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Current estimates suggest that nearly half a billion people worldwide are affected by hearing loss. Because of the major psychological, social, economic, and health ramifications, considerable efforts have been invested in identifying the genes and molecular pathways involved in hearing loss, whether genetic or environmental, to promote prevention, improve rehabilitation, and develop therapeutics. Genomic sequencing technologies have led to the discovery of genes associated with hearing loss. Studies of the transcriptome and epigenome of the inner ear have characterized key regulators and pathways involved in the development of the inner ear and have paved the way for their use in regenerative medicine. In parallel, the immense preclinical success of using viral vectors for gene delivery in animal models of hearing loss has motivated the industry to work on translating such approaches into the clinic. Here, we review the recent advances in the genomics of auditory function and dysfunction, from patient diagnostics to epigenetics and gene therapy.

Sequencing exomes/genomes have been successful for identifying recessive genes; however, discovery of dominant genes including deafness genes (DFNA) remains challenging. We report a new DFNA gene, ATP11A, in a Newfoundland family with a variable form of bilateral sensorineural hearing loss (SNHL). Genome-wide SNP genotyping linked SNHL to DFNA33 (LOD = 4.77), a locus on 13q34 previously mapped in a German family with variable SNHL. Whole-genome sequencing identified 51 unremarkable positional variants on 13q34. Continuous clinical ascertainment identified several key recombination events and reduced the disease interval to 769 kb, excluding all but one variant. ATP11A (NC_000013.11: chr13:113534963G>A) is a novel variant predicted to be a cryptic donor splice site. RNA studies verified in silico predictions, revealing the retention of 153 bp of intron in the 3′ UTR of several ATP11A isoforms. Two unresolved families from Israel were subsequently identified with a similar, variable form of SNHL and a novel duplication (NM_032189.3:c.3322_3327+2dupGTCCAGGT) in exon 28 of ATP11A extended exon 28 by 8 bp, leading to a frameshift and premature stop codon (p.Asn1110Valfs43Ter). ATP11A is a type of P4-ATPase that transports (flip) phospholipids from the outer to inner leaflet of cell membranes to maintain asymmetry. Haploinsufficiency of ATP11A, the phospholipid flippase that specially transports phosphatidylserine (PS) and phosphatidylethanolamine (PE), could leave cells with PS/PE at the extracellular side vulnerable to phagocytic degradation. Given that surface PS can be pharmaceutically targeted, hearing loss due to ATP11A could potentially be treated. It is also likely that ATP11A is the gene underlying DFNA33.

The age of sequencing has provided unprecedented insights into the human genome. The coding region of the genome comprises nearly 20,000 genes, of which approximately 4000 are associated with human disease. Beyond the protein-coding genome, which accounts for only 3% of the genome, lies a vast pool of regulatory elements in the form of promoters, enhancers, RNA species, and other intricate elements. These features undoubtably influence human health and disease, and as a result, a great deal of effort is currently being invested in deciphering their identity and mechanism. While a paucity of material has caused a lag in identifying these elements in the inner ear, the emergence of technologies for dealing with a minimal number of cells now has the field working overtime to catch up. Studies on microRNAs (miRNAs), long non-coding RNAs (lncRNAs), methylation, histone modifications, and more are ongoing. A number of microRNAs and other noncoding elements are known to be associated with hearing impairment and there is promise that regulatory elements will serve as future tools and targets of therapeutics and diagnostics. This review covers the current state of the field and considers future directions for the noncoding genome and implications for hearing loss.

The study was conducted between 2018 and 2020. From a cohort of 113 hearing impaired (HI), five non-DFNB12 probands identified with heterozygous CDH23 variants were subjected to exome analysis. This resolved the etiology of hearing loss (HL) in four South Indian assortative mating families. Six variants, including three novel ones, were identified in four genes: PNPT1 p.(Ala46Gly) and p.(Asn540Ser), MYO15A p.(Leu1485Pro) and p.(Tyr1891Ter), PTPRQ p.(Gln1336Ter), and SLC12A2 p.(Pro988Ser). Compound heterozygous PNPT1 variants were associated with DFNB70 causing prelingual profound sensorineural hearing loss (SNHL), vestibular dysfunction, and unilateral progressive vision loss in one family. In the second family, MYO15A variants in the myosin motor domain, including a novel variant, causing DFNB3, were found to be associated with prelingual profound SNHL. A novel PTPRQ variant was associated with postlingual progressive sensorineural/mixed HL and vestibular dysfunction in the third family with DFNB84A. In the fourth family, the SLC12A2 novel variant was found to segregate with severe-to-profound HL causing DFNA78, across three generations. Our results suggest a high level of allelic, genotypic, and phenotypic heterogeneity of HL in these families. This study is the first to report the association of PNPT1, PTPRQ, and SLC12A2 variants with HL in the Indian population.

