A Rapid Method for Simultaneous Screening of Multi-Gene Mutations Associated with Hearing Loss in the Korean Population

Hearing loss (HL) is a congenital disease with a high prevalence, and patients with hearing loss need early diagnosis for treatment and prevention. The GJB2, MT-RNR1, and SLC26A4 genes have been reported as common causative genes of hearing loss in the Korean population and some mutations of these genes are the most common mutations associated with hearing loss. Accordingly, we developed a method for the simultaneous detection of seven mutations (c.235delC of GJB2, c.439A>G, c.919-2A>G, c.1149+3A>G, c.1229C>T, c.2168A>G of SLC26A4, and m.1555A>G of the MT-RNR1 gene) using multiplex SNaPshot minisequencing to enable rapid diagnosis of hereditary hearing loss. This method was confirmed in patients with hearing loss and used for genetic diagnosis of controls with normal hearing and neonates. We found that 4.06% of individuals with normal hearing and 4.32% of neonates were heterozygous carriers. In addition, we detected that an individual is heterozygous for two different mutations of GJB2 and SLC26A4 gene, respectively and one normal hearing showing the heteroplasmy of m.1555A>G. These genotypes corresponded to those determined by direct sequencing. Overall, we successfully developed a robust and cost-effective diagnosis method that detects common causative mutations of hearing loss in the Korean population. This method will be possible to detect up to 40% causative mutations associated with prelingual HL in the Korean population and serve as a useful genetic technique for diagnosis of hearing loss for patients, carriers, neonates, and fetuses.     

Phenotype and genotype of deaf patients with combined genomic and mitochondrial inheritance models

In most studies, sensorineural hearing loss is reported as a single-gene disease with autosomal dominant or autosomal recessive or with X-linked or maternal inheritance. It is uncommon that the hearing impairment is caused by a combined inheritance model including genomic and mitochondrial models. Here, we report six patients with sensorineural hearing loss caused by co-existing mutations in GJB2 or SLC26A4 and the mitochondrial gene. And there was no significant difference in hearing phenotypes between the six patients and the controls. The results indicate the complicated genetic etiology of, and may impact the diagnostic strategy for, hereditary hearing impairment. All patient siblings will carry mitochondrial DNA A1555G or C1494T mutations, and 25% of siblings may carry the same homozygous or compound heterozygote mutations in GJB2 or SLC26A4. Although this combined inheritance is not common in the Chinese deaf population (0.10%), our findings will have great impact in genetic counseling and risk prediction for deafness.     

Etiology and Audiological Outcomes at 3 Years for 364 Children in Australia

Hearing loss is an etiologically heterogeneous trait with differences in the age of onset, severity and site of lesion. It is caused by a combination of genetic and/or environmental factors. A longitudinal study to examine the efficacy of early intervention for improving child outcomes is ongoing in Australia. To determine the cause of hearing loss in these children we undertook molecular testing of perinatal “Guthrie” blood spots of children whose hearing loss was either detected via newborn hearing screening or detected later in infancy. We analyzed the GJB2 and SLC26A4 genes for the presence of mutations, screened for the mitochondrial DNA (mtDNA) A1555G mutation, and screened for congenital CMV infection in DNA isolated from dried newborn blood spots. Results were obtained from 364 children. We established etiology for 60% of children. One or two known GJB2 mutations were present in 82 children. Twenty-four children had one or two known SLC26A4 mutations. GJB2 or SLC26A4 changes with unknown consequences on hearing were found in 32 children. The A1555G mutation was found in one child, and CMV infection was detected in 28 children. Auditory neuropathy spectrum disorder was confirmed in 26 children whose DNA evaluations were negative. A secondary objective was to investigate the relationship between etiology and audiological outcomes over the first 3 years of life. Regression analysis was used to investigate the relationship between hearing levels and etiology. Data analysis does not support the existence of differential effects of etiology on degree of hearing loss or on progressiveness of hearing loss.     

