Transmission of mitochondrial DNA heteroplasmy in normal pedigrees

The presence of multiple mitochondrial genotypes (heteroplasmy) has been studied in normal individuals. Six multigenerational normal families were screened for heteroplasmy by PCR of the mitochondrial control region and the cytochrome c oxidase intergenic regions. Two individuals from different families exhibited multiple length polymorphisms in a homopolymeric tract at positions 16 184–16 193 and a grandmother in a third family was heteroplasmic for both cytosine and thymidine at position 15 945. Although the 15 945 T variant comprised 28% of the grandmother’s mitochondrial DNA, this sequence was not present in any of her descendants. Heteroplasmy was detected in 2.5% of the 96 mother-offspring pairs, consistent with the possibility that it may not be rare. Received: 18 August 1997 / Accepted: 10 November 1997     

Molecular instability in the COII-tRNA(Lys) intergenic region of the human mitochondrial genome: multiple origins of the 9-bp deletion and heteroplasmy for expanded repeats.

We have identified two individuals from Glasgow in Scotland who have a deletion of one of two copies of the intergenic 9-bp sequence motif CCCCCTCTA, located between the cytochrome oxidase II (COII) and lysine tRNA (tRNA(Lys)) genes 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 the COII-tRNA(Lys) intergenic 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 generated de 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 the COII-tRNA(Lys) intergenic region is unstable owing to slipped-strand mispairing. Although sequences containing four copies of the CCCCCTCTA motif are less stable in 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.     

Evidence for paternal transmission and heteroplasmy in the mitochondrial genome of Silene vulgaris, a gynodioecious plant

Gynodioecy refers to the co-occurrence of females and hermaphrodites in the same population. In many gynodioecious plants, sex is determined by an epistatic interaction between mitochondrial and nuclear genes, resulting in intragenomic evolutionary conflict, should the mitochondrial genome be maternally inherited. While maternal inheritance of the mitochondrial genome is common in angiosperms, few gynodioecious species have been studied. Here, the inheritance of the mitochondrial genes atpA and coxI was studied in 318 Silene vulgaris individuals distributed among 23 crosses. While maternal inheritance was indicated in 96% of the individuals studied, one or more individuals from each of four sib groups displayed a genotype that was identical to the father, or that did not match either parent. Given evidence that inheritance is not strictly maternal, it was hypothesized that some individuals could carry a mixture of maternally and paternally derived copies of the mitochondrial genome, a condition known as heteroplasmy. Since heteroplasmy might be difficult to detect should multiple versions of the mitochondrial genome co-occur in highly unequal copy number, a method was devised to amplify low-copy number forms of atpA differentially. Evidence for heteroplasmy was found in 23 of the 99 individuals studied, including cases in which the otherwise cryptic form of atpA matched the paternal genotype. The distribution of shared nucleotide sequence polymorphism among atpA haplotypes and the results of a population survey of the joint distribution of atpA and coxI haplotypes across individuals supports the hypothesis that heteroplasmy facilitates formation of novel mitochondrial genotypes by recombination.     

The largest unassigned regions of the male- and female-transmitted mitochondrial DNAs in Musculista senhousia (Bivalvia Mytilidae)

Musculista senhousia is a marine mussel with doubly uniparental inheritance (DUI) of mitochondria. In this study we analyzed the largest unassigned region (LUR) of its female- and male-transmitted mitochondrial genomes, described their fine characteristics and searched for shared features. Our results suggest that both LURs contain the control region of their respective mitochondrial genomes. The female-transmitted control region is duplicated in tandem, with the two copies evolving in concert. This makes the F-mtDNA of M. senhousia the first Bivalve mitochondrial genome with this feature. We also compared M. senhousia control regions to that of other Mytilidae, and demonstrated that signals for basic mtDNA functions are retained over evolutionary times even among the fast-evolving mitochondrial genomes of DUI species. Finally, we discussed how similarities between female and male LURs may be explained in the context of DUI evolution and if the duplicated female control region might have influenced the DUI system in this species.     

