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Although variant alleles of hundreds of genes are associated with sensorineural deafness in children, the genes and alleles involved remain largely unknown in the Sub-Saharan regions of Africa. We ascertained 56 small families mainly of Yoruba ethno-lingual ancestry in or near Ibadan, Nigeria, that had at least one individual with nonsyndromic, severe-to-profound, prelingual-onset, bilateral hearing loss not attributed to nongenetic factors. We performed a combination of exome and Sanger sequencing analyses to evaluate both nuclear and mitochondrial genomes. No biallelic pathogenic variants were identified in GJB2, a common cause of deafness in many populations. Potential causative variants were identified in genes associated with nonsyndromic hearing loss (CIB2, COL11A1, ILDR1, MYO15A, TMPRSS3, and WFS1), nonsyndromic hearing loss or Usher syndrome (CDH23, MYO7A, PCDH15, and USH2A), and other syndromic forms of hearing loss (CHD7, OPA1, and SPTLC1). Several rare mitochondrial variants, including m.1555A>G, were detected in the gene MT-RNR1 but not in control Yoruba samples. Overall, 20 (33%) of 60 independent cases of hearing loss in this cohort of families were associated with likely causal variants in genes reported to underlie deafness in other populations. None of these likely causal variants were present in more than one family, most were detected as compound heterozygotes, and 77% had not been previously associated with hearing loss. These results indicate an unusually high level of genetic heterogeneity of hearing loss in Ibadan, Nigeria and point to challenges for molecular genetic screening, counseling, and early intervention in this population.

Mutations of coding regions and splice sites of SLC26A4 cause Pendred syndrome and nonsyndromic recessive hearing loss DFNB4. SLC26A4 encodes pendrin, a transmembrane exchanger of anions and bases. The mutant SLC26A4 phenotype is characterized by inner ear malformations, including an enlarged vestibular aqueduct (EVA), incomplete cochlear partition type II and modiolar hypoplasia, progressive and fluctuating hearing loss, and vestibular dysfunction. A thyroid iodine organification defect can lead to multinodular goiter and distinguishes Pendred syndrome from DFNB4. Pendred syndrome and DFNB4 are each inherited as an autosomal recessive trait caused by biallelic mutations of SLC26A4 (M2). However, there are some EVA patients with only one detectable mutant allele (M1) of SLC26A4. In most European-Caucasian M1 patients, there is a haplotype that consists of 12 variants upstream of SLC26A4, called CEVA (Caucasian EVA), which acts as a pathogenic recessive allele in trans to mutations affecting the coding regions or splice sites of SLC26A4. This combination of an M1 genotype with the CEVA haplotype is associated with a less severe phenotype than the M2 genotype. The phenotype in EVA patients with no mutant alleles of SLC26A4 (M0) has a very low recurrence probability and is likely to be caused by other factors.

Human alphapapillomaviruses (αHPV) infect genital mucosa, and a high-risk subset is a necessary cause of cervical cancer. Licensed L1 virus-like particle (VLP) vaccines offer immunity against the nine most common αHPV associated with cervical cancer and genital warts. However, vaccination with an αHPV L2-based multimer vaccine, α11-88x5, protected mice and rabbits from vaginal and skin challenge with diverse αHPV types. While generally clinically inapparent, human betapapillomaviruses (βHPV) are possibly associated with cutaneous squamous cell carcinoma (CSCC) in epidermodysplasia verruciformis (EV) and immunocompromised patients. Here we show that α11-88x5 vaccination protected wild type and EV model mice against HPV5 challenge. Passive transfer of antiserum conferred protection independently of Fc receptors (FcR) or Gr-1+ phagocytes. Antisera demonstrated robust antibody titers against ten βHPV by L1/L2 VLP ELISA and neutralized and protected against challenge by 3 additional βHPV (HPV49/76/96). Thus, unlike the licensed vaccines, α11-88x5 vaccination elicits broad immunity against αHPV and βHPV.

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.

The TMC genes encode a set of homologous transmembrane proteins whose functions are not well understood. Biallelic mutations in either TMC6 or TMC8 are detected in more than half of cases of the pre-malignant skin disease epidermodysplasia verruciformis (EV). It is controversial whether EV induced by mutations in TMC6 or TMC8 originates from keratinocyte or lymphocyte defects. Quantification of TMC6 and TMC8 RNA levels in various organs revealed that lymphoid tissues have the highest levels of expression of both genes, and custom antibodies confirmed protein expression in mouse lymphocytes. To study the function of these proteins we generated mice with targeted deletion mutant alleles of Tmc6 or Tmc8. Either TMC6 or TMC8 deficiency induced a reduction in apparent molecular weight and/or amount of the other TMC molecule. Co-immunoprecipitation experiments indicated that TMC6 and TMC8 formed a protein complex in mouse and human T cells. MS and biochemical analysis demonstrated that TMC6 and TMC8 additionally interacted with the CIB1 protein to form TMC6-TMC8-CIB1 trimers. We demonstrated that TMC6 and TMC8 regulated CIB1 levels by protecting CIB1 from ubiquitination and proteasomal degradation. Reciprocally, CIB1 was needed for stabilizing TMC6 and TMC8 levels. These results suggest why inactivating mutations in any of the three human genes leads to similar clinical presentations. We also demonstrated that TMC6 and TMC8 levels are drastically lower and the proteins are less active in regulating CIB1 in keratinocytes than in T cells. Our study suggests that defects in lymphocytes may contribute to the etiology and pathogenesis of EV.

