Targeted Resequencing of Deafness Genes Reveals a Founder MYO15A Variant in Northeastern Brazil

Identifying the genetic etiology in a person with hearing loss (HL) is challenging due to the extreme genetic heterogeneity in HL and the population‐specific variability. In this study, after excluding GJB2 variants, targeted resequencing of 180 deafness‐related genes revealed the causative variants in 11 of 19 (58%) Brazilian probands with autosomal recessive HL. Identified pathogenic variants were in MYO15A (10 families) and CLDN14 (one family). Remarkably, the MYO15A p.(Val1400Met) variant was identified in eight families from the city of Monte Santo in the northeast region of Brazil. Haplotype analysis of this variant was consistent with a single founder. No other cases with this variant were detected among 105 simplex cases from other cities of northeastern Brazil, suggesting that this variant is confined to a geographical region. This study suggests that it is feasible to develop population‐specific screening for deafness variants once causative variants are identified in different geographical groups.     

A DFNA5 Mutation Identified in Japanese Families with Autosomal Dominant Hereditary Hearing Loss

Mutations in DFNA5 lead to autosomal dominant nonsyndromic hereditary hearing loss (NSHHL). To date, four different mutations in DFNA5 have been reported to cause hearing loss. A 3 bp deletion mutation (c.991‐15_991‐13del) was identified in Chinese and Korean families with autosomal dominant NSHHL, which suggested that the 3 bp deletion mutation was derived from a single origin. In the present study, we performed genetic screening of mutations in the interval between intron 6 and exon 9 of DFNA5 in 65 Japanese patients with autosomal dominant NSHHL and identified the c.991‐15_991‐13del mutation in two patients. Furthermore, we compared the DFNA5‐linked haplotypes consisting of intragenic SNPs between the reported Chinese and Korean families and found that the Japanese patients showed a shared region spanning 41,874 bp. This is the first report of DFNA5 mutations in Japanese patients with autosomal dominant NSHHL, supporting the suggestion that the 3 bp deletion mutation occurred in their ancestors.     

Targeted Deep Resequencing Identifies MID2 Mutation for X‐Linked Intellectual Disability with Varied Disease Severity in a Large Kindred from India

We report a novel missense mutation (c.1040G>A, p.Arg347Gln) in MID2, which encodes ubiquitin ligase E3, as the likely cause of X‐linked mental retardation in a large kindred. The mutation was observed in all affected and obligate carriers but not in any unaffected males of the family or in population controls (n = 200). When transiently expressed in HEK293T cell line, the mutation was found to abolish the function of the COS domain in the protein. The GFP‐tagged mutant protein accumulated in the cytoplasm instead of binding to the cytoskeleton resulting in its altered subcellular localization. Screening of coding exons of this gene in additional 480 unrelated individuals with idiopathic intellectual disability identified another novel variation p.Asn343Ser. This study highlights the growing role of the ubiquitin pathway in intellectual disability and also, the difference in MID2 determined phenotype observed in this study compared with that of its paralogue MID1 reported in literature.     

Update of the Spectrum of GJB2 Mutations in 107 Patients with Nonsyndromic Hearing Loss in the Fujian Population of China

Mutations in the GJB2 gene, encoding connexin26, which is expressed in the inner ear, have been shown to be responsible for the majority of nonsyndromic hearing loss (NSHL) cases. To update and evaluate the spectrum and prevalence of GJB2 mutations in the Fujian population, we screened exon 2 (coding), exon 1, and flanking introns of GJB2 in 107 NSHL probands and 61 individuals with normal hearing. Twelve different variants were identified, including three pathogenic mutations (c.235delC, c.299_300delAT, and c.508insAACG), one hypomorphic allele (p.V37I), three polymorphic variants (p.V27I, p.E114G, and p.I230T), and five rare variants (p.N62N, p.F115C, p.T123N, p.G21E, and p.F142I). The p.G21E and p.F142I variants were potentially pathogenic as predicted by PolyPhen‐2, SIFT, and PROVEAN. The most common mutation was c.235delC with allele frequency 12.6% (27/214). The most common polymorphisms in the Fujian population were p.V27I and p.E114G, both detected at high frequency in probands and controls. The p.E114G variant was always in cis with p.V27I, and formed the haplotype, p.[V27I; E114G] in the Fujian population. Interestingly, only 17.76% (19/107) of NSHL probands had clearly defined pathogenic mutations in GJB2, indicating that the pathogenesis of NSHL in the Fujian population is heterogenous, and that further analysis of other NSHL genes is necessary.     

