线粒体DNA的异质性

哺乳动物线粒体DNA（mitochondrial DNA, mtDNA）位于线粒体.当细胞中mtDNA发生突变时,就会出现野生型与突变型mtDNA的共存.这种情况被称为mtDNA异质性.从mtDNA异质性的形成到在表型上引起相应的病变是一个复杂的过程.mtDNA异质性是如何形成和其在特异组织的增殖复制,mtDNA异质性的变化对个体的影响,如何提高mtDNA突变负荷检测的精度和灵敏度都是近些年的研究热点.本文对mtDNA异质性的检测、遗传、组织特异性以及其相关的疾病等方面进行了阐述.     

线粒体DNA异质性

线粒体 DNA（mitochondrial DNA,mtDNA）是线粒体内最重要的遗传物质。mtDNA 突变普 遍存在,突变型 mtDNA 与野生型 mtDNA 共存的现象被称为 mtDNA 异质性。mtDNA 异质性与衰老和多种疾病密切相关。mtDNA异质性特性、mtDNA 异质性与衰老和疾病相关性以及线粒体疾病的治疗等都是近年来遗传学研究的热点。本文从 mtDNA 异质性的动态变化、组织特异性、mtDNA 异质性与疾病以及线粒体疾病的治疗等方面对 mtDNA 异质性进行综述。     

Drosophila Mitochondrial Genetics: Evolution of Heteroplasmy through Germ Line Cell Divisions

The mitochondrial genotype of all F₁ female offspring (426 individuals) of a single Drosophila mauritiana female, heteroplasmic for two types of mtDNA (a short and a long genome), was established. All descendants were heteroplasmic. The earliest eggs laid by this female show the cytoplasmic genetic structure of ovariole stem cells at the end of development. Cohorts of females from the eggs laid day after day by this female, throughout the 31 days of its life, provide information on the evolution of the mitochondrial genotypes in the course of successive divisions of stem cells. An increase of the percentage of long DNA in offspring was observed as the female aged. Moreover, the variance of the genotypes increases as rounds of stem cell division progress. These results are supported by observations based on the adults issued from the early and late eggs, for three additional heteroplasmic females.     

动物mtDNA控制区及保守与异质

本文通过文献综述,对动物线粒体DNA控制区进行了阐述.从线粒体控制区（control region）基因组的研究出发,重点介绍了动物线粒体控制区基因组结构特点.主要结论：由于碱基替换、插入和缺失以及重复序列数目的变异致使D-loop成为mtDNA中变异最多的区域,但突变和结构重排并不是发生在整个D-loop区域,而是在高变区;大多研究集中在mtDNA D-loop保守区和异质方面：对D-loop序列分析,能较好地阐明动物的起源,在动物亲缘关系鉴定、系统进化和物种形成方式的研究等领域具有广阔的研究和应用前景.     

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.     

Mitochondrial DNA Heteroplasmy and Purifying Selection in the Mammalian Female Germ Line

Inherited mutations in the mitochondrial (mt)DNA are a major cause of human disease, with approximately 1 in 5000 people affected by one of the hundreds of identified pathogenic mtDNA point mutations or deletions. Due to the severe, and often untreatable, symptoms of many mitochondrial diseases, identifying how these mutations are inherited from one generation to the next has been an area of intense research in recent years. Despite large advances in our understanding of this complex process, many questions remain unanswered, with one of the most hotly debated being whether or not purifying selection acts against pathogenic mutations during germline development.     

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.     

A Method for Measuring the Heteroplasmy Level of Mitochondrial DNA Mutations

This paper presents a method for measuring the heteroplasmy level of mitochondrial DNA mutations, which is based on real-time PCR using TaqMan fluorescent probes. The method makes it possible to detect the heteroplasmy level of mtDNA mutations and has high accuracy and resolution. It shows significant differences between the parameters of heteroplasmy of patients belonging to different groups by the degree of disease. Application of this method, in particular, to determine the predisposition to atherosclerosis, makes it possible to determine whether the patient belongs to a low-, medium-, or high-risk group of atherosclerosis.     

Mitochondrial DNA Transmission Genetics in Crickets

This paper presents the results of a single generation study of the transmission genetics of mitochondrial DNA in the field cricket Gryllus firmus. In this species, individuals heteroplasmic for at least two different-sized mitochondrial genomes can be collected easily from natural populations. The frequencies of mtDNA size variants in heteroplasmic females and samples of their offspring were estimated by densitometry of autoradiographs. The variance in mitochondrial genotype frequencies among the offspring of heteroplasmic females indicates that, through genetic drift, fixation would take several hundred animal generations. Differences between the observations and data on mtDNA transmission in yeast and cows are discussed in light of the differences in organelle sampling regime and early developmental events in these species. Our data also show shifts in genotype frequencies in the transmission from mother to offspring that suggest a bias in favor of smaller genomes. The nature of mtDNA size variation in natural populations of crickets is discussed in reference to a mutation-selection balance.     