Usher syndrome has been historically categorized into one of three classical types based on the patient phenotype. However, the vestibular phenotype does not infallibly predict which Usher genes are mutated. Conversely, the Usher syndrome genotype is not sufficient to reliably predict vestibular function. Here we present a characterization of the vestibular phenotype of 90 patients with clinical presentation of Usher syndrome (59 females), aged 10.9 to 75.5 years, with genetic variants in eight Usher syndromic genes and expand the description of atypical Usher syndrome. We identified unexpected horizontal semicircular canal reactivity in response to caloric and rotational stimuli in 12.5% (3 of 24) and 41.7% (10 of 24), respectively, of our USH1 cohort. These findings are not consistent with the classical phenotypic definition of vestibular areflexia in USH1. Similarly, 17% (6 of 35) of our cohort with USH2A mutations had saccular dysfunction as evidenced by absent cervical vestibular evoked myogenic potentials in contradiction to the classical assumption of normal vestibular function. The surprising lack of consistent genotypic to vestibular phenotypic findings as well as no clear vestibular phenotypic patterns among atypical USH cases, indicate that even rigorous vestibular phenotyping data will not reliably differentiate the three USH types.

Systemically administered aminoglycoside antibiotics can enter inner ear hair cells and trigger apoptosis. However, the in vivo route(s) by which aminoglycoside antibiotics enter hair cells remains controversial. Aminoglycosides can enter mouse hair cells by endocytosis or by permeation through transmembrane ion channels such as sensory mechanoelectrical transduction (MET) channels, transient receptor potential (TRP) channels, P2X channels, Piezo2-containing ion channels, or a combination of these routes. Transmembrane channel-like 1 (TMC1) and TMC2 are essential for sensory MET and appear to be the pore-forming components of sensory MET channels. The present study tested the hypothesis that systemic fluorescent gentamicin enters mouse hair cells predominantly through sensory MET channels. We employed Tmc1(Δ), Tmc2(Δ), and Tmc1::mCherry mice. In Tmc1::mCherry mice, the transgene was integrated on the X chromosome, resulting in mosaic expression of TMC1-mCherry in the hair cells of female heterozygous mice. After systemic administration of gentamicin-conjugated Texas Red (GTTR) into Tmc1(Δ);Tmc2(Δ) mice and wild-type mice at postnatal day 4 (P4), robust GTTR fluorescence was detected in wild-type hair cells, whereas little or no GTTR fluorescence was detected in Tmc1(Δ);Tmc2(Δ) hair cells. When GTTR was injected into developing mice at P0, P2, P4, or P6, the GTTR fluorescent intensity gradually increased from P0 to P4 in wild-type hair cells, whereas the intensity was stably low from P0 through P6 in Tmc1(Δ);Tmc2(Δ) hair cells. The increase in the GTTR intensity coincided with the spatio-temporal onset of sensory MET in wild-type hair cells. In Tmc1::mCherry cochleae, only hair cells that showed a significant uptake of systemic GTTR took up FM1-43. Transmission electron microscopy could detect no disruption of normal endocytosis at the apical surface of Tmc1(Δ);Tmc2(Δ) hair cells in vitro. These results provide substantial novel evidence that in vivo gentamicin enters neonatal mouse hair cells predominantly through sensory MET channels and not via endocytosis.

Dizziness and hearing loss are among the most common disabilities. Many forms of hereditary balance and hearing disorders are caused by abnormal development of stereocilia, mechanosensory organelles on the apical surface of hair cells in the inner ear. The deaf whirler mouse, a model of human Usher syndrome (manifested by hearing loss, dizziness, and blindness), has a recessive mutation in the whirlin gene, which renders hair cell stereocilia short and dysfunctional. In this study, wild-type whirlin cDNA was delivered to the inner ears of neonatal whirler mice using adeno-associated virus serotype 2/8 (AAV8-whirlin) by injection into the posterior semicircular canal. Unilateral whirlin gene therapy injection was able to restore balance function as well as improve hearing in whirler mice for at least 4 months. Our data indicate that gene therapy is likely to become a treatment option for hereditary disorders of balance and hearing.

