Frequency and Pattern of Heteroplasmy in the Control Region of Human Mitochondrial DNA

In this work, we present the results of the screening of human mitochondrial DNA (mtDNA) heteroplasmy in the control region of mtDNA from 210 unrelated Spanish individuals. Both hypervariable regions of mtDNA were amplified and sequenced in order to identify and quantify point and length heteroplasmy. Of the 210 individuals analyzed, 30% were fully homoplasmic and the remaining presented point and/or length heteroplasmy. The prevalent form of heteroplasmy was length heteroplasmy in the poly(C) tract of the hypervariable region II (HVRII), followed by length heteroplasmy in the poly(C) tract of hypervariable region I (HVRI) and, finally, point heteroplasmy, which was found in 3.81% of the individuals analyzed. Moreover, no significant differences were found in the proportions of the different kinds of heteroplasmy in the population when blood and buccal cell samples were compared. The pattern of heteroplasmy in HVRI and HVRII presents important differences. Moreover, the mutational profile in heteroplasmy seems to be different from the mutational pattern detected in population. The results suggest that a considerable number of mutations and, particularly, transitions that appear in heteroplasmy are probably eliminated by drift and/or by selection acting at different mtDNA levels of organization. Taking as a whole the results reported in this work, it is mandatory to perform a broad-scale screening of heteroplasmy to better establish the heteroplasmy profile which would be important for medical, evolutionary, and forensic proposes.     

Mitochondrial genome analysis in penile carcinoma

Penile cancer is a rare neoplasm that seems to be linked to socio-economic differences. Mitochondrial genome alterations are common in many tumors types and are reported as regulating oxidative metabolism and impacting tumorigenesis. In this study, we evaluate for the first time the mitochondrial genome in penile carcinoma (PeCa), aiming to evaluate heteroplasmy, mitochondrial DNA (mtDNA) mutational load and mtDNA content in Penile tumors. Using next generation sequencing (NGS), we sequenced the mitochondrial genome of 13 penile tumors and 12 non-neoplastic tissue samples, which allowed us to identify mtDNA variants and heteroplasmy. We further evaluated variant’s pathogenicity using Mutpred predictive software and calculated mtDNA content using quantitative PCR. Mitochondrial genome sequencing revealed an increase number of non-synonymous variants in the tumor tissue, along with higher frequency of heteroplasmy and mtDNA depletion in penile tumors, suggesting an increased mitochondrial instability in penile tumors. We also described a list of mitochondrial variants found in penile tumor and normal tissue, including five novel variants found in the tumoral tissue. Our results showed an increased mitochondrial genome instability in penile tumors. We also suggest that mitochondrial DNA copy number (mtDNAcn) and mtDNA variants may act together to imbalance mitochondrial function in PeCa. The better understanding of mitochondrial biology can bring new insights on mechanisms and open a new field for therapy in PeCa.     

A thirty million year-old inherited heteroplasmy

Due to essentially maternal inheritance and a bottleneck effect during early oogenesis, newly arising mitochondrial DNA (mtDNA) mutations segregate rapidly in metazoan female germlines. Consequently, heteroplasmy (i.e. the mixture of mtDNA genotypes within an organism) is generally resolved to homoplasmy within a few generations. Here, we report an exceptional transpecific heteroplasmy (predicting an alanine/valine alloacceptor tRNA change) that has been stably inherited in oniscid crustaceans for at least thirty million years. Our results suggest that this heteroplasmy is stably transmitted across generations because it occurs within mitochondria and therefore escapes the mtDNA bottleneck that usually erases heteroplasmy. Consistently, at least two oniscid species possess an atypical trimeric mitochondrial genome, which provides an adequate substrate for the emergence of a constitutive intra-mitochondrial heteroplasmy. Persistence of a mitochondrial polymorphism on such a deep evolutionary timescale suggests that balancing selection may be shaping mitochondrial sequence evolution in oniscid crustaceans.     

