1. We are studying the structure, function, and expression of the transmembrane channel-like (TMC) genes 1 and 2. We have successfully generated mice segregating knockout (null) alleles of Tmc1 and Tmc2. We are characterizing their mutant auditory and vestibular (balance) phenotypes at the functional, cellular and molecular levels. We are studying genetic interactions between Tmc1 and Tmc2 in mice carrrying knockout alleles of both genes. Mice that are homozygous for knockout alleles of both genes are deaf and have abnormal vestibular 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. These results indicate that both Tmc1 and Tmc2 are required for normal vestibular function (balance), whereas only Tmc1 is required for hearing.? ? 2. We previously used a yeast two-hybrid screen to isolate genes encoding proteins that potentially interact with Tmc1 and Tmc2. We narrowed the list to a few candidate genes implicated in vesicular trafficking and in antiapoptosis and cell survival. We are are continuing to use a combination of approaches to determine which interactions might occur in situ in hair cells.? ? 3. We have generated knockout mice for Tmc3, Tmc6, and Tmc8 to better understand the function(s) of Tmc genes and proteins. Tmc3 appears to be expressed primarily in neuroendocrine tissues and organs. Tmc6 and Tmc8 are primarily expressed in lymphoid cells and tissues, and truncating allele of either TMC6 or TMC8 cause the autosomal recessive disease epidermodysplasia verruciformis, characterized by chronic cutaneous HPV infections and susceptibility to non-melanoma skin cancers, and defects in helper T cell function. We are currently expanding the colonies and analyzing the mice for any phenotypic abnormalities.? ? 4. We are continuing our effort to identify the gene mutated in the mouse Twirler strain. Heterozygous Twirler mice have inner ear malformations and obesity, whereas homozygous mice are born with cleft palate and die at birth. The critical interval containing the Twirler gene is approximately 750 kilobases and contains one known gene and two predicted genes. We have identified a probable mutation. We are currently generating a knock-in mouse line with this mutation to confirm its pathogenicity. We are also comprehensively characterizing the Twirler phenotype by detailed analyses of its obesity and inner ear phenotypes. ? ? 5. Enlargement of the vestibular aqueduct (EVA) is the most commonly detected radiologic malformation in temporal bones of individuals with hearing loss. A significant proportion of EVA cases have been reported to be associated with mutations of the SLC26A4 gene, in which mutations cause Pendred syndrome. PS is an autosomal recessive disorder comprised of bilateral sensorineural hearing loss and a defect in the ability of the thyroid gland to organify iodine. EVA is a universal finding in the ears of affected PS individuals. PS is correlated with two mutant SLC26A4 alleles, and nonsyndromic EVA is associated with one or zero mutant SLC26A4 alleles. Based upon our data, we hypothesize that one or more other genetic or environmental factors may act alone or in combination with a single SLC26A4 mutation to cause EVA. We have completed a comparative genome hybridization-microarray analysis to search for deletion and duplication mutations of SLC26A4 that might cause EVA. We found not evidence for such mutations. We are analyzing our genotype and phenotype data to identify clinical features that may guide molecular diagnosis or clinical prognosis. We have completed functional studies of several missense substitutions that have been always or usually been detected as the single SLC26A4 variant in EVA patients and shown that they appear to have wild type trafficking and functional activity, indicating they are benign polymorphic variants. These results have important implications for molecular diagnosis of EVA patients, as well as for categorizing patients according to SLC26A4 genotype for studies to identify other causes of EVA.? ? 6. We ascertained a large North American family segregating progressive, nonsyndromic sensorineural hearing loss in a matrilineal/maternal/mitochondrial pattern of inheritance. We sequenced the entire mitochondrial genome in several affected individuals and have identified a rare mutation in the tRNA-Ser(UCN) gene. We analyzed the mitochondrial haplogroup associated with this mutation and compared it to that associated with this same mutation in a previously reported family. Our results show the haplogroups are different, and we did not find the mutation in haplogroup-matched normal control samples. Our study confirms the pathogenicicty of the tRNA-Ser(UCN) mutation in sensorineural hearing loss.? ? 7. The research laboratory of Dr. Guy Van Camp (University of Antwerp, Belgium) discovered a family with dominant progressive hearing loss caused by the D572N mutation of TMC1, the same mutation causing hearing loss in a family we studied. We compared the mutation-linked genetic marker genotypes and haplotypes, and found they were different between the two families. This indicates that the mutations probably arose independently, not from a common founder.
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