Usher syndrome type I (USH1) is characterized by deafness, vestibular areflexia, and progressive retinal degeneration. The protein-truncating p.Arg245* founder variant of PCDH15 (USH1F) has an ~2% carrier frequency amongst Ashkenazi Jews accounts for ~60% of their USH1 cases. Here, longitudinal phenotyping in 13 USH1F individuals revealed progressive retinal degeneration, leading to severe vision loss with macular atrophy by the sixth decade. Half of the affected individuals were legally blind by their mid-50s. The mouse Pcdh15R250X variant is equivalent to human p.Arg245*. Homozygous Pcdh15R250X mice also have visual deficits and aberrant light-dependent translocation of the phototransduction cascade proteins, arrestin, and transducin. Retinal pigment epithelium (RPE)-specific retinoid cycle proteins, RPE65 and CRALBP, were also reduced in Pcdh15R250X mice, indicating a dual role for protocadherin-15 in photoreceptors and RPE. Exogenous 9-cis retinal improved ERG amplitudes in Pcdh15R250X mice, suggesting a basis for a clinical trial of FDA-approved retinoids to preserve vision in USH1F patients.

Incomplete partition type II (IP-II) is frequently identified in ears with SLC26A4 mutations. Cochleae with IP-II are generally observed to have 1½ turns; the basal turns are normally formed, and the apical turn is dilated or cystic. The objective of this study was to characterize the pathomorphogenesis of the IP-II cochlear anomaly in Slc26a4-null mice. Otic capsules were dissected from Slc26a4(Δ/+) and Slc26a4(Δ/Δ) mice at 1 and 8 days of age and at 1 and 3 months of age. X-ray micro-computed tomography was used to image samples. We used a multiplanar view and three-dimensional reconstructed models to calculate the cochlear duct length, cochlear turn rotation angle, and modiolus tilt angle. The number of inner hair cells was counted, and the length of the cochlear duct was measured in a whole-mount preparation of the membranous labyrinth. X-ray micro-computed tomography mid-modiolar planar views demonstrated cystic apical turns in Slc26a4(Δ/Δ) mice resulting from the loss or deossification of the interscalar septum, which morphologically resembles IP-II in humans. Planes vertical to the modiolus showed a similar mean rotation angle between Slc26a4(Δ/+) and Slc26a4(Δ/Δ) mice. In contrast, the mean cochlear duct length and mean number of inner hair cells in Slc26a4(Δ/Δ) mice were significantly smaller than in Slc26a4(Δ/+) mice. In addition, there were significant differences in the mean tilt angle and mean width of the modiolus. Our analysis of Slc26a4-null mice suggests that IP-II in humans reflects loss or deossification of the interscalar septum but not a decreased number of cochlear turns.

Usher syndrome type I (USH1) is characterized by congenital deafness, vestibular areflexia, and progressive retinal degeneration with age. The protein-truncating p.Arg245* founder variant of PCDH15 has an ~2% carrier frequency among Ashkenazi Jews, accounting for nearly 60% of their USH1 cases. Here, longitudinal ocular phenotyping in thirteen USH1F individuals harboring the p.Arg245* variant revealed progressive retinal degeneration, leading to severe loss of vision with macular atrophy by the sixth decade. Half of the affected individuals met either the visual acuity or visual field loss definition for legal blindness by the middle of their fifth decade of life. Mice homozygous for p.Arg250* (Pcdh15R250X; equivalent to human p.Arg245*) also have early visual deficits evaluated using electroretinography. Light-dependent translocation of phototransduction cascade proteins, arrestin and transducin, was found to be impaired in Pcdh15R250X mice. Retinal pigment epithelium-(RPE) specific visual retinoid cycle proteins, RPE65 which converts all-trans retinoids to 11-cis retinoids and CRALBP that transports retinoids, and key retinoid levels were also reduced in Pcdh15R250X mice, suggesting a dual role for protocadherin-15 in photoreceptors and RPE. Administration of exogenous 9-cis retinal, an analog of the naturally occurring 11-cis retinal, improved ERG amplitudes in these mutant mice, suggesting a basis for a clinical trial of exogenous FDA approved retinoids to preserve vision in USH1F patients.SummaryIn a preclinical setting studying exogenous retinoids using a novel Usher syndrome mouse model, we describe a potential therapy to treat PCDH15-mediated visual dysfunction.

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

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.

Usher syndrome has classically been described as a combination of hearing loss and rod-cone dystrophy; vestibular dysfunction is present in many patients. Three distinct clinical subtypes were documented in the late 1970s. Genotyping efforts have led to the identification of several genes associated with the disease. Recent literature has seen multiple publications referring to "atypical" Usher syndrome presentations. This manuscript reviews the molecular etiology of Usher syndrome, highlighting rare presentations and molecular causes. Reports of "atypical" disease are summarized noting the wide discrepancy in the spectrum of phenotypic deviations from the classical presentation. Guidelines for establishing a clear nomenclature system are suggested.

