Molecular instability in theCOII–tRNALysintergenic region of the human mitochondrial genome: multiple origins of the 9–bp deletion and heteroplasmy for expanded repeats

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Abstract
We have identified two individuals from Glasgow in Scotland who have a deletion of one of two copies of the cytochrome oxidise II (COII) intergenic 9–bp sequence motif CCCCCTCTA, located between theCOIIandtRNALysgenes of the human mitochondrial genome. Although this polymorphism is common in Africa and Asia, it has not been reported in Northern Europe. Analysis of the mitochondrial DNA control region sequences of these two individuals suggests that they belong to a lineage that originated independently of the previously characterized African and Asian 9–bp deleted lineages. Among the Scottish population we have also identified a maternal lineage of three generations exhibiting heteroplasmy for two, three and four copies of the CCCCCTCTA motif. Polymerase chain reaction amplification across theCOII–tRNALysintergenic region of these individuals gives different ratios of the three product lengths that are dependent on the concentration of the DNA–binding dye crystal violet. To investigate whether changes in repeat number were generatedde novo, we constructed clones containing known numbers of the CCCCCTCTA motif. In the presence of high concentrations of crystal violet we obtained two, three and four copies of this motif when the amplification template contained only four copies. Various DNA–binding drugs are known to stabilize bulged structures in DNA and contribute to the process of slipped–strand–mispairing during DNA replication. These results suggest that theCOII–tRNALysintergenic region is unstable owing to slipped–strand mispairing. Although sequences containing four copies of the CCCCCTCTA motif are less stablein vitro, we observed an increase in the proportion of mitochondrial genomes with four repeats between a mother and a daughter in the heteroplasmic lineage. From this we conclude that drift in the germ–line lineage is a main factor in the maintenance or loss of heteroplasmy.

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