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Cadherin 23 (CDH23) is one of the most common genes responsible for hereditary hearing loss; a mutation of CDH23 can cause a wide range of symptoms depending on the variant. In this study, an iPSC line was generated from a patient with late-onset, progressive high frequency hearing loss caused by c.[719C > T];[6085C > T]:p.[P240L];[R2029W] compound heterozygous variants of CDH23. The cells were confirmed to have a normal karyotype, express markers of pluripotency, and have tri-embryonic differentiation potential. This disease-specific iPSC line will further the construction of disease models and the elucidation of the pathophysiology of CDH23 mutations.

Abstract To investigate the association between hereditary hearing loss and vestibular function, we compared vestibular function and symptoms among patients with GJB2, SLC26A4, and CDH23 variants. Thirty-nine patients with sensory neural hearing loss (11 males and 28 females) with biallelic pathogenic variants in either GJB2, SLC26A4, or CDH23 were included in this study (13 GJB2, 15 SLC26A4, and 11 CDH23). The patients were examined using caloric testing and cervical and ocular vestibular-evoked myogenic potentials (cVEMP and oVEMP). We also compared vestibular function and symptoms between patients with these gene variants and 78 normal-hearing ears without vestibular symptoms as controls. The frequency of semicircular canal hypofunction in caloric testing was higher in patients with SLC26A4 variants (47%) than in those with GJB2 (0%) and CDH23 variants (27%). According to the cVEMP results, 69% of patients with GJB2 variants had saccular hypofunction, a significantly higher proportion than in those carrying other variants (SLC26A4, 20%; CDH23, 18%). In oVEMP, which reflects utricular function, no difference was observed in the frequency of hypofunction among the three genes (GJB2, 15%; SLC26A4, 40%; and CDH23, 36%). Hence, discernable trends indicate vestibular dysfunction associated with each gene.

Abstract The MYO7A gene is known to be responsible for both syndromic hearing loss (Usher syndrome type1B:USH1B) and non-syndromic hearing loss including autosomal dominant and autosomal recessive inheritance (DFNA11, DFNB2). However, the prevalence and detailed clinical features of MYO7A-associated hearing loss across a large population remain unclear. In this study, we conducted next-generation sequencing analysis for a large cohort of 10,042 Japanese hearing loss patients. As a result, 137 patients were identified with MYO7A-associated hearing loss so that the prevalence among Japanese hearing loss patients was 1.36%. We identified 70 disease-causing candidate variants in this study, with 36 of them being novel variants. All variants identified in autosomal dominant cases were missense or in-frame deletion variants. Among the autosomal recessive cases, all patients had at least one missense variant. On the other hand, in patients with Usher syndrome, almost half of the patients carried biallelic null variants (nonsense, splicing, and frameshift variants). Most of the autosomal dominant cases showed late-onset progressive hearing loss. On the other hand, cases with autosomal recessive inheritance or Usher syndrome showed congenital or early-onset hearing loss. The visual symptoms in the Usher syndrome cases developed between age 5–15, and the condition was diagnosed at about 6–15 years of age.

A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at −80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (B2M, HPRT1, GAPDH and GUSB). The selected reference genes were then used to examine the effect on the expression outcome of target genes (OTOF and TECTA), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of OTOF and TECTA, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies.

Abstract The STRC gene, located on chromosome 15q15.3, is one of the genetic causes of autosomal recessive mild-to-moderate sensorineural hearing loss. One of the unique characteristics of STRC-associated hearing loss is the high prevalence of long deletions or copy number variations observed on chromosome 15q15.3. Further, the deletion of chromosome 15q15.3 from STRC to CATSPER2 is also known to be a genetic cause of deafness infertility syndrome (DIS), which is associated with not only hearing loss but also male infertility, as CATSPER2 plays crucial roles in sperm motility. Thus, information regarding the deletion range for each patient is important to the provision of appropriate genetic counselling for hearing loss and male infertility. In the present study, we performed next-generation sequencing (NGS) analysis for 9956 Japanese hearing loss patients and analyzed copy number variations in the STRC gene based on NGS read depth data. In addition, we performed Multiplex Ligation-dependent Probe Amplification analysis to determine the deletion range including the PPIP5K1, CKMT1B, STRC and CATSPER2 genomic region to estimate the prevalence of the STRC-CATSPER deletion, which is causative for DIS among the STRC-associated hearing loss patients. As a result, we identified 276 cases with STRC-associated hearing loss. The prevalence of STRC-associated hearing loss in Japanese hearing loss patients was 2.77% (276/9956). In addition, 77.1% of cases with STRC homozygous deletions carried a two copy loss of the entire CKMT1B-STRC-CATSPER2 gene region. This information will be useful for the provision of more appropriate genetic counselling regarding hearing loss and male infertility for the patients with a STRC deletion.

