Identification of a de novo, Novel Pathogenic Variant in the Splice Region of the SOX10 Gene in an Iranian Azeri Turkish Family with Waardenburg Syndrome

Abstract

Background

Waardenburg syndrome (WS) is an inherited heterogeneous auditory pigmentary syndrome, divided into at least four types and characterized by iris heterochromia, white forelock, prominent nasal root, dystopia canthorum, middle eyebrow hypertrichosis, and deafness. Pathogenic variants in the SOX10 gene have been reported to be involved in WS disease.

Methods

Whole exome sequencing (WES) was conducted on a 24-year-old male, who originated from Iranian Azeri Turkish ethnic group, with symptoms of deafness and blue eyes from brown-eyed parents. Web-based tools including Mutation Taster, VarSome, SIFT, Human Splicing Finder (HSF), and I-TASSER, were used for bioinformatics analysis. To verify the WES findings, DNAs taken from the blood samples of all family members were subjected to PCR-Sanger sequencing.

Results

A novel heterozygous pathogenic variant, NC_000022.11 (NM_006941):c.428+1G>T, located in the second intron of the SOX10 gene and disrupting the splicing site, was identified in the proband. Sanger sequencing was applied on the proband and his parents. The results showed that the variant was a de novo pathogenic variant with an autosomal dominant inheritance pattern.

Conclusions

Identification of a novel de novo pathogenic variant, NC_000022.11 (NM_006941):c.428+1G>T, in the second intron of the SOX10 gene with autosomal dominant inheritance pattern.

Established Facts

• Mutations in SOX10 gene result in Waardenburg syndrome.

Novel Insights

• Identification of a de novo and novel pathogenic variant (NC_000022.11 (NM_006941):c.428+1G>T) in SOX10 gene of a patient with Waardenburg syndrome.

Introduction

One of the most prevalent birth defects in developed nations is hearing impairment, which affects over 28 million Americans and occurs in 2–3 per 1,000 live births [Kassebaum et al., 2017]. It is a major contributor to childhood disabilities and a frequent basis for patient referrals to an otolaryngologist. Up to 50–60% of the patients with congenital sensorineural hearing loss (SNHL) have a genetic basis for their conditions [Morton and Nance, 2006; Carey and Palumbos, 2016]. About 30% of hearing loss cases with hereditary etiology is attributed to syndromic hearing loss. To gain further insights into the genetic basis of hearing loss and provide genetic counseling to affected families, screening identified genes for mutations is important [Soares de Lima et al., 2018]. Waardenburg syndrome (WS) is one of the most prevalent disorders among syndromic hearing loss diseases [Cohen and Phillips, 2012].

WS is a rare autosomal dominant disorder with a prevalence of 1 in 42,000 live births [Apaydin et al., 2004], initially introduced by Petrus J. Waardenburg in 1951 [Waardenburg, 1951]. This disease is characterized by congenital sensorineural hearing loss; change in the color of the eyes, hair, and skin; and deafness, with or without dystopia canthorum (a lateral movement of the inner canthi of the eyes) [Baldwin et al., 1995; Eigelshoven et al., 2009; Pardono et al., 2003]. It is grouped into four subgroups (WS1–4). Pathogenic variants in any of several different genes, including PAX3, MITF, EDNRB, EDN3, SNAI2, SOX10, WS2B, and TYR [Pingault et al., 2010; Yu et al., 2020], could result in WS. The presence or absence of one or more of these particular gene mutations will affect phenotypic variance, differentiating the many subtypes of the disorder [Pingault et al., 2010]. Both WS subtypes I and III are linked to the PAX3 gene mutations. Pathogenic variants in MITF and SNAI2 are linked to WS II. EDN3, EDNRB, and SOX10 genes are involved in WS subtype IV (or Shah-Waardenburg) [Li et al., 2019]. Both familial and isolated patients with WS IV have predominantly been found to have heterozygous SOX10 mutations. Ataxia and seizures are only a few examples of the symptoms that carriers of the SOX10 mutation may experience [Amiel and Lyonnet, 2001].

