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The genetic heterogeneity of sensorineural hearing loss (SNHL) is a major hurdle to the detection of disease-causing variants. We aimed to identify underlying causal genes associated with mid-frequency hearing loss (HL), which contributes to less than about 1% of SNHL cases, by whole exome sequencing (WES). Thirty families segregating mid-frequency SNHL, in whom biallelic GJB2 mutations had been previously excluded, were selected from among 851 families in our DNA repository of SNHL. DNA samples from the probands were subjected to WES analysis and searched for candidate variants associated with SNHL. We were able to identify the genetic aetiology in six probands (20%). In total, we found three pathogenic and three likely pathogenic variants in four genes COL4A5, OTOGL, TECTA, TMPRSS3). One proband was a compound heterozygote for a pathogenic variant and a variant of uncertain significance (VUS) in MYO15A gene. To date, MYO15A and TMPRSS3 have not yet been described in association with mid-frequency SNHL. In eight additional probands, eight candidate VUS variants were detected in five genes (DIAPH1, MYO7A, TECTA, TMC1, TSPEAR). Seven of these 16 variants have not yet been published or mentioned in the available databases. The most prevalent gene was TECTA, identified in 23% of all tested families. Furthermore, we confirmed the hypothesis that a substantive portion of cases with this conspicuous audiogram shape is a consequence of a genetic disorder.

Recessive variants in GJB2 are the most common genetic cause of sensorineural hearing impairment. However, in many patients, only one variant in the GJB2 coding region is identified using conventional sequencing strategy (eg, Sanger sequencing), resulting in nonconfirmative diagnosis. Conceivably, there might be other unidentified pathogenic variants in the noncoding region of GJB2 or other deafness-causing genes in these patients. To address this, a next-generation sequencing-based diagnostic panel targeting the entire GJB2 gene and the coding regions of 158 other known deafness-causing genes was designed and applied to 95 patients with nonsyndromic sensorineural hearing impairment (including 81 Han Taiwanese and 14 Mongolian patients) in whom only a single GJB2 variant had been detected using conventional Sanger sequencing. The panel confirmed the genetic diagnosis in 24 patients (25.3%). Twenty-two of them had causative variants in several deafness-causing genes other than GJB2, including MYO15A, MYO7A, TECTA, POU4F3, KCNQ4, SLC26A4, OTOF, MT-RNR1, MITF, WFS1, and USH2A. The other two patients had causative variants in GJB2, including a Taiwanese patient with a mosaic maternal uniparental disomy c.235delC variant (approximately 69% mosaicism) and a Mongolian patient with compound heterozygous c.35dupG and c.35delG variants, which occurred at the same site. This study demonstrates the utility of next-generation sequencing in clarifying the genetic diagnosis of hearing-impaired patients with nonconfirmative GJB2 genotypes on conventional genetic examinations.

Background. Approximately 70% of congenital deafness is attributable to genetic causes. Incidence of congenital deafness is known to be higher in families with consanguineous marriage. In this study, we investigated the genetic causes in three consanguineous Pakistani families segregating with prelingual, severe-to-profound deafness. Results. Through targeted next-generation sequencing of 414 genes known to be associated with deafness, homozygous variants c.536del (p. Leu180Serfs ∗ 20) in TECTA, c.3719 G>A (p. Arg1240Gln) in MYO7A, and c.482+1986_1988del in HGF were identified as the pathogenic causes of enrolled families. Interestingly, in one large consanguineous family, an additional c.706G>A (p. Glu236Lys) variant in the X-linked POU3F4 gene was also identified in multiple affected family members causing deafness. Genotype-phenotype cosegregation was confirmed in all participating family members by Sanger sequencing. Conclusions. Our results showed that the genetic causes of deafness are highly heterogeneous. Even within a single family, the affected members with apparently indistinguishable clinical phenotypes may have different pathogenic variants.

Key points The aim was to determine whether detachment of the tectorial membrane (TM) from the organ of Corti in Tecta/Tectb-/- mice affects the biophysical properties of cochlear outer hair cells (OHCs). Tecta/Tectb-/- mice have highly elevated hearing thresholds, but OHCs mature normally. Mechanoelectrical transducer (MET) channel resting open probability (Po ) in mature OHC is ∼50% in endolymphatic [Ca2+ ], resulting in a large standing depolarizing MET current that would allow OHCs to act optimally as electromotile cochlear amplifiers. MET channel resting Po in vivo is also high in Tecta/Tectb-/- mice, indicating that the TM is unlikely to statically bias the hair bundles of OHCs. Distortion product otoacoustic emissions (DPOAEs), a readout of active, MET-dependent, non-linear cochlear amplification in OHCs, fail to exhibit long-lasting adaptation to repetitive stimulation in Tecta/Tectb-/- mice. We conclude that during prolonged, sound-induced stimulation of the cochlea the TM may determine the extracellular Ca2+ concentration near the OHC's MET channels. Abstract The tectorial membrane (TM) is an acellular structure of the cochlea that is attached to the stereociliary bundles of the outer hair cells (OHCs), electromotile cells that amplify motion of the cochlear partition and sharpen its frequency selectivity. Although the TM is essential for hearing, its role is still not fully understood. In Tecta/Tectb-/- double knockout mice, in which the TM is not coupled to the OHC stereocilia, hearing sensitivity is considerably reduced compared with that of wild-type animals. In vivo, the OHC receptor potentials, assessed using cochlear microphonics, are symmetrical in both wild-type and Tecta/Tectb-/- mice, indicating that the TM does not bias the hair bundle resting position. The functional maturation of hair cells is also unaffected in Tecta/Tectb-/- mice, and the resting open probability of the mechanoelectrical transducer (MET) channel reaches values of ∼50% when the hair bundles of mature OHCs are bathed in an endolymphatic-like Ca2+ concentration (40 μM) in vitro. The resultant large MET current depolarizes OHCs to near -40 mV, a value that would allow optimal activation of the motor protein prestin and normal cochlear amplification. Although the set point of the OHC receptor potential transfer function in vivo may therefore be determined primarily by endolymphatic Ca2+ concentration, repetitive acoustic stimulation fails to produce adaptation of MET-dependent otoacoustic emissions in vivo in the Tecta/Tectb-/- mice. Therefore, the TM is likely to contribute to the regulation of Ca2+ levels around the stereocilia, and thus adaptation of the OHC MET channel during prolonged sound stimulation.

