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

No description available.

Résumé non transmis / SOX10 is a transcription factor with well-known functions in neural crest and oligodendrocyte development. Mutations in SOX10 were first associated with Waardenburg-Hirschsprung disease (WS4; deafness, pigmentation defects and intestinal aganglionosis). However, variable phenotypes that extend beyond the WS4 definition are now reported. The neurological phenotypes associated with some truncating mutations are suggested to be the result of escape from the nonsense-mediated mRNA decay pathway; but, to date, no mechanism has been suggested for missense mutations, of which approximately 20 have now been reported, and about half of which are redistributed in vitro to nuclear bodies of undetermined nature and function. Here, we reported that the paraspeckle protein p54NRB, which plays a crucial role in the regulation of gene expression during many cellular processes including differentiation, and is a member of the Drosophila behavior Human Splicing (DBHS) protein family, interacts and acts synergistically with SOX10 to regulate several target genes. Interestingly, this multifunctional protein, as well as two other members of the DBHS protein family, co-localized with SOX10 mutants in nuclear bodies, suggesting the possible paraspeckle nature of these foci or re-localization of the DBHS members to other subnuclear compartments. Remarkably, the co-transfection of wild-type and mutant SOX10 constructs led to the sequestration of wild-type SOX10 in mutant-induced foci. However, only foci forming mutants exclusively found in the nucleus altered synergistic activity between SOX10 and p54NRB. We proposed that such a dominant negative effect may contribute to or be at the origin of the progressive neurological phenotype observed in affected patients.One of the roles of p54NRB is the regulation of gene expression via nuclear retention, by binding to hyperedited IRAlu sequences this protein blocks their efficient export to the cytoplasm (Zhang and Carmichael., 2001), we then decided to get into the world of editing. Editing, is a molecular mechanism characterized by the deaminase conversion of adenosines into inosines (A-to-I). In mammals, this molecular modification, is performed by a cluster of three enzymes named Adenosine deaminases acting on RNA (ADARs 1-3) (Wagner RW et al., 1989).In order to evaluate the role of ADAR1 in NC development, we decided to conditionally invalidate the expression of this enzyme using the NC specific HtPA-Cre line. Two main crossing strategies were followed, one including the Rosa26R-LacZ marker (RADR crossing) to track the NCCs and one not (CADR crossing). Globally, the Adar1 deficient pups harvested from the CADR crossing presented with 100% mortality within the first three days after birth. The survival rate of the mutants generated using the second strategy (RADR) was higher, however, none of the mutants survived up to P30. In general, the mutants of the latest crossing, presented with pleiotropic NC phenotype: abnormal melanocyte, ENS and sciatic nerve defects were observed.

A síndrome de Waardenburg é uma síndrome geneticamente heterogênea, com uma taxa de penetrância muito alta e expressividade extremamente variável. O objetivo desse estudo foi a caracterização molecular de uma amostra brasileira de pacientes com SW, dando continuidade ao estudo clínico feito em Pardono (2005), por meio do estudo de 48 probandos classificados com a síndrome de Waardenburg tipo 1 ou 2. Foram estudados os genes PAX3, MITF, SOX10, SNAI2, EDN3 e EDNRB, por meio do sequenciamento pelo método de Sanger, e investigadas as microdeleções e microduplicações dos genes PAX3, MITF e SOX10 pela técnica de MLPA (Multiplex Ligationdependent Probe Amplification). Dentre os resultados obtidos, identificou-se 17 mutações potencialmente patogênicas (35,4% dos probandos). Dessas, seis são variações de número de cópias (12,5% dos probandos). Além disso, foi realizado um levantamento na base de dados LOVD (Leiden Open Variation Database), no qual constam 105 mutações não sinônimas exônicas consideradas causativas da SW. Diversos algoritmos foram utilizados para avaliar a possível patogenicidade dessas mutações, os quais levam em conta as frequências das mutações na base de dados do projeto 1000 genomas e 6500 exomas, anotam as previsões dadas pelos programas Polyphen2, MutationTaster, LRT e SIFT e verificam a conservação em mamíferos e primatas. Por meio dessa análise, verificou-se que em 19 mutações desse tipo (18%) faltam evidências de sua patogenicidade, colocando-se em dúvida a sua relação com a síndrome de Waardenburg / Waardenburg syndrome (WS) is a genetically heterogeneous syndrome, with a very high penetrance rate and highly variable expressivity. The focus of this study was the molecular characterization of a Brazilian sample of patients with WS (48 probands classified with Waardenburg syndrome type 1 or 2). The analysis of genes PAX3, MITF, SOX10, SNAI2, EDN3 and EDNRB were performed by the Sanger sequencing method. Microduplications and microdeletions in genes PAX3, MITF and SOX10 were investigated by MLPA technique (Multiplex Ligationdependent Probe Amplification). We detected 17 mutations considered as potentially pathogenic in 17 probands of the sample (35,4 % of probands). Among these, six are copy number variations (12,5% of probands). In addition, we performed a survey using the database of LOVD (Leiden Open Variation Database), which contains 105 non-synonymous exonic mutations considered causative of WS. Several algorithms were used to evaluate the possible pathogenicity of these mutations, taking into account the frequency of mutations in the database project in 1000 genomes and 6500 exomes, and using programs : Polyphen2, MutationTaster, LRT and SIFT. These algorithms also verify the conservation of the variations in mammals and primates. Through this analysis, lack of evidence was found for the pathogenicity of 19 non-synonymous mutations (18%) and association of these with Waardenburg syndrome is questioned