High-throughput detection of mutations responsible for childhood hearing loss using resequencing microarrays

Background  

Despite current knowledge of mutations in 45 genes that can cause nonsyndromic sensorineural hearing loss (SNHL), no unified clinical test has been developed that can comprehensively detect mutations in multiple genes. We therefore designed Affymetrix resequencing microarrays capable of resequencing 13 genes mutated in SNHL (GJB2, GJB6, CDH23, KCNE1, KCNQ1, MYO7A, OTOF, PDS, MYO6, SLC26A5, TMIE, TMPRSS3, USH1C). We present results from hearing loss arrays developed in two different research facilities and highlight some of the approaches we adopted to enhance the applicability of resequencing arrays in a clinical setting.     

Diagnostic Application of Targeted Resequencing for Familial Nonsyndromic Hearing Loss

Identification of causative genes for hereditary nonsyndromic hearing loss (NSHL) is important to decide treatment modalities and to counsel the patients. Due to the genetic heterogeneity in sensorineural genetic disorders, the high-throughput method can be adapted for the efficient diagnosis. To this end, we designed a new diagnostic pipeline to screen all the reported candidate genes for NSHL. For validation of the diagnostic pipeline, we focused upon familial NSHL cases that are most likely to be genetic, rather than to be infectious or environmental. Among the 32 familial NSHL cases, we were able to make a molecular genetic diagnosis from 12 probands (37.5%) in the first stage by their clinical features, characteristic inheritance pattern and further candidate gene sequencing of GJB2, SLC26A4, POU3F4 or mitochondrial DNA. Next we applied targeted resequencing on 80 NSHL genes in the remaining 20 probands. Each proband carried 4.8 variants that were not synonymous and had the occurring frequency of less than three among the 20 probands. These variants were then filtered out with the inheritance pattern of the family, allele frequency in normal hearing 80 control subjects, clinical features. Finally NSHL-causing candidate mutations were identified in 13(65%) of the 20 probands of multiplex families, bringing the total solve rate (or detection rate) in our familial cases to be 78.1% (25/32) Damaging mutations discovered by the targeted resequencing were distributed in nine genes such as WFS1, COCH, EYA4, MYO6, GJB3, COL11A2, OTOF, STRC and MYO3A, most of which were private. Despite the advent of whole genome and whole exome sequencing, we propose targeted resequencing and filtering strategy as a screening and diagnostic tool at least for familial NSHL to find mutations based upon its efficacy and cost-effectiveness.     

Common molecular etiologies are rare in nonsyndromic Tibetan Chinese patients with hearing impairment

Background

Thirty thousand infants are born every year with congenital hearing impairment in mainland China. Racial and regional factors are important in clinical diagnosis of genetic deafness. However, molecular etiology of hearing impairment in the Tibetan Chinese population living in the Tibetan Plateau has not been investigated. To provide appropriate genetic testing and counseling to Tibetan families, we investigated molecular etiology of nonsyndromic deafness in this population.

Methods

A total of 114 unrelated deaf Tibetan children from the Tibet Autonomous Region were enrolled. Five prominent deafness-related genes, GJB2, SLC26A4, GJB6, POU3F4, and mtDNA 12S rRNA, were analyzed. Inner ear development was evaluated by temporal CT. A total of 106 Tibetan hearing normal individuals were included as genetic controls. For radiological comparison, 120 patients, mainly of Han ethnicity, with sensorineural hearing loss were analyzed by temporal CT.

Results

None of the Tibetan patients carried diallelic GJB2 or SLC26A4 mutations. Two patients with a history of aminoglycoside usage carried homogeneous mtDNA 12S rRNA A1555G mutation. Two controls were homozygous for 12S rRNA A1555G. There were no mutations in GJB6 or POU3F4. A diagnosis of inner ear malformation was made in 20.18% of the Tibetan patients and 21.67% of the Han deaf group. Enlarged vestibular aqueduct, the most common inner ear deformity, was not found in theTibetan patients, but was seen in 18.33% of the Han patients. Common molecular etiologies, GJB2 and SLC26A4 mutations, were rare in the Tibetan Chinese deaf population.