The SLC26A4 gene, which encodes the anion exchanger pendrin, is involved in determining syndromic (Pendred syndrome) and non-syndromic (DFNB4) autosomal recessive hearing loss. SLC26A4 c.349C>T, p.L117F is a relatively common allele in the Ashkenazi Jewish community, where its minor allele frequency is increased compared to other populations. Although segregation and allelic data support the pathogenicity of this variant, former functional tests showed characteristics that were indistinguishable from those of the wild-type protein. Here, we applied a triad of cell-based assays, i.e., measurement of the ion transport activity by a fluorometric method, determination of the subcellular localization by confocal microscopy, and assessment of protein expression levels, to conclusively assign or exclude the pathogenicity of SLC26A4 p.L117F. This protein variant showed a moderate, but significant, reduction in ion transport function, a partial retention in the endoplasmic reticulum, and a strong reduction in expression levels as a consequence of an accelerated degradation by the Ubiquitin Proteasome System, all supporting pathogenicity. The functional and molecular features of human pendrin p.L117F were recapitulated by the mouse ortholog, thus indicating that a mouse carrying this variant might represent a good model of Pendred syndrome/DFNB4.

Loss-of-function variants in AP3D1 have been linked to Hermansky–Pudlak syndrome (HPS) 10, a severe multisystem disorder characterized by oculocutaneous albinism, immunodeficiency, neurodevelopmental delay, hearing loss (HL), and neurological abnormalities, fatal in early childhood. Here, we report a consanguineous family who presented with presumably isolated autosomal recessive (AR) HL. Whole-exome sequencing was performed on all core family members, and selected patients were screened using array-based copy-number analysis and karyotyping. Candidate variants were validated by Sanger sequencing and assessed in silico. A homozygous, likely pathogenic p.V711I missense variant in AP3D1 segregated with the HL. The family was characterized by thorough medical and laboratory examination. The HL was consistent across patients and accompanied by neurological manifestations in two brothers. The sole female patient was diagnosed with premature ovarian failure. Further findings, including mild neutropenia and reduced NK-cell cytotoxicity in some as well as brain alterations in all homozygous patients, were reminiscent of HPS10, though milder and lacking the characteristic albinism. Previously unrecognized, milder, isolated HL was identified in all heterozygous carriers. A protein model indicates that the variant interferes with protein–protein interactions. These results suggest that a missense variant alters inner-ear-specific functions leading to HL with mild HPS10-like symptoms of variable penetrance. Milder HL in heterozygous carriers may point towards semi-dominant inheritance of this trait. Since all previously reported HPS10 cases were pediatric, it is unknown whether the observed primary ovarian insufficiency recapitulates the subfertility in Ap3d1-deficient mice.

Objectives To better distinguish NOG-related-symphalangism spectrum disorder (NOG-SSD) from chromosomal 17q22 microdeletion syndromes and to inform surgical considerations in stapes surgery for patients with NOG-SSD. Background Mutations in NOG cause a variety of skeletal syndromes that often include conductive hearing loss. Several microdeletions of chromosome 17q22 lead to severe syndromes with clinical characteristics that overlap NOG-SSD. Isolated deletion of NOG has not been described, and therefore the contribution of NOG deletion in these syndromes is unknown. Methods Two families with autosomal dominant NOG-SSD exhibited stapes ankylosis, facial dysmorphisms, and skeletal and joint anomalies. In each family, NOG was evaluated by genomic sequencing and candidate mutations confirmed as damaging by in vitro assays. Temporal bone histology of a patient with NOG-SSD was compared with temporal bones of 40 patients diagnosed with otosclerosis. Results Family 1 harbors a 555 kb chromosomal deletion encompassing only NOG and ANKFN1. Family 2 harbors a missense mutation in NOG leading to absence of noggin protein. The incus-footplate distance of the temporal bone was significantly longer in a patient with NOG-SSD than in patients with otosclerosis. Conclusion The chromosomal microdeletion of family 1 led to a phenotype comparable to that due to a NOG point mutation and much milder than the phenotypes due to other chromosome 17q22 microdeletions. Severe clinical findings in other microdeletion cases are likely due to deletion of genes other than NOG. Based on temporal bone findings, we recommend that surgeons obtain longer stapes prostheses before stapes surgery in individuals with NOG-SSD stapes ankylosis.

Hearing loss is a hallmark of aging, typically initially affecting the higher frequencies. In echolocating bats, the ability to discern high frequencies is essential. However, nothing is known about age-related hearing loss in bats, and they are often assumed to be immune to it. We tested the hearing of 47 wild Egyptian fruit bats by recording their auditory brainstem response and cochlear microphonics, and we also assessed the cochlear histology in four of these bats. We used the bats’ DNA methylation profile to evaluate their age and found that bats exhibit age-related hearing loss, with more prominent deterioration at the higher frequencies. The rate of the deterioration was ∼1 dB per year, comparable to the hearing loss observed in humans. Assessing the noise in the fruit bat roost revealed that these bats are exposed to continuous immense noise—mostly of social vocalizations—supporting the assumption that bats might be partially resistant to loud noise. Thus, in contrast to previous assumptions, our results suggest that bats constitute a model animal for the study of age-related hearing loss.

Nonsyndromic hearing loss is genetically heterogeneous. Despite comprehensive genetic testing, many cases remain unsolved because the clinical significance of identified variants is uncertain or because biallelic pathogenic variants are not identified for presumed autosomal recessive cases. Common synonymous variants are often disregarded. Determining the pathogenicity of synonymous variants may improve genetic diagnosis. We report a synonymous variant c.9861 C > T/p.(Gly3287=) in MYO15A in homozygosity or compound heterozygosity with another pathogenic or likely pathogenic MYO15A variant in 10 unrelated families with nonsyndromic sensorineural hearing loss. Biallelic variants in MYO15A were identified in 21 affected and were absent in 22 unaffected siblings. A mini-gene assay confirms that the synonymous variant leads to abnormal splicing. The variant is enriched in the Ashkenazi Jewish population. Individuals carrying biallelic variants involving c.9861 C > T often exhibit progressive post-lingual hearing loss distinct from the congenital profound deafness typically associated with biallelic loss-of-function MYO15A variants. This study establishes the pathogenicity of the c.9861 C > T variant in MYO15A and expands the phenotypic spectrum of MYO15A-related hearing loss. Our work also highlights the importance of multicenter collaboration and data sharing to establish the pathogenicity of a relatively common synonymous variant for improved diagnosis and management of hearing loss.

