Molecular Mechanisms and Clinical Phenotypes of GJB2 Missense Variants.

The GJB2 gene is the most common gene responsible for hearing loss (HL) worldwide, and missense variants are the most abundant type. GJB2 pathogenic missense variants cause nonsyndromic HL (autosomal recessive and dominant) and syndromic HL combined with skin diseases. However, the mechanism by which these different missense variants cause the different phenotypes is unknown. Over 2/3 of the GJB2 missense variants have yet to be functionally studied and are currently classified as variants of uncertain significance (VUS). Based on these functionally determined missense variants, we reviewed the clinical phenotypes and investigated the molecular mechanisms that affected hemichannel and gap junction functions, including connexin biosynthesis, trafficking, oligomerization into connexons, permeability, and interactions between other coexpressed connexins. We predict that all possible GJB2 missense variants will be described in the future by deep mutational scanning technology and optimizing computational models. Therefore, the mechanisms by which different missense variants cause different phenotypes will be fully elucidated.

Molecular diagnosis of hereditary deafness and application of stepwise testing strategy.

Hereditary deafness is one of the most common sensory disorders in humans, and exhibits high genetic heterogeneity. At present, the commonly used molecular diagnostic methods include gene chip, Sanger sequencing, targeted enrichment sequencing, and whole-exome sequencing, with diagnosis rates reaching 33.5%-56.67%. However, there are still a considerable number of patients who can not get a timely and definitive molecular diagnosis. Furthermore, considering the economic burden on patients' families and the relatively high cost of whole-exome or whole-genome sequencing, it is vital to provide stepwise strategies combining multiple detection methods according to the phenotypes of patients. In this review, we evaluate and discuss the utility of molecular diagnosis and the application of stepwise testing strategies in hereditary deafness to provide reference for the selection of diagnostic strategies.

Increased diagnostic yield in a cohort of hearing loss families using a comprehensive stepwise strategy of molecular testing.

Hearing loss is one of the most common sensory disorders in humans. This study proposes a stepwise strategy of deafness gene detection using multiplex PCR combined with high-throughput sequencing, Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and whole-exome sequencing (WES) to explore its application in molecular diagnosis of hearing loss families. A total of 152 families with hearing loss were included in this study, the highest overall diagnosis rate was 73% (111/152). The diagnosis rate of multiplex PCR combined with high-throughput sequencing was 52.6% (80/152). One families was diagnosed by Sanger sequencing of GJB2 exon 1. Two families were diagnosed by MLPA analysis of the STRC gene. The diagnosis rate with additional contribution from WES was 18.4% (28/152). We identified 21 novel variants from 15 deafness genes by WES. Combining WES and deep clinical phenotyping, we diagnosed 11 patients with syndromic hearing loss (SHL). This study demonstrated improved diagnostic yield in a cohort of hearing loss families and confirmed the advantages of a stepwise strategy in the molecular diagnosis of hearing loss.

A likely pathogenic POLD1 variant associated with mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome in a Chinese patient.

BACKGROUND: Mandibular hypoplasia, deafness, progeroid features, and lipodystrophy syndrome (MDPL; OMIM# 615381) is a rare autosomal dominant disorder, with only a few reported cases worldwide. Herein, we describe the clinical features and underlying molecular etiology of MDPL syndrome in an 8-year-old Chinese patient. METHODS: We performed otological, endocrine, ultrasound, and radiological examinations, as well as genetic testing. Additionally, the literature concerning MDPL was reviewed to do a retrospective analysis of the pathogenesis, genotype-phenotype correlation, and clinical management. RESULTS: The proband was diagnosed with MDPL, presenting with mandibular hypoplasia, a characteristic facial appearance, lipodystrophy, and sensorineural hearing loss (SNHL). Whole-exome sequencing and bioinformatics analysis revealed a de novo missense variant in the POLD1 gene, NM_002691.4:c.3185A>G (NP_002682.2:p.(Gln1062Arg)). The retrospective analysis showed wide variation in the MDPL phenotype, but the most frequent features included mandibular hypoplasia, characteristic facial appearance, lipodystrophy, and SNHL. CONCLUSIONS: This study supplements the mutational spectrum of POLD1. The genetic analysis contributes to the diagnosis of syndromic deafness, and it has a vital role in clinical management and future genetic consultation.

Genetic and Phenotypic Variability in Chinese Patients With Branchio-Oto-Renal or Branchio-Oto Syndrome.