A síndrome de Waardenburg é uma síndrome geneticamente heterogênea, com uma taxa de penetrância muito alta e expressividade extremamente variável. O objetivo desse estudo foi a caracterização molecular de uma amostra brasileira de pacientes com SW, dando continuidade ao estudo clínico feito em Pardono (2005), por meio do estudo de 48 probandos classificados com a síndrome de Waardenburg tipo 1 ou 2. Foram estudados os genes PAX3, MITF, SOX10, SNAI2, EDN3 e EDNRB, por meio do sequenciamento pelo método de Sanger, e investigadas as microdeleções e microduplicações dos genes PAX3, MITF e SOX10 pela técnica de MLPA (Multiplex Ligationdependent Probe Amplification). Dentre os resultados obtidos, identificou-se 17 mutações potencialmente patogênicas (35,4% dos probandos). Dessas, seis são variações de número de cópias (12,5% dos probandos). Além disso, foi realizado um levantamento na base de dados LOVD (Leiden Open Variation Database), no qual constam 105 mutações não sinônimas exônicas consideradas causativas da SW. Diversos algoritmos foram utilizados para avaliar a possível patogenicidade dessas mutações, os quais levam em conta as frequências das mutações na base de dados do projeto 1000 genomas e 6500 exomas, anotam as previsões dadas pelos programas Polyphen2, MutationTaster, LRT e SIFT e verificam a conservação em mamíferos e primatas. Por meio dessa análise, verificou-se que em 19 mutações desse tipo (18%) faltam evidências de sua patogenicidade, colocando-se em dúvida a sua relação com a síndrome de Waardenburg / Waardenburg syndrome (WS) is a genetically heterogeneous syndrome, with a very high penetrance rate and highly variable expressivity. The focus of this study was the molecular characterization of a Brazilian sample of patients with WS (48 probands classified with Waardenburg syndrome type 1 or 2). The analysis of genes PAX3, MITF, SOX10, SNAI2, EDN3 and EDNRB were performed by the Sanger sequencing method. Microduplications and microdeletions in genes PAX3, MITF and SOX10 were investigated by MLPA technique (Multiplex Ligationdependent Probe Amplification). We detected 17 mutations considered as potentially pathogenic in 17 probands of the sample (35,4 % of probands). Among these, six are copy number variations (12,5% of probands). In addition, we performed a survey using the database of LOVD (Leiden Open Variation Database), which contains 105 non-synonymous exonic mutations considered causative of WS. Several algorithms were used to evaluate the possible pathogenicity of these mutations, taking into account the frequency of mutations in the database project in 1000 genomes and 6500 exomes, and using programs : Polyphen2, MutationTaster, LRT and SIFT. These algorithms also verify the conservation of the variations in mammals and primates. Through this analysis, lack of evidence was found for the pathogenicity of 19 non-synonymous mutations (18%) and association of these with Waardenburg syndrome is questioned