Autosomal dominant non-syndromic hearing loss (ADNSHL) is genetically and clinically heterogeneous. To date, 48 causative genes have been identified. Of these genes, TECTA, WFS1 or KCNQ4 mutations are the most frequently identified in subjects with ADNSHL in Japan. We performed mutation analysis of TECTA, WFS1 and KCNQ4 in a Japanese family with ADNSHL, and identified a heterozygous missense mutation in TECTA. The missense mutation was located in the zonadhesin domain in the alpha-tectorin protein encoded by the gene. The affected family members, including 6-year-old girl, 59-year-old grandmother and 33-year-old father, showed bilateral moderate to severe sensorineural hearing loss. Their audiogram patterns were of the symmetric and descending type. The average annual threshold deterioration of the girl was 0.5 dB/year and that of her father was 0.5 dB/year, indicating that their hearing loss was stable. Their auditory findings were consistent with the established genotype-phenotype correlation of missense mutations in TECTA: missense mutations in the zonadhesin domain cause high-frequency hearing loss; when amino acid residues other than cysteine are affected, the hearing loss is stable., 浜松医科大学医学部耳鼻咽喉科・頭頸部外科学教室 峯田周幸教授退任記念論文集～症例から学ぶ～, Festschrift for Professor Hiroyuki Mineta In Hornor of His Retirement as Chairman of Hamamatsu University School of Medicine

Amphibolidae is one of the most abundant families of gastropods in estuarine environments of south-eastern Australia. However, the range limits of the species of Salinator, one of the family’s two genera in the region, remain unclear partly due to uncertainty of identifications based solely on shell morphology. Insufficient data have been collected to address questions regarding the genetic variability of any of the species of Salinator. Here, DNA sequences from a segment of the cytochrome c oxidase subunit I and 28S ribosomal RNA genes were collected to investigate the distribution and variation of the three Salinator species in the region, these being S. fragilis, S. rhamphidia and S. tecta. The results demonstrate a large range extension in S. rhamphidia and suggest that S. tecta may have limited distribution in Tasmania. In contrast to previously-studied estuarine Mollusca in the south-eastern coasts of the mainland and Tasmania, S. rhamphidia has regional differentiation. There is evidence of genetic disequilibrium within S. fragilis, suggesting that it may presently comprise contributions from two distinct sets of populations.

Non-syndromic autosomal recessive hearing loss is an extremely heterogeneous disease caused by mutations in more than 80 genes. We examined Czech patients with early/prelingual non-syndromic, presumably genetic hearing loss (NSHL) without known cause after GJB2 gene testing. Four hundred and twenty-one unrelated patients were examined for STRC gene deletions with quantitative comparative fluorescent PCR (QCF PCR), 197 unrelated patients with next-generation sequencing by custom-designed NSHL gene panels and 19 patients with whole-exome sequencing (WES). Combining all methods, we discovered the cause of the disease in 54 patients. The most frequent type of NSHL was DFNB16 (STRC), which was detected in 22 patients, almost half of the clarified patients. Other biallelic pathogenic mutations were detected in the genes: MYO15A, LOXHD1, TMPRSS3 (each gene was responsible for five clarified patients, CDH23 (four clarified patients), OTOG and OTOF (each gene was responsible for two clarified patients). Other genes (AIFM1, CABP2, DIAPH1, PTPRQ, RDX, SLC26A4, TBC1D24, TECTA, TMC1) that explained the cause of hearing impairment were further detected in only one patient for each gene. STRC gene mutations, mainly deletions remain the most frequent NSHL cause after mutations in the GJB2.

Background Hearing loss is the most common sensory defect, and it affects over 6% of the population worldwide. Approximately 50–60% of hearing loss patients are attributed to genetic causes. Currently, more than 100 genes have been reported to cause non-syndromic hearing loss. It is possible and efficient to screen all potential disease-causing genes for hereditary hearing loss by whole exome sequencing (WES). Methods We collected 5 consanguineous pedigrees from Pakistan with hearing loss and applied WES in selected patients for each pedigree, followed by bioinformatics analysis and Sanger validation to identify the causal genes. Results Variants in 7 genes were identified and validated in these pedigrees. We identified single candidate variant for 3 pedigrees: GIPC3 (c.937 T > C), LOXHD1 (c.6136G > A) and TMPRSS3 (c.941 T > C). The remaining 2 pedigrees each contained two candidate variants: TECTA (c.4045G > A) and MYO15A (c.3310G > T and c.9913G > C) for one pedigree and DFNB59 (c.494G > A) and TRIOBP (c.1952C > T) for the other pedigree. The candidate variants were validated in all available samples by Sanger sequencing. Conclusion The candidate variants in hearing-loss genes were validated to be co-segregated in the pedigrees, and they may indicate the aetiologies of hearing loss in such patients. We also suggest that WES may be a suitable strategy for hearing-loss gene screening in clinical detection.

Changing consumption patterns and increasing health awareness, especially in Europe, are resulting in an increased demand for sesame seeds. In 2016, Asia imported the highest quantity of sesame seeds, followed by Europe and North America. We examined, for the first time, the effects of treatment with sesame oil and sesamin in hearing impairment models. Sesame oil exhibited an ameliorative effect on auditory impairment in a hair cell line in zebrafish and mice. In ototoxic zebrafish larvae, neuromasts and otic cells increased in numbers because of sesame oil. Furthermore, auditory function in noise-induced hearing loss (NIHL) was studied through auditory brainstem response to evaluate the therapeutic effects of sesame oil. Sesame oil reduced the hearing threshold shift in response to clicks and 8, 16-kHz tone bursts in NIHL mice. Auditory-protective effect of sesame oil was seen in zebrafish and mice; therefore, we used chromatographic analysis to study sesamin, which is the major effective factor in sesame oil. To investigate its effects related to auditory function, we studied the hearing-related gene, Tecta, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) assay. Auditory cell proliferation was induced by treatment with sesame oil and sesamin using Tecta (Tectorin Alpha) regulation. The expression of Tecta increases in the apex area of the cochlear hair cells as they grow, and their activity is enhanced by sesame oil and sesamin. These results provide a novel mechanistic insight into the sesame oil activities and suggest that sesamin, the key constituent in sesame oil, is responsible for its auditory function related benefits, including protection of auditory cells and reversal of their impairments.

