Phylogenetic analysis of mitochondrial DNA:detection of mutations in patients with occipital stroke

Abstract

Abstract A mitochondrial disorder may be one of the rare aetiologies of occipital stroke. Clinical and molecular analysis has suggested that 10% of young patients with occipital stroke have a mitochondrial disorder and 6% harbour the mutation 3243A>G in mitochondrial DNA (mtDNA), causing the MELAS syndrome. To identify other possible mtDNA mutations involved, we studied mtDNA genotypes in patients who had suffered an occipital stroke and in whom the common pathogenic mutations in mtDNA had been excluded. Since one systematic way of comparing mtDNA sequences is through phylogenetic analysis, a phylogenetic network for the Finnish mtDNA haplogroup U was constructed and used to identify differences in mtDNA between patients and controls. The usefulness of conformation sensitive gel electrophoresis (CSGE) for analysing differences within the coding sequence of mtDNA was also estimated. We studied mtDNA genotypes of 29 patients with occipital stroke. The aetiology of the stroke was assessed using the criteria of the Baltimore-Washington Cooperative Young Stroke Study, and migraine was diagnosed in 18 patients according to the International Headache Society criteria. Moreover, we studied the mtDNA genotypes of 42 patients with migraine and a total of 480 population controls who reported that they themselves and their mothers were healthy with respect to common clinical manifestations of mtDNA disease. The mtDNA haplogroups were detected by restriction fragment analysis and the mtDNA structures of 14 patients with occipital stroke and 43 subjects belonging to haplogroup U were examined by CSGE. The data acquired by CSGE were then used to construct a phylogenetic network for the Finnish mtDNA haplogroup U. We found CSGE to be a highly sensitive and specific method for screening mutations and polymorphisms in mtDNA. The sequence data on the 43 subjects belonging to the mtDNA haplogroup U were used to construct a phylogenetic network, which was found to be an unambiguous tree with few homoplasies that pointed to several previously unidentified common polymorphisms. The major branch of the network was U5, which seemed to be quite specific to the Finns. Branches representing haplogroups U2, U4, U7 and K could also be detected. Restriction fragment analysis of the patients with occipital stroke revealed that all those with migraine as a probable aetiology belonged to the mtDNA haplogroup U, suggesting that this genotype confers a risk of occipital stroke. In addition to the five patients with migrainous stroke, we analyzed the complete mtDNA coding sequences of nine other patients with occipital stroke belonging to haplogroup U by CSGE. Analysis of the phylogenetic network revealed an association of migrainous stroke with mtDNA haplogroup U5. Furthermore, the distribution of the mtDNA genotypes in the patients with stroke differed from that found in the controls. Four patients harboured potentially pathogenic mutations. CSGE proved to be an effective method for use in mitochondrial genetics, enabling us to construct an unambiguous network for the Finnish haplogroup U. Similar phylogenetic networks are required for the purposes of both medical genetics and population genetics. Such networks were found to be helpful in deciding between a rare polymorphism and a pathogenic mutation in patients with occipital stroke. Likewise, they enabled more detailed comparisons to be made between and within populations and allowed more accurate phylogenetic relationships to be determined