SEQUENCING AND QUANTITATIVE ASSESSMENT OF MUTANT AND WILD-TYPE MITOCHONDRIAL-DNA IN PARAFFIN SECTIONS FROM CASES OF MELAS

MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and st roke-like episodes) is a clinically devastating disease of children an d young adults. The cause of the stroke-like episodes is not known. We have sequenced the mitochondrial DNA (mtDNA) in archival paraffin-emb edded material from two cases. In only one of these did the mitochondr ial tRNA(Leu(UUR)) gene contain the nucleotide 3243 A-to-G mutation th at is most commonly responsible for MELAS. In this case, we determined the relative proportion of mutant:wild-type mtDNA in sections of the central nervous system and other tissues by PCR amplification, PalI di gestion, DNA electrophoresis, and scanning densitometry of the ethidiu m bromide-stained gels. The technique allowed the proportion of mitoch ondria that contain the mutant genome to be compared with the histolog ical findings in immediately adjacent sections of tissue. The mutant m tDNA was detectable in most tissues, the percentage of mtDNA ranging f rom barely detectable levels to 78 per cent. The relative amount of mu tant mtDNA correlated poorly with the distribution of histological les ions, both within the central nervous system and in other tissues exam ined. The proportion was high in tissues such as liver, kidney, adrena l, and pancreas that appeared histologically normal. Relatively low le vels were present in some regions of the central nervous system, such as the occipital lobe, which contained many of the characteristic infa rct-like lesions. These observations do not support previous speculati on that the distribution of these lesions reflects that of the defecti ve mitochondria. The results emphasize the usefulness of the polymeras e chain reaction in correlative histogenetic studies.