Phylogenetic analysis of mitochondrial DNA

Detection of mutations in patients with occipital stroke

Saara Finnilä

Department of Neurology

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.

Table of Contents
Acknowledgements
Abbreviations
List of original articles
1. Introduction
2. Review of the literature
2.1. Mitochondria
2.1.1. Structure of mitochondria
2.1.2. Energy production by mitochondria
2.2. The mitochondrial genome
2.2.1. Organization of the human mitochondrial genome
2.2.2. Special features of mitochondrial genetics
2.3. Mitochondrial DNA sequence variation in human populations
2.3.1. Mitochondrial DNA haplogroups
2.3.2. Mitochondrial DNA as a phylogenetic tool
2.4. Mutations in mtDNA as causes of diseases
2.4.1. Point mutations
2.4.2. Large-scale rearrangements of mtDNA
2.4.3. MtDNA genotypes conferring increased risk of disease
2.5. Defects in nucleo-mitochondrial signalling and nuclear gene defects
2.6. Migraine
2.7. Occipital stroke
2.7.1. Stroke-like episodes in the MELAS syndrome
3. Aims of the present research
4. Subjects and methods
4.1. Patients (I and IV)
4.2. Population controls (I–IV)
4.3. DNA extraction (I–IV)
4.4. Primers for polymerase chain reactions (I–IV)
4.5. Restriction fragment analysis of mtDNA (I–IV)
4.5.1. Detection of the 5656A>G by NheI digestion and inhibition of the enzyme by NaCl (II)
4.5.2. Detection of common mtDNA mutations (I)
4.5.3. Analysis of mtDNA haplogroups (I–IV)
4.6. Conformation sensitive gel electrophoresis (CSGE) (III and IV)
4.6.1. PCR for CSGE
4.6.2. CSGE
4.7. Sequencing (III and IV)
4.8. Cloning (II and IV)
4.9. Phylogenetic analysis (III and IV)
5. Results
5.1. Association of migrainous occipital stroke with mtDNA haplogroup U (I)
5.2. 5656A>G is not a pathogenic mutation but a common variant in haplogroup U (II)
5.3. Use of CSGE to analyse mtDNA (III)
5.4. Phylogenetic networks for the Finnish haplogroup U based on coding region and HVS-I (III)
5.5. Analysis of mtDNA in patients with occipital stroke using the phylogenetic network for the mtDNA haplogroup UK (IV)
5.6. Possible pathogenic mutations in patients with occipital stroke (IV)
6. Discussion
6.1. CSGE is an effective means of resolving mtDNA sequences
6.2. A phylogenetic network for the Finnish mtDNA haplogroup UK
6.3. Genotypes of the patients with occipital stroke and an association of migrainous stroke with U5
6.4. mtDNA mutations in patients with occipital stroke
7. Conclusions
References
List of Tables
1. Common clinical manifestations of mitochondrial disorders.
2. Identification of European specific mtDNA haplogroups by restriction fragment analysis.
3. Mutations in the coding region of mtDNA found in patients with occipital stroke.
List of Figures
1. The electron transport chain components involved in oxidative phosphorylation are located within the mitochondrial inner membrane. C, cytochrome c; Q, ubiquinone.
2. The human mitochondrial genome encodes 13 subunits of respiratory chain complexes: seven subunits (ND 1–6 and 4L) of complex I, cytochrome b (Cyt b) of complex III, the COX I–III subunits of cytochrome oxidase or complex IV, and the ATPase 6 and 8 subunits of FOF1 ATP synthase. MtDNA also encodes 12S and 16S rRNA genes and 22 tRNA genes. The abbreviated amino acid names indicate the corresponding amino acid tRNA genes. The outer strand is heavy-chain DNA and the inner one light-chain DNA. OH and OL are the replication origins of the light and heavy chain, respectively, while PH and PL indicate the transcription sites.
3. tRNALeu(UUR). The nucleotide transitions 3243A>G, 3252A>G, 3256C>T, 3271T>C and 3291T>C are associated with the MELAS syndrome.
4. Outlines of phylogenetic network of Finnish mtDNA haplogroup UK (see Figure 1, IV). Circles denote subgroups of haplogroup U with the exception of the Saami motif (SM). Ellipses denote patients with occipital stroke. Patients with migrainous stroke are marked by asterisks.
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