This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To develop a primate model of Usher Syndrome by genetic screening. Mutations in connexin 26 (GJB2) cause 10-20% of childhood hearing loss in Americans and Europeans. Usher syndrome, a recessive disorder characterized by retinitis pigmentosa (RP) and sensorineural hearing loss, is the leading cause of combined hearing and vision loss in the industrialized world, affecting 25,000-30,000 people in the United States. Mutations in the USH2A gene account for over half of Usher syndrome cases and 25-50% of autosomal recessive RP cases, which affects about 67,000 people in the United States. Our objectives are to create macaque models of the most prevalent Usher Syndrome (USH2A) and recessive hearing loss (GJB2) genes by screening outbred primates. Segments of USH2A exons plus flanking intronic DNA were amplified and sequenced. Pathology of putative mutations was evaluated based on frequency and predicted alteration of protein structure. We found 28 polymorphic variants in 60% of the Macaca mulatta USH2A coding sequence. More than 83 rare variants were found, 36 of which altered amino acids. An alu insertion in IVS7 was predicted to be nonpathologic based on allele frequency (1.4%). The best candidate for a pathologic allele created a strong splice donor site in exon 8, predicted to lead to a frameshift and stop codon. We tested USH2A mRNA from salivary gland biopsies from the putative splice mutation carrier using nested RT-PCR and sequencing. Both alleles were correctly spliced, indicating that the variant is nonpathologic. The same methods could be used to test human variants predicted to cause aberrant splicing. A macaque model will provide insights into pathologic mechanisms for retinitis pigmentosa, and may be better suited than mice for testing new treatments that could reduce or prevent inherited deafness and blindness. This research used WNPRC Research Services.
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