A murine model for the del(GJB6-D13S1830) deletion recapitulating the phenotype of human DFNB1 hearing impairment: generation and functional and histopathological study.

Inherited hearing impairment is a remarkably heterogeneous monogenic condition, involving hundreds of genes, most of them with very small (< 1%) epidemiological contributions. The exception is GJB2, the gene encoding connexin-26 and underlying DFNB1, which is the most frequent type of autosomal recessive non-syndromic hearing impairment (ARNSHI) in most populations (up to 40% of ARNSHI cases). DFNB1 is caused by different types of pathogenic variants in GJB2, but also by large deletions that keep the gene intact but remove an upstream regulatory element that is essential for its expression. Such large deletions, found in most populations, behave as complete loss-of-function variants, usually associated with a profound hearing impairment. By using CRISPR-Cas9 genetic edition, we have generated a murine model (Dfnb1em274) that reproduces the most frequent of those deletions, del(GJB6-D13S1830). Dfnb1em274 homozygous mice are viable, bypassing the embryonic lethality of the Gjb2 knockout, and present a phenotype of profound hearing loss (> 90 dB SPL) that correlates with specific structural abnormalities in the cochlea. We show that Gjb2 expression is nearly abolished and its protein product, Cx26, is nearly absent all throughout the cochlea, unlike previous conditional knockouts in which Gjb2 ablation was not obtained in all cell types. The Dfnb1em274 model recapitulates the clinical presentation of patients harbouring the del(GJB6-D13S1830) variant and thus it is a valuable tool to study the pathological mechanisms of DFNB1 and to assay therapies for this most frequent type of human ARNSHI.

A novel variant in the GJB6 gene in a large Chinese family with a unique phenotype of Clouston syndrome.

Clouston syndrome (OMIM #129500), also known as hidrotic ectodermal dysplasia type 2, is a rare autosomal dominant skin disorder. To date, four mutations in the GJB6 gene, G11R, V37E, A88V, and D50N, have been confirmed to cause this condition. In previous studies, the focus has been mainly on gene sequencing, and there has been a lack of research on clinical manifestations and pathogenesis. To confirm the diagnosis of this pedigree at the molecular level and summarize and analyse the clinical phenotype of patients and to provide a basis for further study of the pathogenesis of the disease, we performed whole-exome and Sanger sequencing on a large Chinese Clouston syndrome pedigree. Detailed clinical examination included histopathology, hair microscopy, and scanning electron microscopy. We found a novel heterozygous missense variant (c.134G>C:p.G45A) for Clouston syndrome. We identified a new clinical phenotype involving all nail needling pain in all patients and found a special honeycomb hole structure in the patients' hair under scanning electron microscopy. Our data reveal that a novel variant (c.134G>C:p.G45A) plays a likely pathogenic role in this pedigree and highlight that genetic testing is necessary for the diagnosis of Clouston syndrome.

Recent insights into gap junction biogenesis in the cochlea.

In the cochlea, connexin 26 (Cx26) and connexin 30 (Cx30) co-assemble into two types of homomeric and heteromeric gap junctions between adjacent non-sensory epithelial cells. These channels provide a mechanical coupling between connected cells, and their activity is critical to maintain cochlear homeostasis. Many of the mutations in GJB2 or GJB6, which encode Cx26 and Cx30 in humans, impair the formation of membrane channels and cause autosomal syndromic and non-syndromic hearing loss. Thus, deciphering the connexin trafficking pathways in situ should represent a major step forward in understanding the pathogenic significance of many of these mutations. A growing body of evidence now suggests that Cx26/Cx30 heteromeric and Cx30 homomeric channels display distinct assembly mechanisms. Here, we review the most recent advances that have been made toward unraveling the biogenesis and stability of these gap junctions in the cochlea.