Targeted Next‐Generation Sequencing on Hirschsprung Disease: A Pilot Study Exploits DNA Pooling

To adopt an efficient approach of identifying rare variants possibly related to Hirschsprung disease (HSCR), a pilot study was set up to evaluate the performance of a newly designed protocol for next generation targeted resquencing. In total, 20 Chinese HSCR patients and 20 Chinese sex‐matched individuals with no HSCR were included, for which coding sequences (CDS) of 62 genes known to be in signaling pathways relevant to enteric nervous system development were selected for capture and sequencing. Blood DNAs from eight pools of five cases or controls were enriched by PCR‐based RainDance technology (RDT) and then sequenced on a 454 FLX platform. As technical validation, five patients from case Pool‐3 were also independently enriched by RDT, indexed with barcode and sequenced with sufficient coverage. Assessment for CDS single nucleotide variants showed DNA pooling performed well (specificity/sensitivity at 98.4%/83.7%) at the common variant level; but relatively worse (specificity/sensitivity at 65.5%/61.3%) at the rare variant level. Further Sanger sequencing only validated five out of 12 rare damaging variants likely involved in HSCR. Hence more improvement at variant detection and sequencing technology is needed to realize the potential of DNA pooling for large‐scale resequencing projects.     

Congenital Middle Ear Malformation with Common Deafness Gene Mutation Analysis: A Review of 813 Profound Sensorineural Hearing Loss Child Patients

Deafness gene variants play a key role in inner ear malformations. However, the relationship between congenital middle ear malformations and common deafness genes (GJB2, SLC26A4, and mtDNA) in profound sensorineural hearing loss (SNHL) child patients remains poorly investigated. Here we showed that there was no statistical significance in the total mutation frequency of the three common deafness genes in the middle ear malformation group (21.2%, 41/193) in comparison with the normal middle ear and inner ear group (21.0%, 116/553) (χ² = 0.0061, p = 0.940). Moreover, the mutation ratio of GJB2 and SLC26A4 in the middle ear malformation group (18.7%, 36/193; 2.6%, 5/193) was not significantly different from that in the normal middle ear and inner ear group (17.7%, 98/553; 2.4%, 13/553) (χ² = 0.084, p = 0.772; χ² = 0.0000, p = 1.000). The mutation ratio of GJB2 235delC and GJB2 79G>A in the middle ear malformation group (8.8%, 17/193; 8.8%, 17/193) was almost the same to that in the normal middle ear and inner ear group (8.6%, 48/553; 6.7%, 37/553) (χ² = 0.0030, p = 0.957; χ² = 0.9556, p = 0.328). The high jugular bulb subgroup analysis also showed the same results. Our findings suggested that GJB2, SLC26A4, and mtDNA mutations might not be related to the middle ear malformations in profound SNHL child patients. Anat Rec, 303:594–599, 2020. © 2019 American Association for Anatomy     

Autosomal Dominant Hearing Loss resulting from p.R75Q Mutation in the GJB2 Gene: Nonsyndromic presentation in a South Indian Family

Mutations in the GJB2 gene encoding the gap junction protein Connexin 26 have been associated with autosomal recessive as well as dominant nonsyndromic hearing loss. Owing to the involvement of connexins in skin homeostasis, GJB2 mutations have also been associated with syndromic forms of hearing loss showing various skin manifestations. We report an assortatively mating hearing impaired family of south Indian origin with three affected members spread over two generations, having p.R75Q mutation in the GJB2 gene in the heterozygous condition. The inheritance pattern was autosomal dominant with mother and son being affected. Dermatological and histopathologic examinations showed absence of palmoplantar keratoderma. To the best of our knowledge, this is the first report from India on p.R75Q mutation in the GJB2 gene with nonsyndromic hearing loss.     

