Molecular Analysis Of Human Hereditary Deafness

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1. We are studying the transmembrane channel-like (TMC) genes 1 and 2. We have generated mice segregating knockout (null) alleles of Tmc1 and Tmc2. We are characterizing their mutant auditory and vestibular (balance) phenotypes. 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. We use LacZ reporter genes in the knockout mice to study where the genetic interaction might be taking place. The Tmc1 reporter is expressed in hair cells of all of the vestibular end organs whereas the Tmc2 reporter is expressed in hair cells of the cristae ampullaris. We are now using hair cell markers to determine if these genes are expressed in type 1 or type 2 hair cells, or both. We are collaborating with Dr. Charley Della Santina to measure vestibulo-ocular responses in the mice to determine if their abnormal balance and gait is due to abnormal function of the semicircular canals (cristae) or other parts of the vestibular system. 2. We used a yeast two-hybrid screen to isolate genes encoding proteins that potentially interact with TMC1. We narrowed the list to a few candidate genes implicated in vesicular trafficking and in antiapoptosis and cell survival. We are using a combination of approaches to determine which interactions occur in situ in hair cells. 3. 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) 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. 4. We are continuing our work 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 have generated a knock-in mouse line with this mutation to confirm its pathogenicity. We have completed a comprehensive characterization of the Twirler obesity and inner ear phenotypes. The obesity phenotype is associated with hyperphagia of unknown etiology and insulin-resistant diabetes mellitus. The inner ear phenotype is characterized by hypoplasia or dysplasia of the semicircular canals; the lateral canals are most severely affected. 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 have also PCR-amplified and sequenced all noncoding conserved sequences in and around SLC26A4. We found not evidence for such mutations. We have completed segregation analyses of EVA to gain insight into the causes of EVA in patients with one or zero mutant alleles. The results are consistent with inheritance of a second recessive allele in the patients with one detectable SLC26A4 mutation, whereas the results for patients with no mutant alleles of SLC26A4 suggest one or more of the following: autosomal recessive inheritance of alleles at another locus, non-genetic causes, a complex etiology of two or more genetic and/or non-genetic factors, or a variety of different etiologies among different patients. These results yield recurrence risk estimates, dependent upon the number of SLC26A4 mutant alleles, that are very helpful for counseling patients and families with EVA. We analyzed our genotype and phenotype data to identify clinical features that guide molecular diagnosis or clinical prognosis. We comprehensively and quantitatively analyzed the thyroid phenotypes in our EVA cohort. Our study is the first to rigorously analyze and describe the ultrasonographic phenotype of the thyroid gland in EVA patients. Our results show that thyroid gland volume (goiter) is dependent upon the number of mutant alleles in pre-pubescent subjects whereas it is dependent upon age in post-pubescent subjects. Therefore goiter is a more specific sign of biallelic SlC264A4 mutations (Pendred syndrome) in children than in adults. We continue to observe a strong correlation of an abnormal perchlorate discharge test with two mutant alleles of SLC26A4. Our data provides a basis for the surveillance and management of the thyroid gland in EVA subjects. We have completed functional studies of several missense substitutions of SLC26A4 that have always or usually been detected as the single SLC26A4 variant in nonsyndromic 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. 8. We performed a genotypic survey for DFNB7/B11 deafness caused by TMC1 mutations in Pakistan. We identified several novel mutations and showed that TMC1 mutations account for approximately 3.4% of severe to profound, prelingual-onset deafness in Pakistan.