Contains fulltext : 243959.pdf (Publisher’s version ) (Closed access) PURPOSE: The ClinGen Variant Curation Expert Panels (VCEPs) provide disease-specific rules for accurate variant interpretation. Using the hearing loss-specific American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines, the Hearing Loss VCEP (HL VCEP) illustrates the utility of expert specifications in variant interpretation. METHODS: A total of 157 variants across nine HL genes, previously submitted to ClinVar, were curated by the HL VCEP. The curation process involved collecting published and unpublished data for each variant by biocurators, followed by bimonthly meetings of an expert curation subgroup that reviewed all evidence and applied the HL-specific ACMG/AMP guidelines to reach a final classification. RESULTS: Before expert curation, 75% (117/157) of variants had single or multiple variants of uncertain significance (VUS) submissions (17/157) or had conflicting interpretations in ClinVar (100/157). After applying the HL-specific ACMG/AMP guidelines, 24% (4/17) of VUS and 69% (69/100) of discordant variants were resolved into benign (B), likely benign (LB), likely pathogenic (LP), or pathogenic (P). Overall, 70% (109/157) variants had unambiguous classifications (B, LB, LP, P). We quantify the contribution of the HL-specified ACMG/AMP codes to variant classification. CONCLUSION: Expert specification and application of the HL-specific ACMG/AMP guidelines effectively resolved discordant interpretations in ClinVar. This study highlights the utility of ClinGen VCEPs in supporting more consistent clinical variant interpretation.

Objective: While COVID-19 symptoms impact rhinology (anosmia) and laryngology (airways), two major disciplines of the otolaryngology armamentarium, the virus has seemed to spare the auditory system. A recent study, however, reported changes in otoacoustic emission (OAE) signals measured in SARS-COV-2 positive patients. We sought to assess the effect of COVID-19 infection on auditory performance in a cohort of recovered SARS-COV-2 patients and controls. To avoid a potential bias of previous audiological dysfunction not related to SARS-COV-2 infection, the study encompasses patients with normal auditory history. We hypothesized that if SARS-COV-2 infection predisposes to hearing loss, we would observe subtle and early audiometric deficits in our cohort in the form of subclinical auditory changes. Study Design: Cross-sectional study. Setting: Tertiary referral center. Patients: The Institutional Review Board approved the study and we recruited participants who had been positive for SARS-COV-2 infection, according to an Reverse Transcription Polymerase Chain Reaction (RT-PCR) test on two nasopharyngeal swabs. The patients included in this study were asymptomatic for the SARS-COV-2 infection and were evaluated following recovery, confirmed by repeated swab testing. The control group comprised healthy individuals matched for age and sex, and with a normal auditory and otologic history. Intervention(s): The eligibility to participate in this study included a normal audiogram, no previous auditory symptoms, normal otoscopy examination with an intact tympanic membrane, and bilateral tympanometry type A. None of our volunteers reported any new auditory symptoms following SARS-COV-2 infection. Ototacoustic emissions (OAE) and auditory brainstem response (ABR) measurements were used to evaluate the auditory function. Main Outcome Measure(s): OAE and ABR measurements. Results: We have found no significant differences between recovered asymptomatic SARS-COV-2 patients and controls in any of transitory evoked otoacoustic emission (TEOAE), distortion product otoacoustic emissions (DPOAE), or ABR responses. Conclusions: There is no cochlear dysfunction represented by ABR, TEOAE, and DPOAE responses in recovered COVID-19 asymptomatic patients. Retrocochlear function was also preserved as evident by the ABR responses. A long-term evaluation of a larger cohort of SARS-COV-2 patients will help to identify a possible contribution of SARS-COV-2 infection to recently published anecdotal auditory symptoms associated with COVID-19.

The auditory system comprises the auditory periphery, engaged in sound transduction and the central auditory system, implicated in auditory information processing and perception. Recently, evidence mounted that the mammalian peripheral and central auditory systems share a number of genes critical for proper development and function. This bears implication for auditory rehabilitation and evolution of the auditory system. To analyze to which extent microRNAs (miRNAs) belong to genes shared between both systems, we characterize the expression pattern of 12 cochlea-abundant miRNAs in the central auditory system. Quantitative real-time PCR (qRT-PCR) demonstrated expression of all 12 genes in the cochlea, the auditory hindbrain and the non-auditory prefrontal cortex (PFC) at embryonic stage (E)16 and postnatal stages (P)0 and P30. Eleven of them showed differences in expression between tissues and nine between the developmental time points. Hierarchical cluster analysis revealed that the temporal expression pattern in the auditory hindbrain was more similar to the PFC than to the cochlea. Spatiotemporal expression analysis by RNA in situ hybridization demonstrated widespread expression throughout the cochlear nucleus complex (CNC) and the superior olivary complex (SOC) during postnatal development. Altogether, our data indicate that miRNAs represent a relevant class of genetic factors functioning across the auditory system. Given the importance of gene regulatory network (GRN) components for development, physiology and evolution, the 12 miRNAs provide promising entry points to gain insights into their molecular underpinnings in the auditory system. Electronic supplementary material The online version of this article (10.1007/s00441-020-03290-x) contains supplementary material, which is available to authorized users.