Conclusion

The mutation spectrum of hearing loss differs significantly between Chinese Tibetan patients and Han patients. The incidence of inner ear malformation in Tibetans is almost as high as that in Han deaf patients, but the types of malformation vary greatly. Hypoxia and special environment in plateau may be one cause of developmental inner ear deformity in this population.     

Results of molecular genetic testing in Russian patients with Pendred syndrome and allelic disorders

Pendred syndrome is an autosomal recessive inherited disorder characterized by a combination of sensorineural hearing impairment and euthyroid goiter; its clinical manifestation in children is hardly distinguishable from nonsyndromic hearing loss. Pendred syndrome is one of the most frequent types of syndromic hearing loss. Hearing impairment is accompanied by abnormal development of the bony labyrinth—enlarged vestibular aqueduct (EVA) and occasionally combined with Mondini dysplasia. Mutations in the SLC26A4 gene, which encodes the pendrin protein, are responsible for both Pendred syndrome and for allelic disorder (nonsyndromic enlarged vestibular aqueduct). The present study for the first time conducted molecular genetic analysis in 20 Russian patients with Pendred syndrome, EVA and/or Mondini dysplasia. As a result, six pathogenic mutations in the SLC26A4 gene were revealed in four patients. The mutation c.222G>T (p.Trp74Cys) was detected for the first time. Mutations were found in patients with Pendred syndrome and nonsyndromic EVA with or without Mondini dysplasia. Mutations were not detected in patients with isolated Mondini dysplasia. One proband with clinical diagnosis Pendred syndrome was homozygous for the c.35delG mutation in the GJB2 gene. The absence of frequent mutations, including well-known ones or “hot” exons in the SLC26A4 gene, was reported. Therefore, the optimal method to search for mutations in the SLC26A4 gene in Russian patients is Sanger sequencing of all exons and exon-intron boundaries in the SLC26A4 gene.     

Evaluation of the pathogenicity of GJB3 and GJB6 variants associated with nonsyndromic hearing loss

A number of genes responsible for hearing loss are related to ion recycling and homeostasis in the inner ear. Connexins (Cx26 encoded by GJB2, Cx31 encoded by GJB3 and Cx30 encoded by GJB6) are core components of gap junctions in the inner ear. Gap junctions are intercellular communication channels and important factors that are associated with hearing loss. To date, a molecular genetics study of GJB3 and GJB6 as a causative gene for hearing loss has not been performed in Korea. This study was therefore performed to elucidate the genetic characteristics of Korean patients with nonsyndromic sensorineural hearing loss and to determine the pathological mechanism of hearing loss by analyzing the intercellular communication function of Cx30 and Cx31 variants. Sequencing analysis of the GJB3 and GJB6 genes in our population revealed a total of nine variants, including four novel variants in the two genes. Three of the novel variants (Cx31-p.V27M, Cx31-p.V43M and Cx-30-p.I248V) and two previously reported variants (Cx31-p.V84I and Cx30-p.A40V) were selected for functional studies using a pathogenicity prediction program and assessed for whether the mutations were located in a conserved region of the protein. The results of biochemical and ionic coupling tests showed that both the Cx31-p.V27M and Cx31-p.V84I variants did not function normally when each was expressed as a heterozygote with the wild-type Cx31. This study demonstrated that two variants of Cx31 were pathogenic mutations with deleterious effect. This information will be valuable in understanding the pathogenic role of GJB3 and GJB6 mutations associated with hearing loss.     