Objectives Hereditary hearing loss exhibits high degrees of genetic and clinical heterogeneity. To elucidate the population-specific and age-related genetic and clinical spectra of hereditary hearing loss, we investigated the sequencing data of causally associated hearing loss genes in a large cohort of hearing-impaired probands with a balanced age distribution from a single center in Southwest Germany. Design Genetic testing was applied to 305 hearing-impaired probands/families with a suspected genetic hearing loss etiology and a balanced age distribution over a period of 8 years (2011-2018). These individuals were representative of the regional population according to age and sex distributions. The genetic testing workflow consisted of single-gene screening (n = 21) and custom-designed hearing loss gene panel sequencing (n = 284) targeting known nonsyndromic and syndromic hearing loss genes in a diagnostic setup. Retrospective reanalysis of sequencing data was conducted by applying the current American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines. Results A genetic diagnosis was established for 75 (25%) of the probands that involved 75 causal variants in 35 genes, including 16 novel causal variants and 9 medically significant variant reclassifications. Nearly half of the solved cases (47%; n = 35) were related to variants in the five most frequently affected genes: GJB2 (25%), MYO15A, WFS1, SLC26A4, and COL11A1 (all 5%). Nearly one-quarter of the cases (23%; n = 17) were associated with variants in seven additional genes (TMPRSS3, COL4A3, LOXHD1, EDNRB, MYO6, TECTA, and USH2A). The remaining one-third of single cases (33%; n = 25) were linked to variants in 25 distinct genes. Diagnostic rates and gene distribution were highly dependent on phenotypic characteristics. A positive family history of autosomal-recessive inheritance in combination with early onset and higher grades of hearing loss significantly increased the solve rate up to 60%, while late onset and lower grades of hearing loss yielded significantly fewer diagnoses. Regarding genetic diagnoses, autosomal-dominant genes accounted for 37%, autosomal-recessive genes for 60%, and X-linked genes for 3% of the solved cases. Syndromic/nonsyndromic hearing loss mimic genes were affected in 27% of the genetic diagnoses. Conclusions The genetic epidemiology of the largest German cohort subjected to comprehensive targeted sequencing for hereditary hearing loss to date revealed broad causal gene and variant spectra in this population. Targeted hearing loss gene panel analysis proved to be an effective tool for ensuring an appropriate diagnostic yield in a routine clinical setting including the identification of novel variants and medically significant reclassifications. Solve rates were highly sensitive to phenotypic characteristics. The unique population-adapted and balanced age distribution of the cohort favoring late hearing loss onset uncovered a markedly large contribution of autosomal-dominant genes to the diagnoses which may be a representative for other age balanced cohorts in other populations.

Cochlear function depends on the operation of a coupled feedback loop, incorporating outer hair cells (OHCs), and structured to assure that inner hair cells (IHCs) convey frequency specific acoustic information to the brain, even at very low sound levels. Although our knowledge of OHC function and its contribution to cochlear amplification has expanded, the importance of the tectorial membrane (TM) to the processing of mechanical inputs has not been fully elucidated. In addition, there are a surprising number of genetic mutations that affect TM structure and that produce hearing loss in humans. By synthesizing old and new results obtained on several mouse mutants, we learned that animals with abnormal TMs are prone to generate spontaneous otoacoustic emissions (SOAE), which are uncommon in most wildtype laboratory animals. Because SOAEs are not produced in TM mutants or in humans when threshold shifts exceed approximately 25 dB, some degree of cochlear amplification is required. However, amplification by itself is not sufficient because normal mice are rarely spontaneous emitters. Since SOAEs reflect active cochlear operation, TM mutants are valuable for studying the oscillatory nature of the amplification process and the structures associated with its stabilization. Inasmuch as the mouse models were selected to mirror human auditory disorders, using SOAEs as a noninvasive clinical tool may assist the classification of individuals with genetic defects that influence the active mechanisms responsible for sensitivity and frequency selectivity, the hallmarks of mammalian hearing.

Studies of genetic disorders of sensorineural hearing loss have been instrumental in delineating mechanisms that underlie the remarkable sensitivity and selectivity that are hallmarks of mammalian hearing. For example, genetic modifications of TECTA and TECTB, which are principal proteins that comprise the tectorial membrane (TM), have been shown to alter auditory thresholds and frequency tuning in ways that can be understood in terms of changes in the mechanical properties of the TM. Here we investigate effects of genetic modification targeting CEACAM16, a third important TM protein. Loss of CEACAM16 has been recently shown to lead to progressive reductions in sensitivity. While age-related hearing losses have previously been linked to changes in sensory receptor cells, the role of the TM in progressive hearing loss is largely unknown. Here, we show that TM stiffness and viscosity are significantly reduced in adult mice that lack functional CEACAM16 relative to age-matched wild-type controls. By contrast, these same mechanical properties of TMs from juvenile mice that lack functional CEACAM16 are more similar to those of wild-type mice. Thus changes in hearing phenotype align with changes in TM material properties and can be understood in terms of the same TM wave properties that were previously used to characterize modifications of TECTA and TECTB. These results demonstrate that CEACAM16 is essential for maintaining TM mechanical and wave properties, which in turn, are necessary for sustaining the remarkable sensitivity and selectivity of mammalian hearing with increasing age.

Hearing loss (HL) is one of the most frequent birth defects, and genetic factors contribute to the pathogenesis of the disorder in about half of the patients. In the present study, we performed whole-exome sequencing (WES) based on Next-generation sequencing (NGS) in an Iranian family with hereditary HL. Then, Sanger sequencing was used to verify the segregation of the variant recognized in affected family members. A novel homozygous frameshift variation, c.649-650insC, in TECTA was found in the family, which might lead to a truncated TECTA protein (p. Asn218Gln fsX31). Our findings propose that the homozygous TECTA-p.N218QfsX31 mutation is the pathogenic variant for ARNSHL. To the best of our knowledge, this mutation has not been described in patients with the HL phenotype and so far has not to be reported in any of the mutation databases. Our data expand the spectrum of mutations in the TECTA gene in nonsyndromic hearing loss.

