Inheritance of mitochondrial DNA recombinants in double-heteroplasmic families: potential implications for phylogenetic analysis

Abstract

Recently, somatic recombination of human mitochondrial DNA (mtDNA) was discovered in skeletal muscle. To determine whether recombinant mtDNA molecules can be transmitted through the germ line, we investigated two families, each harboring two inherited heteroplasmic mtDNA mutations. Using allele-specific polymerase chain reaction and single-cell and single-molecule mutational analyses, we discovered, in both families, all four possible allelic combinations of the two heteroplasmic mutations (tetraplasmy), the hallmark of mtDNA recombination. We strongly suggest that these recombinant mtDNA molecules were inherited rather than de novo generated somatically, because they (1) are highly abundant and (2) are present in different tissues of maternally related family members, including young individuals. Moreover, the comparison of the complete mtDNA sequence of one of the families with database sequences revealed an irregular, nontreelike pattern of mutations, reminiscent of a reticulation. We therefore propose that certain reticulations of the human mtDNA phylogenetic tree might be explained by recombination of coexisting mtDNA molecules harboring multiple mutations.

Figure 1.

Family with A8344G and A16182C heteroplasmic mutations. A, Pedigree. The first number indicates the percentage of 8344G; the second number indicates the percentage of 16182C in blood samples of the family members. B, Apparent cosegregation of the 16182A allele with the 8344G allele in tissue samples of double-heteroplasmic individuals. Mutation loads were determined by RFLP and were confirmed by sequencing. Circle = blood; square = buccal cells; triangle = urine sediment; diamond = skeletal-muscle biopsy sample; hexagon = fibroblasts; inverse triangle = brain biopsy sample. Black indicates the sister of the patient’s mother; red indicates the patient’s mother; white indicates the patient; green indicates the patient’s sister. The 8344G mutant allele primarily co-occurs with the 16182A wild-type allele (and the 8344A wild-type allele with the d-loop mutant 16182C). A third allelic combination, the putative “original” wild-type 8344A/16182A, is present in the tissue samples of the sister of the patient’s mother (the data points are below the line). Data points above the line in the tissue samples of the patient’s sister indicate that 8344G also occurs in combination with the 16182C allele (the fourth allelic combination, double mutant, and the potential recombinant). C, The recombinant 8344G/16182C allele, present in the blood of the patient’s mother. Note the faint band indicating the presence of 8344G in the blood of the patient’s mother, whereas 16182A is virtually absent in this sample (lane 3), which confirms the presence of the recombinant 8344G/16182C. Lane 1, DNA ladder. Lane 2, Patient’s muscle biopsy (MB) sample. Lane 3, Blood of the patient’s mother (BL). Lane 4, Undigested PCR product (U). D, Distribution of the two heteroplasmic mtDNA mutations in single fibroblasts of the patient’s sister. Data points populate all four corners of the graph, which indicates tetraplasmy.

Figure 2.

Family with A3243G and G16428A heteroplasmic mutations. A, Pedigree. The first number indicates the percentage of 3243G; the second number indicates the percentage of 16428A in blood samples of the family members. B, Apparent association of the 16428A allele with the 3243G allele in tissue samples of double-heteroplasmic individuals. The mutation loads were determined by RFLP and were confirmed by sequencing. Circle = blood; triangle = urine sediment; diamond = skeletal-muscle biopsy sample; inverse triangle = brain biopsy sample. Black = patient; red = daughter of the patient’s aunt; white = the patient’s aunt. C, Distribution of the heteroplasmic mutations in different tissue samples from the family members. Note the clear presence of 16428G in the samples of the patient’s maternal aunt (lane 7). Lane 1, DNA ladder. Lanes 2–5, Patient muscle biopsy (MB) sample, brain (BR), blood (BL), and urine sediment (UR). Lanes 6–7, Patient’s aunt's blood (BL) and urine sediment (UR). Lane 8, Patient’s cousin's blood (BL). Lane 9, Homoplamic 16428A control (C) (isolated PCR fragment). D, Tetraplasmic distribution of the two heteroplasmic mutations, A3243G and G16428A, in skeletal muscle (MB) and brain (BR) of the patient. Note the presence of both digested and undigested bands in the allele-specific reaction pairs. The potential recombinant 16428G/3243G is represented by the digested PCR product in the M lanes. M = mutant 3243G allele-specific PCR; W = wild-type 3243A allele-specific PCR. T = triplasmic control DNA mixture harboring wild-type, single-mutant 16428A and double-mutant 3243G/16428A mtDNA molecules. E, Tetraplasmy of the A3243G/G16428A alleles in urine sediments from the patient and his maternal aunt. Note the mtDNA tetraplasmy in both urine sediments, with the potential recombinant 16428G/3243G (digested PCR product in lanes M). U = undigested PCR product; C = control PCR product with the 16428A mutation.

Figure 3.

Schematic representation of different theoretical scenarios that result in reticulation. A, Standard phylogenetic tree. Two sequential mutations create three different types of genomes (no reticulation). In some cases, all four possible combinations of the two mutations are present in a population (B). Then they have to be formally related by a nontree network structure (reticulation). The square-shaped reticulations can be easily recognized within typical mtDNA phylogenetic trees.^, Reticulations are usually “resolved” into tree structures by the assumption of either recurrent mutations (C) or reverse mutations (D). We propose that, alternatively, reticulations can result from recombination of mtDNA in double-heteroplasmic individuals (E). The persistent heteroplasmy (double line) is switched to a triplasmic double-heteroplasmic state (triple line) by a second mutational event. Recombination can then create the fourth allelic combination that leads to reticulation.