Complete Mitochondrial Genome Analysis and Clinical Documentation of a Five‐Generational Indian Family with Mitochondrial 1555A>G Mutation and Postlingual Hearing Loss

Hearing loss is the most common sensory disorder and is genetically heterogeneous. Apart from nuclear gene mutations, a number of inherited mitochondrial mutations have also been implicated. The m.1555A>G mutation in the mitochondrial MT‐RNR1 gene is reported as the most common mutation causing nonsyndromic hearing loss in various ethnic populations. We report here for the first time the clinical, genetic and molecular characterisation of a single large five‐generational Tamil‐speaking South Indian family with maternally inherited nonsyndromic postlingual hearing loss. Molecular analysis led to identification of m.1555A>G in 28 maternal relatives with variable degree of phenotypic expression. The penetrance of hearing loss among the maternal relatives in this family was 55%. Sequence analysis of the complete mitochondrial genome in 36 members of this pedigree identified 25 known variants and one novel variant co‐transmitted along with m.1555A>G mutation. The mtDNA haplotype analysis revealed that the maternal relatives carry the R*T₂ haplotype similar to Europeans and South Asians. Sequencing of the coding exon of GJB2 nuclear gene did not show any pathogenic mutations. The results suggest that other nuclear or environmental modifying factors could have played a role in the differential expression of mutation m.1555A>G in postlingual hearing loss in this family.     

A Case of Dysplastic Nevus of the External Auditory Canal Presenting with Conductive Hearing Loss

A nevus which is a benign melanocytic neoplasm rarely occurs within the external auditory canal (EAC). A dysplastic nevus presents atypical features both clinically and histologically, and is important as a potential precursor for melanoma. We present a case of a 33-year-old female patient with a dysplastic nevus in her EAC. Physical examination revealed a protruding mass arising from the posterior wall of the left cartilaginous EAC. The mass showed clinically characteristic findings of a melanocytic nevus. The patient underwent excisional biopsy via a transcanal approach under local anesthesia. Histopathological examination revealed an intradermal nevus with atypical melanocytes without pleomorphism. There was no evidence of recurrence two years after surgical excision.     

Functional Analysis of a Novel Connexin30 Mutation in a Large Family with Hearing Loss,Pesplanus, Ichthyosis,Cutaneous Nodules,and Keratoderma

Mutations in the gap‐junction gene Cx30 (Connexin30, GJB6) are a known cause of hearing loss. Here, we report our findings on a large multigeneration family in which severe to profound sensorineural hearing impairment is associated with a variety of skin‐related anomalies. Genome‐wide analysis of the family showed that the locus maps to chromosome region 13ptel‐q12.1 and that a novel mutation, p.N54K, in Cx30, cosegregates with the phenotype. Unlike wild‐type Cx30, p.N54K Cx30 is predominantly localized in the cytoplasm and does not permit transfer of neurobiotin, suggesting improper cellular localization and abolishment of gap‐junction activity.     