The auditory system is a complex sensory network with an orchestrated multilayer regulatory program governing its development and maintenance. Accumulating evidence has implicated long non-coding RNAs (lncRNAs) as important regulators in numerous systems, as well as in pathological pathways. However, their function in the auditory system has yet to be explored. Using a set of specific criteria, we selected four lncRNAs expressed in the mouse cochlea, which are conserved in the human transcriptome and are relevant for inner ear function. Bioinformatic characterization demonstrated a lack of coding potential and an absence of evolutionary conservation that represent properties commonly shared by their class members. RNAscope analysis of the spatial and temporal expression profiles revealed specific localization to inner ear cells. Sub-cellular localization analysis presented a distinct pattern for each lncRNA and mouse tissue expression evaluation displayed a large variability in terms of level and location. Our findings establish the expression of specific lncRNAs in different cell types of the auditory system and present a potential pathway by which the lncRNA Gas5 acts in the inner ear. Studying lncRNAs and deciphering their functions may deepen our knowledge of inner ear physiology and morphology and may reveal the basis of as yet unresolved genetic hearing loss-related pathologies. Moreover, our experimental design may be employed as a reference for studying other inner ear-related lncRNAs, as well as lncRNAs expressed in other sensory systems.

Periodic organization of cells is required for the function of many organs and tissues. The development of such periodic patterns is typically associated with mechanisms based on intercellular signaling such as lateral inhibition and Turing patterning. Here we show that the transition from disordered to ordered checkerboard-like pattern of hair cells and supporting cells in the mammalian hearing organ, the organ of Corti, is likely based on mechanical forces rather than signaling events. Using time-lapse imaging of mouse cochlear explants, we show that hair cells rearrange gradually into a checkerboard-like pattern through a tissue-wide shear motion that coordinates intercalation and delamination events. Using mechanical models of the tissue, we show that global shear and local repulsion forces on hair cells are sufficient to drive the transition from disordered to ordered cellular pattern. Our findings suggest that mechanical forces drive ordered hair cell patterning in a process strikingly analogous to the process of shear-induced crystallization in polymer and granular physics., The periodic organization of cells is typically associated with mechanisms based on intercellular signaling such as lateral inhibition and Turing patterning. Here the authors show that hair cells in the inner ear rearrange gradually into a checkerboard-like pattern through a tissue-wide shear motion that coordinates intercalation and delamination events.

Since 1999, the COCH gene encoding cochlin, has been linked to the autosomal dominant non-syndromic hearing loss, DFNA9, with or without vestibular abnormalities. The hearing impairment associated with the variants affecting gene function has been attributed to a dominant-negative effect. Mutant cochlin was seen to accumulate intracellularly, with the formation of aggregates both inside and outside the cells, in contrast to the wild-type cochlin that is normally secreted. While additional recessive variants in the COCH gene (DFNB110) have recently been reported, the mechanism of the loss-of-function (LOF) effect of the COCH gene product remains unknown. In this study, we used COS7 cell lines to investigate the consequences of a novel homozygous frameshift variant on RNA transcription, and on cochlin translation. Our results indicate a LOF effect of the variant and a major decrease in cochlin translation. This data have a dramatic impact on the accuracy of genetic counseling for both heterozygote and homozygote carriers of LOF variants in COCH.

Mutations in more than 150 genes are responsible for inherited hearing loss, with thousands of different, severe causal alleles that vary among populations. The Israeli Jewish population includes communities of diverse geographic origins, revealing a wide range of deafness-associated variants and enabling clinical characterization of the associated phenotypes. Our goal was to identify the genetic causes of inherited hearing loss in this population, and to determine relationships among genotype, phenotype, and ethnicity. Genomic DNA samples from informative relatives of 88 multiplex families, all of self-identified Jewish ancestry, with either non-syndromic or syndromic hearing loss, were sequenced for known and candidate deafness genes using the HEar-Seq gene panel. The genetic causes of hearing loss were identified for 60% of the families. One gene was encountered for the first time in human hearing loss: ATOH1 (Atonal), a basic helix-loop-helix transcription factor responsible for autosomal dominant progressive hearing loss in a five-generation family. Our results demonstrate that genomic sequencing with a gene panel dedicated to hearing loss is effective for genetic diagnoses in a diverse population. Comprehensive sequencing enables well-informed genetic counseling and clinical management by medical geneticists, otolaryngologists, audiologists, and speech therapists and can be integrated into newborn screening for deafness.

