The broad, long-term objectives of this proposal are: (i) to elucidate the molecular basis of susceptibility to aminoglycoside ototoxicity, (2) to generate diagnostic DNA tests to prevent the use of aminoglycosides in genetically susceptible individuals, and (3) to design therapeutic approaches to prevent, correct, or circumvent aminoglycoside ototoxicity in those susceptible individuals in whom the use of the antibiotic cannot be avoided.
The specific aims are: (1) Identification of 200 individuals from different ethnic backgrounds with aminoglycoside ototoxicity. (2) Characterization of all the mitochondrial DNA defects predisposing to aminoglycoside ototoxicity. (3) Identification of nuclear-encoded genes implicated in aminoglycoside sensitivity. (4) Analysis of all individuals with aminoglycoside ototoxicity, who do not have mitochondrial DNA defects, for mutations in the candidate genes identified in 3. (5) Study the biochemical effects of the predisposing mutations in vitro. The health-relatedness of the project is in its ability to prevent aminoglycoside ototoxicity in genetically susceptible individuals. The experimental design and methodology will include the collection of blood samples, and the establishment of lymphoblastoid cell lines, from individuals with clinically well documented aminoglycoside ototoxicity. The DNA extracted from those samples will be analyzed by molecular biological techniques, including single-stranded conformation analysis, heteroduplex analysis, and DNA sequencing, for the presence of mitochondrial ribosomal RNA mutations. Nuclear-encoded candidate genes for aminoglycoside susceptibility mutations will be identified through a combination of approaches involving yeast genetics and cloning methods based on yeast-human homologies. Candidate genes will be screened for the presence of susceptibility mutations with methods similar to the ones used for the mitochondrial ribosomal RNA gene. The biochemical effects of susceptibility mutations will be evaluated in the lymphoblastoid cell lines by biochemical and molecular biological methods.
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