Factors Affecting the Relative Abundance of Nuclear Copies of Mitochondrial DNA (Numts) in Hominoids

Although nuclear copies of mitochondrial DNA (numts) can originate from any portion of the mitochondrial genome, evidence from humans suggests that more variable parts of the mitochondrial genome, such as the mitochondrial control region (MCR), are under-represented in the nucleus. This apparent deficit might arise from the erosion of sequence identity in numts originating from rapidly evolving mitochondrial sequences. However, the extent to which mitochondrial sequence properties impacts the number of numts detected in genomic surveys has not been evaluated. In order to address this question, we: (1) conducted exhaustive BLAST searches of MCR numts in three hominoid genomes; (2) assessed numt prevalence across the four MCR sub-domains (HV1, CCD, HV2, and MCR_F); (3) estimated their insertion rates in great apes (Hominoidea); and (4) examined the relationship between mitochondrial DNA variability and numt prevalence in sequences originating from MCR and coding regions of the mitochondrial genome. Results indicate a marked deficit of numts from HV2 and MCR_F MCR sub-domains in all three species. These MCR sub-domains exhibited the highest proportion of variable sites and the lowest number of detected numts per mitochondrial site. Variation in MCR insertion rate between lineages was also observed with a pronounced burst in recent integrations within chimpanzees and orangutans. A deficit of numts from HV2/MCR_F was observed regardless of age, whereas HV1 is under-represented only in older numts (>25 million years). Finally, more variable mitochondrial genes also exhibit a lower identity with nuclear copies and because of this, appear to be under-represented in human numt databases.     

Individual human hair mitochondrial DNA control region heteroplasmy proportions in mothers and children

Due to maternal inheritance, lack of recombination and a high polymorphic density, the mtDNA control region hypervariable (HV) regions are well suited for forensic identification using a maternal relative as the known sample. This analysis can be performed in hair, however, heteroplasmy in this tissue is not rare and can result in an apparent sequence mismatch that complicates this application. There is little data comparing mother and child mtDNA-CR heteroplasmic proportions in hair. In this study, we assayed four hairs per individual in 26 mother-child pairs by TTGE for heteroplasmy across HV1. Single nucleotide heteroplasmy was detected in seven families, and in four families at least two hairs were heteroplasmic. In each of the latter families, sequencing and PCR-RFLP confirmed single nucleotide heteroplasmy in proportions of the variant ranging from < or =10 to > or =90% in the mothers, with far less variability in their children. Sequencing alone would have revealed apparent homoplasmic differences at one nucleotide in these families, possibly resulting in an 'inconclusive' verdict for relatedness of child and mother. However, mother-child heteroplasmic variability did not exceed intra-individual variability in the mothers alone.     

Rapid evolution of a heteroplasmic repetitive sequence in the mitochondrial DNA control region of carnivores

We describe a repetitive DNA region at the 3 end of the mitochondrial DNA (mtDNA) control region and compare it in 21 carnivore species representing eight carnivore families. The sequence and organization of the repetitive motifs can differ extensively between arrays; however, all motifs appear to be derived from the core motif ACGT. Sequence data and Southern blot analysis demonstrate extensive heteroplasmy. The general form of the array is similar between heteroplasmic variants within an individual and between individuals within a species (varying primarily in the length of the array, though two clones from the northern elephant seal are exceptional). Within certain families, notably ursids, the array structure is also similar between species. Similarity between species was not apparent in other carnivore families, such as the mustelids, suggesting rapid changes in the organization and sequence of some arrays. The pattern of change seen within and between species suggests that a dominant mechanism involved in the evolution of these arrays is DNA slippage. A comparative analysis shows that the motifs that are being reiterated or deleted vary within and between arrays, suggesting a varying rate of DNA turnover. We discuss the evolutionary implications of the observed patterns of variation and extreme levels of heteroplasmy.By acceptance of this article, the publisher acknowledges the right of the US Government to retain non-exclusive, royalty-free license in and to any copyright covering the article. Correspondence to: A.R. Hoetzel     

Molecular mechanisms of extensive mitochondrial gene rearrangement in plethodontid salamanders

