ENLARGED VESTIBULAR AQUEDUCTS (EVA) We ascertain families with multiple members with nonsyndromic EVA that is not associated with detectable SLC26A4 mutations or Pendred syndrome. Our hypothesis is that these families segregate recessive alleles at one or more other genetic loci that cause nonsyndromic EVA. We are using those families in a linkage-based exome sequencing strategy to identify other genetic causes of EVA. We used recombination breakpoint mapping to define a region of shared linkage overlap containing the SLC26A4 gene on chromosome 7 to search for occult (unidentified) mutations of SLC26A4 in families segregating nonsyndromic EVA with only one detectable mutant allele of SLC26A4. Our hypothesis is that these families segregate a second, unidentified, mutation of SLC26A4. We have used massively parallel sequencing to sequence the entire region. We are using a combination of genetic and functional expression approaches identify pathogenic variants. We completed an analysis of vestibular (balance) signs and symptoms in our cohort of EVA patients, and an analysis of the results of vestibular testing (Ref. 1). We concluded that 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 vestibu- lar dysfunction in patients with EVA. We previously generated a doxycycline-inducible Slc26a4-expression mouse line. This transgenic mouse line allows us to manipulate Slc26a4 expression (on an Slc26a4-knockout background) by the administration of doxycycline in drinking water. We manipulated doxycycline administration to generate (DE17.5) mice in which there is significant residual hearing and isolated EVA at the age of one month. Longitudinal analysis of DE17.5 mice revealed large fluctuations of hearing from 1-3 months of age, followed by progressive hearing loss from 9-12 months of age. We previously reported our findings between 1-3 months of age in a previous report. In the past year, we published a paper showing that the hearing loss from 6-12 months of age is irreversible and associated with reduction of the endocochlear potential, edema of the stria vascular followed by atrophy, and morphological degeneration of the marginal layer of the stria associated with reduced expression of critical genes (e.g., KCNQ1) (Ref. 2). The pattern of hearing loss remarkably resembles that observed in many human patients with EVA and validates the potential of this animal model to further explore the pathophysiology and potential therapeutic interventions to prevent hearing loss fluctuation and progression. We have tested the ability of Slc26a4 expression to stabilize hearing and prevent fluctuations in mature ears of our mouse model. Rescue of expression Slc26a4 at postnatal day 6 prevents fluctuation of hearing. Waiting until 1 month to resume Slc26a4 expression is not effective at preventing fluctuation. The prevention of fluctuation is correlated with expression of Slc26a4 in spindle-shaped cells in the stria vascularis. We are defining the cellular and molecular transcriptomic architecture of the mouse endolymphatic sac using RNA-seq analysis of single cells isolated from the endolymphatic sac epithelium. TMC GENES We previously generated and reported mice with knockout (null) alleles of Tmc1 and Tmc2. We had shown that Tmc1 and Tmc2 are functionally redundant and required for mechanotransduction in the stereocilia of postnatal cochlear and vestibular sensory hair cells (reviewed in refs. 3 and 4). The results suggested that TMC1 and TMC2 may comprise the hair cell mechanoelectrical transduction channel, or are intimately involved in its development and/or function. We tested this hypothesis by localization of TMC1 and TMC2 proteins in hair cells. We showed that TMC1 and TMC2 are localized to the tips of shorter rows of sensory hair cell stereocilia (Ref. 5). These are the sites of mechanotransduction in inner ear hair cells. These results support our hypothesis that TMC1 and TMC2 are components of the mechanotransduction channel complex of inner ear hair cells. We generated knockout mice for Tmc6 and Tmc8 to better understand the function(s) of Tmc genes and proteins. Mutations in human TMC6 or TMC8 genes cause epidermodysplasia verruciformis, a recessive disease resulting in chronic cutaneous HPV infections (papillomas or warts) with increased susceptibility to non-melanoma skin cancers. We have done extensive RNA expression analyses to show that Tmc6 and Tmc8 are primarily expressed in lymphoid cells and tissues and lung and skin, and primarily during development. The homozygous knockout mice have no obvious phenotypic abnormalities, so we are collaborating with Dr. Paul Lambert to determine if these mice have alterations in their susceptibility or response to papillomavirus infection and progression to non-melanoma skin cancers. We are collaborating with Dr. Larry Samelson to explore the role of TMC6 and TMC8 in T-lymphocytes. DFNA34 HEARING LOSS We mapped a novel nonsyndromic hearing loss locus, DFNA34, in a single large family. We used recombinations to define a critical map interval in which the gene and mutation must be located. We identified a likely mutation in a gene (NLRP3) in which other mutations cause hearing loss associated with autoinflammatory disease. In order to confirm this mutation as causative, we used massively parallel sequencing as well as conventional Sanger dideoxy sequencing to rule out mutations in any of the other genes in the critical map interval. We detect expression of the candidate gene in the inner ear. We collaborated with Drs. Daniel Kastner. Paola Pinto-Patarroyo and Raphaela Goldbach-Mansky to study the patients for evidence of cochlear and systemic auto-inflammation on magnetic resonance imaging studies at the NIH Clinical Center. We have detected evidence of systemic and cochlear auto-inflammation, providing conclusive proof of the pathogenic nature of the mutation we have detected. We have also shown the existence of macrophage/monocyte-like cells in the normal resting mouse cochlea. We have shown that these cells are capable of expressing NLRP3 and secreting interleukin-1beta. Therefore the mouse cochlea has resident cells capable of mounting an innate immune response. We hypothesize that DFNA34 causes cochlear hearing loss by abnormal activation of the NLRP3 inflammasome pathway within the cochlea. COLLABORATIVE PROJECTS We collaborated with Dr. Thomas Friedman and others in a project that generated and characterized an Ildr1-null mouse model of DFNB42 deafness (ref. 6). We collaborated with the NIDCD Audiology Unit, NIDCD Section on Human Genetics, and the National Eye Institute to characterize ERG findings in a cohort of subjects with Usher syndrome (ref. 7).
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