of Sox10 in regulating cranial NCCs contribution to CVG glia. In our laboratory we have generated a mouse mutant Sox10EGFP in which the Sox10 N-terminal domain was fused to the EGFP reporter. To investigate the function of Sox10 in NCCs invasion and gliogenesis of CVG, phenotypic analysis of Sox10NGFP mutant mouse were performed. EGFP expression in the CVG and inner ear epithelium of Sox10NGFP/+ embryos recapitulated the dynamic expression pattern of Sox10. Sox10NGFP/NGFP mutants displayed a reduced number of migrating NCCs and lacked NCCs or glia in their CVG. Moreover, loss of glial cell in the developing spiral ganglia of Sox10NGFP/NGFP mice led to disorganized fasciculation and degeneration of axonal filaments. These data suggest that Sox10 is required for maintaining the cranial NC stem cell pool, and is also essential for CVG gliogenesis and normal growth and innervation of spiral ganglion neurons. To study the function of Sox10 in regulating cochlear morphogenesis, morphological and histological analysis of mutant cochlear were performed. As illustrated by paint-filling analysis, Sox10NGFP/NGFP mice developed a shortened cochlear duct, reduced cochlear turning and enlarged endolymph lumen. Sensory hair cell patterning in the organ of Corti was normal in the Sox10 mutant as shown by immunohistochemistry analysis, suggesting that cochlear lumen enlargement was not due to disrupted planar cell polarity (PCP) pathway. To explore the molecular basis of Sox10-mediated cochlear morphogenic defect, expression of genes related to cochlear development were examined by qRT-PCR. Candidate genes included those involved in fluid homeostasis, which are known to affect the size of cochlear lumen. Up-regulated expression of Aquaporin 3, a water channel protein in the cochlear epithelium that facilitates water transport across the cell membrane, was observed in Sox10NGFP/NGFP cochlear. These results suggest that Sox10 may regulate cochlear morphogenesis by controlling endolymph homeostasis. In conclusion, Sox10 is required in multiple processes during inner ear development including NCC invasion, gliogenesis and cochlear morphogenesis, and their abnormal development can lead to sensorineural deafness in WS4 syndrome. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy