A de novo deletion mutation in SOX10 in a Chinese family with Waardenburg syndrome type 4

Waardenburg syndrome type 4 (WS4) or Waardenburg-Shah syndrome is a rare genetic disorder with a prevalence of <1/1,000,000 and characterized by the association of congenital sensorineural hearing loss, pigmentary abnormalities, and intestinal aganglionosis. There are three types of WS4 (WS4A–C) caused by mutations in endothelin receptor type B, endothelin 3, and SRY-box 10 (SOX10), respectively. This study investigated a genetic mutation in a Chinese family with one WS4 patient in order to improve genetic counselling. Genomic DNA was extracted, and mutation analysis of the three WS4 related genes was performed using Sanger sequencing. We detected a de novo heterozygous deletion mutation [c.1333delT (p.Ser445Glnfs*57)] in SOX10 in the patient; however, this mutation was absent in the unaffected parents and 40 ethnicity matched healthy controls. Subsequent phylogenetic analysis and three-dimensional modelling of the SOX10 protein confirmed that the c.1333delT heterozygous mutation was pathogenic, indicating that this mutation might constitute a candidate disease-causing mutation.

Scientific RepoRts | 7:41513 | DOI: 10.1038/srep41513 Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All procedures were performed in accordance with the approved guidelines.
Mutation screening. Peripheral blood was collected, and genomic DNA was extracted using a DNeasy blood and tissue kit from Qiagen (Hilden, Germany). Polymerase chain reaction (PCR) was performed to amplify all coding exons and intron/exon boundaries of the EDNRB, EDN3, SOX10, PAX3, MITF, and SNAI2 genes. Some of the primers used in the study were referenced from a master's thesis (title here, Dong Siqi; Chinese PLA General Hospital, Beijing, China), and other primers were designed using Primer 5. Primers are shown in Table 1. PCR of the SOX10 exons was performed in a total volume of 50 μ L containing 60 ng of genomic DNA, 400 nM each of the forward and reverse primers, 40 mM dNTPs, and 2.5 U LA Taq DNA polymerase with GC buffer I from TAKARA (Tokyo, Japan). The amplification consisted of an initial denaturation stage at 94 °C for 3 min, followed by 35 cycles consisting of denaturation at 94 °C for 30 s, annealing for 30 s at 60 °C, and extension at 72 °C for 50 s, with an extension step performed at 72 °C for 3 min. Amplification of exons for the remaining genes was performed using 2× PCR master mix under similar conditions, except for annealing at 57 °C. PCR products were purified and sequenced using an ABI 3500 Dx genetic analyser with a BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA, USA), and the sequences were analysed using NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Protein structure prediction. Both the wild-type and mutated SOX10 protein sequences were used to perform protein structure prediction using I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) as previously reported [16][17][18][19] . In I-TASSER, the B-factor, which indicates the extent of the inherent thermal mobility of residues/atoms in proteins, is calculated from threading template proteins from the Protein Data Bank along with sequence profiles derived from sequence databases. The normalized B-factor of the target protein was defined by B = (B′ − u)/s, where B′ represents the raw B-factor value, and u and s represent the mean and standard deviation of the raw B-factors along the sequence, respectively.

Results
Clinical findings. A 1-year-old male patient was referred to our hospital with the chief complaint of Hirschsprung disease accompanied by heterochromia iridis and congenital hearing loss. Based on these clinical features, he was first suspected to be a WS4 patient. Neither parent of the patient exhibited similar symptoms (Fig. 1).