人类线粒体DNA异质性研究进展及其法医学应用

线粒体DNA(mitochondrial DNA mtDNA)的异质性自从被发现以来,一直被遗传学、进化学、发育遗传学以及法医遗传学、分子生物学领域所重视。由于线粒体异质性的存在,使得很多涉及疾病、进化、系统发育线粒体基因组与核基因组的相互作用关系、线粒体DNA复制机制以及法医学运用线粒体DNA进行实际案件评估的问题变得复杂化。此外线粒体DNA异质性的发生原因以及对线粒体异质性的检测方法标准化问题还没有一个统一的答案。针对线粒体DNA异质性带来的种种问题,近年来国内外取得了不少研究进展。     

动物线粒体基因组研究进展

对动物线粒体分子生物学的最新研究进展进行了较详细的阐述.从线粒体基因组(mtDNA)的研究背景出发,重点介绍了动物线粒体基因组的组成和结构特点,以及目前动物mtDNA与核基因组的关系、线粒体基因的遗传、起源和进化研究中的热点问题.     

Extensive paternal mtDNA leakage in natural populations of Drosophila melanogaster

Strict maternal inheritance is considered a hallmark of animal mtDNA. Although recent reports suggest that paternal leakage occurs in a broad range of species, it is still considered an exceptionally rare event. To evaluate the impact of paternal leakage on the evolution of mtDNA, it is essential to reliably estimate the frequency of paternal leakage in natural populations. Using allele‐specific real‐time quantitative PCR (RT‐qPCR), we show that heteroplasmy is common in natural populations with at least 14% of the individuals carrying multiple mitochondrial haplotypes. However, the average frequency of the minor mtDNA haplotype is low (0.8%), which suggests that this pervasive heteroplasmy has not been noticed before due to a lack of power in sequencing surveys. Based on the distribution of mtDNA haplotypes in the offspring of heteroplasmic mothers, we found no evidence for strong selection against one of the haplotypes. We estimated that the rate of paternal leakage is 6% and that at least 100 generations are required for complete sorting of mtDNA haplotypes. Despite the high proportion of heteroplasmic individuals in natural populations, we found no evidence for recombination between mtDNA molecules, suggesting that either recombination is rare or recombinant haplotypes are counter‐selected. Our results indicate that evolutionary studies using mtDNA as a marker might be biased by paternal leakage in this species.     

Direct evidence for homologous recombination in mussel (Mytilus galloprovincialis) mitochondrial DNA.

The assumption that animal mitochondrial DNA (mtDNA) does not undergo homologous recombination is based on indirect evidence, yet it has had an important influence on our understanding of mtDNA repair and mutation accumulation (and thus mitochondrial disease and aging) and on biohistorical inferences made from population data. Recently, several studies have suggested recombination in primate mtDNA on the basis of patterns of frequency distribution and linkage associations of mtDNA mutations in human populations, but others have failed to produce similar evidence. Here, we provide direct evidence for homologous mtDNA recombination in mussels, where heteroplasmy is the rule in males. Our results indicate a high rate of mtDNA recombination. Coupled with the observation that mammalian mitochondria contain the enzymes needed for the catalysis of homologous recombination, these findings suggest that animal mtDNA molecules may recombine regularly and that the extent to which this generates new haplotypes may depend only on the frequency of biparental inheritance of the mitochondrial genome. This generalization must, however, await evidence from animal species with typical maternal mtDNA inheritance.     

Genetic Diversity,Heteroplasmy, and Recombination in Mitochondrial Genomes of Daphnia pulex,Daphnia pulicaria,and Daphnia obtusa

Genetic variants of mitochondrial DNA at the individual (heteroplasmy) and population (polymorphism) levels provide insight into their roles in multiple cellular and evolutionary processes. However, owing to the paucity of genome-wide data at the within-individual and population levels, the broad patterns of these two forms of variation remain poorly understood. Here, we analyze 1,804 complete mitochondrial genome sequences from Daphnia pulex, Daphnia pulicaria, and Daphnia obtusa. Extensive heteroplasmy is observed in D. obtusa, where the high level of intraclonal divergence must have resulted from a biparental-inheritance event, and recombination in the mitochondrial genome is apparent, although perhaps not widespread. Global samples of D. pulex reveal remarkably low mitochondrial effective population sizes, <3% of those for the nuclear genome. In addition, levels of population diversity in mitochondrial and nuclear genomes are uncorrelated across populations, suggesting an idiosyncratic evolutionary history of mitochondria in D. pulex. These population-genetic features appear to be a consequence of background selection associated with highly deleterious mutations arising in the strongly linked mitochondrial genome, which is consistent with polymorphism and divergence data suggesting a predominance of strong purifying selection. Nonetheless, the fixation of mildly deleterious mutations in the mitochondrial genome also appears to be driving positive selection on genes encoded in the nuclear genome whose products are deployed in the mitochondrion.     