Vertigo is the leading symptom of vestibular disorders and a major risk factor for falls. In a genome-wide association study of vertigo (Ncases = 48,072, Ncontrols = 894,541), we uncovered an association with six common sequence variants in individuals of European ancestry, including missense variants in ZNF91, OTOG, OTOGL, and TECTA, and a cis-eQTL for ARMC9. The association of variants in ZNF91, OTOGL, and OTOP1 was driven by an association with benign paroxysmal positional vertigo. Using previous reports of sequence variants associating with age-related hearing impairment and motion sickness, we found eight additional variants that associate with vertigo. Although disorders of the auditory and the vestibular system may co-occur, none of the six genome-wide significant vertigo variants were associated with hearing loss and only one was associated with age-related hearing impairment. Our results uncovered sequence variants associating with vertigo in a genome-wide association study and implicated genes with known roles in inner ear development, maintenance, and disease., Astros Skuladottir et al. conducted a genome-wide association study on 48,072 vertigo cases and 894,541 controls from four populations with European ancestries. They identified six common variants associated with vertigo, thereby providing further insight into the etiology of vestibular disorders.

The global standing-wave model for generation of spontaneous otoacoustic emissions (SOAEs) suggests that they are amplitude-stabilized standing waves and that the spacing between SOAEs corresponds to the interval over which the phase changes by one cycle as determined from the phase-gradient delays of stimulus frequency otoacoustic emissions (SFOAEs). Because data characterizing the relationship between spontaneous and evoked emissions in nonhuman mammals are limited, we examined SOAEs and SFOAEs in tectorial membrane (TM) mutants and their controls. Computations indicate that the spacing between adjacent SOAEs is predicted by the SFOAE phase-gradient delays for TM mutants lacking Ceacam16, where SOAE frequencies are greater than ~20 kHz and the mutants retain near-normal hearing when young. Mice with a missense mutation in Tecta (TectaY1870C/+), as well as mice lacking Otoancorin (Otoa−/−), were also examined. Although these mutants exhibit hearing loss, they generate SOAEs with average frequencies of 11 kHz in TectaY1870C/+ and 6 kHz in Otoa−/−. In these animals, the spacing between adjacent SOAEs is larger than predicted by the SFOAE phase delays. It is also demonstrated that mice do not exhibit the strong frequency-dependence in signal coding that characterizes species with good low-frequency hearing. In fact, a transition occurs near the apical end of the mouse cochlea rather than at the mid-point along the cochlear partition. Hence, disagreements with the standing-wave model are not easily explained by a transition in tuning ratios between apical and basal regions of the cochlea, especially for SOAEs generated in TectaY1870C/+mice.

Background The extremely high genetic heterogeneity of hearing loss due to diverse group of genes encoding proteins required for development, function, and maintenance of the complex auditory system makes the genetic diagnosis of this disease challenging. Up to now, 121 different genes have been identified for nonsyndromic hearing loss (NSHL), of which 76 genes are responsible for the most common forms of NSHL, autosomal recessive nonsyndromic hearing loss (ARNSHL). Methods After excluding mutations in the most common ARNSHL gene, GJB2, by Sanger sequencing, genetic screening for a panel of genes responsible for hereditary hearing impairment performed in 9 individuals with ARNSHL from unrelated Iranian consanguineous pedigrees. Results One compound heterozygote and eight homozygote variants, of which five are novel, were identified: CDH23:p.(Glu1970Lys), and p.(Ala1072Asp), GIPC3:p.(Asn82Ser), and (p.Thr41Lys), MYO7A:p.[Phe456Phe]; p.[Met708Val], and p.(Gly163Arg), TECTA:p.(Leu17Leufs*19), OTOF:c.1392+1G>A, and TRIOBP:p.(Arg1068*). Sanger sequencing confirmed the segregation of the variants with the disease in each family. Conclusion Finding more variants and expanding the spectrum of hearing impairment mutations can increase the diagnostic value of molecular testing in the screening of patients and can improve counseling to minimize the risk of having affected children for at risk couples., In the present study, nine unrelated Iranian consanguineous families with at least one affected individual were tested by next‐generation sequencing (NGS) of 127 known deafness genes. In this report, one compound heterozygote and eight homozygote variants, of which five are novel, were identified: CDH23:p.(Glu1970Lys), and p.(Ala1072Asp), GIPC3:p.(Asn82Ser), and (p.Thr41Lys), MYO7A:p.[Phe456Phe]; p.[Met708Val], and p.(Gly163Arg),TECTA:p.(Leu17Leufs*19), OTOF:c.1392+1G>A, and TRIOBP:p.(Arg1068*). Sanger sequencing confirmed the segregation of the variants with the disease in each family.

Purpose Usher syndrome (USH) is a rare disorder characterized by retinitis pigmentosa (RP) and sensorineural hearing loss. Several genes are responsible for the disease, but not all cases are explained by mutations in any of these, supporting the fact that there remain other unknown genes that have a role in the syndrome. We aimed to find the genetic cause of presumed USH patients lacking pathogenic mutations in the known USH genes. Methods Whole exome sequencing was performed on a priori USH-diagnosed subjects from nine unrelated families, which had shown negative results for an USH-targeted panel in a previous study. Results We identified possible pathogenic variants in six of the studied families. One patient harbored mutations in REEP6 and TECTA, each gene tentatively causative of one of the two main symptoms of the disease, mimicking the syndrome. In three patients, only the retinal degeneration causative mutations were detected (involving EYS, WDR19, and CNGB1 genes). Another family manifested a dementia-linked retinal dystrophy dependent on an allele dosage in the GRN gene. Last, another case presented a homozygous mutation in ASIC5, a gene not yet associated with USH. Conclusions Our findings demonstrate that pending cases should be clinically and genetically carefully assessed, since more patients than expected may be either related phenocopies or affected by a more complex disease encompassing additional symptoms rather than classical USH.

