Blaydon, D.C.; (2004) Molecular genetics of Usher syndrome type 1C. Doctoral thesis , University of London.

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Abstract

Usher syndrome type 1C is an autosomal recessive condition in which profound, congenital sensorineural deafness is found in association with vestibular hypofunction and childhood onset retinitis pigmentosa. The gene responsible for Usher type 1C, USH1C, codes for a PDZ domain-containing protein, harmonin, of unknown function. In addition, the locus for a form of non- syndromic autosomal recessive deafness, DFNB18, overlaps with the USH1C gene. In this thesis the USH1C gene is studied in more detail, both at the molecular level and at the protein level. Two cohorts of patients, individuals diagnosed with Usher type 1, and a group of sibs with recessive non-syndromic deafness concordant for markers flanking the DFNB18 locus, were screened for mutations in USH1C. One Usher type 1 patient was homozygous for a recurrent mutation, and the possibility of a founder effect was investigated by analysing intragenic SNPs. Another Usher type 1 patient had two novel coding mutations that were studied in more detail to establish whether they were likely to be disease-causing or represent rare polymorphisms.USH1C is an alternatively spliced gene with evidence for tissue-specific isoforms of the protein. The repertoire of alternative isoforms and their tissue distributions were studied in human foetal tissues using non-quantitative RT- PCR. Particular attention was paid to a putative isoform thought to utilize an alternative start site in the centre of the gene. This isoform may have importance in other tissues when a mutation at the 5' end of USH1C results in a non-functional protein from the usual start site. The sub-cellular localization of harmonin was investigated in individual human epithelial cells using fluorescent immunocytochemistry, and fluorescent immunohistochemistry was used to study the localization in mouse inner ear sections. Finally, to understand more about the possible role of harmonin in the ear and the eye, an in vitro GST pull-down assay was set up to investigate the interaction of harmonin with another Usher type 1 protein, protocadherin 15.

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