Background: Branchio-oto-renal syndrome (BOR) and branchio-oto syndrome (BOS) are rare autosomal dominant disorders defined by varying combinations of branchial, otic, and renal anomalies. Here, we characterized the clinical features and genetic etiology of BOR/BOS in several Chinese families and then explored the genotypes and phenotypes of BOR/BOS-related genes, as well as the outcomes of auditory rehabilitation in different modalities. Materials and Methods: Probands and all affected family members underwent detailed clinical examinations. Their DNA was subjected to whole-exome sequencing to explore the underlying molecular etiology of BOR/BOS; candidate variants were validated using Sanger sequencing and interpreted in accordance with the American College of Medical Genetics guidelines. In addition, a literature review concerning EYA1 and SIX1 alterations was performed to explore the genotypes and phenotypes of BOR/BOS-related genes. Results: Genetic testing identified the novel deletion (c.1425delC, p(Asp476Thrfs*4); NM_000,503.6), a nonsense variant (c.889C > T, p(Arg297*)), and two splicing variants in the EYA1 gene (c.1050+1G > T and c.1140+1G > A); it also identified one novel missense variant in the SIX1 gene (c.316G > A, p(Val106Met); NM_005,982.4). All cases exhibited a degree of phenotypic variability between or within families. Middle ear surgeries for improving bone-conduction component hearing loss had unsuccessful outcomes; cochlear implantation (CI) contributed to hearing gains. Conclusion: This is the first report of BOR/BOS caused by the SIX1 variant in China. Our findings increase the numbers of known EYA1 and SIX1 variants. They also emphasize the usefulness of genetic testing in the diagnosis and prevention of BOR/BOS while demonstrating that CI for auditory rehabilitation is a feasible option in some BOR/BOS patients.

High Genetic Heterogeneity in Chinese Patients With Waardenburg Syndrome Revealed by Next-Generation Sequencing.

OBJECTIVE: This study aimed to explore the genetic causes of probands who were diagnosed with Waardenburg syndrome (WS) or congenital sensorineural hearing loss. METHODS: A detailed physical and audiological examinations were carried out to make an accurate diagnosis of 14 patients from seven unrelated families. We performed whole-exome sequencing in probands to detect the potential genetic causes and further validated them by Sanger sequencing in the probands and their family members. RESULTS: The genetic causes for all 14 patients with WS or congenital sensorineural hearing loss were identified. A total of seven heterozygous variants including c.1459C > T, c.123del, and c.959-409_1173+3402del of PAX3 gene (NM_181459.4), c.198_262del and c.529_556del of SOX10 gene (NM_006941.4), and c.731G > A and c.970dup of MITF gene (NM_000248.3) were found for the first time. Of these mutations, we had confirmed two (c.1459C > T and c.970dup) are de novo by Sanger sequencing of variants in the probands and their parents. CONCLUSION: We revealed a total of seven novel mutations in PAX3, SOX10, and MITF, which underlie the pathogenesis of WS. The clinical and genetic characterization of these families with WS elucidated high heterogeneity in Chinese patients with WS. This study expands the database of PAX3, SOX10, and MITF mutations and improves our understanding of the causes of WS.

Increased diagnosis of enlarged vestibular aqueduct by multiplex PCR enrichment and next-generation sequencing of the SLC26A4 gene.

BACKGROUND: The enlarged vestibular aqueduct (EVA) is the commonest malformation of inner ear accompanied by sensorineural hearing loss in children. Three genes SLC26A4, FOXI1, and KCNJ10 have been associated with EVA, among them SLC26A4 being the most common. Yet, hotspot mutation screening can only diagnose a small number of patients. METHODS: Thus, in this study, we designed a new molecular diagnosis panel for EVA based on multiplex PCR enrichment and next-generation sequencing of the exon and flanking regions of SLC26A4. A total of 112 hearing loss families with EVA were enrolled and the pathogenicity of the rare variants detected was interpreted according to the American College of Medical Genetics and Genomics (ACMG) guidelines. RESULTS: Our results showed that 107/112 (95.54%) families carried SLC26A4 biallelic mutations, 4/112 (3.57%) carried monoallelic variants, and 1/112 (0.89%) had none variant, resulting in a diagnostic rate of 95.54%. A total of 49 different variants were detected in those patients and we classified 30 rare variants as pathogenic/likely pathogenic, of which 13 were not included in the Clinvar database. CONCLUSION: Our diagnostic panel has an increased diagnostic yield with less cost, and the curated list of pathogenic variants in the SLC26A4 gene can be directly used to aid the genetic counseling to patients.

Early truncation of the N-terminal variable region of EYA4 gene causes dominant hearing loss without cardiac phenotype.

BACKGROUND: Autosomal dominant hearing loss (ADHL) accounts for about 20% of all hereditary non-syndromic HL. Truncating mutations of the EYA4 gene can cause either non-syndromic ADHL or syndromic ADHL with cardiac abnormalities. It has been proposed that truncations of the C-terminal Eya domain lead to non-syndromic HL, whereas early truncations of the N-terminal variable region cause syndromic HL with cardiac phenotype. METHODS: The proband and all the other hearing impaired members of the family underwent a thorough clinical and audiological evaluation. The cardiac phenotype was examined by ECG and echocardiography. Their DNA was subjected to target exome sequencing of 129 known deafness genes. The sequencing data were analyzed and the candidate variants were interpreted following the ACMG guidelines for clinical sequence interpretation. The effect of candidate variant on EYA4 gene expression was assessed by quantitative PCR and western blot of gene production in blood. RESULTS: We report a Chinese family cosegregating post-lingual onset, progressive ADHL with a novel nonsense mutation NM_004100.4:c.543C>G (p.Tyr181Ter) of EYA4. Two affected members show no cardiac abnormalities at least until now revealed by electrocardiography and echocardiography. The overall expression level of the EYA4 gene in the proband was lower than that in his unaffected relative. CONCLUSION: This report expands the mutational spectrum of the EYA4 gene and highlights the fact that more data are needed to elucidate the complex genotype-phenotype correlation of EYA4 mutations.