Abstract A number of genes are reportedly responsible for hereditary hearing loss, which accounts for over 50% of all congenital hearing loss cases. Recent advances in genetic testing have enabled the identification of pathogenic variants in many cases, and systems have been developed to provide personalized treatment based on etiology. Gene therapy is expected to become an unprecedented curative treatment. Several reports have demonstrated the successful use of cochlear gene therapy to restore auditory function in mouse models of genetic deafness; however, many hurdles remain to its clinical application in humans. Herein, we focus on the frequency of deafness genes in patients with congenital and late‐onset progressive hearing loss and discuss the following points regarding which genes need to be targeted to efficiently proceed with clinical application: (a) which cells' genes are expressed within the cochlea, (b) whether gene transfer to the targeted cells is possible using vectors such as adeno‐associated virus, (c) what phenotype of hearing loss in patients is exhibited, and (d) whether mouse models exist to verify the effectiveness of treatment. Moreover, at the start of clinical application, gene therapy in combination with cochlear implantation may be useful for cases of progressive hearing loss.

While biallelic mutations of the SLC26A4 gene cause non-syndromic hearing loss with enlarged vestibular aqueducts or Pendred syndrome, a considerable number of patients carry mono-allelic mutations. Here the authors identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4.

Abstract More than 400 syndromes associated with hearing loss and other symptoms have been described, corresponding to 30% of cases of hereditary hearing loss. In this study we aimed to clarify the mutation spectrum of syndromic hearing loss patients in Japan by using next-generation sequencing analysis with a multiple syndromic targeted resequencing panel (36 target genes). We analyzed single nucleotide variants, small insertions, deletions and copy number variations in the target genes. We enrolled 140 patients with any of 14 syndromes (BOR syndrome, Waardenburg syndrome, osteogenesis imperfecta, spondyloepiphyseal dysplasia congenita, Stickler syndrome, CHARGE syndrome, Jervell and Lange-Nielsen syndrome, Pendred syndrome, Klippel-Feil syndrome, Alport syndrome, Norrie disease, Treacher-Collins syndrome, Perrault syndrome and auditory neuropathy with optic atrophy) and identified the causative variants in 56% of the patients. This analysis could identify the causative variants in syndromic hearing loss patients in a short time with a high diagnostic rate. In addition, it was useful for the analysis of the cases who only partially fulfilled the diagnostic criteria.

The OTOF gene (Locus: DFNB9), encoding otoferlin, is reported to be one of the major causes of non-syndromic recessive sensorineural hearing loss, and is also reported to be the most common cause of non-syndromic recessive auditory neuropathy spectrum disorder (ANSD). In the present study, we performed OTOF mutation analysis using massively parallel DNA sequencing (MPS). The purpose of this study was to reveal the frequency and precise genetic and clinical background of OTOF-related hearing loss in a large hearing loss population. A total of 2,265 Japanese sensorineural hearing loss (SNHL) patients compatible with autosomal recessive inheritance (including sporadic cases) from 53 otorhinolaryngology departments nationwide participated in this study. The mutation analysis of 68 genes, including the OTOF gene, reported to cause non-syndromic hearing loss was performed using MPS. Thirty-nine out of the 2,265 patients (1.72%) carried homozygous or compound heterozygous mutations in the OTOF gene. It is assumed that the frequency of hearing loss associated with OTOF mutations is about 1.72% of autosomal recessive or sporadic SNHL cases. Hearing level information was available for 32 of 39 patients with biallelic OTOF mutations; 24 of them (75.0%) showed profound hearing loss, 7 (21.9%) showed severe hearing loss and 1 (3.1%) showed mild hearing loss. The hearing level of patients with biallelic OTOF mutations in this study was mostly severe to profound, which is consistent with the results of past reports. Eleven of the 39 patients with biallelic OTOF mutations had been diagnosed with ANSD. The genetic diagnosis of OTOF mutations has significant benefits in terms of clinical decision-making. Patients with OTOF mutations would be good candidates for cochlear implantation; therefore, the detection of OTOF mutations is quite beneficial for patients, especially for those with ANSD.

