Mitochondrial DNA transfer RNA gene sequence variations in patients with mitochondrial disorders

Many different pathogenic mutations in the mitochondrial (mt) transfer RNA (tRNA) genes have been reported for patients with mitochondrial encephalomy opathy. Although some of them are recurrent, most have only been described once and appear to be restricted to one patient or to one family. The incid ence of mt tRNA gene alterations is not known, even though the frequency of some recurrent mutations has been analysed both in patients and in the gen eral population. In this study, we describe the results of stepwise screeni ng for sequence variations in the mt tRNA genes of 166 patients selected ac cording to several criteria. Extensive sequence analysis of the tRNA genes was performed using denaturing gradient gel electrophoresis. A total of 31 patients (19%) were found to harbour significant levels of a pathogenic mut ation, thus confirming the importance of mt tRNA mutations in human patholo gy. Forty-three different sequence variations were found, illustrating the great diversity of the mtDNA sequence in humans. The functional assessment of all these sequence variations represented a difficult task; it was mostl y based on indirect data, such as the phylogenetic conservation of modified nucleotides and the proportions of variant species in different tissues of the index case or in blood of maternal relatives. Direct demonstration of a correlation between the proportion of heteroplasmic sequence variations a nd the cytochrome c oxidase defect was performed at the single muscle-fibre level. Eleven heteroplasmic sequence variations were found, six of which a re new mutations. One is a known Caucasian polymorphism but the other 10 ar e pathogenic. Two of them are the well-known pathogenic MELAS (mitochondria l myopathy, encephalopathy, lactic acidosis and stroke-like episodes) (A324 3G) and MERRF (myoclonic epilepsy with ragged-red fibres) (A8344G) point mu tations. They were found in 23 patients. The eight other mutations were res tricted to one patient. The pathogenic nature of these mutations was demons trated directly for five of them and hypothesized from indirect arguments f or the other three. Thirty-two homoplasmic sequence variations were found. Twenty-nine were considered to be polymorphisms, even though 15 of these we re found for the first time in our patients and two others had been reporte d previously as pathogenic. The pathogenic nature of three homoplasmic vari ants remains questionable.