Very Low-Level Heteroplasmy mtDNA Variations Are Inherited in Humans

Little is known about the inheritance of very low heteroplasmy mitochondria DNA （mtDNA） variations. Even with the development of new next-generation sequencing methods, the practical lower limit of measured heteroplasmy is still about 1% due to the inherent noise level of the sequencing. In this study, we sequenced the mitochondrial genome of 44 individuals using Illumina high-throughput sequencing technology and obtained high-coverage mitochondria sequencing data. Our study population contains many mother-offspring pairs. This unique study design allows us to bypass the usual heteroplasmy limitation by analyzing the correlation of mutation levels at each position in the mtDNA sequence between maternally related pairs and non-related pairs. The study showed that very low heteroplasmy variants, down to almost 0.1%, are inherited maternally and that this inheritance begins to decrease at about 0.5%, cor- resnondin to abottleneck of about 200 mtDNA.     

Linkage disequilibrium and phylogenetic congruence between chloroplast and mitochondrial haplotypes in Silene vulgaris

Both the chloroplast and mitochondrial genomes are used extensively in studies of plant population genetics and systematics. In the majority of angiosperms, the chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) are each primarily transmitted maternally, but rare biparental transmission is possible. The extent to which the cpDNA and mtDNA are in linkage disequilibrium is argued to be dependent on the fidelity of co-transmission and the population structure. This study reports complete linkage disequilibrium between cpDNA and mtDNA haplotypes in 86 individuals from 17 populations of Silene vulgaris, a gynodioecious plant species. Phylogenetic analysis of cpDNA and mtDNA haplotypes within 14 individuals supports a hypothesis that the evolutionary histories of the chloroplasts and mitochondria are congruent within S. vulgaris, as might be expected if this association persists for long periods. This provides the first documentation of the evolutionary consequences of long-term associations between chloroplast and mitochondrial genomes within a species. Factors that contribute to the phylogenetic and linkage associations, as well as the potential for intergenomic hitchhiking resulting from selection on genes in one organellar genome are discussed.     

Investigation of Heteroplasmy in the Human Mitochondrial DNA Control Region: A Synthesis of Observations from More Than 5000 Global Population Samples

Instances of point and length heteroplasmy in the mitochondrial DNA control region were compiled and analyzed from over 5,000 global human population samples. These data represent observations from a large and broad population sample, representing nearly 20 global populations. As expected, length heteroplasmy was frequently observed in the HVI, HVII and HVIII C-stretches. Length heteroplasmy was also observed in the AC dinucleotide repeat region, as well as other locations. Point heteroplasmy was detected in approximately 6% of all samples, and while the vast majority of heteroplasmic samples comprised two molecules differing at a single position, samples exhibiting two and three mixed positions were also observed in this data set. In general, the sites at which heteroplasmy was most commonly observed correlated with reported control region mutational hotspots. However, for some sites, observations of heteroplasmy did not mirror established mutation rate data, suggesting the action of other mechanisms, both selective and neutral. Interestingly, these data indicate that the frequency of heteroplasmy differs between particular populations, perhaps reflecting variable mutation rates among different mtDNA lineages and/or artifacts of particular population groups. The results presented here contribute to our general understanding of mitochondrial DNA control region heteroplasmy and provide additional empirical information on the mechanisms contributing to mtDNA control region mutation and evolution. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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).     