Abstract DIAPH1 encodes human DIA1, a formin protein that elongates unbranched actin. The c.3634+1G>T DIAPH1 mutation causes autosomal dominant nonsyndromic sensorineural hearing loss, DFNA1, characterized by progressive deafness starting in childhood. The mutation occurs near the C‐terminus of the diaphanous autoregulatory domain (DAD) of DIA1, which interacts with its N‐terminal diaphanous inhibitory domain (DID), and may engender constitutive activation of DIA1. However, the underlying pathogenesis that causes DFNA1 is unclear. We describe a novel patient‐derived DIAPH1 mutation (c.3610C>T) in two unrelated families, which results in early termination prior to a basic amino acid motif (RRKR1204–1207) at the DAD C‐terminus. The mutant DIA1(R1204X) disrupted the autoinhibitory DID‐DAD interaction and was constitutively active. This unscheduled activity caused increased rates of directional actin polymerization movement and induced formation of elongated microvilli. Mice expressing FLAG‐tagged DIA1(R1204X) experienced progressive deafness and hair cell loss at the basal turn and had various morphological abnormalities in stereocilia (short, fused, elongated, sparse). Thus, the basic region of the DAD mediates DIA1 autoinhibition; disruption of the DID‐DAD interaction and consequent activation of DIA1(R1204X) causes DFNA1.

A heterozygous mutation in the Wolfram syndrome type 1 gene (WFS1) causes autosomal dominant nonsyndromic hereditary hearing loss, DFNA6/14/38, or Wolfram-like syndrome. To date, more than 40 different mutations have been reported to be responsible for DFNA6/14/38. In the present study, WFS1 variants were screened in a large series of Japanese hearing loss (HL) patients to clarify the prevalence and clinical characteristics of DFNA6/14/38 and Wolfram-like syndrome. Massively parallel DNA sequencing of 68 target genes was performed in 2,549 unrelated Japanese HL patients to identify genomic variations responsible for HL. The detailed clinical features in patients with WFS1 variants were collected from medical charts and analyzed. We successfully identified 13 WFS1 variants in 19 probands: eight of the 13 variants were previously reported mutations, including three mutations (p.A684V, p.K836N, and p.E864K) known to cause Wolfram-like syndrome, and five were novel mutations. Variants were detected in 15 probands (2.5%) in 602 families with presumably autosomal dominant or mitochondrial HL, and in four probands (0.7%) in 559 sporadic cases; however, no variants were detected in the other 1,388 probands with autosomal recessive or unknown family history. Among the 30 individuals possessing variants, marked variations were observed in the onset of HL as well as in the presence of progressive HL and tinnitus. Vestibular symptoms, which had been rarely reported, were present in 7 out of 30 (23%) of the affected individuals. The most prevalent audiometric configuration was low-frequency type; however, some individuals had high-frequency HL. Haplotype analysis in three mutations (p.A716T, p.K836T, and p.E864K) suggested that the mutations occurred at these mutation hot spots. The present study provided new insights into the audiovestibular phenotypes in patients with WFS1 mutations.

A variant in a transcription factor gene, POU4F3, is responsible for autosomal dominant nonsyndromic hereditary hearing loss, DFNA15. To date, 14 variants, including a whole deletion of POU4F3, have been reported to cause HL in various ethnic groups. In the present study, genetic screening for POU4F3 variants was carried out for a large series of Japanese hearing loss (HL) patients to clarify the prevalence and clinical characteristics of DFNA15 in the Japanese population. Massively parallel DNA sequencing of 68 target candidate genes was utilized in 2,549 unrelated Japanese HL patients (probands) to identify genomic variations responsible for HL. The detailed clinical features in patients with POU4F3 variants were collected from medical charts and analyzed. Novel 12 POU4F3 likely pathogenic variants (six missense variants, three frameshift variants, and three nonsense variants) were successfully identified in 15 probands (2.5%) among 602 families exhibiting autosomal dominant HL, whereas no variants were detected in the other 1,947 probands with autosomal recessive or inheritance pattern unknown HL. To obtain the audiovestibular configuration of the patients harboring POU4F3 variants, we collected audiograms and vestibular symptoms of the probands and their affected family members. Audiovestibular phenotypes in a total of 24 individuals from the 15 families possessing variants were characterized by progressive HL, with a large variation in the onset age and severity with or without vestibular symptoms observed. Pure-tone audiograms indicated the most prevalent configuration as mid-frequency HL type followed by high-frequency HL type, with asymmetry observed in approximately 20% of affected individuals. Analysis of the relationship between age and pure-tone average suggested that individuals with truncating variants showed earlier onset and slower progression of HL than did those with non-truncating variants. The present study showed that variants in POU4F3 were a common cause of autosomal dominant HL.