OBJECTIVE: To characterize the audiometric phenotype of autosomal dominant DFNA34 hearing loss (HL) caused by a missense substitution in the NLRP3 gene. NLRP3 encodes a critical component of the NLRP3 inflammasome that is activated in innate immune responses. STUDY DESIGN: This study was conducted under protocol 01-DC-0229 approved by the NIH Combined Neurosciences IRB. We performed medical and developmental history interviews and physical and audiological examinations of affected individuals with DFNA34 HL caused by the p.Arg918Gln mutation of NLRP3. We retrospectively reviewed audiological reports, when available, from other health care centers. SETTING: Federal biomedical research facility. SUBJECTS: Eleven members of a North American family segregating p.Arg918Gln. MAIN OUTCOME MEASURES: Pure-tone thresholds, rates of pure-tone threshold progression, and speech discrimination scores. RESULTS: Eight subjects had bilateral sensorineural HL with an onset in the late 2nd to 4th decade of life. Slowly progressive HL initially primarily affected high frequencies. Low and middle frequencies were affected with advancing age, resulting in moderate HL with a downsloping audiometric configuration. The average annual threshold deterioration was 0.9 to 1.5 dB/year. Speech recognition scores ranging from 60-100% were consistent with cochlear, but not retrocochlear, etiology. Three subjects (16, 22, and 32 years old) had normal hearing thresholds. CONCLUSIONS: DFNA34 HL has an onset during early adulthood and progresses approximately 1.2 dB/year.

Background Enlargement of the vestibular aqueduct (EVA) is the most common radiological abnormality in children with sensorineural hearing loss. Mutations in coding regions and splice sites of the SLC26A4 gene are often detected in Caucasians with EVA. Approximately one-fourth of patients with EVA have two mutant alleles (M2), one-fourth have one mutant allele (M1) and one-half have no mutant alleles (M0). The M2 genotype is correlated with a more severe phenotype. Methods We performed genotype–haplotype analysis and massively parallel sequencing of the SLC26A4 region in patients with M1 EVA and their families. Results We identified a shared novel haplotype, termed CEVA (Caucasian EVA), composed of 12 uncommon variants upstream of SLC26A4 . The presence of the CEVA haplotype on seven of ten ‘mutation-negative’ chromosomes in a National Institutes of Health M1 EVA discovery cohort and six of six mutation-negative chromosomes in a Danish M1 EVA replication cohort is higher than the observed prevalence of 28 of 1006 Caucasian control chromosomes (p Conclusions The CEVA haplotype causally contributes to most cases of Caucasian M1 EVA and, possibly, some cases of M0 EVA. The CEVA haplotype of SLC26A4 defines the most common allele associated with hereditary hearing loss in Caucasians. The diagnostic yield and prognostic utility of sequence analysis of SLC26A4 exons and splice sites will be markedly increased by addition of testing for the CEVA haplotype.

Sensorineural hearing loss can result from dysfunction of the inner ear, auditory nerve, or auditory pathways in the central nervous system. Sensorineural hearing loss can be associated with age, exposure to ototoxic drugs or noise, or mutations in nuclear or mitochondrial genes. However, it is idiopathic in some patients. Although these disorders are mainly caused by dysfunction of the inner ear, little of the pathophysiology in sensorineural hearing loss is known due to inaccessibility of the living human inner ear for biopsy and pathological analysis. The inner ear has previously been thought of as an immune-privileged organ. We recently showed that a missense mutation of the NLRP3 gene is associated with autosomal-dominant sensorineural hearing loss with cochlear autoinflammation in two unrelated families. NLRP3 encodes the NLRP3 protein, a key component of the NLRP3 inflammasome that is expressed in immune cells, including monocytes and macrophages. Gain-of-function mutations of NLRP3 cause abnormal activation of the NLRP3 inflammasome leading to IL-1β secretion in a spectrum of autosomal dominant systemic autoinflammatory phenotypes termed cryopyrin-associated periodic syndromes. The affected subjects of our two families demonstrated atypical phenotypes compared with those reported for subjects with cryopyrin-associated periodic syndromes. These observations led us to test the hypothesis that macrophage/monocyte-like cells in the cochlea can mediate local autoinflammation via activation of the NLRP3 inflammasome. The inflammasome can indeed be activated in macrophage/monocyte-like cells of the mouse cochlea, with secretion of IL-1β. The macrophage/monocyte-like cells in the cochlea were also found to be associated with hearing loss in a Slc26a4-insufficient mouse model of human deafness. This review addresses our understanding of genetic hearing loss mediated by autoinflammation and macrophage/monocyte-like cells in the cochlea.