Here we report a novel pathogenic variant, NC_000022.11 (NM_006941):c.428+1G>T, in heterozygous state in the SOX10 gene identified in a patient with syndromic hearing loss from the Iranian Azeri Turkish population. This variant was neither observed in the other members of the family nor in the four hundred healthy individuals from the same ethnic group, confirming that it was a de novo pathogenic variant with a dominant inheritance pattern.

Materials and Methods

Patient

All the work described has been carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki). The project was ethically approved by the Committee of the University of Tabriz [IR.TABRIZU.REC.1401/008]. A 24-year-old man with blue eyes and hearing impairment from a family with non-consanguineous marriage was referred to the genetics laboratory. Both parents were healthy and had brown eyes with no other indications of disease. None of the other family members was diagnosed with the same illness.

Written informed consents were obtained from the patient and his parents prior to collecting blood samples. Genomic DNAs were extracted, quantified, and subjected to WES analysis, as outlined previously [Li and Durbin, 2010; Li et al., 2009]. In brief, each eluted-enriched DNA sample was sequenced on an Illumina NovaSeq 6000 platform, and 150 bp sequence reads were mapped to the UCSC (University of California, Santa Cruz) human reference genome (GRCh37/hg19 and GRCh38/hg38) using Burrows-Wheeler Aligner (BWA) and the CLC desktop genomic software [Li and Durbin. 2009]. Low-quality reads and duplicates (Qbase<20) were removed using Picard and Trimmomatics V0.39 tools, respectively [Bolger et al., 2014]. Sam-tools were used to sort and index BAM files [Danecek et al., 2021]. Variants were annotated using the web tool wANNOVAR [Chang and Wang, 2012]. In order to remove polymorphic variants, filtration was performed by eliminating variants located in deep interonic sites, upstream, downstream, and ncRNA regions. Afterward, variants reported in different populations with frequency greater than 0.05 were excluded. The VCF file was taken to the CADD database [Kircher et al., 2014] for further analysis. We conducted the relevant filtration with phred ≥10, and then pulled the data from the CADD database to complete the filtration in the Mutation Taster database [Schwarz et al., 2014]. The genes obtained in this step were applied to further exploration and evaluation. It was revealed that the genes related to Waardenburg syndrome were got from the Online Mendelian Inheritance in Man (OMIM) database based on disease symptoms [Yang et al., 2015]. Also, based on the ACMG guideline (American College of Medical Genetics and Genomics) and the genes from the final analysis in the mutation taster database, we compared the reference genes and the candidate genes. At this stage, the candidate genes were procured for additional examination. Genes related to the WS were identified and screened for the potential to cause disease. This was achieved by employing VarSome [Kopanos et al., 2019], SIFT [Vaser R et al., 2016], and Mutation Taster, three web-based tools. Primers were designed for the part of the genome sequence that underwent pathogenic variants. Sanger sequencing was used after designing primers for the patient and parents.

Results

The proband belonged to a family with non-consanguineous marriage from Iranian Azeri Turkish ethnic group. A 24-year-old man with hearing loss was referred to the genetic laboratory for further genetic analysis using WES. His 2-year-old son also had hearing loss, blue eyes, and a pigmentary problem in his hair. A novel and de novo pathogenic heterozygous variant, NC_000022.11 (NM_006941):c.428+1G>T, in the splice site region of the SOX10 gene has been identified (shown in Fig. 1). Sanger sequencing for the proband and his parents was carried out for further evaluation (shown in Fig. 1). The findings have shown that the parents’ genotypes were homozygous for the wild type, while the patient was heterozygous for NC_000022.11 (NM_006941):c.428+1G>T pathogenic variant (shown in Fig. 1). His son with the condition also carried the same mutation in heterozygous state. Bioinformatics analysis using several web-based tools demonstrated that this variant is a pathogenic variant. Four hundred healthy individuals from the same ethnic group were screened for the variant, and no carrier was identified. The HSF web-based tool reported score of 93.12 for a wild type and a score of 65.98 for a mutant type. The splicing mechanism has most likely been impaired by variant NC_000022.11 (NM_006941):c.428+1G>T in the donor site of intron 2, according to the web-based tools Human Splicing Finder (HSF) [Desmet et al., 2009] and MaxEnt [Phillips et al., 2017]. Applying bioinformatics tools showed that this variant resulted in production of truncated protein (shown in Fig. 2).

