Molecular Analysis Of Human Hereditary Deafness

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TMC GENES We have generated mice with knockout (null) alleles of Tmc1 and Tmc2. We are characterizing 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. These results indicate that both Tmc1 and Tmc2 are required for normal balance function, whereas only Tmc1 is required for hearing. We are currently working to identify the location and function of TMC1 and TMC2 proteins in sensory hair cells of the hearing and balance organs. 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. TWIRLER MOUSE We have identified the Twirler mutation in the first intron of the Zeb1 gene on chromosome 18. The phenotype of a knock-in mouse line with this mutation confirmed its pathogenicity. The resulting line has all of the phenotypic features of Twirler, thus proving the pathogenic role of the candidate mutation. The mutation alters the binding of C-Myb protein to a consensus Myb binding site that is disrupted by the mutation. The mutant mice show increased levels of Zeb1 RNA and protein expression. Our collaborators Dr. Ronna Hertzano and her colleagues showed that Zeb1 is a regulator of expression of other genes that are important for maintaining mesenchymal versus epithelial cell identity in the developing mouse ear. It does this by repressing the mesenchymal expression of genes that are normally expressed in epithelial cells. The alteration of Zeb1 expression in Twirler ears results in abnormal expression of its target genes with disruption of the development of the inner ear structures, leading to inner ear malformations and loss of hearing and balance. ENLARGED VESTIBULAR AQUEDUCTS (EVA) We ascertain families with multiple members with nonsyndromic EVA that is not associated with detectable SLC26A4 mutations or Pendred syndrome. We are using those families in a genetic linkage-based strategy to identify other genetic causes of EVA. We are evaluating several candidate regions of potential linkage. 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 hearing status of these mice since human patients with EVA have incrementally progressive or fluctuating hearing loss. COLLABORATIVE PROJECTS We collaborated with Dr. Thomas Friedman to characterize the inner ear structural phenotypes associated with deafness in Pakistani patients with LAMM syndrome caused by FGF3 mutations. We collaborated with Dr. Tsutomu Nakashima to evaluate the effect of dietary iodine deficiency on the development of goiter in the Slc26a4-knockout mouse model of Pendred syndrome.