人工耳蜗植入聋儿术前基因检测及家系分析

摘要: 国内外研究表明GJB2、SLC26A4(PDS)和线粒体DNA(Mitochondrial DNA, mtDNA)的病理性突变导致了大部分的遗传性聋。 文章收集了2006年4月~2007年9月接受人工耳蜗(Cochlear implant, CI)植入的14 例患儿及其父母的外周血, 应用基因诊断方法进行 GJB2、SLC26A4(PDS)和mtDNA 1555位点突变检测。结果显示, 35.7%的患儿检测到致病突变, 其中28.6%为GJB2基因突变, 类型均为235delC纯和突变, 其父母为携带GJB2 235delC的杂和子; 7.1%为mtDNA A1555G突变, 其母亲亦携带mtDNA A1555G突变。这表明CI 植入聋儿最常见的基因突变是GJB2 235delC突变, 其次是mtDNA A1555G突变, 通过对耳聋家系常见致病基因的检测和家系分析, 可以对优生优育及减少耳聋发病率提供科学准确的遗传信息。     

Application of deafness diagnostic screening panel based on deafness mutation/gene database using invader assay

Despite progress in identification of deafness genes, clinical application has lagged due to the genetic heterogeneity of deafness. We designed and tested a comprehensive and simple diagnostic strategy to simultaneously detect deafness gene mutations based on a mutation/gene database followed by Invader assay screening of 41 known mutations of nine known deafness genes. Three hundred thirty-eight Japanese patients with congenital or childhood-onset (up to age 10) bilateral sensorineural hearing loss participated in this study. A total of 100 (29.6%) subjects had at least one mutation in GJB2, SLC26A4, and/or the mitochondrial 12S rRNA, indicating that these are the three major causative genes in Japanese deafness patients. The present study demonstrated that the Invader assay has excellent sensitivity and accuracy, and its application to deafness mutation screening will greatly improve medical management and facilitate extensive genetic counseling for hearing impairment.     

Spectrum of the GJB2 mutations in Belarussian patients with hearing loss. Findings of pilot genetic screening of hearing impairment in newborns

A total of 111 unrelated probands and their 8 sibs from Grodno oblast (Belarus) with bilateral isolated sensorineural hearing impairment were studied for the presence of mutations in the connexin 26 (GJB2) gene. Mutations were detected in 51 probands (46% of the sample). A significantly higher frequency of the GJB2 gene mutations was observed in familial cases of the disease with the autosomal recessive mode of inheritance (in 78% of families). Detected characteristics of the GJB2 gene mutation spectrum demonstrated that the using the algorithm, which was designed for Russian patients, is optimal for the molecular study of patients from Belarus. In the sample of patients with hearing loss, the highest (among other similar samples studied in the world) allele frequency of c.313_326del14 mutation (7% of all pathological GJB2 alleles) was registered; Polish origin of this deletion was suggested. It was demonstrated that detection of the GJB2 gene mutation on one patient’s chromosome only is insufficient to confirm a molecular genetic diagnosis of hearing loss of the DFNB1 genetic type (autosomal recessive hearing loss caused by the GJB2 gene mutations). Pilot screening for the GJB2 gene mutations in newborns from Grodno oblast was performed. The material from 235 children was studied during the screening; nine heterozygous carriers of the mutation were found. The c.35delG mutation was detected in a homozygous state in a single newborn (hearing loss of moderate severity was subsequently audiologically confirmed in this child).     

表观遗传学——耳聋研究的新视野

耳聋是一种常见的人类感觉系统缺陷, 新生儿发病率可达1/1000～3/1000。耳蜗感觉神经上皮毛细胞的结构或功能异常可导致耳聋,遗传因素在其中起重要作用。虽然一些与遗传性耳聋相关的基因及染色体位点已经被定位或克隆, 仍有很多耳聋的病因尚不清楚。人们发现, 除了常见的热点基因突变(GJB2、SLC26A4、线粒体DNA C1494T和A1555G等)外, 一些表观遗传学的改变也在耳聋的发生中起重要作用。例如, miR-96 突变会导致人和小鼠的渐进性失聪, 异常的CpG岛甲基化与一些耳聋综合征的发生有关等。文章着重对表观遗传学在耳聋领域的研究现状和进展进行了综述。     

Application of a New Genetic Deafness Microarray for Detecting Mutations in the Deaf in China

Objective

The aim of this study was to evaluate the GoldenGate microarray as a diagnostic tool and to elucidate the contribution of the genes on this array to the development of both nonsyndromic and syndromic sensorineural hearing loss in China.