Background To date, 102 genes have been reported as responsible for non-syndromic hearing loss, some of which are associated with specific audiogram features. Four genes have been reported as causative for mid-frequency sensorineural hearing loss (MFSNHL), among which TECTA is the most frequently reported; however, the prevalence of TECTA mutations is unknown. To elucidate the prevalence of TECTA mutation in MFSNHL and clarify genotype-phenotype correlations, we analyzed the genetic and clinical features of patients with MFSNHL. Methods Subjects with bilateral non-syndromic hearing loss were prescreened for GJB2 and m.1555A > G and m.3243A > G mitochondrial DNA mutations, and patients with inner ear malformations were excluded. We selected MFSNHL patients whose audiograms met the U-shaped criterion proposed by the GENDEAF study group, along with those with shallow U-shaped audiograms, for TECTA analysis. All TECTA exons were analyzed by Sanger sequencing. Novel missense variants were classified as possibly pathogenic, non-pathogenic, and variants of uncertain significance, based on genetic data. To evaluate novel possibly pathogenic variants, we predicted changes in protein structure by molecular modeling. Results Pathogenic and possibly pathogenic variants of TECTA were found in 4 (6.0%) of 67 patients with MFSNHL. In patients with U-shaped audiograms, none (0%) of 21 had pathogenic or possibly pathogenic variants. In patients with shallow U-shaped audiograms, four (8.7%) of 46 had pathogenic or possibly pathogenic variants. Two novel possibly pathogenic variants were identified and two previously reported mutations were considered as variant of unknown significance. The clinical features of patients with pathogenic and possibly pathogenic variants were consistent with those in previous studies. Pathogenic or possibly pathogenic variants were identified in 3 of 23 families (13.0%) which have the family histories compatible with autosomal dominant and 1 of 44 families (2.3%) which have the family histories compatible with sporadic or autosomal recessive. Conclusions TECTA mutations were identified in 6.0% of MFSNHL. These mutations were more frequent in patients with shallow U-shaped audiograms than those with U-shaped audiograms, and in families which have the family histories compatible with autosomal dominant than those with the family histories compatible with sporadic or autosomal recessive. Electronic supplementary material The online version of this article (10.1186/s13023-017-0708-z) contains supplementary material, which is available to authorized users.

We present detailed comparative analyses to assess population-level differences in patterns of genetic deafness between European/American and Japanese cohorts with non-syndromic hearing loss. One thousand eighty-three audiometric test results (921 European/American and 162 Japanese) from members of 168 families (48 European/American and 120 Japanese) with non-syndromic hearing loss secondary to pathogenic variants in one of three genes (KCNQ4, TECTA, WFS1) were studied. Audioprofile characteristics, specific mutation types, and protein domains were considered in the comparative analyses. Our findings support differences in audioprofiles driven by both mutation type (non-truncating vs. truncating) and ethnic background. The former finding confirms data that ascribe a phenotypic consequence to different mutation types in KCNQ4; the latter finding suggests that there are ethnic-specific effects (genetic and/or environmental) that impact gene-specific audioprofiles for TECTA and WFS1. Identifying the drivers of ethnic differences will refine our understanding of phenotype-genotype relationships and the biology of hearing and deafness.

TECTA is well known as a causative gene for autosomal dominant mid-frequency hearing loss observed in various populations. In this study, we performed next-generation sequencing analysis of a large Japanese hearing loss cohort, including eight hundred and twelve (812) subjects from unrelated autosomal dominant hearing loss families, to estimate the prevalence and phenotype-genotype correlations in patients with TECTA mutations. The prevalence of TECTA mutations in Japanese autosomal dominant sensorineural hearing loss families was found to be 3.2%. With regard to the type of hearing loss, the patients with mutations in the nidogen-like domain or ZA domain of TECTA showed varied audiograms. However, most of the patients with mutations in the ZP domain showed mid-frequency hearing loss. The rate of hearing deterioration in TECTA-associated hearing loss patients and in the normal hearing Japanese control population were the same and regression lines for each group were parallel. We carried out haplotype analysis for four families which had one recurring missense variant, c.5597C&gt, T (p.Thr1866Met). Our results revealed four different haplotypes, suggesting that this mutation occurred independently in each family. In conclusion, TECTA variants represent the second largest cause of autosomal dominant sensorineural hearing loss in Japan. The hearing loss progression observed in the patients with TECTA mutations might reflect presbycusis. The c.5597C&gt, T mutation occurred in a mutational hot spot and is observed in many ethnic populations.

Inzwischen sind über 150 Gene im Zusammenhang mit hereditärer Schwerhörigkeit beschrieben. Die Ausprägung des Phänotyps genetischer Schwerhörigkeit (Hörvermögen, Beginn, Progredienz, assoziierte Symptome) ist in Abhängigkeit vom Genotyp sehr heterogen. Auch die Sprachverständlichkeit mit Cochlea Implantat (CI) ist für die einzelnen Gene unterschiedlich. Ob die Expressionsorte der Gene, wie zum Beispiel das Spiralganglion und der Hörnerv, einen Einfluss darauf haben, wird diskutiert. Hochdurchsatzsequenzierung lässt die genetische Diagnostik in sogenannten Panel-Untersuchungen zu. In dieser Arbeit wurde eine Datenbank zur Phänotyp-Genotyp-Korrelation entworfen. Es wurde ein Kollektiv von 284 schwerhörigen Patienten für die retrospektive Analyse auf genetische Schwerhörigkeit ausgewählt. Bei über 80% der Patienten fanden sich Mutationen, die mit Schwerhörigkeit in Verbindung stehen. Davon waren 54% von einer hereditären Form der Schwerhörigkeit betroffen. Es erfolgte eine Auswertung bezüglich Häufigkeitsverteilung der ursächlichen Gene und die häufigsten Gene (GJB2 17%, MYO7A 8%, MYO15A 7%, TECTA 6% und MYO6 3%) wurden mit klinischem Verlauf, audiologischen Befunden und der CI-Performance korreliert. Zusammenfassend zeigte die Versorgung mit CI für die häufigsten Gene GJB2, MYO15A, TECTA, und MYO6 gute Ergebnisse. Bei MYO7A-Patienten hingegen war die CI-Performance unterdurchschnittlich. Des Weiteren weisen die Ergebnisse daraufhin, dass der Expressionsort alleine nicht auf die CI- Performance schließen lässt, sondern jedes Gen einen eigenen prognostischen Faktor für die Implantation darstellt.