Population genetics of the cytoplasm and the units of selection on mitochondrial DNA in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Biological variation exists across a nested set of hierarchical levels from nucleotides within genes to populations within species to lineages within the tree of life. How selection acts across this hierarchy is a long-standing question in evolutionary biology. Recent studies have suggested that genome size is influenced largely by the balance of selection, mutation and drift in lineages with different population sizes. Here we use population cage and maternal transmission experiments to identify the relative strength of selection at an individual and cytoplasmic level. No significant trends were observed in the frequency of large (L) and small (S) mtDNAs across 14 generations in population cages. In all replicate cages, new length variants were observed in heteroplasmic states indicating that spontaneous length mutations occurred in these experimental populations. Heteroplasmic flies carrying L genomes were more frequent than those carrying S genomes suggesting an asymmetric mutation dynamic from larger to smaller mtDNAs. Mother-offspring transmission of heteroplasmy showed that the L mtDNA increased in frequency within flies both between and within generations despite sampling drift of the same intensity as occurred in population cages. These results suggest that selection for mtDNA size is stronger at the cytoplasmic than at the organismal level. The fixation of novel mtDNAs within and between species requires a transient intracellular heteroplasmic stage. The balance of population genetic forces at the cytoplasmic and individual levels governs the units of selection on mtDNA, and has implications for evolutionary inference as well as for the effects of mtDNA mutations on fitness, disease and aging.     

Endotherms,ectotherms, and mitochondrial genome-size variation

The patterns of mitochondrial genomesize variation were investigated in endothermic and ectothermic species to examine the role that thermal habit might play in the evolution of animal mitochondrial DNA (mtDNA). Data on mtDNA size (the modal, largest, and smallest mtDNA reported within a species), the percent variation in mtDNA size (the difference in size between the largest and smallest mtDNAs divided by the model genome size for a given species), and the frequency of heteroplasmic individuals (those carrying more than one mtDNA length variant) were tabulated from the literature. Endotherms showed significantly less variation in mtDNA size and tended to have smaller mtDNAs than ectotherms. Further comparisons between endothermic and ectothermic vertebrates revealed that the largest genome and the percent variation in genome size were significantly smaller in the former than the latter. There was no difference between endothermic and ectotherms in the frequency of heteroplasmy. These data are discussed in light of two hypotheses: (1) more intense directional and purifying selection for small genome size in the cytoplasms of species with higher metabolic rates and (2) reduced mutation pressures generating mtDNA size variants in endotherms relative to those in ectotherms. The general trends are consistent with the selection hypothesis but in certain species mtDNA size variation appears to be governed by mutational pressures. To test these competing hypotheses further, comparative studies are proposed where mitochondrial genome size is quantified in sister taxa and tissue types with very different metabolic rates.     

Recurrent Tissue-Specific mtDNA Mutations Are Common in Humans

Mitochondrial DNA (mtDNA) variation can affect phenotypic variation; therefore, knowing its distribution within and among individuals is of importance to understanding many human diseases. Intra-individual mtDNA variation (heteroplasmy) has been generally assumed to be random. We used massively parallel sequencing to assess heteroplasmy across ten tissues and demonstrate that in unrelated individuals there are tissue-specific, recurrent mutations. Certain tissues, notably kidney, liver and skeletal muscle, displayed the identical recurrent mutations that were undetectable in other tissues in the same individuals. Using RFLP analyses we validated one of the tissue-specific mutations in the two sequenced individuals and replicated the patterns in two additional individuals. These recurrent mutations all occur within or in very close proximity to sites that regulate mtDNA replication, strongly implying that these variations alter the replication dynamics of the mutated mtDNA genome. These recurrent variants are all independent of each other and do not occur in the mtDNA coding regions. The most parsimonious explanation of the data is that these frequently repeated mutations experience tissue-specific positive selection, probably through replication advantage.     

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.     