Extensive gene rearrangement is reported in the mitochondrial genomes of lungless salamanders (Plethodontidae). In each genome with a novel gene order, there is evidence that the rearrangement was mediated by duplication of part of the mitochondrial genome, including the presence of both pseudogenes and additional, presumably functional, copies of duplicated genes. All rearrangement-mediating duplications include either the origin of light-strand replication and the nearby tRNA genes or the regions flanking the origin of heavy-strand replication. The latter regions comprise nad6, trnE, cob, trnT, an intergenic spacer between trnT and trnP and, in some genomes, trnP, the control region, trnF, rrnS, trnV, rrnL, trnL1, and nad1. In some cases, two copies of duplicated genes, presumptive regulatory regions, and/or sequences with no assignable function have been retained in the genome following the initial duplication; in other genomes, only one of the duplicated copies has been retained. Both tandem and nontandem duplications are present in these genomes, suggesting different duplication mechanisms. In some of these mitochondrial DNAs, up to 25% of the total length is composed of tandem duplications of noncoding sequence that includes putative regulatory regions and/or pseudogenes of tRNAs and protein-coding genes along with the otherwise unassignable sequences. These data indicate that imprecise initiation and termination of replication, slipped-strand mispairing, and intramolecular recombination may all have played a role in generating repeats during the evolutionary history of plethodontid mitochondrial genomes.     

Phylogeography of shy and white-capped albatrosses inferred from mitochondrial DNA sequences: implications for population history and taxonomy

The evolutionary relationship between shy (Thalassarche cauta) and white-capped (T. steadi) albatrosses was examined using mitochondrial control region sequences. Results were interpreted in the context of a recent and contentious taxonomic revision that recommended full species status for shy and white-capped albatrosses. Low sequence divergence between shy and white-capped albatrosses (1.8%) and between their close relatives, Salvin's and Chatham albatrosses (2.9%), was observed. Much higher sequence divergence was found between the shy/white-capped pair and the Salvin's/Chatham pair (7.0%). Phylogenetic analyses confirmed the separation of the shy/white-capped pair from the Salvin's/Chatham pair but did not provide species-level resolution. Phylogeographic analyses, including a nested clade analysis, FST estimates and an analysis of molecular variance, indicated unambiguous genetic structuring between shy and white-capped albatrosses, thus confirming the demographic isolation of the species, but showed little to no structuring within each species. The geographical distribution of mtDNA haplotypes and other evidence suggest that shy albatrosses arose through range expansion by white-capped albatrosses.     

Heteroplasmy and evidence for recombination in the mitochondrial control region of the flatfish Platichthys flesus

The general assumption that mitochondrial DNA (mtDNA) does not undergo recombination has been challenged recently in invertebrates. Here we present the first direct evidence for recombination in the mtDNA of a vertebrate, the flounder Platichthys flesus. The control region in the mtDNA of this flatfish is characterized by the presence of a variable number of tandem repeats and a high level of heteroplasmy. Two types of repeats were recognized, differing by two C-T point mutations. Most individuals carry a pure "C" or a pure "T" array, but one individual showed a compound "CT" array. Such a compound array is evidence for recombination in the mtDNA control region from the flounder.     

Comparative and evolutionary analysis of mitochondrial genes in Indian major carps

Direct sequencing of mitochondrial DNA regions such as cytochrome b, ATPase 6/8 and control region was performed to study comparative and evolutionary status of the three mitochondrial genes in Labeo rohita, Catla catla and Cirrhinus mrigala. DNA sequence alignment among species using specific software revealed comparative rates of divergence with considerably faster and more heterogeneous substitution rate for control region as compared to cytochrome b and ATPase 6/8. Despite the relatively high variability of control region, the overall levels of sequence divergence were low in coding regions. Two protein coding genes and the control region with varying degree of sequence divergence established two distinct groups which are genetically distant from each other exhibiting identical phylogenetic structure in IMCs. Closest relationship was between Labeo rohita and Catla catla indicating that they might have diverged from a common ancestral stock in genealogical lineage whereas Cirrhinus mrigala showed greater divergence with all the three DNA regions studied. Findings of this study will help to understand evolution of mitochondrial DNA genes in carps and facilitate future investigations on phylogeographic structure of Indian carps.     