Spontaneous otoacoustic emissions (SOAEs) recorded from the ear canal in the absence of sound reflect cochlear amplification, an outer hair cell (OHC) process required for the extraordinary sensitivity and frequency selectivity of mammalian hearing. Although wild-type mice rarely emit, those with mutations that influence the tectorial membrane (TM) show an incidence of SOAEs similar to that in humans. In this report, we characterized mice with a missense mutation inTecta,a gene required for the formation of the striated-sheet matrix within the core of the TM. Mice heterozygous for the Y1870C mutation (TectaY1870C/+) are prolific emitters, despite a moderate hearing loss. Additionally, Kimura’s membrane, into which the OHC stereocilia insert, separates from the main body of the TM, except at apical cochlear locations. Multimodal SOAEs are also observed inTectaY1870C/+mice where energy is present at frequencies that are integer multiples of a lower-frequency SOAE (the primary). Second-harmonic SOAEs, at twice the frequency of a lower-frequency primary, are the most frequently observed. These secondary SOAEs are found in spatial regions where stimulus-evoked OAEs are small or in the noise floor. Introduction of high-level suppressors just above the primary SOAE frequency reduce or eliminate both primary and second-harmonic SOAEs. In contrast, second-harmonic SOAEs are not affected by suppressors, either above or below the second-harmonic SOAE frequency, even when they are much larger in amplitude. Hence, second-harmonic SOAEs do not appear to be spatially separated from their primaries, a finding that has implications for cochlear mechanics and the consequences of changes to TM structure.

This study was designed to delineate genetic contributions, if any, to sporadic forms of mild to moderate sensorineural hearing loss (SNHL) not related to GJB2 mutations (DFNB1) in a pediatric population. We recruited 11 non-DFNB1 simplex cases of mild to moderate SNHL in children. We applied whole-exome sequencing to all 11 probands. We used a filtering strategy assuming that de novo variants of known autosomal dominant (AD) deafness genes, biallelic mutations in autosomal recessive (AR) genes, monoallelic mutations in X chromosome genes for males, and digenic inheritance could be associated. Candidate variants first were prioritized with allele frequency in public databases and confirmed by a phase or a segregation test in each family. Additional information from the literature or public databases was used to identify strong candidate variants. Strong candidate variants were detected in 5 of 11 probands (45.4%). A diverse mode of inheritance implicated the sporadic occurrence of the phenotype. AR mutations in OTOGL and SERPINB6 and digenic inheritance involving two deafness genes, GPR98 and PDZ7, were detected. A de novo AD mutation also was detected in TECTA and MYH14. No syndromic feature was detected in individuals with GPR98/PDZ7 or MYH14 variants in our cohort at this moment. Mild to moderate pediatric SNHL, even if sporadic, features a strong genetic etiology and can manifest via diverse modes of inheritance. In addition, a multidisciplinary approach should be used for a correct diagnosis. Genet Med 17 11, 901–911.

Background Many mutations in the α-tectorin gene (TECTA) have been reported to cause non-syndromic hearing loss (NSHL) in either a dominant or recessive inheritance pattern. Among the identified TECTA mutations, H1400Y has been associated with NSHL in two independent studies. However, its exact role in contributing to genetic hearing loss remains elusive. Case presentation We herein report the whole-exome sequencing of a proband presenting with prelingual, non-progressive, mild-to-moderate hearing loss in a simplex family. By using trio-based whole-exome sequencing, we found two heterozygous mutations of R1890C and H1400Y in the ZP and ZA domains of TECTA, respectively. R1890C, previously reported as a pathogenic autosomal dominant mutation of genetic hearing loss, was found to be inherited in a de novo pattern, causing hearing loss in the proband. By contrast, H1400Y was not segregated in this family, and one family member with normal hearing also carried the H1400Y mutation. Conclusion According to the hearing loss-specific American College of Medical Genetics and Genomics (ACMG) guidelines, we conclude that H1400Y should be disqualified as a cause of genetic hearing loss. True pathogenic variants causing genetic hearing loss should be more deliberately reported in accordance with ACMG guidelines.

Detecting associations between genomic changes and phenotypic differences is fundamental to understanding how phenotypes evolved. By systematically screening for parallel amino acid substitutions, we detected known as well as novel cases (Strc, Tecta, Cabp2) of parallelism between echolocating bats and toothed whales in proteins that could contribute to high frequency hearing adaptations. Interestingly, our screen also showed that echolocating mammals exhibit an unusually high number of parallel substitutions in fast-twitch muscle fiber proteins. Both bats and dolphins produce an extremely rapid call rate when homing in on their prey, which was shown in bats to be powered by specialized superfast muscles. We show that these genes with parallel substitutions (Casq1, Atp2a1, Myh2, Myl1) are expressed in the superfast sound-producing muscle of bats. Furthermore, we found that the calcium storage protein calsequestrin 1 of bats and dolphins functionally converged in its ability to form calcium-sequestering polymers at lower calcium concentrations, which may contribute to rapid calcium transients required for superfast muscle physiology. The proteins that our genomic screen detected could be involved in the convergent evolution of vocalization in echolocating mammals by potentially contributing to both rapid Ca2+transients and increased shortening velocities in superfast muscles.AbbreviationsSRsarcoplasmic reticulum

AIMS: West syndrome (WS) is a classic form of early infantile epileptic encephalopathy (EIEE) characterized by tonic spasms with clustering, arrest of psychomotor development, and hypsarrhythmia on electroencephalography. Genetic defects play a critical role in the pathology of WS, and 54 EIEE genes have been identified till date. This study was designed to uncover new candidate genes for West syndrome. METHODS: In this study, we recruited 56 Chinese families with WS of unknown etiology. Whole exome sequencing (WES) was performed to identify Mendelian inheritance rare or novel variants. The association between candidate genes and WS was analyzed from many aspects, including recurrent genes in patients, predicted variant effect on genes, human tolerance to deficient genes, gene expression in the nervous system, coexpression with EIEE genes, mutual interaction with known EIEE proteins, genes related to ion channel or fragile X mental retardation protein function, and mouse models with manifestation of seizures. Genes with supporting evidence from those aspects were defined as highlight candidate genes. RESULTS: Whole exome sequencing identified 112 candidate variants in 89 genes. Among the candidate genes, 33 were autosomal dominant, 22 were autosomal recessive, and 34 were X‐linked. Complex bioinformatic analysis revealed 17 highlight candidate genes: ATP2A2, CD99L2, CLCN6, CYFIP1, CYFIP2, GNB1, GPT2, HUWE1, KMT2D, MYO18A, NOS3, RYR1, RYR2, RYR3, TAF1, TECTA, and UBA1. The majority of highlight candidate genes are calcium‐signaling pathway and mental retardation genes. CONCLUSIONS: This is the first WES study of Chinese WS patients with unknown etiology. This combination of phenotypic and genomic data will enable further testing to elucidate mechanisms underlying the pathogenesis of WS.