Non-coding RNAs (ncRNAs) play a critical role in the entire body, and their mis-regulation is often associated with disease. In parallel with the advances in high-throughput sequencing technologies, there is a great deal of focus on this broad class of RNAs. Although these molecules are not translated into proteins, they are now well established as significant regulatory components in many biological pathways and pathological conditions. ncRNAs can be roughly divided into two main sub-groups based on the length of the transcript, with both the small and long non-coding RNAs having diverse regulatory functions. The smaller length group includes ribosomal RNAs (rRNA), transfer RNAs (tRNA), small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNA), microRNAs (miRNA), small interfering RNAs (siRNA), and PIWI-associated RNAs (piRNA). The longer length group includes linear long non-coding RNAs (lncRNA) and circular RNAs (circRNA). This review is designed to present the different classes of small and long ncRNA molecules and describe some of their known roles in physiological and pathological conditions, as well as methods used to assess the validity and function of miRNAs and lncRNAs, with a focus on their role and functions in the inner ear, hearing and deafness.

The study was conducted between 2018 and 2020. From a cohort of 113 hearing impaired (HI), five non-DFNB12 probands identified with heterozygous CDH23 variants were subjected to exome analysis. This resolved the etiology of hearing loss (HL) in four South Indian assortative mating families. Six variants, including three novel ones, were identified in four genes: PNPT1 p.(Ala46Gly) and p.(Asn540Ser), MYO15A p.(Leu1485Pro) and p.(Tyr1891Ter), PTPRQ p.(Gln1336Ter), and SLC12A2 p.(Pro988Ser). Compound heterozygous PNPT1 variants were associated with DFNB70 causing prelingual profound sensorineural hearing loss (SNHL), vestibular dysfunction, and unilateral progressive vision loss in one family. In the second family, MYO15A variants in the myosin motor domain, including a novel variant, causing DFNB3, were found to be associated with prelingual profound SNHL. A novel PTPRQ variant was associated with postlingual progressive sensorineural/mixed HL and vestibular dysfunction in the third family with DFNB84A. In the fourth family, the SLC12A2 novel variant was found to segregate with severe-to-profound HL causing DFNA78, across three generations. Our results suggest a high level of allelic, genotypic, and phenotypic heterogeneity of HL in these families. This study is the first to report the association of PNPT1, PTPRQ, and SLC12A2 variants with HL in the Indian population.

PURPOSE Pathogenic variants in GJB2 are the most common cause of autosomal recessive sensorineural hearing loss. The classification of c.101T>C/p.Met34Thr and c.109G>A/p.Val37Ile in GJB2 are controversial. Therefore, an expert consensus is required for the interpretation of these two variants. METHODS The ClinGen Hearing Loss Expert Panel collected published data and shared unpublished information from contributing laboratories and clinics regarding the two variants. Functional, computational, allelic, and segregation data were also obtained. Case-control statistical analyses were performed. RESULTS The panel reviewed the synthesized information, and classified the p.Met34Thr and p.Val37Ile variants utilizing professional variant interpretation guidelines and professional judgment. We found that p.Met34Thr and p.Val37Ile are significantly overrepresented in hearing loss patients, compared to population controls. Individuals homozygous or compound heterozygous for p.Met34Thr or p.Val37Ile typically manifest mild to moderate hearing loss. Several other types of evidence also support pathogenic roles for these two variants. CONCLUSION Resolving controversies in variant classification requires coordinated effort among a panel of international multi-institutional experts to share data, standardize classification guidelines, review evidence, and reach a consensus. We concluded that p.Met34Thr and p.Val37Ile variants in GJB2 are pathogenic for autosomal recessive nonsyndromic hearing loss with variable expressivity and incomplete penetrance.

Genetic variants account for approximately half the cases of congenital and early‐onset deafness. Methods and technologies for viral delivery of genes into the inner ear have evolved over the past decade to render gene therapy a viable and attractive approach for treatment. Variants in SYNE4, encoding the protein nesprin‐4, a member of the linker of nucleoskeleton and cytoskeleton (LINC), lead to DFNB76 human deafness. Syne4 −/− mice have severe‐to‐profound progressive hearing loss and exhibit mislocalization of hair cell nuclei and hair cell degeneration. We used AAV9‐PHP.B, a recently developed synthetic adeno‐associated virus, to deliver the coding sequence of Syne4 into the inner ears of neonatal Syne4 −/− mice. Here we report rescue of hair cell morphology and survival, nearly complete recovery of auditory function, and restoration of auditory‐associated behaviors, without observed adverse effects. Uncertainties remain regarding the durability of the treatment and the time window for intervention in humans, but our results suggest that gene therapy has the potential to prevent hearing loss in humans with SYNE4 mutations., Syne4 deficiency leads to hearing loss in humans. In this work, auditory function was rescued in Syne4 knockout mice by a synthetic AAV that enables safe and efficient transduction of hair cells in the cochlea.

PurposeThe ClinGen Variant Curation Expert Panels (VCEPS) provide disease-specific rules for accurate variant interpretation. Using hearing loss-specific American College of Medical Genetics/Association for Molecular Pathology (HL-specific ACMG/AMP) guidelines, the ClinGen Hearing Loss VCEP (HL VCEP) illustrates the utility of expert specifications in resolving conflicting variant interpretations.MethodsA total of 157 variants across nine hearing loss genes were curated and submitted to ClinVar by the HL VCEP. The curation process consisted of collecting published and unpublished data for each variant by biocurators, followed by bi-monthly meetings of an expert curation subgroup that reviewed all evidence and applied the HL-specific ACMG/AMP guidelines to reach a final classification.ResultsBefore expert curation, 75% (117/157) of variants had single or multiple VUS submissions (17/157) or had conflicting interpretations in ClinVar (100/157). After applying the HL-specific ACMG/AMP guidelines, 24% (4/17) of VUS variants and 69% (69/100) of discordant variants were resolved into Benign (B), Likely Benign (LB), Likely Pathogenic (LP), or Pathogenic (P). Overall, 70% (109/157) variants had unambiguous classifications (B, LB, LP, P). We quantify the contribution of the HL-specified ACMG/AMP codes to variant interpretation.ConclusionExpert specification and application of the HL-specific ACMG/AMP guidelines effectively resolved discordant interpretations in ClinVar. This study supports the utility of ClinGen VCEPs in helping the community move towards more consistent variant interpretations, which will improve the care of patients with genetic disorders.