Methods

We developed a microarray to detect 240 mutations underlying syndromic and nonsyndromic sensorineural hearing loss. The microarray was then used for analysis of 382 patients with nonsyndromic sensorineural hearing loss (including 15 patients with enlarged vestibular aqueduct syndrome), 21 patients with Waardenburg syndrome, and 60 unrelated controls. Subsequently, we analyzed the sensitivity, specificity, and reproducibility of this new approach after Sanger sequencing-based verification, and also determined the contribution of the genes on this array to the development of distinct hearing disorders.

Results

The sensitivity and specificity of the microarray chip were 98.73% and 98.34%, respectively. Genetic defects were identified in 61.26% of the patients with nonsyndromic sensorineural hearing loss, and 9 causative genes were identified. The molecular etiology was confirmed in 19.05% and 46.67% of the patients with Waardenburg syndrome and enlarged vestibular aqueduct syndrome, respectively.

Conclusion

Our new mutation-based microarray comprises an accurate and comprehensive genetic tool for the detection of sensorineural hearing loss. This microarray-based detection method could serve as a first-pass screening (before next-generation-sequencing screening) for deafness-causing mutations in China.     

Contribution of GJB2 Mutations to Hearing Loss in the Hazara Division of Pakistan

Mutations of GJB2, which encodes connexin 26, are the most common cause of hereditary hearing loss in many human populations. This study was initiated to determine the prevalence of GJB2 mutations in individuals with hearing loss from the Hazara Division in Pakistan. We recruited 70 participants with nonsyndromic deafness segregating as an apparently recessive trait and directly sequenced the GJB2 coding region from their DNA. The homozygous mutations c.71 G→A (p.W24X), c.104 T→G (p.I35S), and c.35delG (p.G12VfsX1) were identified as the cause of hearing loss in three participants (4.28%); in populations from other areas of Pakistan, frequencies of 6–7% have been observed. The mutations c.104 T→G and c.35delG were identified in Pakistan for the first time. These results confirm the low prevalence of GJB2 mutations in Hazara and suggest that mutations in other genes may play a significant role in the etiology of deafness in this population.     

Unexpected heterogeneity due to recessive and de novo dominant mutations of GJB2 in an Iranian family with nonsyndromic hearing loss: implication for genetic counseling

Mutations in the GJB2 gene are the most common cause of nonsyndromic autosomal recessive sensorineural hearing loss (HL). A few mutations in GJB2 have also been reported to cause dominant nonsyndromic HL. Here we report a large inbred family including two individuals with nonsyndromic sensorineural hearing loss. A dominant GJB2 mutation, c.551G>A (p.R184Q), was detected in the proband, yet his parents were negative for the mutation. The second affected person had heterozygous c.35delG mutation, which was inherited from his father. Large deletions of the GJB6 gene were not detected in this family. This study highlights the importance of mutation analysis in all affected cases within a pedigree.     

Mutation Spectrum of Common Deafness-Causing Genes in Patients with Non-Syndromic Deafness in the Xiamen Area,China