Tight junctions are cellular junctions that play a major role in the epithelial barrier function. In the inner ear, claudins, occludin, tricellulin, and angulins form the bicellular or tricellular binding of membrane proteins. In these, one type of claudin gene, CLDN14, was reported to be responsible for human hereditary hearing loss, DFNB29. Until now, nine pathogenic variants have been reported, and most phenotypic features remain unclear. In the present study, genetic screening for 68 previously reported deafness causative genes was carried out to identify CLDN14 variants in a large series of Japanese hearing loss patients, and to clarify the prevalence and clinical characteristics of DFNB29 in the Japanese population. One patient had a homozygous novel variant (c.241C>T: p.Arg81Cys) (0.04%: 1/2549). The patient showed progressive bilateral hearing loss, with post-lingual onset. Pure-tone audiograms indicated a high-frequency hearing loss type, and the deterioration gradually spread to other frequencies. The patient showed normal vestibular function. Cochlear implantation improved the patient’s sound field threshold levels, but not speech discrimination scores. This report indicated that claudin-14 is essential for maintaining the inner ear environment and suggested the possible phenotypic expansion of DFNB29. This is the first report of a patient with a tight junction variant receiving a cochlear implantation.

In general, autosomal dominant inherited hearing loss does not have a founder mutation, with the causative mutation different in each family. For this reason, there has been a strong need for efficient diagnosis methods for autosomal dominant sensorineural hearing loss (ADSNHL) patients. This study sought to verify the effectiveness of our analysis algorithm for the screening of ADSNHL patients as well as the usefulness of the massively parallel DNA sequencing (MPS).Seventy-five Japanese ADSNHL patients from 53 ENT departments nationwide participated in this study. We conducted genetic analysis of 75 ADSNHL patients using the Invader assay, TaqMan genotyping assay and MPS-based genetic screening.A total of 46 (61.3%) ADSNHL patients were found to have at least one candidate gene variant.We were able to achieve a high mutation detection rate through the combination of the Invader assay, TaqMan genotyping assay and MPS. MPS could be used to successfully identify mutations in rare deafness genes.

Sensorineural hearing loss is one of the most common neurosensory disorders in humans. The incidence of SNHL is estimated to be 1 in 500-1000 newborns. In more than half of these patients, the hearing loss is associated with genetic causes. In Japan, genetic testing for the patients with SNHL using the Invader assay to screen for 46 mutations in 13 deafness genes was approved by the Ministry of Health, Labour and Welfare for inclusion in social health insurance coverage in 2012. Furthermore, from August 2015, this genetic testing has been expanded to screen for 154 mutations in 19 deafness genes using targeted genomic enrichment with massively parallel DNA sequencing combined with the Invader assay and TaqMan genotyping. For this study we analyzed 717 unrelated Japanese hearing loss patients. The total allele frequency of 154 mutations in 19 deafness genes was 32.64% (468/1434) and the total numbers of cases associated with at least one mutation was 44.07% (316/717). Among these, we were able to diagnose 212 (30%) patients, indicating that the present screening could efficiently identify causative mutations in hearing loss patients. It is noteworthy that 27 patients (3.8%) had coexistent multiple mutations in different genes. Five of these 27 patients (0.7%, 5/717 overall) were diagnosed with genetic hearing loss affected by concomitant with responsible mutations in more than two different genes. For patients identified with multiple mutations in different genes, it is necessary to consider that several genes might have an impact on their phenotypes.