Hereditary hearing loss (HL) is a common sensory disorder, with an incidence of 1–2 per 1000 newborns, and has a genetic etiology in over 50% of cases. It occurs either as part of a syndrome or in isolation and is genetically very heterogeneous which poses a challenge for clinical and molecular diagnosis. We used exome sequencing to seek a genetic cause in a group of 56 subjects (49 probands) with HL: 32 with non-syndromic non-GJB2 HL and 17 with syndromic HL. Following clinical examination and clinical exome sequencing, an etiological diagnosis was established in 15 probands (15/49; 30%); eight (8/17;47%) from the syndromic group and seven (7/32; 21%) from the non-syndromic non-GJB2 subgroup. Fourteen different (half of them novel) non-GJB2 variants causing HL were found in 10 genes (CHD7, HDAC8, MITF, NEFL, OTOF, SF3B4, SLC26A4, TECTA, TMPRSS3, USH2A) among 13 probands, confirming the genetic heterogeneity of hereditary HL. Different genetic causes for HL were found in a single family while three probands with apparent syndromic HL were found to have HL as a separate clinical feature, distinct from the complex phenotype. Clinical exome sequencing proved to be an effective tool used to comprehensively address the genetic heterogeneity of HL, to detect clinically unrecognized HL syndromes, and to decipher complex phenotypes in which HL is a separate feature and not part of a syndrome.

Spontaneous otoacoustic emissions (SOAEs) are a signature of cochlear amplification, a process associated with outer hair cell (OHC) function and required for the sensitivity and frequency selectivity of cochlear responses. Although normal mice rarely exhibit these signals, those with mutations that influence the structure of the tectorial membrane (TM) show a greater incidence of these phenomena. In this report, we study mouse models with mutations affecting the striated-sheet matrix that forms the body of this accessory structure. In addition to CEACAM16, the matrix is composed of both α- and β-tectorin (TECTA and TECTB respectively) and these latter two noncollagenous proteins are the focus of this study. Of all the mutants tested to date, mice heterozygous (het) for a missense mutation (c.5609A>G, p.Tyr1870Cys) in Tecta (TectaY1870C/+ mice) are prolific emitters with an average of ∼7 SOAEs per cochlea, which is higher than in the very few normal mice with SOAEs where the average is 2-3 per cochlea. A small number of homozygous Tectb−/- mice lacking TECTB are also emitters, but they produce only one SOAE in any given ear. Although both mouse mutants have hearing loss, SOAE frequencies coincide with frequency regions where some degree of amplification is retained. The larger number of SOAEs in TectaY1870C/+ mice appears to correlate with an increase in porosity (1), which controls the spread of excitation of tectorial membrane traveling waves. Sellon and colleagues reported that this change in the material properties of the TM was associated with the larger size of nanoscale pores linked to the Y1870C missense mutation in TECTA. In mice lacking Tectb, where porosity is wild-type like, the number of SOAEs per cochlea is small and only a few of these animals present with this phenomenon. Characterization of traveling-wave properties in other emitting TM mutants will be required to know if the correlation between increased porosity and increased numbers of SOAEs reliably causes the change in phenotype.

Hereditary hearing loss (HHL) is a common disorder characterized by a huge genetic heterogeneity. The definition of a correct molecular diagnosis is essential for proper genetic counseling, recurrence risk estimation, and therapeutic options. From 20 to 40% of patients carry mutations in GJB2 gene, thus, in more than half of cases it is necessary to look for causative variants in the other genes so far identified (~100). In this light, the use of next-generation sequencing technologies has proved to be the best solution for mutational screening, even though it is not always conclusive. Here we describe a combined approach, based on targeted re-sequencing (TRS) of 96 HHL genes followed by high-density SNP arrays, aimed at the identification of the molecular causes of non-syndromic HHL (NSHL). This strategy has been applied to study 103 Italian unrelated cases, negative for mutations in GJB2, and led to the characterization of 31% of them (i.e., 37% of familial and 26.3% of sporadic cases). In particular, TRS revealed TECTA and ACTG1 genes as major players in the Italian population. Furthermore, two de novo missense variants in ACTG1 have been identified and investigated through protein modeling and molecular dynamics simulations, confirming their likely pathogenic effect. Among the selected patients analyzed by SNP arrays (negative to TRS, or with a single variant in a recessive gene) a molecular diagnosis was reached in ~36% of cases, highlighting the importance to look for large insertions/deletions. Moreover, copy number variants analysis led to the identification of the first case of uniparental disomy involving LOXHD1 gene. Overall, taking into account the contribution of GJB2, plus the results from TRS and SNP arrays, it was possible to reach a molecular diagnosis in ~51% of NSHL cases. These data proved the usefulness of a combined approach for the analysis of NSHL and for the definition of the epidemiological picture of HHL in the Italian population.

Morphometric study was conducted on Indian roofed turtle, Pangshura tecta, between January 1997 and December 2000 in several district of Bangladesh. The mean weight of adult male was 92.6 ± 13.3 g and of female 441.1 ± 185.4 g. The length and width of carapace as well as those of the plastron and the height of the shell varied with the body weight of the turtle. The percentage of hard parts of the body weight of P. tecta was 35.8 ± 2.5 %, whereas percentage of soft parts of the body weight was 64.2 ± 2.5 %. The average weight of the female P. tecta was 4.8 times higher than that of the male.Bangladesh J. Zool. 41(2): 207-215, 2013

The middle-frequency sensorineural hearing loss (MFSNHL) is rare among hereditary non-syndromic hearing loss. To date, only three genes are reported to be associated with MFSNHL, including TECTA, EYA4 and COL11A2. In this report, we analyzed and explored the clinical audiological characteristics and the causative gene of a Chinese family named HG-Z087 with non-syndromic autosomal dominant inherited MFSNHL. Clinical audiological characteristics and inheritance pattern of a family were evaluated, and pedigree was drawn based on medical history investigation. Our results showed that the Chinese family was characterized by late onset, progressive, non-sydromic autosomal dominant MFSNHL. Targeted exome sequencing, conducted using DNA samples of an affected member in this family, revealed a novel heterozygous missense mutation c.1643C>G in exon 18 of EYA4, causing amino-acid (aa) substitution Arg for Thr at a conserved position aa-548. The p.T548R mutation related to hearing loss in the selected Chinese family was validated by Sanger sequencing. However, the mutation was absent in control group containing 100 DNA samples from normal Chinese families. In conclusion, we identified the pathogenic gene and found that the novel missense mutation c.1643C>G (p.T548R) in EYA4 might have caused autosomal dominant non-syndromic hearing impairment in the selected Chinese family.

