EXHAUSTIVE SCANNING APPROACH TO SCREEN ALL THE MITOCHONDRIAL TRANSFER-RNA GENES FOR MUTATIONS AND ITS APPLICATION TO THE INVESTIGATION OF 35 INDEPENDENT PATIENTS WITH MITOCHONDRIAL DISORDERS

To gain a better understanding of the molecular basis of mitochondrial (mt) encephalomyopathies, a highly heterogeneous condition, we develo ped a denaturing gradient gel electrophoresis-based approach that allo ws rapid and exhaustive screening for mutations of all 22 mt tRNA-enco ding genes and their flanking regions in large cohorts of patients, Th is method, that detects heteroplasmy (i.e. co-existence of mutant and wild-type mtDNA species in various ratios) directly, was applied to th e investigation of 35 independent patients with a disease phenotype co mpatible with a mitochondrial encephalomyopathy. Twenty-five of the 35 patients investigated displayed a sequence variation in at least one tRNA gene, A total of 46 different sequence variations (41 point mutat ions, four short insertions and one short deletion), among which 20 ar e new, were characterized, Forty of them were present in a homoplasmic state, whereas six were heteroplasmic, Twenty-two were located in tRN A genes, among which 10 are new homoplasmic or heteroplasmic sequence variations; 24 were located in flanking regions (12 in mRNA-encoding g enes, seven of them leading to missense sequence variations; two in rR NA genes; and 10 in non-coding regions), This study demonstrates (i) t he high frequency of homoplasmic tRNA gene sequence variations in our patient sample, and (ii) the existence of several polymorphic sites in tRNA gene regions that may be helpful for defining haplogroups in dif ferent populations, It relies on a screening method that can now be ap plied easily to other population samples.