A novel variant in the GJB6 gene in a large Chinese family with a unique phenotype of Clouston syndrome / 医学前沿

Clouston syndrome (OMIM #129500), also known as hidrotic ectodermal dysplasia type 2, is a rare autosomal dominant skin disorder. To date, four mutations in the GJB6 gene, G11R, V37E, A88V, and D50N, have been confirmed to cause this condition. In previous studies, the focus has been mainly on gene sequencing, and there has been a lack of research on clinical manifestations and pathogenesis. To confirm the diagnosis of this pedigree at the molecular level and summarize and analyse the clinical phenotype of patients and to provide a basis for further study of the pathogenesis of the disease, we performed whole-exome and Sanger sequencing on a large Chinese Clouston syndrome pedigree. Detailed clinical examination included histopathology, hair microscopy, and scanning electron microscopy. We found a novel heterozygous missense variant (c.134G>C:p.G45A) for Clouston syndrome. We identified a new clinical phenotype involving all nail needling pain in all patients and found a special honeycomb hole structure in the patients' hair under scanning electron microscopy. Our data reveal that a novel variant (c.134G>C:p.G45A) plays a likely pathogenic role in this pedigree and highlight that genetic testing is necessary for the diagnosis of Clouston syndrome.

Connexin 30 Deficiency Ameliorates Disease Progression at the Early Phase in a Mouse Model of Amyotrophic Lateral Sclerosis by Suppressing Glial Inflammation.

Connexin 30 (Cx30), which forms gap junctions between astrocytes, regulates cell adhesion and migration, and modulates glutamate transport. Cx30 is upregulated on activated astroglia in central nervous system inflammatory lesions, including spinal cord lesions in mutant superoxide dismutase 1 (mSOD1) transgenic amyotrophic lateral sclerosis (ALS) model mice. Here, we investigated the role of Cx30 in mSOD1 mice. Cx30 was highly expressed in the pre-onset stage in mSOD1 mice. mSOD1 mice with knockout (KO) of the Cx30 gene (Cx30KO-mSOD1 mice) showed delayed disease onset and tended to have an extended survival period (log-rank, p = 0.09). At the progressive and end stages of the disease, anterior horn cells were significantly preserved in Cx30KO-mSOD1 mice. In lesions of these mice, glial fibrillary acidic protein/C3-positive inflammatory astroglia were decreased. Additionally, the activation of astrocytes in Cx30KO-mSOD1 mice was reduced compared with mSOD1 mice by gene expression microarray. Furthermore, expression of connexin 43 at the pre-onset stage was downregulated in Cx30KO-mSOD1 mice. These findings suggest that reduced expression of astroglial Cx30 at the early disease stage in ALS model mice protects neurons by attenuating astroglial inflammation.

A Gap-Junction Mutation Reveals That Outer Hair Cell Extracellular Receptor Potentials Drive High-Frequency Cochlear Amplification.