The GLUN2D subunit of the N-methyl D-aspartate receptor (NMDAR) is encoded by the GRIN2D gene. Mutations in GRIN2D have been associated with neurodevelopmental and epileptic encephalopathies. Access to patient samples harboring mutations in GRIN2D can contribute to understanding the role of NMDAR in neuronal development and function. We report the generation of induced pluripotent stem cell (iPSC) lines from a GRIN2D-developmental and epileptic encephalopathy (DEE) patient, carrying a de novo c.1999G>A heterozygous pathogenic variant, and his healthy parent. Generated lines highly expressed pluripotency markers, spontaneously differentiated into the three germ layers, retained the deficiency-causing mutation, and displayed normal karyotypes.

Many government websites and mobile content are inaccessible for people with vision, hearing, and cognitive disabilities. The COVID-19 pandemic highlighted these disparities when health authority website information, critical in providing resources for curbing the spread of the virus, remained inaccessible for disabled populations. The Web Content Accessibility Guidelines provide comparatively universally accepted guidelines for website accessibility. We utilized these parameters to examine the number of countries with or without accessible health authority websites. The resulting data indicate a dearth of countries with websites accessible for persons with disabilities. Methods of information dissemination must take into consideration individuals with disabilities, particularly in times of global health crises.

The genetic characterization of a common phenotype for an entire population reveals both the causes of that phenotype for that place and the power of family-based, population-wide genomic analysis for gene and mutation discovery. We characterized the genetics of hearing loss throughout the Palestinian population, enrolling 2,198 participants from 491 families from all parts of the West Bank and Gaza. In Palestinian families with no prior history of hearing loss, we estimate that 56% of hearing loss is genetic and 44% is not genetic. For the great majority (87%) of families with inherited hearing loss, panel-based genomic DNA sequencing, followed by segregation analysis of large kindreds and transcriptional analysis of participant RNA, enabled identification of the causal genes and mutations, including at distant noncoding sites. Genetic heterogeneity of hearing loss was striking with respect to both genes and alleles: The 337 solved families harbored 143 different mutations in 48 different genes. For one in four solved families, a transcription-altering mutation was the responsible allele. Many of these mutations were cryptic, either exonic alterations of splice enhancers or silencers or deeply intronic events. Experimentally calibrated in silico analysis of transcriptional effects yielded inferences of high confidence for effects on splicing even of mutations in genes not expressed in accessible tissue. Most (58%) of all hearing loss in the population was attributable to consanguinity. Given the ongoing decline in consanguineous marriage, inherited hearing loss will likely be much rarer in the next generation.

Since 1999, the COCH gene encoding cochlin, has been linked to the autosomal dominant non-syndromic hearing loss, DFNA9, with or without vestibular abnormalities. The hearing impairment associated with the variants affecting gene function has been attributed to a dominant-negative effect. Mutant cochlin was seen to accumulate intracellularly, with the formation of aggregates both inside and outside the cells, in contrast to the wild-type cochlin that is normally secreted. While an additional recessive variant in the COCH gene (DFNB110) has recently been reported, the mechanism of the loss-of-function (LOF) effect of the COCH gene product remains unknown. In this study, we used COS7 cell lines to investigate the consequences of a novel homozygous frameshift variant on RNA transcription, and on cochlin translation. Our results indicate a LOF effect of the variant and a major decrease in cochlin translation. This data has a dramatic impact on the accuracy of genetic counseling for both heterozygote and homozygote carriers of LOF variants in COCH.

Mutations in more than 150 genes are responsible for inherited hearing loss, with thousands of different, severe causal alleles that vary among populations. The Israeli Jewish population includes communities of diverse geographic origins, revealing a wide range of deafness-associated variants and enabling clinical characterization of the associated phenotypes. Our goal was to identify the genetic causes of inherited hearing loss in this population, and to determine relationships among genotype, phenotype, and ethnicity. Genomic DNA samples from informative relatives of 88 multiplex families, all of self-identified Jewish ancestry, with either non-syndromic or syndromic hearing loss, were sequenced for known and candidate deafness genes using the HEar-Seq gene panel. The genetic causes of hearing loss were identified for 60% of the families. One gene was encountered for the first time in human hearing loss:ATOH1(Atonal), a basic helix-loop-helix transcription factor responsible for autosomal dominant progressive hearing loss in a five-generation family. Our results demonstrate that genomic sequencing with a gene panel dedicated to hearing loss is effective for genetic diagnoses in a diverse population. Comprehensive sequencing enables well-informed genetic counseling and clinical management by medical geneticists, otolaryngologists, audiologists, and speech therapists and can be integrated into newborn screening for deafness.