Otoliths are biomineralised structures important for balance and hearing in fish. Their counterparts in the mammalian inner ear, otoconia, have a primarily vestibular function. Otoliths and otoconia form over sensory maculae and are attached to the otolithic membrane, a gelatinous extracellular matrix that provides a physical coupling between the otolith and the underlying sensory epithelium. In this study, we have identified two proteins required for otolith tethering in the zebrafish ear, and propose that there are at least two stages to this process: seeding and maintenance. The initial seeding step, in which otolith precursor particles tether directly to the tips of hair cell kinocilia, fails to occur in the einstein (eis) mutant. The gene disrupted in eis is otogelin (otog); mutations in the human OTOG gene have recently been identified as causative for deafness and vestibular dysfunction (DFNB18B). At later larval stages, maintenance of otolith tethering to the saccular macula is dependent on tectorin alpha (tecta) function, which is disrupted in the rolling stones (rst) mutant. α-Tectorin (Tecta) is a major constituent of the tectorial membrane in the mammalian cochlea. Mutations in the human TECTA gene can cause either dominant (DFNA8/12) or recessive (DFNB21) forms of deafness. Our findings indicate that the composition of extracellular otic membranes is highly conserved between mammals and fish, reinforcing the view that the zebrafish is an excellent model system for the study of deafness and vestibular disease., Summary: The tethering of zebrafish otoliths, which are important for balance and hearing in fish, occurs in two stages that are dependent on genes that have been implicated in human deafness.

Aim Hearing loss is the most common sensory disorder that affects approximately one per 1000 live births. With this project, we aimed to identify gene variants that were common causes of hearing loss in Turkey to contribute to the planning of genetic screening programs for hearing loss, as well as to improve genetic counseling to affected families. Material and methods Twenty-one families with at least two affected individuals and parental consanguinity who presented with non-syndromic severe-to-profound sensorineural hearing loss were included in this study. We first screened for mutations in GJB2 and mitochondrial DNA 12S RNA genes. Subsequently, we genotyped the TMIE c.250C>T and SNP markers flanking the SLC26A4, MYO7A, MYO15A, OTOF, CDH23, TMIE, TECTA, PCDH15, TMC1, TMPRSS3, TMHS genes in the remaining twelve families without mutations in GJB2. Results Screening for mutations in GJB2 gene showed c.[35delG];[35delG] mutation in four families, c.[35delG];[507C>A] mutation in two families, c.[35delG];[-23+1G>A] mutation in one family, and c.457G>A heterozygous mutation in one family. Genotyping SNP markers showed the c.[250C>T];[250C>T] mutation in TMIE in one family. A homozygous region with SNP genotypes was detected with the OTOF gene in one family, the TMPRSS3 gene in another family, and also a homozygous region was detected with TMHS, OTOF, and TMPRSS3 genes in another family. Conclusions Further research will be required to determine the genetic bases of hearing loss in families with non-syndromic hearing loss.

Chemotherapy induced peripheral neuropathy (CIPN) is a dose limiting toxicity of paclitaxel, termed here paclitaxel induced peripheral neuropathy (PIPN). PIPN affects a minority of patients and cannot be predicted from clinical patient characteristics. Therefore, a phamacogenomic basis has been proposed for PIPN (1). Genome wide association studies (GWAS) provide an unbiased approach for the identification of novel genetic loci associated with complex traits (2). Schneider et al. performed the first PIPN GWAS in 2011, (3) finding that the risk of PIPN was linked to two single nucleotide variants (SNV) in two genes that had previously not been known to be associated with this condition or with CIPN due to any other drug. The SNV rs2296308 in RWDD3 was reported to be associated with PIPN with a HR=1.5 and a significance level of p=8.5 × 10−8. The rs1829 SNV in TECTA was reported to be associated with PIPN with a HR= 2.1 and a significance level of p=3.2×10−7. Both SNV thereby passed the threshold set by the authors for genome-wide significance thereby representing important novel PIPN gene candidates. Bergmann et al. investigated rs2296308 and rs1829 in a Scandinavian ovarian cancer patient cohort (4). Their study failed to corroborate the report by Schneider et al., thereby calling that GWAS result into question. The original GWAS consisted of a very large cohort, 2204 patients, while the validation attempt by Bergmann et al. was limited to a smaller cohort of 241 patients. Both studies used similar phenotyping and analyses relying on Common Toxicity Criteria Adverse Effects (CTCAE) reporting of toxicity and time-to-grade 2 or worse scores as the primary endpoint. These conflicting results thereby created uncertainty in the field regarding the role of RWWD3 and TECTA. We recently reported a new PIPN cohort, N08C1, for which improved phenotyping was available using serial assessments of PIPN symptoms with the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ) CIPN-20 (CIPN20) (5). Here, we report testing of the association of rs2296308 in RWWD3 and rs1829 in TECTA with PIPN in N08C1.

Autosomal dominant sensorineural hearing loss (ADSNHL) is extremely genetically heterogeneous, making it difficult to molecularly diagnose. We identified a multiplex (n=28 affected) family from the genetic isolate of Newfoundland, Canada with variable SNHL and used a targeted sequencing approach based on population-specific alleles in WFS1, TMPRSS3 and PCDH15; recurrent mutations in GJB2 and GJB6; and frequently mutated exons of KCNQ4, COCH and TECTA. We identified a novel, in-frame deletion (c.806_808delCCT: p.S269del) in the voltage-gated potassium channel KCNQ4 (DFNA2), which in silico modeling predicts to disrupt multimerization of KCNQ4 subunits. Surprisingly, 10/23 deaf relatives are non-carriers of p.S269del. Further molecular characterization of the DFNA2 locus in deletion carriers ruled out the possibility of a pathogenic mutation other than p.S269del at the DFNA2A/B locus and linkage analysis showed significant linkage to DFNA2 (maximum LOD=3.3). Further support of genetic heterogeneity in family 2071 was revealed by comparisons of audio profiles between p.S269del carriers and non-carriers suggesting additional and as yet unknown etiologies. We discuss the serious implications that genetic heterogeneity, in this case observed within a single family, has on molecular diagnostics and genetic counseling.