The aetiological diagnosis of recessive non-syndromic hearing loss poses a challenge owing to marked heterogeneity and the lack of identifying clinical features. The finding that up to 50% of recessive non-syndromal genetic hearing loss among Caucasians was due to mutations in GJB2, the gene encoding Connexin 26 (Cx26) was a breakthrough, whose value as a diagnostic tool has been limited by the significant variation in the prevalence of deafness genes and loci among population groups. The significant association of the GJB6-D13S1830 deletion among individuals with one mutant GJB2 allele highlighted the need to explore population specific genetic mutations for NSHL. Although data from Sub-Saharan Africa is limited, reported studies found a high prevalence of R143W GJB2 mutation among Ghanaian, the 35delG mutation in 5 out of 139 Sudanese and a low prevalence of GJB2 variations among 385 Kenyan deaf children. The mutation spectrum of Waardenburg Syndrome (WS) in Africans has not been documented. During a visit to a School for the Deaf in the Limpopo Province of South Africa in 1997, it was noted that a high number of students came from Nzhelele sub-district. All had childhood onset hearing loss with no associated anomalies or disorders. The question arose as to whether there was a high-risk area for deafness in the Limpopo Province and what the aetiology of this hearing loss was.The main aim of this study was to investigate the role of GJB2, the GJB6-D13S1830 deletion, and the four common mitochondrial mutations, A1555G, A3243G, A7511C and A7445G, in the African hearing-impaired population of Limpopo province in South Africa, and to identify the mutation spectrum of the deafness genes found. The type and degree of hearing loss in this hearing impaired population would also be assessed. Secondly, this study sought to identify the mutations in a sibling pair with 2 clinical WS and to use the findings in a future study to establish the mutation spectrum of WS in the African population of the Limpopo province and of South Africa in general. The study was designed as a two phase study, in which phase 1 was used for hypothesis formulation and phase 2 was for hypothesis testing. While phase 1 was a descriptive retrospective case study, phase 2 was a combination of sample survey and prospective descriptive case study. In phase 1, demographic data of 361 students in two schools of the deaf in the Limpopo province was analyzed for evidence of areas of high risk populations for deafness in the province. In phase 2, a group of 182 individuals with genetic non-syndromic hearing loss (NSHL) and two siblings with clinical WS from two schools for the Deaf in the Limpopo Province of South Africa were investigated. A thorough clinical examination, audiological evaluation and urinalysis were done. Mutational screening was carried out in all 184 subjects using genomic DNA using single-strand conformation polymorphism (SSCP), multiplex polymerase chain reaction (PCR), and direct sequencing for GJB2, and Restriction Fragment-Length Polymorphism (PCR–RFLP) analysis for GJB6, and SSCP, hetero-duplex analysis, and direct sequencing of the first 8 exons of PAX3 and all of MITF for Waarenburg syndrome. Data analysis was by geographical mapping, frequency tables, tests of association with calculation of odds ratios, and binary logistic regression analysis using STATA and GIS mapping systems. The results indicate that there seem to be areas of genuine populations at risk for hearing loss in the Limpopo province of South Africa, namely Mutale and parts of Makhado and Thulamela municipalities. In Thulamela (NP343) wards 11-15, 26-30 and 31-35, and in Mutale (NP 344) wards 6-10, together accounted for 67 (18%) of participants in phase 1, and 33 (18%) of the participants in phase 2 of the study. Mutale municipality in the Vhembe 3 district gave with a projected prevalence of at least 13.14 deaf children per 100,000 African population attending the local school for the deaf. The observed hearing loss is a genetic, non-syndromic form, which is mainly severe and severe to profound, although without any clear defining configuration or shape. It is a stable, non-progressive and prelingual form of hearing loss, implying that this may be a recessive form of deafness. No identifiable environmental confounding factors or associations were identified. The deafness is not linked the common known auditory gene mutations in GJB2, the GJB6-D13S1830 deletion, or the common mitochondrial mutations A1555G, A3243G, A7511C and A7445G. Severe and profound levels of hearing loss were found in 22.8% and 75% of the cohort respectively, with the majority exhibiting flat (70.1%) or sloping (23.4%) audiograms that were commonly symmetrical (81.5%). However, as indicated, there was no clear pattern in the audiological findings overall. None of the 184 hearing impaired individuals exhibited any of the reported disease causing mutations of GJB2, including 35delG. There was, however, a high prevalence of two variants, the C>T variant at position g.3318-15 and the C>T variant at position g.3318-34, occurring in 21.4% and 46.2% of the deaf cohort respectively. The same variants were found to occur in 35% and 42.6% of a normal hearing control group (n = 63) respectively, indicating that these variations are polymorphisms. In three subjects (1.63% of the cohort), a T>A homozygous variation at position g.3318-6 was detected. Its significance in the causation of NSSNHL is yet to be determined. The GJB6-D13S1830 deletion was not detected in any of the participants. None of the four mitochondrial mutations screened for were found. 4 These results indicate that GJB2 is not a significant deafness gene in the African population of the Limpopo Province of South Africa and that significant genes for non-syndromic recessive hearing loss in this population are yet to be found. The geographical clustering of deafness found in this study, combined with the lack of identifiable common associated clinical features among the subjects of this study (excluding the WS sibling pair), suggests that these subjects have a genetic recessive non-syndromal type of hearing loss. In the context of historical and cultural evidence of consanguinity in this population, a founder effect cannot be ruled out. A rare mutation, R223X, previously identified only once out of 470 WS patients, was identified in the PAX3 gene among the WS sibling pair. A novel silent change GGG>GGT at amino acid 293, was also identified. These identical findings document, for the first time, a molecular defect in WS in an African sibling pair, and confirm WS Type I in this family, which could be found in other WS type I South Africans in the Limpopo Province of South Africa. The current study demonstrated that parents of genetically hearing impaired children in these areas are able to detect hearing loss at an early age, with over 60% suspecting their children’s hearing loss below 6 months of age. A child-centered management model encompassing all the areas relevant to childhood deafness/hearing impairment, which takes into consideration the prevailing logistical and financial constraints of the available healthcare system, is proposed. The implementation of this model requires a paradigm shift from the current fragmented model of service delivery to a cohesive patient-centered approach, based on concrete data from appropriate community based research, in which all the relevant parties communicate and share resources. 5 It would achieve the goals of early detection and intervention, as well as inclusive education for all. The relevant health and education policies are already in place and the posts funded. Equitable implementation of these policies would require appropriate community based research, as well as improved communication and consultation between the various stakeholders to ensure an efficient and affordable quality healthcare service for all hearing impaired South Africans.