Absolute quantitation of a heteroplasmic mitochondrial DNA deletion using a multiplex three-primer real-time PCR assay

Quantitation of wild-type and deleted mitochondrial DNA (mtDNA) coexisting within the same cell (a.k.a., heteroplasmy) is important in mitochondrial disease and aging. We report the development of a multiplex three-primer PCR assay that is capable of absolute quantitation of wild-type and deleted mtDNA simultaneously. Molecular beacons were designed to hybridize with either type of mtDNA molecule, allowing real-time detection during PCR amplification. The assay is specific and can detect down to six copies of mtDNA, making it suitable for single-cell analyses. The relative standard deviation in the threshold cycle number is approximately 0.6%. Heteroplasmy was quantitated in individual cytoplasmic hybrid cells (cybrids), containing a large mtDNA deletion, and bulk cell samples. Individual cybrid cells contained 100-2600 copies of wild-type mtDNA and 950-4700 copies of deleted mtDNA, and the percentage of heteroplasmy ranged from 43+/-16 to 95+/-16%. The average amount of total mtDNA was 3800+/-1600 copies/cybrid cell, and the average percentage of heteroplasmy correlated well with the bulk cell sample. The single-cell analysis also revealed that heteroplasmy in individual cells is highly heterogeneous. This assay will be useful for monitoring clonal expansions of mtDNA deletions and investigating the role of heteroplasmy in cell-to-cell heterogeneity in cellular models of mitochondrial disease and aging.     

Sequence analysis of the complete mitochondrial DNA molecule of the hedgehog,Erinaceus europaeus,and the phylogenetic position of the Lipotyphla

The sequence of the mitochondrial DNA (mtDNA) molecule of the European hedgehog (Erinaceus europaeus) was determined. The length of the sequence presented is 17,442 nucleotides (nt). The molecule is thus the largest eutherian mtDNA molecule so far reported. The organization of the molecule conforms with that of other eutherians, but the control region of the molecule is exceptionally long, 1,988 nt, due to the presence of repeated motifs at two different positions in the 3 part of the control region. The length of the control region is not absolute due to pronounced heteroplasmy caused by variable numbers of the motif TACGCA in one of the repetitive regions. The sequence presented includes 46 repeats of this type. The other repeated region is composed of different AT-rich repeats. This region was identical among four clones studied. Comparison of mitochondrial peptide-coding genes identified a separate position of the hedgehog among several mammalian orders. The concatenated protein sequence of the 13 peptide-coding genes was used in a phylogenetic study using the opossum as outgroup. The position of the hedgehog sequence was basal among the other eutherian sequences included: human, rat, mouse, cow, blue whale, harbor seal, and horse. The analysis did not resolve the relationship among carnivores, perissodactyls, and artiodactyls/cetaceans, suggesting a closer relationship among these orders than acknowledged by classical approaches. Correspondence to: U. Arnason     

Length and restriction site heteroplasmy in the mitochondrial DNA of american shad (alosa sapidissima)

Restriction endonuclease analysis was used to assess mitochondrial DNA (mtDNA) variation in American shad (Alosa sapidissima) collected from 14 rivers ranging from Florida to Quebec. Two types of heteroplasmy were observed, one involving a major length polymorphism and the other a single restriction site. Shad mtDNA occurred in two principal size classes, 18.3 and 19.8 kb. Of 244 shad examined, 30 were heteroplasmic and carried both size classes of mtDNA in varying proportions; the remainder were homoplasmic for the smaller size class of mtDNA. The large mtDNA variant occurred most frequently at the southern end of the range, and except for two individuals from Nova Scotia, was not detected among shad from rivers north of the Delaware. In contrast, ten shad heteroplasmic for a SalI restriction site originated from rivers ranging from South Carolina to Nova Scotia. DNA mapping and hybridization experiments indicated that the length polymorphism is in the D-loop-containing region and consists of a tandemly repeated 1.5-kb DNA sequence occurring in two and three copies, respectively, in the two major size classes of shad mtDNA. Continuous length variation up to approximately 40 bp occurs among copies of the repeat both within and among individuals. Restriction site data support the conclusion that both forms of heteroplasmy in shad mtDNA have originated more than once.     