Exome Sequencing Identifies a Novel Frameshift Mutation of MYO6 as the Cause of Autosomal Dominant Nonsyndromic Hearing Loss in a Chinese Family

Autosomal dominant types of nonsyndromic hearing loss (ADNSHL) are typically postlingual in onset and progressive. High genetic heterogeneity, late onset age, and possible confounding due to nongenetic factors hinder the timely molecular diagnoses for most patients. In this study, exome sequencing was applied to investigate a large Chinese family segregating ADNSHL in which we initially failed to find strong evidence of linkage to any locus by whole‐genome linkage analysis. Two affected family members were selected for sequencing. We identified two novel mutations disrupting known ADNSHL genes and shared by the sequenced samples: c.328C>A in COCH (DFNA9) resulting in a p.Q110K substitution and a deletion c. 2814_2815delAA in MYO6 (DFNA22) causing a frameshift alteration p.R939Tfs*2. The pathogenicity of novel coding variants in ADNSHL genes was carefully evaluated by analysis of co‐segregation with phenotype in the pedigree and in light of established genotype–phenotype correlations. The frameshift deletion in MYO6 was confirmed as the causative variant for this pedigree, whereas the missense mutation in COCH had no clinical significance. The results allowed us to retrospectively identify the phenocopy in one patient that contributed to the negative finding in the linkage scan. Our clinical data also supported the emerging genotype–phenotype correlation for DFNA22.     

A Dominant Mutation in the Stereocilia‐Expressing Gene TBC1D24 is a Probable Cause for Nonsyndromic Hearing Impairment

Mutations in TBC1D24 have been linked to a variety of epileptic syndromes and recently to syndromic hearing impairment DOORS syndrome and nonsyndromic hearing impairment DFNB86. All TBC1D24 mutations reported so far were inherited in the recessive mode. In a dominant family segregated with late‐onset, progressive, nonsyndromic hearing impairment, linkage analysis revealed a 2.07 Mb candidate region on chromosome 16p13.3 that contains TBC1D24. Whole‐exome sequencing identified a heterozygous p.Ser178Leu variant of TBC1D24 as the only candidate mutation segregating with the hearing loss within the family. In perinatal mouse cochlea, we detected a restricted expression of Tbc1d24 in the stereocilia of the hair cells as well as in the spiral ganglion neurons. Our study suggested that the p.Ser178Leu mutation of TBC1D24 is a probable cause for dominant, nonsyndromic hearing impairment. Identification of TBC1D24 as the stereocilia‐expressing gene may shed new light on its specific function in the inner ear.     

Prevalence of Mutations in Deafness‐Causing Genes in Cochlear Implanted Patients with Profound Nonsyndromic Sensorineural Hearing Loss in Shandong Province,China

The mutations of GJB2, SLC26A4, and mtDNA12SrRNA are the most common inherited causes of nonsyndromic sensorineural hearing loss (NSHL) in China, yet previous genetic screenings were mainly carried on patients with moderate‐to‐profound impairment. We aimed to detect the mutation frequencies in NSHL population within a more specified range of severity. Patients with profound NSHL who had undergone cochlear implantation in the Shandong Provincial Hospital (Shandong, China) were recruited. The majority (n = 472) were between 0.7 and 6 years old, and the remaining (n = 63) were between 6 and 70 years old. In total, 115 mutation alleles of the three genes were screened with SNP scan assay. Of the patients, 19.44% (104/535) were found to have GJB2 mutations, and the most common allele was c.235delC, followed by c.299_300delAT and c.109G>A. SLC26A4 mutations were detected in 13.46% patients (72/535), and the most common allele was c.919‐2A>G (IVS7‐2A>G), followed by c.1174A>T and c.2168A>G. Seven patients (1.31%) carried mutations in mtDNA12SrRNA, with the alleles of m.1555A>G and m.1494C>T. We found the allele frequency of c.109G>A (GJB2) was relatively lower in the profound NSHL population in comparison to the moderate‐to‐profound ones, and the c.1174A>T (SLC26A4) relatively higher. It suggests those mutations may be connected with the degree of deafness, which needs more observations and analyses to support.     