Cochlear amplification enables the enormous dynamic range of hearing through amplifying cochlear responses to low- to moderate-level sounds and compressing them to loud sounds. Amplification is attributed to voltage-dependent electromotility of mechanosensory outer hair cells (OHCs) driven by changing voltages developed across their cell membranes. At low frequencies, these voltage changes are dominated by intracellular receptor potentials (RPs). However, OHC membranes have electrical low-pass filter properties that attenuate high-frequency RPs, which should potentially attenuate amplification of high-frequency cochlear responses and impede high-frequency hearing. We made in vivo intracellular and extracellular electrophysiological measurements from the organ of Corti of male and female mice of the CBA/J strain, with excellent high-frequency hearing, and from the CD-1 mouse strain, which has sensitive hearing below 12 kHz but loses high-frequency hearing within a few weeks postpartum. The CD-1 mouse strain was transfected with an A88V mutation of the connexin 30 gap-junction protein. By blocking the action of the GJ protein to reduce input resistance, the mutation increased the OHC extracellular RP (ERP) magnitude and rescued high-frequency hearing. However, by increasing the organ of Corti resistance, the mutation rescued high-frequency hearing through preserving the OHC extracellular RP (ERP) magnitude. We measured the voltage developed across the basolateral membranes of OHCs, which controls their electromotility, for low- to high-frequency sounds in male and female mice of the CD-1 strain that expressed the A88V mutation. We demonstrate that ERPs, not RPs, drive OHC motility and cochlear amplification at high frequencies because at high frequencies, ERPs are not frequency attenuated, exceed RPs in magnitude, and are appropriately timed to provide cochlear amplification.SIGNIFICANCE STATEMENT Cochlear amplification, which enables the enormous dynamic range of hearing, is attributed to voltage-dependent electromotility of the mechanosensory outer hair cells (OHCs) driven by sound-induced voltage changes across their membranes. OHC intracellular receptor potentials are electrically low-pass filtered, which should hinder high-frequency hearing. We measured the intracellular and extracellular voltages that control OHC electromotility in vivo in a mouse strain with impaired high-frequency hearing. A gap-junction mutation of the strain rescued high-frequency hearing, increased organ of Corti resistance, and preserved large OHC extracellular receptor potentials but reduced OHC intracellular receptor potentials and impaired low-frequency hearing. We concluded intracellular potentials drive OHC motility at low frequencies and extracellular receptor potentials drive OHC motility and cochlear amplification at high frequencies.

Dynamic Spatiotemporal Expression Changes in Connexins of the Developing Primate's Cochlea.

Connexins are gap junction components that are essential for acquiring normal hearing ability. Up to 50% of congenital, autosomal-recessive, non-syndromic deafness can be attributed to variants in GJB2, the gene that encodes connexin 26. Gene therapies modifying the expression of connexins are a feasible treatment option for some patients with genetic hearing losses. However, the expression patterns of these proteins in the human fetus are not fully understood due to ethical concerns. Recently, the common marmoset was used as a primate animal model for the human fetus. In this study, we examined the expression patterns of connexin 26 and connexin 30 in the developing cochlea of this primate. Primate-specific spatiotemporal expression changes were revealed, which suggest the existence of primate-specific control of connexin expression patterns and specific functions of these gap junction proteins. Moreover, our results indicate that treatments for connexin-related hearing loss established in rodent models may not be appropriate for human patients, underscoring the importance of testing these treatments in primate models before applying them in human clinical trials.

Astrocyte-derived neurons provide excitatory input to the adult striatal circuitry.

Astrocytes have emerged as a potential source for new neurons in the adult mammalian brain. In mice, adult striatal neurogenesis can be stimulated by local damage, which recruits striatal astrocytes into a neurogenic program by suppression of active Notch signaling (J. P. Magnusson et al., Science 346, 237-241 [2014]). Here, we induced adult striatal neurogenesis in the intact mouse brain by the inhibition of Notch signaling in astrocytes. We show that most striatal astrocyte-derived neurons are confined to the anterior medial striatum, do not express established striatal neuronal markers, and exhibit dendritic spines, which are atypical for striatal interneurons. In contrast to striatal neurons generated during development, which are GABAergic or cholinergic, most adult astrocyte-derived striatal neurons possess distinct electrophysiological properties, constituting the only glutamatergic striatal population. Astrocyte-derived neurons integrate into the adult striatal microcircuitry, both receiving and providing synaptic input. The glutamatergic nature of these neurons has the potential to provide excitatory input to the striatal circuitry and may represent an efficient strategy to compensate for reduced neuronal activity caused by aging or lesion-induced neuronal loss.

Efnb2 haploinsufficiency induces early gap junction plaque disassembly and endocytosis in the cochlea.