PurposeProper interpretation of genomic variants is critical to successful medical decision making based on genetic testing results. A fundamental prerequisite to accurate variant interpretation is the clear understanding of the clinical validity of gene-disease relationships. The Clinical Genome Resource (ClinGen) has developed a semi-quantitative framework to assign clinical validity to gene-disease relationships.MethodsThe ClinGen Hearing Loss Gene Curation Expert Panel (HL GCEP) uses this framework to perform evidence-based curations of genes present on testing panels from 17 clinical laboratories in the Genetic Testing Registry. The HL GCEP curated and reviewed 142 genes and 164 gene-disease pairs, including 105 nonsyndromic and 59 syndromic forms of hearing loss.ResultsThe final outcome included 82 Definitive (50%), 12 Strong (7%), 25 Moderate (15%), 32 Limited (20%), 10 Disputed (6%), and 3 Refuted (2%) classifications. The summary of each curation is date stamped with the HL GCEP approval, is live, and will be kept up-to-date on the ClinGen website (https://search.clinicalgenome.org/kb/gene-validity).ConclusionThis gene curation approach serves to optimize the clinical sensitivity of genetic testing while reducing the rate of uncertain or ambiguous test results caused by the interrogation of genes with insufficient evidence of a disease link.

Objectives/Hypothesis To characterize the severity and natural history of hearing loss, and the prevalence of having a cochlear implant in a maturing cohort of individuals with enlarged vestibular aqueduct (EVA) and zero or one mutant allele of SLC26A4. Study Design Prospective cohort study of subjects ascertained between 1998 and 2015 at the National Institutes of Health Clinical Center. Methods Study subjects were 127 individuals (median age, 8 years; range, 0–59 years) with EVA in at least one ear. Results Ears with EVA and zero or one mutant allele of SLC26A4 had mean 0.5/1/2/4-kHz pure-tone averages of 62.6 and 52.9 dB HL, respectively, in contrast to EVA ears with two mutant alleles of SLC26A4 (88.1 dB HL; P < .01). This association was independent of age, sex, or side of EVA (P < .001). Natural history of hearing loss was not associated with number of mutant alleles (P = .94). The prevalence of having a cochlear implant was nine (12%) of 76, two (13%) of 15, and 12 (38%) of 32 subjects with zero, one, and two mutant alleles, respectively (P = .00833). This association was not independent (P = .534) but reflected underlying correlations with age at time of first audiogram (P = .003) or severity of hearing loss (P = .000). Conclusions Ears with EVA and zero or one mutant allele of SLC26A4 have less severe hearing loss, no difference in prevalence of fluctuation, and a lower prevalence of cochlear implantation in comparison to ears with two mutant alleles of SLC26A4. Level of Evidence NA Laryngoscope, 127:E238–E243, 2017

SLC26A4 mutations cause fluctuating and progressive hearing loss associated with enlargement of the vestibular aqueduct (EVA). SLC26A4 encodes a transmembrane anion exchanger called pendrin expressed in nonsensory epithelial cells of the lateral wall of cochlea, vestibular organs and endolymphatic sac. We previously described a transgenic mouse model of EVA with doxycycline (dox)-inducible expression of Slc26a4 in which administration of dox from conception to embryonic day 17.5 (DE17.5) resulted in hearing fluctuation between 1 and 3 months of age. In the present study, we hypothesized that Slc26a4 is required to stabilize hearing in DE17.5 ears between 1 and 3 months of age. We tested our hypothesis by evaluating the effect of postnatal re-induction of Slc26a4 expression on hearing. Readministration of dox to DE17.5 mice at postnatal day 6 (P6), but not at 1 month of age, resulted in reduced click-evoked auditory brainstem response (ABR) thresholds, less fluctuation of hearing and a higher surface density of pendrin expression in spindle-shaped cells of the stria vascularis. Pendrin expression in spindle-shaped cells was inversely correlated with ABR thresholds. These findings suggest that stabilization of hearing by readministration of dox at P6 is mediated by pendrin expression in spindle-shaped cells. We conclude that early re-induction of Slc26a4 expression can prevent fluctuation of hearing in our Slc26a4-insufficient mouse model. Restoration of SLC26A4 expression and function could reduce or prevent fluctuation of hearing in EVA patients.