Striatin, a subunit of the serine/threonine phosphatase PP2A, is a core member of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complexes. The protein is expressed in the cell junctions between epithelial cells, which play a role in maintaining cell-cell junctional integrity. Since adhesion is crucial for the function of the mammalian inner ear, we examined the localization and function of striatin in the mouse cochlea. Our results show that in neonatal mice, striatin is specifically expressed in the cell-cell junctions of the inner hair cells, the receptor cells in the mammalian cochlea. Auditory brainstem response measurements of striatin-deficient mice indicated a progressive, high-frequency hearing loss, suggesting that striatin is essential for normal hearing. Moreover, scanning electron micrographs of the organ of Corti revealed a moderate degeneration of the outer hair cells in the middle and basal regions, concordant with the high-frequency hearing loss. Importantly, striatin-deficient mice show aberrant ribbon synapse maturation that may lead to the observed auditory impairment. Together, these results suggest a novel function for striatin in the mammalian auditory system.

Deafness is the most common sensory disability in humans affecting all aspects of life. Approximately 50% of congenital deafness is hereditary and about half of genetic deafness is still unsolved. To date, more than 150 genes are known to cause hearing loss worldwide, with specific genes contributing to deafness in distinct populations. Of these, more than 20 genes are involved in deafness among the Jewish Israeli hearing-impaired population. The most common gene in many worldwide populations, including Israel, is GJB2, which encodes the connexin 26 protein. The second most common gene among Jews is TMC1, with most pathogenic variants found only among Jews of Moroccan origin. Most other pathogenic variants found in the Jewish population are origin-specific and not found in other Jewish ethnic groups or in other worldwide populations. In patients where hereditary deafness is suspected, known variants in the specific ethnicity are routinely examined. In Israel, the GJB2 gene is screened in all cases of hereditary deafness and the TMC1 gene is screened in deaf persons of Jewish Moroccan origin. In cases where no variant is found in a known gene, more comprehensive diagnostic tests should be used. Since the beginning of the deep sequencing era, less than a decade ago, the number of deafness-related genes in the Jewish population has increased by threefold. Identifying the pathogenic variant makes it possible to study molecular pathogenesis, to anticipate and understand the prognosis, to calculate probability of concomitant morbidity, to offer prenatal diagnosis, prevent recurrence of deafness in the family and early rehabilitation. Currently, cochlear implant offers the greatest chance for rehabilitation. The hope is that understanding the molecular pathogenesis will in the future lead to personalized medical treatment. We review the genetics of deafness, with an emphasis on the Jewish population in Israel, new diagnostic methods and suggest a diagnostic algorithm and future treatment methods.

Abnormal formation of otoconia, the biominerals of the inner ear, results in balance disorders. The inertial mass of otoconia activates the underlying mechanosensory hair cells in response to change in head position primarily during linear and rotational acceleration. Otoconia associate exclusively with the two gravity receptors, the utricle and saccule. The cristae sensory epithelium is associated with an extracellular gelatinous matrix known as cupula, equivalent to otoconia. During head rotation, the inertia of endolymphatic fluids within the semicircular canals deflect the cupula of the corresponding crista and activates the underlying mechanosensory hair cells. It is believed that detached free floating otoconia particles travel ectopically to the semicircular canal and cristae and are the culprit for benign paroxysmal positional vertigo (BPPV). The Slc26a4 mouse mutant harbors a missense mutation in pendrin. This mutation leads to impaired transport activity of pendrin and to defects in otoconia composition and distribution. All Slc26a4(loop/loop) homozygous mutant mice are profoundly deaf but show inconsistent vestibular deficiency. A panel of behavioral tests was utilized in order to generate a scoring method for vestibular function. A pathological finding of displaced otoconia was identified consistently in the inner ears of mutant mice with severe vestibular dysfunction. In this work, we present a mouse model with a genetic predisposition for ectopic otoconia with a clinical correlation to BPPV. This unique mouse model can serve as a platform for further investigation of BPPV pathophysiology, and for developing novel treatment approaches in a live animal model.

Background: Many government websites and mobile content are inaccessible for people with vision, hearing, and cognitive disabilities. The COVID-19 pandemic highlighted these disparities when health authority website information, critical in providing resources for curbing the spread of the virus, remained inaccessible for disabled populations. Methods: The Web Content Accessibility Guidelines (WCAG) provide comparatively universally accepted guidelines for website accessibility. We utilized these parameters to examine the number of countries with or without accessible health authority websites. Findings:The resulting data indicate a dearth of countries with websites accessible for persons with disabilities. Fewer than 5% of all countries had health authority websites fully accessible according to the WCAG. Interpretation: Methods of information dissemination must take into consideration individuals with disabilities, particularly in times of global health crises. Government health websites can be considered the most trustworthy sources of preventative healthcare information and therefore must be accessible to all, regardless of ability. Nations should seek to improve website compliance with the WCAG. Funding Statement: The authors report no funding sources. Declaration of Interests: The authors have no other competing interests to declare.