Chromosome 13q deletions encompassing EFNB2, which encodes the transmembrane protein ephrin-B2, are likely to cause syndromic forms of sensorineural hearing loss of unclear origin. Thus, unravelling the pathogenic mechanisms could help to improve therapeutic strategies. In the cochlea, adjacent non-sensory epithelial cells are connected via gap junction channels, the activity of which is critical to maintain cochlear homeostasis. Here we show that ephrin-B2 promotes the assembly of connexin 30 (Cx30) gap junction plaques (GJPs) between adjacent non-sensory Deiters' cells. An in situ proximity ligation assay revealed that ephrin-B2 preferentially interacts with Cx30 in the periphery of the GJPs, i.e. where newly synthesized connexin hemichannels accrue to the GJP. Moreover, we observed that heterozygous mice encoding an Efnb2 null allele display excessive clathrin-mediated internalization of Cx30 GJPs in early postnatal stages. Finally, an in vitro organotypic assay revealed that ectopic activation of ephrin-B2 reverse signalling promotes the internalization of Cx30 GJPs. These data argue in favor of a cell-autonomous, Eph receptor-independent role of ephrin-B2 in the assembly of Cx30 GJPs. According to recent observations, early GJP degradation could certainly play a role in the pathogenic process leading to progressive sensorineural hearing loss due to Efnb2/EFNB2 haploinsufficiency.

Astroglial Cx30 differentially impacts synaptic activity from hippocampal principal cells and interneurons.

Astrocytes play important roles in brain function via dynamic structural and functional interactions with neurons. Yet the underlying mechanisms remain poorly defined. A typical feature of astrocytes is the high expression of connexins, which mediate their extensive intercellular communication and regulate their structural properties. In particular, connexin 30 (Cx30), one of the two connexins abundantly expressed by astrocytes, was recently shown to be a critical regulator of excitatory synaptic transmission by controlling the astroglial coverage of synapses. However, the role of Cx30 in the regulation of inhibitory synaptic transmission and excitatory/inhibitory balance remains elusive. Here, we investigated the role of astroglial Cx30 on the electrophysiological and morphological properties of five classes of hippocampal CA1 stratum oriens and pyramidale neurons, defined by the unsupervised Ward's clustering. Using Cx30 knockout mice, we found that Cx30 alters specific properties of some subsets of CA1 interneurons, such as resting membrane potential and sag ratio, while other parameters, such as action potential threshold and saturation frequency, were more frequently altered among the different classes of neurons. The excitation-inhibition balance was also differentially and selectively modulated among the different neuron subtypes. Only slight morphological differences were observed on reconstructed neurons. Altogether, these data indicate that Cx30 differentially alters the electrophysiological and morphological properties of hippocampal cell populations, and modulates both their excitatory and inhibitory inputs. Astrocytes, via Cx30, are thus active modulators of both excitatory and inhibitory synapses in the hippocampus.

Is it time to report carrier state for recessive disorders in every microarray analysis?-A pilot model based on hearing loss genes deletions.

This study aimed to examine the implications of reporting heterozygous losses of recessive genes in Chromosomal Microarray Analysis (CMA), based on the incidence of microdeletions of three common hearing impairment genes in the local cohort and the prevalence of sequence variants in these genes in worldwide databases. Prevalence of heterozygous microdeletions in OTOA and STRC genes, as well as deletions in the DFNB1 locus encompassing GJB6 gene, was determined using electronic database of Rabin Medical Center. ClinVar archive and Deafness Variation Database were used to generate a list of clinically significant sequence variants in these three genes, as well as GJB2 gene, and estimation of the frequency of sequence variants was performed. Of the 19,189 CMA tests were performed in our laboratory, 107 STRC microdeletions were found (0.56%), followed in frequency by OTOA deletions (39, 0.2%), and DFNB1 locus deletions (10, 0.05%). The estimated risk for a hearing loss in the examined individual carrying the microdeletion was estimated as 0.11-0.67% for STRC, 0.016-0.13% for OTOA, and 1.9-7.5% in the DFNB1 locus (including double heterozygocity with GJB2 clinically significant sequence variants). The risks were higher in specific populations. In conclusion, we believe that that general decision whether to report or to disregard such incidental findings cannot be part of a uniform policy, but rather based on a detailed evaluation of origin-specific variants for each gene, with a careful consideration and discussion whether to include the microdeletion in the final report for each patient.