Human hearing loss is a common neurosensory disorder about which many basic research and clinically relevant questions are unresolved. This review on hereditary deafness focuses on three examples considered at first glance to be uncomplicated, however, upon inspection, are enigmatic and ripe for future research efforts. The three examples of clinical and genetic complexities are drawn from studies of (i) Pendred syndrome/DFNB4 (PDS, OMIM 274600), (ii) Perrault syndrome (deafness and infertility) due to mutations of CLPP (PRTLS3, OMIM 614129), and (iii) the unexplained extensive clinical variability associated with TBC1D24 mutations. At present, it is unknown how different mutations of TBC1D24 cause non-syndromic deafness (DFNB86, OMIM 614617), epilepsy (OMIM 605021), epilepsy with deafness, or DOORS syndrome (OMIM 220500) that is characterized by deafness, onychodystrophy (alteration of toenail or fingernail morphology), osteodystrophy (defective development of bone), mental retardation, and seizures. A comprehensive understanding of the multifaceted roles of each gene associated with human deafness is expected to provide future opportunities for restoration as well as preservation of normal hearing.

Mouse Tmc1 and Tmc2 are required for sensory transduction in cochlear and vestibular hair cells. Homozygous Tmc1∆/∆ mice are deaf, Tmc2∆/∆ mice have normal hearing, and double homozygous Tmc1∆/∆; Tmc2∆/∆ mice have deafness and profound vestibular dysfunction. These phenotypes are consistent with their different spatiotemporal expression patterns. Tmc1 expression is persistent in cochlear and vestibular hair cells, whereas Tmc2 expression is transient in cochlear hair cells but persistent in vestibular hair cells. On the basis of these findings, we hypothesized that persistent Tmc2 expression in mature cochlear hair cells could restore auditory function in Tmc1∆/∆ mice. To express Tmc2 in mature cochlear hair cells, we generated a transgenic mouse line, Tg[PTmc1::Tmc2], in which Tmc2 cDNA is expressed under the control of the Tmc1 promoter. The Tg[PTmc1::Tmc2] transgene slightly but significantly restored hearing in young Tmc1∆/∆ mice, though hearing thresholds were elevated with age. The elevation of hearing thresholds was associated with deterioration of sensory transduction in inner hair cells and loss of outer hair cell function. Although sensory transduction was retained in outer hair cells, their stereocilia eventually degenerated. These results indicate distinct roles and requirements for Tmc1 and Tmc2 in mature cochlear hair cells.

Objectives/Hypothesis Hearing loss and enlarged vestibular aqueduct (EVA) can be inherited as an autosomal recessive trait caused by mutant alleles of the SLC26A4 gene. In some other families, EVA does not segregate in a typical autosomal recessive pattern. The goal of this study was to characterize the SLC26A4 genotypes and phenotypes of extended families with atypical segregation of EVA. Study Design Prospective study of cohort of families ascertained between 1998 and 2014 at the National Institutes of Health Clinical Center. Methods Study subjects were members of eight families segregating EVA in at least two members who were not related as siblings. Evaluations included pure-tone audiometry, temporal bone imaging, SLC26A4 nucleotide sequence analysis, SLC26A4-linked marker genotype and haplotype analysis, and pedigree analysis. Results One family had members with EVA caused by different etiologies, and two families had pseudodominant inheritance of recessive mutations of SLC26A4. In five families, the etiology remained unknown and could include inheritance of mutant alleles at another genetic locus, nongenetic influences, or a combination of these factors. Conclusions Familial EVA can demonstrate a variety of atypical segregation patterns. Pseudodominant inheritance of SLC26A4 mutations or recessive alleles of other hearing loss genes may be more likely to occur in families in which deaf individuals have intermarried. The etiologic basis of atypical segregation of EVA without detectable SLC26A4 mutations remains unknown. Future studies of these families may reveal novel genes for EVA. Level of Evidence NA Laryngoscope, 2015

OBJECTIVE. Enlarged vestibular aqueduct (EVA) is the most common inner ear malformation. While a strong correlative relationship between EVA and hearing loss is well established, its association with vestibular dysfunction is less well understood. In this study, we examine the effects of EVA on the vestibular system in patients with EVA. STUDY DESIGN. Prospective, cross-sectional study of a cohort ascertained between 1999 and 2013. SETTING. National Institutes of Health Clinical Center, a federal biomedical research facility. SUBJECTS AND METHODS. In total, 106 patients with unilateral or bilateral EVA, defined as a midpoint diameter greater than 1.5 mm, were referred or self-referred to participate in a study of the clinical and molecular aspects of EVA. Clinical history was ascertained with respect to the presence or absence of various vestibular signs and symptoms and history of head trauma. Videonystagmography (VNG), cervical vestibular evoked myogenic potential (cVEMP), and rotational vestibular testing (RVT) were performed to assess the vestibular function. RESULTS. Of the patients with EVA, 45% had vestibular signs and symptoms, and 44% of tested patients had abnormal VNG test results. An increased number of vestibular signs and symptoms was correlated with the presence of bilateral EVA (P = .008) and a history of head injury (P < .001).Abnormal VNG results also correlated with a history of head injury (P = .018). CONCLUSION. Vestibular dysfunction is common in patients with EVA. However, not all patients with vestibular signs and symptoms have abnormal vestibular test results. Clinicians should be aware of the high prevalence of vestibular dysfunction in patients with EVA.

