Molecular Analysis Of Human Hereditary Deafness

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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 are also using recombination breakpoint mapping and exome sequencing 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 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 defined a time window of embryonic day 16.5 to postnatal day 2 during which Slc26a4 is required for auditory development and function. We can manipulate doxycycline administration to generate mice in which there is significant residual hearing and isolated EVA, a phenotype which models human EVA more closely than the existing knockout mouse. We collaborated with Dr. Philine Wangemann to show the cochleae of these mice have acidic endolymph (the fluid filling the cochlear duct) and a reduction of the endocochlear potential (the electrochemical gradient required for normal inner ear sensory cell function). We are currently evaluating the long-term natural history and molecular-cellular-physiologic pathogenesis of hearing loss in these mice since human patients with EVA have incrementally progressive or fluctuating hearing loss. TMC GENES We generated mice with knockout (null) alleles of Tmc1 and Tmc2. We characterized their hearing and balance function. Mice that are homozygous for the Tmc1 knockout allele are deaf. Mice that are homozygous for the Tmc2 knockout allele have normal hearing and balance. Mice that are homozygous for knockout alleles of both genes are deaf and have abnormal balance function. We showed that Tmc1 and Tmc2 are functionally redundant and required for mechanotransduction in the stereocilia of postnatal cochlear and vestibular sensory hair cells. Fluorophore-tagged TMC1 or TMC2 localized at or near tips of stereocilia, suggesting these proteins either comprise and integral component of the hair cell mechanoelectrical transduction channel, or are intimately involved in its development and/or function. We observed that both Tmc1 and Tmc2 mRNA is expressed in both developing and mature vestibular hair cells, whereas Tmc2 is expressed only transiently in developing cochlear hair cells whereas Tmc1 expression persists in mature cochlear hair cells. These results suggest a model for why humans and mice with Tmc1 mutant alleles have deafness but not vestibular dysfunction: Tmc2 is available to compensate for loss of Tmc1 function in vestibular but not cochlear hair cells. We are currently testing this hypothesis using transgenic mice. We are also pursuing further studies to definitively localize TMC1 and TMC2 proteins in the auditory system, as well as to test the hypothesis that they are integral components of the hair cell mechanoelectrical transduction channel. 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. COLLABORATIVE PROJECTS We collaborated with Dr. Thomas Friedman to CDH23 genotypes and auditory and ocular phenotypes in patients with nonsyndromic DFNB12 deafness or type I Usher syndrome. The results support a correlation of a nonsyndromic deafness phenotype (i.e. with normal vision) with mutations that preserve residual cadherin-23 function.