Mutant Cx30-A88V mice exhibit hydrocephaly and sex-dependent behavioral abnormalities, implicating a functional role for Cx30 in the brain.

Connexin 30 (Cx30; also known as Gjb6 when referring to the mouse gene) is expressed in ependymal cells of the brain ventricles, in leptomeningeal cells and in astrocytes rich in connexin 43 (Cx43), leading us to question whether patients harboring GJB6 mutations exhibit any brain anomalies. Here, we used mice harboring the human disease-associated A88V Cx30 mutation to address this gap in knowledge. Brain Cx30 levels were lower in male and female Cx30A88V/A88V mice compared with Cx30A88V/+ and Cx30+/+ mice, whereas Cx43 levels were lower only in female Cx30 mutant mice. Characterization of brain morphology revealed a disrupted ependymal cell layer, significant hydrocephalus and enlarged ventricles in 3- to 6-month-old adult male and female Cx30A88V/A88V mice compared with Cx30A88V/+ or Cx30+/+ sex-matched littermate mice. To determine the functional significance of these molecular and morphological changes, we investigated a number of behavioral activities in these mice. Interestingly, only female Cx30A88V/A88V mice exhibited abnormal behavior compared with all other groups. Cx30A88V/A88V female mice demonstrated increased locomotor and exploratory activity in both the open field and the elevated plus maze. They also exhibited dramatically reduced ability to learn the location of the escape platform during Morris water maze training, although they were able to swim as well as other genotypes. Our findings suggest that the homozygous A88V mutation in Cx30 causes major morphological changes in the brain of aging mice, possibly attributable to an abnormal ependymal cell layer. Remarkably, these changes had a more pronounced consequence for cognitive function in female mice, which is likely to be linked to the dysregulation of both Cx30 and Cx43 levels in the brain.

Lights and Shadows in the Genetics of Syndromic and Non-Syndromic Hearing Loss in the Italian Population.

Hearing loss (HL), both syndromic (SHL) and non-syndromic (NSHL), is the most common sensory disorder, affecting ~460 million people worldwide. More than 50% of the congenital/childhood cases are attributable to genetic causes, highlighting the importance of genetic testing in this class of disorders. Here we applied a multi-step strategy for the molecular diagnosis of HL in 125 patients, which included: (1) an accurate clinical evaluation, (2) the analysis of GJB2, GJB6, and MT-RNR1 genes, (3) the evaluation STRC-CATSPER2 and OTOA deletions via Multiplex Ligation Probe Amplification (MLPA), (4) Whole Exome Sequencing (WES) in patients negative to steps 2 and 3. Our approach led to the characterization of 50% of the NSHL cases, confirming both the relevant role of the GJB2 (20% of cases) and STRC deletions (6% of cases), and the high genetic heterogeneity of NSHL. Moreover, due to the genetic findings, 4% of apparent NSHL patients have been re-diagnosed as SHL. Finally, WES characterized 86% of SHL patients, supporting the role of already know disease-genes. Overall, our approach proved to be efficient in identifying the molecular cause of HL, providing essential information for the patients' future management.

GJB2 and GJB6 Genetic Variant Curation in an Argentinean Non-Syndromic Hearing-Impaired Cohort.

Genetic variants in GJB2 and GJB6 genes are the most frequent causes of hereditary hearing loss among several deaf populations worldwide. Molecular diagnosis enables proper genetic counseling and medical prognosis to patients. In this study, we present an update of testing results in a cohort of Argentinean non-syndromic hearing-impaired individuals. A total of 48 different sequence variants were detected in genomic DNA from patients referred to our laboratory. They were manually curated and classified based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology ACMG/AMP standards and hearing-loss-gene-specific criteria of the ClinGen Hearing Loss Expert Panel. More than 50% of sequence variants were reclassified from their previous categorization in ClinVar. These results provide an accurately interpreted set of variants to be taken into account by clinicians and the scientific community, and hence, aid the precise genetic counseling to patients.