The inner ear is a complex structure responsible for hearing and balance, and organ pathology is associated with deafness and balance disorders. To evaluate the role of epigenomic dynamics, we performed whole genome bisulfite sequencing at key time points during the development and maturation of the mouse inner ear sensory epithelium (SE). Our single-nucleotide resolution maps revealed variations in both general characteristics and dynamics of DNA methylation over time. This allowed us to predict the location of non-coding regulatory regions and to identify several novel candidate regulatory factors, such as Bach2, that connect stage-specific regulatory elements to molecular features that drive the development and maturation of the SE. Constructing in silico regulatory networks around sites of differential methylation enabled us to link key inner ear regulators, such as Atoh1 and Stat3, to pathways responsible for cell lineage determination and maturation, such as the Notch pathway. We also discovered that a putative enhancer, defined as a low methylated region (LMR), can upregulate the GJB6 gene and a neighboring non-coding RNA. The study of inner ear SE methylomes revealed novel regulatory regions in the hearing organ, which may improve diagnostic capabilities, and has the potential to guide the development of therapeutics for hearing loss by providing multiple intervention points for manipulation of the auditory system.

For multiple generations, much of the Arab population of Northern Israel has lived in communities with consanguineous marriages and large families. These communities have been particularly cooperative and informative for understanding the genetics of recessive traits. We studied the genetics of hearing loss in this population, evaluating 168 families from 46 different villages. All families were screened for founder variants by Sanger sequencing and 13 families were further evaluated by sequencing all known genes for hearing loss using our targeted gene panel HEar-Seq. Deafness in 34 of 168 families (20%) was explained by founder variants in GJB2, SLC26A4, or OTOF. In 6 of 13 families (46%) evaluated using HEar-Seq, deafness was explained by damaging alleles of SLC26A4, MYO15A, OTOG, LOXHD1, and TBC1D24. In some genes critical to hearing, it is particularly difficult to interpret variants that might affect splicing, because the genes are not expressed in accessible tissue. To address this problem for possible splice-altering variants of MYO15A, we evaluated minigenes transfected into HEK293 cells. Results revealed exon skipping in the message of MYO15A c.9083+6T>A, and intron retention in the message of MYO15A c.8340G>A, in each case leading to a premature stop and consistent with co-segregation of homozygosity for each variant with hearing loss. The profile of genetics of hearing loss in this population reflects the genetic heterogeneity of hearing loss and the usefulness of synthetic technologies to evaluate potentially causal variants in genes not expressed in accessible tissues.

Background Hearing loss is a major cause of disability worldwide, impairing communication, health, and quality of life. Emerging methods of gene therapy aim to address this morbidity, which can be employed to fix a genetic problem causing hair cell dysfunction and to promote the proliferation of supporting cells in the cochlea and their transdifferentiation into hair cells. In order to extend the applicability of gene therapy, the scientific community is focusing on discovery of additional deafness genes, identifying new genetic variants associated with hearing loss, and revealing new factors that can be manipulated in a coordinated manner to improve hair cell regeneration. Here, we addressed these challenges via genome-wide measurement and computational analysis of transcriptional profiles of mouse cochlea and vestibule sensory epithelium at embryonic day (E)16.5 and postnatal day (P)0. These time points correspond to developmental stages before and during the acquisition of mechanosensitivity, a major turning point in the ability to hear. Results We hypothesized that tissue-specific transcription factors are primarily involved in differentiation, while those associated with development are more concerned with proliferation. Therefore, we searched for enrichment of transcription factor binding motifs in genes differentially expressed between the tissues and between developmental ages of mouse sensory epithelium. By comparison with transcription factors known to alter their expression during avian hair cell regeneration, we identified 37 candidates likely to be important for regeneration. Furthermore, according to our estimates, only half of the deafness genes in human have been discovered. To help remedy the situation, we developed a machine learning classifier that utilizes the expression patterns of genes to predict how likely they are to be undiscovered deafness genes. Conclusions We used a novel approach to highlight novel additional factors that can serve as points of intervention for enhancing hair cell regeneration. Given the similarities between mouse and human deafness, our predictions may be of value in prioritizing future research on novel human deafness genes. Electronic supplementary material The online version of this article (10.1186/s40246-018-0161-7) contains supplementary material, which is available to authorized users.

The peripheral deafness gene Mir96 is expressed in both the cochlea and central auditory circuits. To investigate whether it plays a role in the auditory system beyond the cochlea, we characterized homozygous Dmdo/Dmdo mice with a point mutation in miR-96. Anatomical analysis demonstrated a significant decrease in volume of auditory nuclei in Dmdo/Dmdo mice. This decrease resulted from decreased cell size. Non-auditory structures in the brainstem of Dmdo/Dmdo mice or auditory nuclei of the congenital deaf Cldn14-/- mice revealed no such differences. Electrophysiological analysis in the medial nucleus of the trapezoid body (MNTB) showed that principal neurons fired preferentially multiple action potentials upon depolarization, in contrast to the single firing pattern prevalent in controls and Cldn14-/- mice. Immunohistochemistry identified significantly reduced expression of two predicted targets of the mutated miR-96, Kv1.6 and BK channel proteins, possibly contributing to the electrophysiological phenotype. Microscopic analysis of the Dmdo/Dmdo calyx of Held revealed a largely absent compartmentalized morphology, as judged by SV2-labeling. Furthermore, MNTB neurons from Dmdo/Dmdo mice displayed larger synaptic short-term depression, slower AMPA-receptor decay kinetics and a larger NMDA-receptor component, reflecting a less matured stage. Again, these synaptic differences were not present between controls and Cldn14-/- mice. Thus, deafness genes differentially affect the auditory brainstem. Furthermore, our study identifies miR-96 as an essential gene regulatory network element of the auditory system which is required for functional maturation in the peripheral and central auditory system alike.