Fig. 1.

Whole exome sequencing results (upper figure) and Sanger sequencing results in the proband and his parents, respectively. One of the mutations in the SOX10 gene (c.428+1G>T) located on the splicing site is shown in the IGV figure of the BAM file of the proband (upper figure). The mutation is shown in heterozygous state in the Sanger sequencing result from the proband (lower figure).

Fig. 2.

A normal and mutant SOX10 protein: on the left, wild-type SOX10 protein with C-score = −0.99 and TM-score = 0.59 ± 0.14 (a), and on the right, the mutant truncated SOX10 protein with C-score = −3.03 and TM-score = 0.37 ± 0.13 (b). An analysis of the 3-D model showed that the novel mutation of c.428 G>T in SOX10 gene led to a short protein (143 residues) with serine instead of arginine at the C-terminal end.

Discussion

Here we report a novel pathogenic variant (NC_000022.11 (NM_006941):c.428+1G>T) in the SOX10 gene with an autosomal dominant pattern in a case who originated from Iranian Azeri Turkish ethnic group. Our patient presented with clinical characteristics of Waardenburg syndrome, namely, severe hearing loss and blue eyes. However, there were no other typical manifestations such as abnormalities of the eyes or face, Hirschsprung disease, or developmental delays. His 2-year-old son, who also carried the same mutation, had hearing loss and blue eyes, as well as pigmentary disturbances in his hair. Based on these clinical features, it is likely that the patient has Waardenburg syndrome type 2 (WS2). This is because WS2 is typically characterized by congenital sensorineural hearing loss and pigmentary disturbances of the skin, hair, and/or eyes, including blue eyes. WS2 can be caused by mutations in several genes, including SOX10, which is mutated in our patient [Bondurand et al., 2007]. It is worth noting that there can be significant variation in the clinical presentation of Waardenburg syndrome [Pingault et al., 2010], even among individuals with the same genetic mutation, as observed in the case of our patient and his son. Therefore, a definitive diagnosis of WS2 would require a thorough evaluation of the patient’s medical history, physical examination, and genetic testing, as well as consideration of other potential causes of hearing loss and pigmentary disturbances. The identified variant (NC_000022.11 (NM_006941):c.428+1G>T) in the case and his affected son was not observed in any of the other family members, confirming that the variant was a de novo pathogenic variant. Additionally, a population study on our cohort from the same ethnic group showed that the variant was absent among healthy individuals, further supporting the pathogenicity of the variant.

Pathogenic variants in the SOX10 gene provide an outstanding example for studying genetic variation and phenotypic pleiotropy. Mutations in this gene can lead to the clinical manifestations of either the WS2 or the WS4 phenotype [Masood et al., 2020]. The SOX10 gene encodes the protein SOX10 (SRY [sex determining region Y] box 10), a transcription factor that regulates the neural crest [Chaoui et al., 2011]. In addition, SOX10 plays an essential role as a transcription factor in glial cell development and myelin formation and maintenance, as suggested in the mouse model [Kuhlbrodt et al., 1998; Britsch et al., 2001; Stolt et al., 2002; Bremer et al., 2011]. The human and mouse SOX10 genes have 466 amino acids in their open reading frames, with 92% nucleotide and 98% amino acid sequence similarities [Pingault et al., 1998]. Two distinct exon numbering systems have been suggested for this gene. In the first system, exons number one and two are considered non-coding exons, whereas on the base of the second system, only exon one is a non-coding exon (NM_006941) [Pingault et al., 1998]. In our case, the variant was identified at intron 2 in the splice donor site region. As per the ClinVar web tool [Landrum et al., 2018], out of the seven pathogenic variants reported on the ClinVar site in the splice site region of the SOX10 gene, three of them were located in intron 2 with nucleotide substitutions. The first variant, NM_006941.4 (SOX10): c.429-1G>A, was found at the splice acceptor site [Landrum et al., 2018; National Center for Biotechnology Information, 2023a], the second variant, NM_006941.4 (SOX10): c.428+2T>C, was detected at the splice donor site [Landrum et al., 2018; National Center for Biotechnology Information, 2023b], and the third variant, NM_006941.4 (SOX10): c.428+1G>A was identified at the splice donor site [Landrum et al., 2018; National Center for Biotechnology Information, 2023c] of the second intron. All these pathogenic variants including our novel pathogenic variant, confirm the existence of the second system with four exons.