Variants in the unconventional myosin gene, MYO1A, have been reported to cause non-syndromic sensorineural hearing loss with a pattern of autosomal dominant inheritance. Others have challenged this association. We used a genotypic ascertainment study design to test the association of MYO1A variants with hearing loss. We evaluated MYO1A variants from a cohort of 951 individuals with exome sequencing who were not ascertained for hearing loss. Five individuals had one of two variants claimed to be associated with sensorineural hearing loss in a prior study and 33 individuals had one of 13 predicted deleterious variants. We obtained audiology evaluations for 12 individuals with these variants of interest. The hearing acuity of the participants was compared with age- and sex-matched controls and published age- and sex-specific reference ranges from a large population of otologically screened adults. None of the participants had bilateral sensorineural hearing loss of moderate or greater severity. These data do not support a causal relationship of variants in MYO1A to sensorineural hearing loss. We suggest that the genotypic ascertainment method is useful to objectively evaluate gene-phenotype associations.

Mutations of SLC26A4 are a common cause of hearing loss associated with enlargement of the endolymphatic sac (EES). Slc26a4 expression in the developing mouse endolymphatic sac is required for acquisition of normal inner ear structure and function. Here, we show that the mouse endolymphatic sac absorbs fluid in an SLC26A4-dependent fashion. Fluid absorption was sensitive to ouabain and gadolinium but insensitive to benzamil, bafilomycin and S3226. Single-cell RNA-seq analysis of pre- and postnatal endolymphatic sacs demonstrates two types of differentiated cells. Early ribosome-rich cells (RRCs) have a transcriptomic signature suggesting expression and secretion of extracellular proteins, while mature RRCs express genes implicated in innate immunity. The transcriptomic signature of mitochondria-rich cells (MRCs) indicates that they mediate vectorial ion transport. We propose a molecular mechanism for resorption of NaCl by MRCs during development, and conclude that disruption of this mechanism is the root cause of hearing loss associated with EES.

The NLRP3 inflammasome is an intracellular innate immune sensor that is expressed in immune cells, including monocytes and macrophages. Activation of the NLRP3 inflammasome leads to IL-1β secretion. Gain-of-function mutations of NLRP3 result in abnormal activation of the NLRP3 inflammasome, and cause the autosomal dominant systemic autoinflammatory disease spectrum, termed cryopyrin-associated periodic syndromes (CAPS). Here, we show that a missense mutation, p.Arg918Gln (c.2753G > A), of NLRP3 causes autosomal-dominant sensorineural hearing loss in two unrelated families. In family LMG446, hearing loss is accompanied by autoinflammatory signs and symptoms without serologic evidence of inflammation as part of an atypical CAPS phenotype and was reversed or improved by IL-1β blockade therapy. In family LMG113, hearing loss segregates without any other target-organ manifestations of CAPS. This observation led us to explore the possibility that resident macrophage/monocyte-like cells in the cochlea can mediate local autoinflammation via activation of the NLRP3 inflammasome. The NLRP3 inflammasome can indeed be activated in resident macrophage/monocyte-like cells in the mouse cochlea, resulting in secretion of IL-1β. This pathway could underlie treatable sensorineural hearing loss in DFNA34, CAPS, and possibly in a wide variety of hearing-loss disorders, such as sudden sensorineural hearing loss and Meniere’s disease that are elicited by pathogens and processes that stimulate innate immune responses within the cochlea.

Enlargement of the vestibular aqueduct (EVA) is the most common radiological abnormality in children with sensorineural hearing loss. Mutations in coding regions and splice sites of theWe performed genotype-haplotype analysis and massively parallel sequencing of theWe identified a shared novel haplotype, termed CEVA (Caucasian EVA), composed of 12 uncommon variants upstream ofThe CEVA haplotype causally contributes to most cases of Caucasian M1 EVA and, possibly, some cases of M0 EVA. The CEVA haplotype of

International audience; Background: Pendrin, the chloride/bicarbonate exchanger of β-intercalated cells of the renal connecting tubule and the collecting duct, plays a key role in NaCl reabsorption by the distal nephron. Therefore, pendrin may be important for the control of extracellular fluid volume and blood pressure.Methods: Here, we have used a genetic mouse model in which the expression of pendrin can be switched-on in vivo by the administration of doxycycline. Pendrin can also be rapidly removed when doxycycline administration is discontinued. Therefore, our genetic strategy allows us to test selectively the acute effects of loss of pendrin function.Results: We show that acute loss of pendrin leads to a significant decrease of blood pressure. In addition, acute ablation of pendrin did not alter significantly the acid-base status or blood K + concentration.Conclusion: By using a transgenic mouse model, avoiding off-target effects related to pharmacological compounds, this study suggests that pendrin could be a novel target to treat hypertension.