Identification of a novel SOX10 heterozygous deletion mutation.
A heterozygous deletion mutation (c.1333delT) in SOX10 was identified in the patient, resulting in replacement of the 445 th Ser with Gln and a shift in the reading frame to produce a longer protein consisting of 501 amino acids (p.Ser445Glnfs*57) as compared with the wild-type SOX10 protein (467 amino acids; Fig. 2, Table 2). We subsequently verified that this mutation did not exist in any of the widely used genomic databases, confirming that c.1333delT constitutes a novel deletion mutation. Moreover, this mutation was not found in the unaffected parents or in 40 unrelated healthy control subjects. However, a heterozygous missense mutation (c.1363C > A) in MITF was found in both the patient and his father, but not in his mother (Fig. 2). This mutation was found in the dbSNP (https://www. ncbi.nlm.nih.gov/projects/SNP/) and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) databases (rs78962087) and is reportedly benign. Furthermore, no mutation was found in the EDN3, EDNRB, PAX3, or SANI2 genes. These results suggested that the heterozygous deletion mutation (c.1333delT) in SOX10 might be associated with the WS4 phenotype of the patient.
Paternity testing and haplotype analysis. SOX10c.1333delT is located in chr22:38369570. To confirm the paternity of the father, five STRs (D22S283, D22S1177, D22S1045, D22S272, and D22S423) ranging from chr22:36750705 to chr:40382524 and five SNPs (rs139873, rs139885, rs4821733, rs3952, and rs5756908) ranging from chr22:38359666 to chr:38476579 were selected from the UCSC Genome Browser (http://genome.ucsc.edu/) based on their proximity to the mutation site. Paternity testing by haplotype analysis confirmed that these were the biological parents of the patient with WS4 (Figs 3 and 4).
Protein structure prediction. The wild-type SOX10 protein consists of 467 amino acids and contains three helices, whereas the SOX10 deletion mutation (c.1333delT) results in a protein consisting of 501 amino acids with four helices (Fig. 5). The wild-type and mutant variants shared identical sequences in the first 444 amino acids, with differences occurring after this point.

Discussion
WS is classified into four primary phenotypes. WS1 is caused by mutations in PAX3 and distinguished by the presence of dystopia canthorum (lateral displacement of the inner canthi). WS2 is caused by mutations in MITF, SOX10, or SNAI2 and distinguished from type 1 by the absence of dystopia canthorum. WS3 is caused by mutations in PAX3, with patients presenting both dystopia canthorum and upper limb abnormalities. WS4 is caused by mutations in EDNRB, EDN3, or SOX10, with patients presenting with phenotypes associated with Hirschsprung disease 1,20-23 . Here, we described a Chinese patient with clinical features of WS4 and identified a novel heterozygous deletion mutation [c.1333delT (p.Ser445Glnfs*57)] in SOX10 that was absent in his unaffected parents and 40 ethnicity matched healthy controls. To the best of our knowledge, this constitutes the first report of this mutation, suggesting it as a candidate disease-causing mutation. SOX10 is located on chromosome 22 and encodes an essential DNA-binding nuclear transcription factor consisting of 467 amino acids and belonging to the SOX family involved in modulating embryonic development and determining cell fate. SOX10 may act as a transcriptional activator upon forming a complex with other proteins and/or as a nucleocytoplasmic shuttle protein critical for neural crest and peripheral nervous system development [24][25][26][27] . Mutations in this gene are associated with WS4 and are present in ~50% of WS4 patients 6,28 .
SOX10 contains a highly conserved high mobility group (HMG) DNA-binding domain and a C-terminal transactivation (TA) domain that is enriched in serine, proline, and acidic residues 29,30 . Additionally, SOX10 contains two separate TA domains, with one localized in the C-terminal region and the other in the central region of the structure. The C-terminal TA domain is frequently involved in various interactions, whereas the TA domain located in the centre of the structure is only involved in TA-related activity in certain cell types and under certain developmental conditions 31 . SOX10 binds to the promoters of its target genes via the HMG domain, with several studies reporting the importance of the TA domain for inducing transcriptional activation of its target genes 32 . Wang et al. 32 identified a c.1063C > T (p.Q355*) mutation in SOX10 in a family with WS4 and reported that the mutated SOX10 variant retained nuclear localization and DNA-binding capabilities comparable to those observed in wild-type SOX10; however, the mutated SOX10 variant was unable to activate transcription of MITF via its promoter and acted as a dominant-negative repressor as compared with activity associated with wild-type SOX10 7,33 . In this study, we detected a c.1333delT (p.Ser-445Glnfs*57) mutation in SOX10 in a family with WS4, with the mutated SOX10 variant sharing sequence homology with only the N-terminal 444 amino acids of the wild-type protein. Furthermore, we identified an additional helix in the C-terminal region of the mutated SOX10 variant (Fig. 4), which may affect its normal biological function.  Table 2. Genetic variants found in this family with WS4. Figure 3. Paternity testing and haplotype analysis. 601, patient; 602, father; 603, mother.
In conclusion, here, we described a de novo heterozygous deletion mutation [c.1333delT (p.Ser445Glnfs*57)] in SOX10 identified in a Chinese family with WS4. Our analyses indicated that this mutation might constitute a candidate disease-causing mutation associated with WS4.