RESUMO
Two genes for components of the mitochondrial translational apparatus, mitochondrial seryl-tRNA synthetase (SARSM) and mitoribosomal protein S12 (RPMS12) lie adjacent to one another on human chromosome 19, within the critical interval for the autosomal dominant deafness locus DFNA4. Both genes are plausible candidates for DFNA4, based on the fact that deafness mutations in mtDNA have been mapped both to tRNA-ser(UCN) and to the accuracy domain of the small subunit rRNA. We have sequenced the coding regions, proximal promoters, 5' and 3' UTR and splice junctional regions of both genes in two families with DFNA4-linked deafness and in controls. Novel polymorphisms 84425C>T, 83907A>G, 79485T>G, 79406C>T, 71755A>C and 68686C>G (numbered as in GenBank AC011455) were found in one or both families, but none is a plausible disease-causing mutation. Although regulatory mutations affecting either gene could still be involved in the phenotype, structural gene mutations affecting SARSM or RPMS12 can be excluded from consideration as the cause of DFNA4-linked deafness, at least in the families identified thus far.
Assuntos
Éxons/genética , Genes Dominantes/genética , Perda Auditiva Neurossensorial/genética , Mitocôndrias , Polimorfismo Genético/genética , Proteínas Ribossômicas/genética , Serina-tRNA Ligase/genética , Cromossomos Humanos Par 19/genética , Análise Mutacional de DNA , Primers do DNA/genética , DNA Intergênico/genética , Família , Células HeLa , Perda Auditiva Neurossensorial/congênito , Humanos , Mitocôndrias/química , Mitocôndrias/enzimologia , Países Baixos , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Sítios de Splice de RNA/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Estados UnidosRESUMO
The nucleotide pair (np) 7472 insC mitochondrial DNA mutation in the tRNA(Ser)(UCN) gene is associated with sensorineural deafness, combined in some individuals with a wider syndrome including ataxia and myo-clonus. Previous studies in osteosarcoma cell cybrids revealed only a mild respiratory defect linked to the mutation. We have investigated the biochemical and molecular consequences of the mutation, using a panel of seven osteosarcoma cell cybrids containing 100% mutant mtDNA, plus two cybrids carrying 100% wild-type mtDNA from the same patient. The mutation is associated with a mild growth deficit in selective (galactose) medium that is only significant in combination with a reduced mtDNA copy number, suggesting a mechanism that might modulate clinical phenotype. The mutation results in a 65% drop in the steady-state level of tRNA(Ser)(UCN), but causes at most only a very mild and quantitative abnormality of mitochondrial protein synthesis, associated with modest hypersensitivity to doxycyclin. No evidence for a specific defect in aminoacylation was obtained, and unlike the case with the np 7445 mutation, the pattern of RNA processing of light strand transcripts of the ND6 region was not systematically altered. Comparing the np 7472 and np 7445 mutant phenotypes in cultured cells suggests that sensorineural deafness can result from a functional insufficiency of mitochondrial tRNA(Ser)(UCN), to which some cells of the auditory system are especially vulnerable.