Abstract
Usher Syndrome is an inherited human disease of progressive visual impairment diagnosed as retinitis pigmentosa (RP) combined with congenital sensorineural hearing loss and manifests as one of 3 recognized clinical subtypes. Usher I patients (USHI) are deaf with no vestibular responses. Usher II patients (USHII) have moderate to severe hearing loss with normal vestibular responses. Usher type III patients (USHIII) suffer progressive hearing loss with variable vestibular abnormalities. Molecular genetic studies have linked the clinical subtypes to 11 loci in the human genome and eight genes have been found mutated in Usher syndrome patients. This dissertation mainly concerns the identification of mutations that cause Usher syndrome. An accurate molecular diagnosis of the causative gene defect is an important variable in evaluating the benefit of modalities for slowing and/or arresting retinal deterioration. Genetic counseling of recurrence risk estimations will be more accurately assessed by knowledge of the distribution and frequency of mutations. As a prerequisite for mutation screening, the USH2A gene intron/exon organization was determined. Fifty seven USHII probands were screened for USH2A mutations. Fifty eight of 114 independent mutant USH2A alleles (51%) were identified; 38/57 (66%) of cases had at least one mutation. 2299delG was the most frequent mutant USH2A allele observed (31/192=16%). C319Y, N346H, C419F mutations affect the laminin type VI domain. To define the spectrum of Usher phenotypes attributable to USH2A mutations, DNA from patients with a referring diagnosis of USHI, USHII, USHIII, USH-atypical and autosomal recessive retinitis pigmentosa (ARRP) were analyzed by a 2299delG ARMS™ assay. Eight hundred and forty seven patients were screened. No USHI diagnosed patients carried 2299delG. Twenty five 22299delG homozygotes and 110 2299delG heterozygotes were diagnosed as USH2. Six heterozygous Usher type III patients, 3 heterozygous Usher- atypical and 1 heterozygous ARRP patient were also found with 2299delG. The prevalence of USH2A 2299delG in diverse RP phenotypes argues for USH2A mutation screening in all patients with RP, regardless of hearing status. With respct to gene discovery, the very large G-coupled receptor 1 ( VLGR1) gene was deemed a suitable candidate for USH2C on 5ql4- q21 with cochlear and retinal EST representation. VLGR1 is a 90 exon alternatively spliced gene with 3 mRNA isoforms. Four VLGR1 mutations in 3/3 original sibships with the highest probability of linkage to 5ql4-q21 were identified, proving it as the USH2C gene. VLGR1 mutations have previously been identified in both man and mouse and are associated with a seizure phenotype in both species. The identification of additional VLGR1 mutations to test whether a phenotype/genotype correlation exists, akin to that shown for other Usher syndrome disease genes, is warranted.