In China, approximately 30,000 babies are born with hearing impairment each year. However, the molecular factors causing congenital hearing impairment in the Xiamen area of Fujian province have not been evaluated. To provide accurate genetic testing and counseling in the Xiamen area, we investigated the molecular etiology of non-syndromic deafness in a deaf population from Xiamen. Unrelated students with hearing impairment (n = 155) who attended Xiamen Special Education School in Fujian Province were recruited for this study. Three common deafness-related genes, GJB2, SLC26A4, and mtDNA12SrRNA, were analyzed using all-exon sequencing. GJB2 mutations were detected in 27.1% (42/155) of the entire cohort. The non-syndromic hearing loss (NSHL) hotspot mutations c.109G>A (p.V37I) and c.235delC were found in this population, whereas the Caucasian hotspot mutation c.35delG was not. The allelic frequency of the c.109G>A mutation was 9.03% (28/310), slightly higher than that of c.235delC (8.39%, 26/310), which is the most common GJB2 mutation in most areas of China. The allelic frequency of the c.109G>A mutation was significantly higher in this Xiamen’s deaf population than that in previously reported cohorts (P = 0.00). The SLC26A4 mutations were found in 16.77% (26/155) of this cohort. The most common pathogenic allele was c.IVS7-2A>G (6.13%, 19/310), and the second most common was the c.1079C>T (p.A360V) mutation (1.94%, 6/310) which has rarely been reported as a hotspot mutation in other studies. The mutation rate of mtDNA12SrRNA in this group was 3.87% (6/155), all being the m.A1555G mutation. These findings show the specificity of the common deaf gene-mutation spectrum in this area. According to this study, there were specific hotspot mutations in Xiamen deaf patients. Comprehensive sequencing analysis of the three common deaf genes can help portray the mutation spectrum and develop optimal testing strategies for deaf patients in this area.     

Forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in DNA diagnostics?

Hearing impairment is the most common sensory disorder, present in 1 of every 500 newborns. With 46 genes implicated in nonsyndromic hearing loss, it is also an extremely heterogeneous trait. Here, we categorize for the first time all mutations reported in nonsyndromic deafness genes, both worldwide and more specifically in Caucasians. The most frequent genes implicated in autosomal recessive nonsyndromic hearing loss are GJB2, which is responsible for more than half of cases, followed by SLC26A4, MYO15A, OTOF, CDH23 and TMC1. None of the genes associated with autosomal dominant nonsyndromic hearing loss accounts for a preponderance of cases, although mutations are somewhat more frequently reported in WFS1, KCNQ4, COCH and GJB2. Only a minority of these genes is currently included in genetic diagnostics, the selection criteria typically reflecting: (1) high frequency as a cause of deafness (i.e. GJB2); (2) association with another recognisable feature (i.e. SLC26A4 and enlarged vestibular aqueduct); or (3) a recognisable audioprofile (i.e. WFS1). New and powerful DNA sequencing technologies have been developed over the past few years, but have not yet found their way into DNA diagnostics. Implementing these technologies is likely to happen within the next 5 years, and will cause a breakthrough in terms of power and cost efficiency. It will become possible to analyze most - if not all - deafness genes, as opposed to one or a few genes currently. This ability will greatly improve DNA diagnostics, provide epidemiological data on gene-based mutation frequencies, and reveal novel genotype-phenotype correlations.     

Simultaneous screening of multiple mutations by invader assay improves molecular diagnosis of hereditary hearing loss: a multicenter study

Although etiological studies have shown genetic disorders to be a common cause of congenital/early-onset sensorineural hearing loss, there have been no detailed multicenter studies based on genetic testing. In the present report, 264 Japanese patients with bilateral sensorineural hearing loss from 33 ENT departments nationwide participated. For these patients, we first applied the Invader assay for screening 47 known mutations of 13 known deafness genes, followed by direct sequencing as necessary. A total of 78 (29.5%) subjects had at least one deafness gene mutation. Mutations were more frequently found in the patients with congenital or early-onset hearing loss, i.e., in those with an awareness age of 0-6 years, mutations were significantly higher (41.8%) than in patients with an older age of awareness (16.0%). Among the 13 genes, mutations in GJB2 and SLC26A4 were mainly found in congenital or early-onset patients, in contrast with mitochondrial mutations (12S rRNA m.1555A>G, tRNA(Leu(UUR)) m.3243A>G), which were predominantly found in older-onset patients. The present method of simultaneous screening of multiple deafness mutations by Invader assay followed by direct sequencing will enable us to detect deafness mutations in an efficient and practical manner for clinical use.