In 1896, Vaughan Pendred, MD, wrote a case report about two siblings that presented with hearing loss and goiter. This initial report was followed over the next 100 years with additional cases, and the condition became known as Pendred syndrome. The underlying gene, SLC26A4, which codes for the protein pendrin, was discovered in 1997, and mutations of SLC26A4 have since been recognized to underlie not only Pendred syndrome but also nonsyndromic hearing loss associated with an enlargement of the vestibular aqueduct (EVA) and variable deficits in vestibular function. In 2001, Dr. Lorraine Everett, in a team led by Dr. Eric Green, reported the first mouse model that recapitulates EVA, deafness, and vestibular dysfunction. This and other mouse models have proven to be tremendously valuable in the quest to understand the role of pendrin in hearing and vestibular function. This chapter summarizes work on these mouse models that are revealing the role of pendrin in the inner ear.

PURPOSE Progressive decline of psychophysical cone-mediated measures has been reported in type 1 (USH1) and type 2 (USH2) Usher syndrome. Conventional cone electroretinogram (ERG) responses in USH demonstrate poor signal-to-noise ratio. We evaluated cone signals in USH1 and USH2 by recording microvolt level cycle-by-cycle (CxC) ERG. METHODS Responses of molecularly genotyped USH1 (n = 18) and USH2 (n = 24) subjects (age range, 15-69 years) were compared with those of controls (n = 12). A subset of USH1 (n = 9) and USH2 (n = 9) subjects was examined two to four times over 2 to 8 years. Photopic CxC ERG and conventional 30-Hz flicker ERG were recorded on the same visits. RESULTS Usher syndrome subjects showed considerable cone flicker ERG amplitude losses and timing phase delays (P < 0.01) compared with controls. USH1 and USH2 had similar rates of progressive logarithmic ERG amplitude decline with disease duration (-0.012 log μV/y). Of interest, ERG phase delays did not progress over time. Two USH1C subjects retained normal response timing despite reduced amplitudes. The CxC ERG method provided reliable responses in all subjects, whereas conventional ERG was undetectable in 7 of 42 subjects. CONCLUSIONS Cycle-by-cycle ERG showed progressive loss of amplitude in both USH1 and USH2 subjects, comparable to that reported with psychophysical measures. Usher subjects showed abnormal ERG response latency, but this changed less than amplitude with time. In USH syndrome, CxC ERG is more sensitive than conventional ERG and warrants consideration as an outcome measure in USH treatment trials.

Mutations of transmembrane channel-like 1 gene (TMC1) can cause dominant (DFNA36) or recessive (DFNB7/B11) deafness. In this article, we describe the characteristics of DFNA36 and DFNB7/B11 deafness, the features of the Tmc1 mutant mouse strains, and recent advances in our understanding of TMC1 function.Publications related to TMC1, DFNA36, or DFNB7/B11 were identified through PubMed.All affected DFNA36 subjects showed post-lingual, progressive, sensorineural hearing loss (HL), initially affecting high frequencies. In contrast, almost all affected DFNB7/B11 subjects demonstrated congenital or prelingual severe to profound sensorineural HL. The mouse Tmc1 gene also has dominant and recessive mutant alleles that cause HL in mutant strains, including Beethoven, deafness, and Tmc1 knockout mice. These mutant mice have been instrumental for revealing that Tmc1 and its closely related paralog Tmc2 are expressed in cochlear and vestibular hair cells, and are required for hair cell mechanoelectrical transduction (MET). Recent studies suggest that TMC1 and TMC2 may be components of the long-sought hair cell MET channel.TMC1 mutations disrupt hair cell MET.

SummarySensory transduction in auditory and vestibular hair cells requires expression of transmembrane channel-like (Tmc) 1 and 2 genes, but the function of these genes is unknown. To investigate the hypothesis that TMC1 and TMC2 proteins are components of the mechanosensitive ion channels that convert mechanical information into electrical signals, we recorded whole-cell and single-channel currents from mouse hair cells that expressed Tmc1, Tmc2, or mutant Tmc1. Cells that expressed Tmc2 had high calcium permeability and large single-channel currents, while cells with mutant Tmc1 had reduced calcium permeability and reduced single-channel currents. Cells that expressed Tmc1 and Tmc2 had a broad range of single-channel currents, suggesting multiple heteromeric assemblies of TMC subunits. The data demonstrate TMC1 and TMC2 are components of hair cell transduction channels and contribute to permeation properties. Gradients in TMC channel composition may also contribute to variation in sensory transduction along the tonotopic axis of the mammalian cochlea.