线粒体DNA A1555G突变的异质性研究进展

线粒体DNA 12S rRNA基因A1555G突变是最早发现的、最常见的与非综合征性耳聋相关的人类线粒体DNA突变。线粒体DNA A1555G突变大多以均质性突变的形式出现,但近来有报道指在不同的家系中也存在异质性的突变形式,且突变负荷与耳聋程度相关。鉴于线粒体DNA A1555G的临床表型在不同个体间存在显著差异,该突变异质性的存在为解释其表型多样性提供了依据。     

Construction of a multiplex allele-specific PCR-based universal array (ASPUA) and its application to hearing loss screening

We demonstrate a new method, using a universal array approach termed multiplex allele-specific PCR-based universal array (ASPUA), and applied it to the mutation detection of hereditary hearing loss. Mutations in many different genes may be the cause of hereditary hearing loss, a sensory defect disorder. Effective methods for genetic diagnosis are clearly needed to provide clinical management. Owing to the broad genetic basis of this condition, clinical assay of such a highly heterogeneous disorder is expensive and time consuming. In ASPUA, the allele discrimination reaction is carried out in solution by multiplex allele-specific PCR and a universal solid phase array with different tag probes is used to display the PCR result. The purpose of developing the ASPUA platform was to utilize the rapidity and simplicity of the amplification refractory mutation system (ARMS) with the detection power of microarray hybridization. This is the first report of the combination of these two technologies, which allow for the completion of allele-specific detection of 11 of the most frequent mutations causing hereditary hearing loss in under 5 hr. The ASPUA platform was validated by accurately analyzing 141 patient samples that had been previously genotyped for GJB2, GJB3, SLC26A4, and MTRNR1. In addition, we also developed a simplified assay by using streptavidin-coated magnetic beads instead of fluorescence for signal display that can be assessed through a conventional light microscope. We demonstrate that the ASPUA platform is rapid, cost-effective, and easily-used, and is especially appropriate for mutation detection in clinical genetic diagnostics.     

七个非综合征型耳聋家系患者的mtDNA A1555G突变性质与特点

目的 探讨非综合征型耳聋家系患者mtDNA A1555G突变性质及其特点,探索临床表型多样性的分子遗传学基础.方法 应用聚合酶链反应-限制性片段长度多态和实时荧光-扩增阻碍突变系统-定量PCR(real time-amplification refractory mutation system-quantitative PCR,RT-ARMS-qPCR)检测7个非综合征型耳聋家系71个成员的mtDNA A1555G突变,并收集、分析其临床资料.结果 7个家系中所有受检的母系成员mtDNA A1555G突变均为阳性,突变性质含同质性和异质性两种;非母系成员及配偶该突变为阴性.7个家系mtDNA A1555G同质性突变的拷贝数与耳聋轻重程度相关(R=0.341,P=0.022);mtDNA A1555G异质性突变的拷贝数与耳聋轻重程度相关(R=0.85,P=0.015).结论 mtDNA A1555G突变可导致非综合征型耳聋和氨基糖甙类抗生素致聋,其突变性质含同质性和异质性两种,且含mtDNA A1555G位点的突变型与野生型的比例与耳聋的严重程度密切相关.     

SOD2 V16A SNP in the Mitochondrial Targeting Sequence is Associated with Noise Induced Hearing Loss in Chinese Workers

Objective: To investigate whether single nucleotide polymorphisms (SNPs) in the Mn-superoxide dismutase gene (SOD2) underlie the susceptibility to noise-induced hearing loss (NIHL). Methods: Audiometric data from 2400 Chinese Han workers who exposed to occupational noise were analyzed. DNA samples were collected from the 10% most susceptible and the 10% most resistant individuals, and five SNPs (SOD2 rs2842980, rs5746136, rs2758331, rs4880 and rs5746092) were genotyped by Taqman SNP Genotyping Kits. The SNP main effects and interactions between noise exposure and SNP were analyzed using logistic regression. Haplotypes were analyzed by using Haploview software. Results: The CT genotype of rs4880 (SOD2 V16A SNP) was associated with a higher risk of NIHL (covariates-adjusted OR, 2.18; 95% CI, 1.34–3.54, P = 0.002). Haplotype analysis revealed that the frequency of AGCCG at the five SNP loci was significantly higher in the susceptible group (P = 0.020). With AGCTG as the reference, the OR (95% CI) was 2.63 (1.14, 6.06). The rs4880 polymorphisms imposed larger effects when the carriers were exposed to higher levels of noise, indicating the interaction between SNP and noise exposure. Conclusions: Our results suggest that SOD2 V16A SNP in the mitochondrial targeting sequence is associated with noise induced hearing loss in Chinese workers, and this effect was enhanced by higher levels of noise exposure.     