Mitochondrial DNA in the Bark Weevils: Size, Structure and Heteroplasmy

Mitochondrial DNA of higher animals has been described as an example of extreme efficiency in genome structure and function. Where exceptionally large size molecules have been found (greater than 20 kb), most have occurred as rare variants within a species, suggesting that these variants arise infrequently and do not persist for long periods in evolutionary time. In contrast, all individuals of at least three species of bark weevil (Curculionidae: Pissodes) possess a mitochondrial genome of unusually large size (30-36 kb). The molecule owes its large size to a dramatically enlarged A + T-rich region (9-13 kb). Gene content and order outside of this region appear to be identical to that found in Drosophila. A series of 0.8-2.0-kb repeated sequences occur adjacent to the large A + T rich region and have perhaps played a role in the generation of the large size as well as an unprecedented frequency of size variant heteroplasmy. Every weevil sampled in all three species (n = 219) exhibits anywhere from two to five distinct size classes of mtDNA. The persistence of this large amount of size polymorphism through two speciation events combined with the abundant size variation within individuals suggests that these molecules may not be subject to strong selection for small overall size and efficiency of replication. This pattern of variation contrasts strongly with the conservation of gene content and arrangement in the coding region of the molecule.     

Paternal transmission of mitochondrial DNA as an integral part of mitochondrial inheritance in metapopulations of Drosophila simulans

Maternal inheritance is one of the hallmarks of animal mitochondrial DNA (mtDNA) and central to its success as a molecular marker. This mode of inheritance and subsequent lack of heterologous recombination allows us to retrace evolutionary relationships unambiguously down the matriline and without the confounding effects of recombinant genetic information. Accumulating evidence of biparental inheritance of mtDNA (paternal leakage), however, challenges our current understanding of how this molecule is inherited. Here, using Drosophila simulans collected from an East African metapopulation exhibiting recurring mitochondrial heteroplasmy, we conducted single fly matings and screened F1 offspring for the presence of paternal mtDNA using allele-specific PCR assays (AS–PCR). In all, 27 out of 4092 offspring were identified as harboring paternal mtDNA, suggesting a frequency of 0.66% paternal leakage in this species. Our findings strongly suggest that recurring mtDNA heteroplasmy as observed in natural populations of Drosophila simulans is most likely caused by repeated paternal leakage. Our findings further suggest that this phenomenon to potentially be an integral part of mtDNA inheritance in these populations and consequently of significance for mtDNA as a molecular marker.     

Selective sweeps of mitochondrial DNA can drive the evolution of uniparental inheritance

Although the uniparental (or maternal) inheritance of mitochondrial DNA (mtDNA) is widespread, the reasons for its evolution remain unclear. Two main hypotheses have been proposed: selection against individuals containing different mtDNAs (heteroplasmy) and selection against “selfish” mtDNA mutations. Recently, uniparental inheritance was shown to promote adaptive evolution in mtDNA, potentially providing a third hypothesis for its evolution. Here, we explore this hypothesis theoretically and ask if the accumulation of beneficial mutations provides a sufficient fitness advantage for uniparental inheritance to invade a population in which mtDNA is inherited biparentally. In a deterministic model, uniparental inheritance increases in frequency but cannot replace biparental inheritance if only a single beneficial mtDNA mutation sweeps through the population. When we allow successive selective sweeps of mtDNA, however, uniparental inheritance can replace biparental inheritance. Using a stochastic model, we show that a combination of selection and drift facilitates the fixation of uniparental inheritance (compared to a neutral trait) when there is only a single selective mtDNA sweep. When we consider multiple mtDNA sweeps in a stochastic model, uniparental inheritance becomes even more likely to replace biparental inheritance. Our findings thus suggest that selective sweeps of beneficial mtDNA haplotypes can drive the evolution of uniparental inheritance.     

Distribution of foreign mitochondrial DNA during the first splitting of transmitochondrial mouse embryos

The distribution of human mitochondrial DNA (mtDNA) among single murine blastomeres was analyzed during the splitting of embryos injected with a suspension of human mitochondria at the one- or two-cell stage. Human mtDNA was detected by PCR with species-specific primers. The total amount of the- and four-cell murine embryos analyzed in the study was 315. In all embryos examined together with murine mtDNA copies of human mitochondrial genome were revealed indicating the phenomenon of an artificially modeled heteroplasmy. Foreign mtDNA was not ubiquitous in blastomeres of transmitochondrial embryos. Mathematical treatment of the results showed that, in the period between the injection of human mitochondria and the subsequent embryo cleavage, an uneven distribution of human mtDNA occurred in the cytoplasm. These results also indicate the presence of more than two to three segregation units of mtDNA in the entire pool of mitochondria (about 500) introduced into an embryo by microinjection.