Altered Gap Junction Network Topography in Mouse Models for Human Hereditary Deafness.

Anisotropic gap junctional coupling is a distinct feature of astrocytes in many brain regions. In the lateral superior olive (LSO), astrocytic networks are anisotropic and oriented orthogonally to the tonotopic axis. In CaV1.3 knock-out (KO) and otoferlin KO mice, where auditory brainstem nuclei are deprived from spontaneous cochlea-driven neuronal activity, neuronal circuitry is disturbed. So far it was unknown if this disturbance is also accompanied by an impaired topography of LSO astrocyte networks. To answer this question, we immunohistochemically analyzed the expression of astrocytic connexin (Cx) 43 and Cx30 in auditory brainstem nuclei. Furthermore, we loaded LSO astrocytes with the gap junction-permeable tracer neurobiotin and assessed the network shape and orientation. We found a strong elevation of Cx30 immunoreactivity in the LSO of CaV1.3 KO mice, while Cx43 levels were only slightly increased. In otoferlin KO mice, LSO showed a slight increase in Cx43 as well, whereas Cx30 levels were unchanged. The total number of tracer-coupled cells was unaltered and most networks were anisotropic in both KO strains. In contrast to the WTs, however, LSO networks were predominantly oriented parallel to the tonotopic axis and not orthogonal to it. Taken together, our data demonstrate that spontaneous cochlea-driven neuronal activity is not required per se for the formation of anisotropic LSO astrocyte networks. However, neuronal activity is required to establish the proper orientation of networks. Proper formation of LSO astrocyte networks thus necessitates neuronal input from the periphery, indicating a critical role of neuron-glia interaction during early postnatal development in the auditory brainstem.

Polydisperse molecular architecture of connexin 26/30 heteromeric hemichannels revealed by atomic force microscopy imaging.

Connexin (Cx) protein forms hemichannels and gap junctional channels, which play diverse and profound roles in human physiology and diseases. Gap junctions are arrays of intercellular channels formed by the docking of two hemichannels from adjacent cells. Each hexameric hemichannel contains the same or different Cx isoform. Although homomeric Cxs forms have been largely described functionally and structurally, the stoichiometry and arrangement of heteromeric Cx channels remain unknown. The latter, however, are widely expressed in human tissues and variation might have important implications on channel function. Investigating properties of heteromeric Cx channels is challenging considering the high number of potential subunit arrangements and stoichiometries, even when only combining two Cx isoforms. To tackle this problem, we engineered an HA tag onto Cx26 or Cx30 subunits and imaged hemichannels that were liganded by Fab-epitope antibody fragments via atomic force microscopy. For Cx26-HA/Cx30 or Cx30-HA/Cx26 heteromeric channels, the Fab-HA binding distribution was binomial with a maximum of three Fab-HA bound. Furthermore, imaged Cx26/Cx30-HA triple liganded by Fab-HA showed multiple arrangements that can be derived from the law of total probabilities. Atomic force microscopy imaging of ringlike structures of Cx26/Cx30-HA hemichannels confirmed these findings and also detected a polydisperse distribution of stoichiometries. Our results indicate a dominant subunit stoichiometry of 3Cx26:3Cx30 with the most abundant subunit arrangement of Cx26-Cx26-Cx30-Cx26-Cx30-Cx30. To our knowledge, this is the first time that the molecular architecture of heteromeric Cx channels has been revealed, thus providing the basis to explore the functional effect of these channels in biology.

A recurrent mutation of GJB6 in a big Chinese family with Hidrotic ectodermal dysplasia.