Length and Sequence Variability in Mitochondrial Control Region of the Milkfish, <Emphasis Type="BoldItalic">Chanos chanos</Emphasis>

Extensive length variability was observed in the mitochondrial control region of the milkfish, Chanos chanos. The nucleotide sequence of the control region and flanking regions was determined. Length variability and heteroplasmy was due to the presence of varying numbers of a 41-bp tandemly repeated sequence and a 48-bp insertion/deletion (indel). The structure and organization of the milkfish control region is similar to that of other teleost fish and vertebrates. However, extensive variation in the copy number of tandem repeats (4–20 copies) and the presence of a relatively large (48-bp) indel, are apparently uncommon in teleost fish control region sequences reported to date. High sequence variability of control region peripheral domains indicates the potential utility of selected regions as markers for population-level studies. Received March 14, 2001; accepted June 29, 2001     

Heteroplasmy and Ancient Translocation of Mitochondrial DNA to the Nucleus in the Chinese Horseshoe Bat (Rhinolophus sinicus) Complex

The utility and reliability of mitochondrial DNA sequences in phylogenetic and phylogeographic studies may be compromised by widespread and undetected nuclear mitochondrial copies (numts) as well as heteroplasmy within individuals. Both numts and heteroplasmy are likely to be common across diverse taxa yet few studies have characterised their frequencies and variation at the intra-specific level. Here we report the presence of both numts and heteroplasmy in the mitochondrial control region of the Chinese horseshoe bat Rhinolophus sinicus. In total we generated 123 sequences from 18 bats, which contained two different numt clades (i.e. Numt-1 and Numt-2) and one mtDNA clade. The sequence divergence between Numt-1 and Numt-2 was 16.8% and each numt type was found in all four R. sinicus taxa, suggesting either two ancient translocations of mitochondrial DNA into the nucleus from the same source taxon, or a single translocation from different source taxa that occurred before the split of R. sinicus into different lineages. Within the mtDNA clade, phylogenetic relationships among the four taxa of R. sinicus were similar to those seen in previous results. Based on PCR comparisons, heteroplasmy was inferred between almost all individuals of R. sinicus with respect to sequence variation. Consistent with introgression of mtDNA between Central sinicus and septentrionalis, individuals from these two taxa exhibited similar signatures of repeated sequences in the control region. Our study highlights the importance of testing for the presence of numts and heteroplasmy when applying mtDNA markers to phylogenetic studies.     

Segregation of mitochondrial genomes in a heteroplasmic lineage with Leber hereditary optic neuroretinopathy.

Relatively little is known about the factors maintaining mitochondrial DNA (mtDNA) sequence diversity in humans. A detailed understanding of the transmission genetics of mtDNA has been partly hampered by the lack of evidence for heteroplasmic individuals. Among families with Leber hereditary optic neuroretinopathy, we found a maternal lineage with individuals heteroplasmic for a single nucleotide change, and we were able to follow the segregation of polymorphic mitochondrial genomes over 3 generations. The results show that rapid segregation can occur but also that the level of heteroplasmy can be maintained from one generation to another. In this family the disease phenotype is associated with the mtDNA sequence change, confirming the involvement of the mutation in the disease.     

Mitochondrial DNA recombination in a free-ranging Australian lizard

Mitochondrial DNA (mtDNA) is the traditional workhorse for reconstructing evolutionary events. The frequent use of mtDNA in such analyses derives from the apparent simplicity of its inheritance: maternal and lacking bi-parental recombination. However, in hybrid zones, the reproductive barriers are often not completely developed, resulting in the breakdown of male mitochondrial elimination mechanisms, leading to leakage of paternal mitochondria and transient heteroplasmy, resulting in an increased possibility of recombination. Despite the widespread occurrence of heteroplasmy and the presence of the molecular machinery necessary for recombination, we know of no documented example of recombination of mtDNA in any terrestrial wild vertebrate population. By sequencing the entire mitochondrial genome (16761bp), we present evidence for mitochondrial recombination in the hybrid zone of two mitochondrial haplotypes in the Australian frillneck lizard (Chlamydosaurus kingii).