SOX10 has a HMG (high mobility group) domain that binds to DNA, a dimerization area that is upstream of the HMG domain, a conserved domain in the core, and a TA (trans-activation) domain at the extreme [Pingault et al., 2010]. The HMG domain involves a conserved sequence of 80 amino acids that is involved in controlling intracellular transport and multiple transcriptional regulations, which are important for the differentiation of oligodendrocytes and the initiation of myelinization and remyelination of the CNS (central nervous system) [He et al., 2016]. Pathogenic missense changes to SOX10 mainly, if not solely, impact the HMG domain. This highlights the significance of the HMG domain in SOX10’s role, as changes to the amino acid side chain are typically not tolerated. This idea is corroborated by population studies that show there are very few nonsynonymous variants in the HMG domain of SOX10 in comparison to other areas (The Genome Aggregation Database) [Moldenæs et al., 2021].

In our scenario, c.428+1G>T, the variant’s pathogenicity is interpreted based on the variant’s splicing effect, which is comparable to one of the previously reported pathogenic variant, c.428+1G>A. Our bioinformatics analysis shows that this variant could disrupt normal splicing process (Fig. 2a) and cause a long mRNA that contains intron 2 (Fig. 2b). Presence of the second intron in mRNA sequence causes a premature termination process, which results in a short protein (143 residues) in which Arg is replaced with Ser at the C-terminal end. The truncated protein would have 4 N-terminal α-helix structures missing 323 terminal amino acids. Therefore, the produced immature protein lacks HMG domain, which consequently causes misfunction of the protein and abnormal phonotype in the case.

However, there are limitations to this study. The splicing effect of the detected variant requires evaluation using a minigene assay, and the postulated hypothesis may be validated using SOX10 cDNA sequencing and qPCR.

Conclusion

This study identified a novel de novo pathogenic variant, NC_000022.11 (NM_006941):c.428+1G>T, in the second intron of the SOX10 gene in a 24-year-old male presenting with some symptoms of Waardenburg syndrome. The variant disrupted the splicing site and was found to have an autosomal dominant inheritance pattern as it caused the disease in a heterozygous state. It was absent among healthy individuals from the same ethnic group, and all the data suggest that this variant is pathogenic.

Acknowledgments

Authors would like to thank all the participants.

Statement of Ethics

The University of Tabriz ethically approved this project, and written informed consent was taken from all participants. Written informed consent was obtained from the patients for publication of the details of their medical case and any accompanying images. The approval reference number from the Ethics Committee of the University of Tabriz is IR.TABRIZU.REC.1401/008.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

There was no funding for the project.

Author Contribution

Faranak Roudbari analyzed WES data and performed bioinformatics analysis. She also designed primers for this family. Amir-Reza Dallal Amandi and Leyla Sadeghi contributed to the bioinformatics analysis of the encoded protein. Neda Jabbarpour performed population screening for the identified mutation. Mortaza Bonyadi designed the project, performed clinical examination on the patient, contributed to analyzing the WES results, and was a main contributor to manuscript preparation.

Data Availability Statement

All data generated or analyzed during this study are included in this published article. Further inquiries can be directed to the corresponding author.