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Recombination of mitochondrial DNA in skeletal muscle of individuals with multiple mitochondrial DNA heteroplasmy

Abstract

Experimental evidence for human mitochondrial DNA (mtDNA) recombination was recently obtained in an individual with paternal inheritance of mtDNA1 and in an in vitro cell culture system2. Whether mtDNA recombination is a common event in humans remained to be determined. To detect mtDNA recombination in human skeletal muscle, we analyzed the distribution of alleles in individuals with multiple mtDNA heteroplasmy using single-cell PCR and allele-specific PCR. In all ten individuals who carried a heteroplasmic D-loop mutation and a distantly located tRNA point mutation or a large deletion, we observed a mixture of four allelic combinations (tetraplasmy), a hallmark of recombination. Twelve of 14 individuals with closely located heteroplasmic D-loop mutation pairs contained a mixture of only three types of mitochondrial genomes (triplasmy), consistent with the absence of recombination between adjacent markers. These findings indicate that mtDNA recombination is common in human skeletal muscle.

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Figure 1: Tetraplasmic allelic distributions of double heteroplasmic mutations in individuals p1–p4 prove the presence of recombination.
Figure 2: Tetraplasmic distribution of alleles is confirmed using pre-PCR separation by gel electrophoresis, which is not subject to PCR artifacts.
Figure 3: Cosegregation of closely spaced alleles in skeletal muscle of double heteroplasmic individuals argues against recurrence of mutations.

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Acknowledgements

We thank A. de Grey for his comments on the manuscript and U. Strube for technical assistance. This study was supported by research grants of the Deutsche Forschungsgemeinschaft and the Bundesministerium für Bildung und Forschung to W.S.K. and by grants from the US National Institutes of Health to K.K.

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Correspondence to Konstantin Khrapko or Wolfram S Kunz.

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Supplementary information

Supplementary Fig. 1

Schematic representation of positions of deletions, D-loop point mutations and used PCR primers. (PDF 64 kb)

Supplementary Fig. 2

Comparison of mtDNA heteroplasmy determined by SYBR Green staining and radioactive detection ('last cycle hot' PCR). (PDF 13 kb)

Supplementary Fig. 3

Purity of agarose gel electrophoresis-separated genomic DNA samples determined by multiplex PCR. (PDF 81 kb)

Supplementary Table 1

Comparison of D-loop mutant loads as determined in allele-specific PCR products and gel-separated DNA fractions. (PDF 23 kb)

Supplementary Table 2

Frequency of heteroplasmic D-loop mutations in post-mitotic tissues. (PDF 8 kb)

Supplementary Table 3

Overview of patients with multiple heteroplasmic mutations in the coding region and the D-loop. (PDF 8 kb)

Supplementary Table 4

Primer sequences and mutation detection. (PDF 15 kb)

Supplementary Note

Proof of the in vivo origin of allelic tetraplasmy. (PDF 10 kb)

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Zsurka, G., Kraytsberg, Y., Kudina, T. et al. Recombination of mitochondrial DNA in skeletal muscle of individuals with multiple mitochondrial DNA heteroplasmy. Nat Genet 37, 873–877 (2005). https://doi.org/10.1038/ng1606

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