Deletions at the SOX10 gene locus cause Waardenburg syndrome types 2 and 4

Waardenburg syndrome (WS) is an auditory-pigmentary disorder that exhibits varying combinations of sensorineural hearing loss and abnormal pigmentation of the hair and skin. Depending on additional symptoms, WS is classified into four subtypes, WS1-WS4. Absence of additional features characterizes WS2. The association of facial dysmorphic features defines WS1 and WS3, whereas the association with Hirschsprung disease (aganglionic megacolon) characterizes WS4, also called "Waardenburg-Hirschsprung disease." Mutations within the genes MITF and SNAI2 have been identified in WS2, whereas mutations of EDN3, EDNRB, and SOX10 have been observed in patients with WS4. However, not all cases are explained at the molecular level, which raises the possibility that other genes are involved or that some mutations within the known genes are not detected by commonly used genotyping methods. We used a combination of semiquantitative fluorescent multiplex polymerase chain reaction and fluorescent in situ hybridization to search for SOX10 heterozygous deletions. We describe the first characterization of SOX10 deletions in patients presenting with WS4. We also found SOX10 deletions in WS2 cases, making SOX10 a new gene of WS2. Interestingly, neurological phenotypes reminiscent of that observed in WS4 (PCWH syndrome [peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, WS, and Hirschsprung disease]) were observed in some WS2-affected patients with SOX10 deletions. This study further characterizes the molecular complexity and the close relationship that links the different subtypes of WS.     

Screening of MITF and SOX10 Regulatory Regions in Waardenburg Syndrome Type 2

Waardenburg syndrome (WS) is a rare auditory-pigmentary disorder that exhibits varying combinations of sensorineural hearing loss and pigmentation defects. Four subtypes are clinically defined based on the presence or absence of additional symptoms. WS type 2 (WS2) can result from mutations within the MITF or SOX10 genes; however, 70% of WS2 cases remain unexplained at the molecular level, suggesting that other genes might be involved and/or that mutations within the known genes escaped previous screenings. The recent identification of a deletion encompassing three of the SOX10 regulatory elements in a patient presenting with another WS subtype, WS4, defined by its association with Hirschsprung disease, led us to search for deletions and point mutations within the MITF and SOX10 regulatory elements in 28 yet unexplained WS2 cases. Two nucleotide variations were identified: one in close proximity to the MITF distal enhancer (MDE) and one within the U1 SOX10 enhancer. Functional analyses argued against a pathogenic effect of these variations, suggesting that mutations within regulatory elements of WS genes are not a major cause of this neurocristopathy.     

The value of MLPA in Waardenburg syndrome

Waardenburg syndrome (WS) is an autosomal-dominant neurocristopathy characterized by sensorineural hearing loss, pigmentary abnormalities of the iris, hair, and skin, and is responsible for about 3% of congenital hearing loss. Point mutations in PAX3 have been identified in more than 90% of affected individuals with WS Type 1/WS Type 3. MITF point mutations have been identified in 10-15% of individuals affected with WS Type 2 (lacking dystopia canthorum). Multiplex ligation-dependent probe amplification (MLPA) is now a standard technology in the molecular genetics laboratory to detect copy number changes in targeted genes. We employed MLPA for PAX3 and MITF in a cohort of patients submitted with a diagnosis of WS1, 2 or 3 who were sequence negative for PAX3 and/or MITF. All coding exons of PAX3 and exons 1, 2, 3, and 10 of MITF were included in the MLPA assay. MLPA on 48 patients with WS 1 or 3 revealed 3 PAX3 whole gene deletions (2 WS1; 1 WS3), 2 PAX3 partial gene deletions [WS1, exon 1 and promoter (1st report); WS1, exons 5-9], and 1 partial MITF deletion ("WS1", exons 3-10) (6/48 approximately 12.5%). MLPA on 41 patients with WS2 and 20 patients submitted with a diagnosis of either WS1 or WS2 revealed no copy number changes. The detection of both partial and whole gene deletions of PAX3/MITF in this clinical cohort increases the mutation detection yield by at least 6% and supports integrating MLPA into clinical molecular testing primarily for patients with WS1 and 3.     