A systematic review of the monogenic causes of Non-Syndromic Hearing Loss (NSHL) and discussion of Current Diagnosis and Treatment options

Hearing impairment is one of the most widespread inheritable sensory disorder affecting at least 1 in every 1000 born. About two-third of hereditary hearing loss (HHL) disorders are non-syndromic. To provide comprehensive update of monogenic causes of non-syndromic hearing loss (NSHL), literature search has been carried out with appropriate keywords in the following databases–PubMed, Google Scholar, Cochrane library, and Science Direct. Out of 2214 papers, 271 papers were shortlisted after applying inclusion and exclusion criterion. Data extracted from selected papers include information about gene name, identified pathogenic variants, ethnicity of the patient, age of onset, gender, title, authors' name, and year of publication. Overall, pathogenic variants in 98 different genes have been associated with NSHL. These genes have important role to play during early embryonic development in ear structure formation and hearing development. Here, we also review briefly the recent information about diagnosis and treatment approaches. Understanding pathogenic genetic variants are helpful in the management of affected and may offer targeted therapies in future.     

Genetic Epidemiology of Mitochondrial Pathogenic Variants Causing Nonsyndromic Hearing Loss in a Large Cohort of South Indian Hearing Impaired Individuals

Mitochondria play a critical role in the generation of metabolic energy in the form of ATP. Tissues and organs that are highly dependent on aerobic metabolism are involved in mitochondrial disorders including nonsyndromic hearing loss (NSHL). Seven pathogenic variants leading to NSHL have so far been reported on two mitochondrial genes: MT‐RNR1 encoding 12SrRNA and MT‐TS1 encoding tRNA for Ser^(UCN). We screened 729 prelingual NSHL subjects to determine the prevalence of MT‐RNR1 variants at position m.961, m.1555A>G and m.1494C>T, and MT‐TS1 m.7445A>G, m.7472insC m.7510T>C and m.7511T>C variants. Mitochondrial pathogenic variants were found in eight probands (1.1%). Five of them were found to have the m.1555A>G variant, two others had m.7472insC and one proband had m.7444G>A. The extended relatives of these probands showed variable degrees of hearing loss and age at onset. This study shows that mitochondrial pathogenic alleles contribute to about 1% prelingual hearing loss. This study will henceforth provide the reference for the prevalence of mitochondrial pathogenic alleles in the South Indian population, which to date has not been estimated. The m.1555A>G variant is a primary predisposing genetic factor for the development of hearing loss. Our study strongly suggests that mitochondrial genotyping should be considered for all hearing impaired individuals and particularly in families where transmission is compatible with maternal inheritance, after ruling out the most common variants.     

近亲结婚所致非综合征型耳聋家系的线粒体基因突变分析

目的对一个呈母系遗传的非综合征型耳聋大家系进行线粒体DNA的突变检测。方法收集湖南省一个非综合征型遗传性耳聋家系成员外周静脉血，提取基因组DNA（含线粒体DNA），设计特异性引物对目的片段进行PCR扩增，直接测序检测突变类型。结果测序结果显示，线粒体12S rRNA基因A1555G突变为该家系的致病突变，非母系成员不存在这一突变。结论该家系中部分成员使用氨基糖甙类抗生素后出现耳聋，可能是因为药物与线粒体12S rRNA基因的A1555G突变共同参与了听力损伤过程。