Tonotopy is one of the most fundamental principles of auditory function. While gradients in various morphological and physiological characteristics of the cochlea have been reported, little information is available on gradient patterns of gene expression. In addition, the audiograms in autosomal dominant non syndromic hearing loss can be distinctive, however, the mechanism that accounts for that has not been clarified. We thought that it is possible that tonotopic gradients of gene expression within the cochlea account for the distinct audiograms.We compared expression profiles of genes in the cochlea between the apical, middle, and basal turns of the mouse cochlea by microarray technology and quantitative RT-PCR. Of 24,547 genes, 783 annotated genes expressed more than 2-fold. The most remarkable finding was a gradient of gene expression changes in four genes (Pou4f3, Slc17a8, Tmc1, and Crym) whose mutations cause autosomal dominant deafness. Expression of these genes was greater in the apex than in the base. Interestingly, expression of the Emilin-2 and Tectb genes, which may have crucial roles in the cochlea, was also greater in the apex than in the base.This study provides baseline data of gradient gene expression in the cochlea. Especially for genes whose mutations cause autosomal dominant non syndromic hearing loss (Pou4f3, Slc17a8, Tmc1, and Crym) as well as genes important for cochlear function (Emilin-2 and Tectb), gradual expression changes may help to explain the various pathological conditions.

Usher syndrome is an autosomal recessive disorder manifesting hearing loss, retinitis pigmentosa and vestibular dysfunction, and having three clinical subtypes. Usher syndrome type 1 is the most severe subtype due to its profound hearing loss, lack of vestibular responses, and retinitis pigmentosa that appears in prepuberty. Six of the corresponding genes have been identified, making early diagnosis through DNA testing possible, with many immediate and several long-term advantages for patients and their families. However, the conventional genetic techniques, such as direct sequence analysis, are both time-consuming and expensive. Targeted exon sequencing of selected genes using the massively parallel DNA sequencing technology will potentially enable us to systematically tackle previously intractable monogenic disorders and improve molecular diagnosis. Using this technique combined with direct sequence analysis, we screened 17 unrelated Usher syndrome type 1 patients and detected probable pathogenic variants in the 16 of them (94.1%) who carried at least one mutation. Seven patients had the MYO7A mutation (41.2%), which is the most common type in Japanese. Most of the mutations were detected by only the massively parallel DNA sequencing. We report here four patients, who had probable pathogenic mutations in two different Usher syndrome type 1 genes, and one case of MYO7A/PCDH15 digenic inheritance. This is the first report of Usher syndrome mutation analysis using massively parallel DNA sequencing and the frequency of Usher syndrome type 1 genes in Japanese. Mutation screening using this technique has the power to quickly identify mutations of many causative genes while maintaining cost-benefit performance. In addition, the simultaneous mutation analysis of large numbers of genes is useful for detecting mutations in different genes that are possibly disease modifiers or of digenic inheritance.

A cochlear implant is an indispensable apparatus for a profound hearing loss patient. But insertion of the electrode entails a great deal of stress to the cochlea, and may cause irreversible damage to hair cells and related nerve structure. Although damage prevention effects of dexamethasone have been reported, long-term administration is difficult. In this study, we used a dexamethasone-eluting electrode in the guinea pig cochlea, and compared the gene expression after 7 days insertion with that of a normal electrode and non-surgically treated control by microarray. 40 genes were up-regulated 2-fold or more in the normal electrode group compared to the non-surgically treated group. Most of the up-regulated genes were associated with immune response and inflammation. In the dexamethasone-eluting group, compared to the normal electrode group, 7 of the 40 genes were further up-regulated, while 12 of them were down-regulated and there was a tendency to return to the non-surgical condition. 9 genes were down-regulated 2-fold or less with normal electrode insertion, and 4 of the 9 tended to return to the non-surgical condition in the dexamethasone-eluting group. These genes are certainly involved in the maintenance of the physiological functions of the cochlea. Our results indicate that the dexamethasone-eluting electrode will have an effect on the normalization of homeostasis in the cochlea.