Locus Heterogeneity for Waardenburg Syndrome is Predictive of Clinical Subtypes

Waardenburg syndrome (WS) is a dominantly inherited and clinically variable syndrome of deafness, pigmentary changes, and distinctive facial features. Clinically, WS type I (WS1) is differentiated from WS type II (WS2) by the high frequency of dystopia canthorum in the family. In some families, WS is caused by mutations in the PAX3 gene on chromosome 2q. We have typed microsatellite markers within and flanking PAX3 in 41 WS1 kindreds and 26 WS2 kindreds in order to estimate the proportion of families with probable mutations in PAX3 and to study the relationship between phenotypic and genotypic heterogeneity. Evaluation of heterogeneity in location scores obtained by multilocus analysis indicated that WS is linked to PAX3 in 60% of all WS families and in 100% of WS1 families. None of the WS2 families were linked. In those families in which equivocal lod scores (between −2 and +1) were found, PAX3 mutations have been identified in 5 of the 15 WS1 families but in none of the 4 WS2 families. Although preliminary studies do not suggest any association between the phenotype and the molecular pathology in 20 families with known PAX3 mutations and in four patients with chromosomal abnormalities in the vicinity of PAX3, the presence of dystopia in multiple family members is a reliable indicator for identifying families likely to have a defect in PAX3.     

Novel mutations in the SOX10 gene in the first two Chinese cases of type IV Waardenburg syndrome

Objective: We analyzed the clinical features and family-related gene mutations for the first two Chinese cases of type IV Waardenburg syndrome (WS4). Methods: Two families were analyzed in this study. The analysis included a medical history, clinical analysis, a hearing test and a physical examination. In addition, the EDNRB, EDN3 and SOX10 genes were sequenced in order to identify the pathogenic mutation responsible for the WS4 observed in these patients. Results: The two WS4 cases presented with high phenotypic variability. Two novel heterozygous mutations (c.254G>A and c.698-2A>T) in the SOX10 gene were detected. The mutations identified in the patients were not found in unaffected family members or in 200 unrelated control subjects. Conclusions: This is the first report of WS4 in Chinese patients. In addition, two novel mutations in SOX10 gene have been identified.     

Waardenburg综合征Ⅱ型患者MITF基因突变分析

Waardenburg综合征(WS)是临床上常见的常染色体显性遗传性耳聋综合征, MITF基因突变与部分Waardenburg 综合征Ⅱ型(WS2)病例的发病有关。MITF属于碱性螺旋-环-螺旋亮氨酸拉链转录因子家族, 能调节酪氨酸酶基因, 参与黑色素细胞的分化。文章报道了1个携带MITF基因点突变的3代Waardenburg综合征Ⅱ型中国家系。先证者表现为先天性重度感音神经性聋、虹膜异色、面部雀斑; 其他家系成员除一名仅表现为先天性耳聋外, 均表现为颜面、上肢雀斑和/或早白发。患者可检测到c.639delA杂合突变, 该突变在MITF基因第7外显子上产生了终止密码子(p.I220X), 突变产生的截短的MITF蛋白没有DNA结合活性。该突变是WS2病例中第3个位于MITF第7外显子的突变, 尚未见报道。该突变与已报道的位于第7外显子其他两个突变仅相差1个碱基, 氨基酸改变十分相似, 但在表型上有显著差别, 提示遗传背景对WS临床表型有重要影响。     

Mutations in the paired domain of the human PAX3 gene cause Klein-Waardenburg syndrome (WS-III) as well as Waardenburg syndrome type I (WS-I).

Waardenburg syndrome type I (WS-I) is an autosomal dominant disorder characterized by sensorineural hearing loss, dystopia canthorum, pigmentary disturbances, and other developmental defects. Klein-Waardenburg syndrome (WS-III) is a disorder with many of the same characteristics as WS-I and includes musculoskeletal abnormalities. We have recently reported the identification and characterization of one of the first gene defects, in the human PAX3 gene, which causes WS-I. PAX3 is a DNA-binding protein that contains a structural motif known as the paired domain and is believed to regulate the expression of other genes. In this report we describe two new mutations, in the human PAX3 gene, that are associated with WS. One mutation was found in a family with WS-I, while the other mutation was found in a family with WS-III. Both mutations were in the highly conserved paired domain of the human PAX3 gene and are similar to other mutations that cause WS. The results indicate that mutations in the PAX3 gene can cause both WS-I and WS-III.     

SOX10 mutations in chronic intestinal pseudo-obstruction suggest a complex physiopathological mechanism

The type IV Waardenburg syndrome (WS4), also referred to as Shah-Waardenburg syndrome or Waardenburg-Hirschsprung disease, is characterised by the association of Waardenburg features (WS, depigmentation and deafness) and the absence of enteric ganglia in the distal part of the intestine (Hirschsprung disease). Mutations in the EDN3, EDNRB, and SOX10 genes have been reported in this syndrome. Recently, a new SOX10 mutation was observed in a girl with a neural crest disorder without evidence of depigmentation, but with severe constipation due to a chronic intestinal pseudo-obstruction and persistence of enteric ganglia. To refine the nosology of WS, we studied patients with typical WS4 (including Hirschsprung disease) or with WS and intestinal pseudo-obstruction. We found three SOX10 mutations, one EDNRB and one EDN3 mutations in patients presenting with the classical form of WS4, and two SOX10 mutations in patients displaying chronic intestinal pseudo-obstruction and WS features. These results show that chronic intestinal pseudo-obstruction may be a manifestation associated with WS, and indicate that aganglionosis is not the only mechanism underlying the intestinal dysfunction of patients with SOX10 mutations.     

