Genetic study of autosomal dominant progressive external ophthalmoplegia and familial myasthenia gravis : linkage analysis, candidate gene cloning and mutation detection

Sammanfattning: Identification of genes responsible for familial human diseases is a major task of medical genetics. In this process, linkage analysis, candidate gene screening and mutation detection are the three major steps (Paper I-VI). The purpose of this study was to elucidate the genetic backgrounds of autosomal dominant progressive external ophthalmoplegia (adPEO) and familial inyasthenia gravis (FMG). The methods applied in this study for linkage analysis and repeat expansion were tested in a family with oculopharyngeal muscular dystrophy (OMPD). The results supported the linkage of this family to chromosome 14q I 1.2-q 13, which had been reported, and did not reveal any expanded CAG/CTG repeats in this family. (Paper I) A genome wide linkage screening was first applied to map the disease locus in a large Pakistani family with adPEO, commonly considered as a mitochondrial disorder because of the finding of multiple deletions of mtDNA. The disease locus was assigned to an I I cM region in chromosome 1 Oq23 -24, and taken together with previously published findings in a single Finnish family a critical region of 7 cM between DlOS198 and DIOS1795 was revealed. (Paper II) One EST (expressed sequence tag) homologous to yeast mitochondrial RNA splice 4 (MRS4) within the critical region was identified. The gene, hMRS314, was cloned and found to have two splicing forms corresponding to a 364 aa and a 177 aa form. Northern blot analysis showed that both forms are ubiquitously expressed, the 177 aa form at comparable levels in the different tissues and the 364 aa form at a relative abundance in skeletal muscle, heart and liver. The mitochondrial protein localization was demonstrated in targeting experiments and in yeast compensation experiments the growth defects of double knockouts of mrs3 degrees and 4degrees growth was restored. The yeast homologues MRS3 and 4, identified by BLAST searching, are members of the gene superfamily of mitochondrial carrier family (MCF). The conservative structure of MCF is well kept in hMRS314 as revealed by multi-alignment of hMRS3/4 with other members of MCF. DNA sequencing of this gene did not show any differences between the adPEO patients and the control. (Paper III) Another gene, Twinkle was cloned from the critical region. Twinkle encodes 684 aa while Twinky, the splice form, encodes 582 aa. BLAST searching showed homology to T7 primase/helicase, and the mitochondrial nucleoid localization demonstrated in targeting experiments. At Western blotting Twinkle was found to form high order multimers while Twinky was detected as a monomer. Multimerization is a feature of the family of ring helicase, which T7 primase/helicase belongs to. A total of 11 mutations were found in 12 families with adPEO including the two reported chromosome 10q-linked families. Targeting and multimerization experiments did not show any differences between the mutants and the wildtype. (Paper IV) A unique Hungarian family was genetically characterized, in which ten members were affected by dominantly inherited myasthenia gravis (MG). The possible involvement of known candidate genes was excluded by mutation screening and linkage analyses of gene loci reported to be mutated in the closely related congenital myasthenia syndrome or linked to MG from association studies. A variant of anticipation was observed in this family RED analysis was therefore carried out, which detected long CAG/CTG repeats in the affecteds, however the repeats were located on the normally expanded ERDA1 locus (Paper V). By genotyping a total of 695 microsatellite markers genome-widely a maximum lod score of 3.31 was obtained for D13S1265 at 0% recombination fraction, thus assigning the disease locus to 13q34 between D13S778 and D13SI315 with 5 cM distance. DNA sequencing of the coding regions of three candidate genes did not show any difference between affected patient and the control. (Paper VI)