Hidrotic ectodermal dysplasia (HED) is a rare inherited syndrome characterised by nail dystrophy, palmoplantar hyperkeratosis and alopecia. Four mutations (p.G11R, p.A88V, p.V37E and p.D50N) in gap junction beta 6 (GJB6) gene, which codes connexin30 protein, have been found to cause HED in different populations. Here, we reported a big Chinese family in which 24 patients over five generations were suffered with HED. Sequence analysis identified all 24 patients carry a recurrent missense mutation c.263C > T (p.A88V) in GJB6. Our results reveal gene testing of GJB6 is important for diagnosis, prenatal diagnosis and future gene treatment of HED.

A potent antagonist antibody targeting connexin hemichannels alleviates Clouston syndrome symptoms in mutant mice.

BACKGROUND: Numerous currently incurable human diseases have been causally linked to mutations in connexin (Cx) genes. In several instances, pathological mutations generate abnormally active Cx hemichannels, referred to also as "leaky" hemichannels. The goal of this study was to assay the in vivo efficacy of a potent antagonist antibody targeting Cx hemichannels. METHODS: We employed the antibody to treat Cx30A88V/A88V adult mutant mice, the only available animal model of Clouston syndrome, a rare orphan disease caused by Cx30 p.A88V leaky hemichannels. To gain mechanistic insight into antibody action, we also performed patch clamp recordings, Ca2+ imaging and ATP release assay in vitro. FINDINGS: Two weeks of antibody treatment sufficed to repress cell hyperproliferation in skin and reduce hypertrophic sebaceous glands (SGs) to wild type (wt) levels. These effects were obtained whether mutant mice were treated topically, by application of an antibody cream formulation, or systemically, by intraperitoneal antibody injection. Experiments with mouse primary keratinocytes and HaCaT cells revealed the antibody blocked Ca2+ influx and diminished ATP release through leaky Cx30 p.A88V hemichannels. INTERPRETATION: Our results show anti-Cx antibody treatment was effective in vivo and sufficient to counteract the effects of pathological connexin expression in Cx30A88V/A88V mice. In vitro experiments suggest antibodies gained control over leaky hemichannels and contributed to restoring epidermal homeostasis. Therefore, regulating cell physiology by antibodies targeting the extracellular domain of Cxs may enforce an entirely new therapeutic strategy. These findings support the further development of antibodies as drugs to address unmet medical needs for Cx-related diseases. FUND: Fondazione Telethon, GGP19148; University of Padova, SID/BIRD187130; Consiglio Nazionale delle Ricerche, DSB.AD008.370.003\TERABIO-IBCN; National Science Foundation of China, 31770776; Science and Technology Commission of Shanghai Municipality, 16DZ1910200.

Frequency of Usher gene mutations in non-syndromic hearing loss: higher variability of the Usher phenotype.

Non-syndromic hearing loss (NSHL) is characterized by a vast genetic heterogeneity; some syndromic forms as Usher syndrome (USH) have onset as isolated deafness and then evolve later in life. We developed an NGS targeted gene-panel containing 59 genes and a customized bioinformatic pipeline for the analysis of DNA samples from clinically highly selected subjects with sensorineural hearing loss, previously resulted negative for GJB2 mutations/GJB6 deletions. Among the 217 tested subjects, 24 (11.1%) were found to carry mutations in genes involved both in NSHL and USH. For 6 out of 24 patients a diagnosis of USH was performed. Eleven subjects out of 24 had hearing loss without vestibular or ocular dysfunction and, due to their young age, it was not possible to establish whether their phenotype could be NSHL or USH. Seven subjects were diagnosed with NSHL, due to their age and phenotype. A total of 41 likely pathogenic/pathogenic mutations were identified, among which 17 novel ones. We report a high frequency of mutations in genes involved both in NSHL and in USH in a cohort of individuals tested for seemingly isolated deafness. Our data also highlight a wider than expected phenotypic variability in the USH phenotype.