Phenotypic similarities in pigs with SOX10c.321dupC and SOX10c.325A>T mutations implied the correlation of SOX10 haploinsufficiency with Waardenburg syndrome

SOX10 is a causative gene of Waardenburg syndrome (WS) that is a rare genetic disorder characterized by hearing loss and pigment disturbance. More than 100 mutations of SOX10 have been found in patients with Type 2 WS (WS2), Type 4 WS (WS4), and more complex syndromes. However, no mutation hotspot has been detected in SOX10, and most cases are sporadic, making it difficult to establish a correlation between the high phenotypic and genetic variability. In this study, a duplication of the 321th cytosine (c.321dupC) was introduced into SOX10 in pigs, which induced premature termination of the translation of SOX10 (p.K108QfsX45). The premature stop codon in Exon 3 triggered the degradation of mutant mRNA through nonsense-mediated mRNA decay. However, SOX10^c.321dupC induced a highly similar phenotype of WS2 with heterogeneous inner ear malformation compared with its adjacent missense mutation SOX10^c.325A>T. In addition, a site-saturation mutation analysis of the SOX10 N-terminal nuclear localization signal (n-NLS), where these two mutations located, revealed the correlation between SOX10 haploinsufficiency and WS by an in vitro reporter assay. The analysis combining the in vitro assay with clinical cases may provide a clue to clinical diagnoses.     

Correlation between Waardenburg syndrome phenotype and genotype in a population of individuals with identified PAX3 mutations

Waardenburg syndrome (WS) type 1 is an autosomal dominant disorder characterized by sensorineural hearing loss, pigmentary abnormalities of the eye, hair, and skin, and dystopia canthorum. The phenotype is variable and affected individuals may exhibit only one or a combination of several of the associated features. To assess the relationship between phenotype and gene defect, clinical and genotype data on 48 families (271 WS individuals) collected by members of the Waardenburg Consortium were pooled. Forty-two unique mutations in the PAX3 gene, previously identified in these families, were grouped in five mutation categories: amino acid (AA) substitution in the paired domain, AA substitution in the homeodomain, deletion of the Ser-Thr-Pro-rich region, deletion of the homeodomain and the Ser-Thr-Pro-rich region, and deletion of the entire gene. These mutation classes are based on the structure of the PAX3 gene and were chosen to group mutations predicted to have similar defects in the gene product. Association between mutation class and the presence of hearing loss, eye pigment abnormality, skin hypopigmentation, or white forelock was evaluated using generalized estimating equations, which allowed for incorporation of a correlation structure that accounts for potential similarity among members of the same family. Odds for the presence of eye pigment abnormality, white forelock, and skin hypopigmentation were 2, 8, and 5 times greater, respectively, for individuals with deletions of the homeodomain and the Pro-Ser-Thr-rich region compared to individuals with an AA substitution in the homeodomain. Odds ratios that differ significantly from 1.0 for these traits may indicate that the gene products resulting from different classes of mutations act differently in the expression of WS. Although a suggestive association was detected for hearing loss with an odds ratio of 2.6 for AA substitution in the paired domain compared with AA substitution in the homeodomain, this odds ratio did not differ significantly from 1.0. Received: 27 July 1997 / Accepted: 9 December 1997     

Identification and functional analysis of a novel mutation in the SOX10 gene associated with Waardenburg syndrome type IV

Waardenburg syndrome type IV (WS4) is a rare genetic disorder, characterized by auditory–pigmentary abnormalities and Hirschsprung disease. Mutations of the EDNRB gene, EDN3 gene, or SOX10 gene are responsible for WS4. In the present study, we reported a case of a Chinese patient with clinical features of WS4. In addition, the three genes mentioned above were sequenced in order to identify whether mutations are responsible for the case. We revealed a novel nonsense mutation, c.1063C>T (p.Q355*), in the last coding exon of SOX10. The same mutation was not found in three unaffected family members or 100 unrelated controls. Then, the function and mechanism of the mutation were investigated in vitro. We found both wild-type (WT) and mutant SOX10 p.Q355* were detected at the expected size and their expression levels are equivalent. The mutant protein also localized in the nucleus and retained the DNA-binding activity as WT counterpart; however, it lost its transactivation capability on the MITF promoter and acted as a dominant-negative repressor impairing function of the WT SOX10.