Target exon resequencing using Massively Parallel DNA Sequencing (MPS) is a new powerful strategy to discover causative genes in rare Mendelian disorders such as deafness. We attempted to identify genomic variations responsible for deafness by massive sequencing of the exons of 112 target candidate genes. By the analysis of 216randomly selected Japanese deafness patients (120 early-onset and 96 late-detected), who had already been evaluated for common genes/mutations by Invader assay and of which 48 had already been diagnosed, we efficiently identified causative mutations and/or mutation candidates in 57 genes. Approximately 86.6% (187/216) of the patients had at least one mutation. Of the 187 patients, in 69 the etiology of the hearing loss was completely explained. To determine which genes have the greatest impact on deafness etiology, the number of mutations was counted, showing that those in GJB2 were exceptionally higher, followed by mutations in SLC26A4, USH2A, GPR98, MYO15A, COL4A5 and CDH23. The present data suggested that targeted exon sequencing of selected genes using the MPS technology followed by the appropriate filtering algorithm will be able to identify rare responsible genes including new candidate genes for individual patients with deafness, and improve molecular diagnosis. In addition, using a large number of patients, the present study clarified the molecular epidemiology of deafness in Japanese. GJB2 is the most prevalent causative gene, and the major (commonly found) gene mutations cause 30-40% of deafness while the remainder of hearing loss is the result of various rare genes/mutations that have been difficult to diagnose by the conventional one-by-one approach. In conclusion, target exon resequencing using MPS technology is a suitable method to discover common and rare causative genes for a highly heterogeneous monogenic disease like hearing loss.

Genetic factors, the most common etiology in severe to profound hearing loss, are one of the key determinants of Cochlear Implantation (CI) and Electric Acoustic Stimulation (EAS) outcomes. Satisfactory auditory performance after receiving a CI/EAS in patients with certain deafness gene mutations indicates that genetic testing would be helpful in predicting CI/EAS outcomes and deciding treatment choices. However, because of the extreme genetic heterogeneity of deafness, clinical application of genetic information still entails difficulties. Target exon sequencing using massively parallel DNA sequencing is a new powerful strategy to discover rare causative genes in Mendelian disorders such as deafness. We used massive sequencing of the exons of 58 target candidate genes to analyze 8 (4 early-onset, 4 late-onset) Japanese CI/EAS patients, who did not have mutations in commonly found genes including GJB2, SLC26A4, or mitochondrial 1555A>G or 3243A>G mutations. We successfully identified four rare causative mutations in the MYO15A, TECTA, TMPRSS3, and ACTG1 genes in four patients who showed relatively good auditory performance with CI including EAS, suggesting that genetic testing may be able to predict the performance after implantation.

The present study of KCNQ4 mutations was carried out to 1) determine the prevalence by unbiased population-based genetic screening, 2) clarify the mutation spectrum and genotype/phenotype correlations, and 3) summarize clinical characteristics. In addition, a review of the reported mutations was performed for better understanding of this deafness gene. The screening using 287 probands from unbiased Japanese autosomal dominant nonsyndromic hearing loss (ADNSHL) families identified 19 families with 7 different disease causing mutations, indicating that the frequency is 6.62% (19/287). While the majority were private mutations, one particular recurrent mutation, c.211delC, was observed in 13 unrelated families. Haplotype analysis in the vicinity of c.211delC suggests existence of a common ancestor. The majority of the patients showed all frequency, but high-frequency predominant, sensorineural hearing loss. The present study adds a new typical audiogram configuration characterized by mid-frequency predominant hearing loss caused by the p.V230E mutation. A variant at the N-terminal site (c. 211delC) showed typical ski-slope type audiogram configuration. Concerning clinical features, onset age was from 3 to 40 years old, and mostly in the teens, and hearing loss was gradually progressive. Progressive nature is a common feature of patients with KCNQ4 mutations regardless of the mutation type. In conclusion, KCNQ4 mutations are frequent among ADNSHL patients, and therefore screening of the gene and molecular confirmation of these mutations have become important in the diagnosis of these conditions.

Screening for gene mutations in CDH23, which has many exons, has lagged even though it is likely to be an important cause for hearing loss patients. To assess the importance of CDH23 mutations in non-syndromic hearing loss, two-step screening was applied and clinical characteristics of the patients with CDH23 mutations were examined in this study. As a first screening, we performed Sanger sequencing using 304 probands compatible with recessive inheritance to find the pathologic mutations. Twenty-six possible mutations were detected to be pathologic in the first screening. For the second screening, using the probes for these 26 mutations, a large cohort of probands (n = 1396) was screened using Taqman amplification-based mutation analysis followed by Sanger sequencing. The hearing loss in a total of 52 families (10 homozygous, 13 compound heterogygous, and 29 heterozygous) was found to be caused by the CDH23 mutations. The majority of the patients showed congenital, high frequency involved, progressive hearing loss. Interestingly, some particular mutations cause late onset moderate hearing loss. The present study is the first to demonstrate the prevalence of CDH23 mutations among non-syndromic hearing loss patients and indicated that mutations of the CDH23 gene are an important cause of non-syndromic hearing loss.

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.