中华鲟(Acipenser sinensis)间的长度变异与个体内的长度异质性

用PCR技术扩增中华鲟（Acipensersinensis）线粒体DNA（mtDNA）控制区（D－loop）时,发现中华鲟天然群体内存在个体间和个体内的mtDNA长度变异现象。DNA测序表明,长度变异发生在mtDNAryloop靠近tRANpro的位置,由长约82碱基对（bp）的重复序列串联形成的。由个体内mtDNA长度变异造成的异质性个体比例为57.4％,非异质性（同质性）个体的比例为426％。非异质性个体间的mtDNA的大小也不一样,存在长度变异。在非异质性个体中,有2、3、4、5个串联重复序列形成的4种分子类型的情况,其重复序列出现的频率从高到低的循序是3→2→4→5。在异质性个体中,同一个体由2种不同分子组合的异质体最普通,占77.78％3种不同分子组合的频率次之,占18.520。4种不同分子组合的异质体比例最少,占3．70％。没有发现由5种不同分子组合的异质体。对所有异质体混合分析表明,各种类型的重复序列出现的比例与非异质体的类似,即分子大小（含重复序列数）从高到低的顺序为3→2→4→5→1。对47尾中华鲟的个体内和个体间的遗传多样性指数分析发现,有65．3％遗传变异表现在群体内的个体间,有347％的遗传变异表现在个体内。由mtDNA长度异质性造成的个体内的多样性是中华鳍物种遗传多样性的另一途径。     

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。     

Detecting Heteroplasmy from High-Throughput Sequencing of Complete Human Mitochondrial DNA Genomes

Heteroplasmy, the existence of multiple mtDNA types within an individual, has been previously detected by using mostly indirect methods and focusing largely on just the hypervariable segments of the control region. Next-generation sequencing technologies should enable studies of heteroplasmy across the entire mtDNA genome at much higher resolution, because many independent reads are generated for each position. However, the higher error rate associated with these technologies must be taken into consideration to avoid false detection of heteroplasmy. We used simulations and phiX174 sequence data to design criteria for accurate detection of heteroplasmy with the Illumina Genome Analyzer platform, and we used artificial mixtures and replicate data to test and refine the criteria. We then applied these criteria to mtDNA sequence reads for 131 individuals from five Eurasian populations that had been generated via a parallel tagged approach. We identified 37 heteroplasmies at 10% frequency or higher at 34 sites in 32 individuals. The mutational spectrum does not differ between heteroplasmic mutations and polymorphisms in the same individuals, but the relative mutation rate at heteroplasmic mutations is significantly higher than that estimated for all mutable sites in the human mtDNA genome. Moreover, there is also a significant excess of nonsynonymous mutations observed among heteroplasmies, compared to polymorphism data from the same individuals. Both mutation-drift and negative selection influence the fate of heteroplasmies to determine the polymorphism spectrum in humans. With appropriate criteria for avoiding false positives due to sequencing errors, next-generation technologies can provide novel insights into genome-wide aspects of mtDNA heteroplasmy.     

Length Heteroplasmy of Sturgeon Mitochondrial DNA: An Illegitimate Elongation Model

Extensive length polymorphism and heteroplasmy (multiple forms within an individual) of the D-loop region are observed in mitochondrial DNA of the white sturgeon (Acipenser transmontanus). The nucleotide sequence of this region, for both a short and a long form, shows that the differences are due to variable numbers of a perfect 82-bp direct repeat. We propose a model for the replicative origin of length differences, involving a competitive equilibrium between the heavy strand and the D-loop strand. This model suggests that frequent misalignment in the repeat region prior to elongation, facilitated by a stable secondary structure in the displaced strand, can explain both the polymorphism and heteroplasmy in this species.     

Mitochondrial DNA Evolution in Mice

This study extends knowledge of mitochondrial DNA (mtDNA) diversity in mice to include 208 animals belonging to eight species in the subgenus Mus. Highly purified mtDNA from each has been subjected to high-resolution restriction mapping with respect to the known sequence of one mouse mtDNA. Variation attributed to base substitutions was encountered at about 200 of the 300 cleavage sites examined, and a length mutation was located in or near the displacement loop. The variability of different functional regions in this genome was as follows, from least to most: ribosomal RNA, transfer RNA, known proteins, displacement loop and unidentified reading frames.—Phylogenetic analysis confirmed the utility of the Sage and Marshall revision of mouse classification, according to which there are at least four species of commensal mice and three species of aboriginal mice in the complex that was formerly considered to be one species. The most thoroughly studied of these species is Mus domesticus, the house mouse of Western Europe and the Mediterranean region, which is the mitochondrial source of all 50 of the laboratory strains examined and of the representatives of wild house mice introduced by Europeans to North and South America during the past few hundred years.—The level of mtDNA variation among wild representatives of (M. musculus) and several other mammalian species. By contrast, among the many laboratory strains that are known or suspected to stem from the pet mouse trade, there is little interstrain variation, most strains having the "old inbred" type of domesticus mtDNA, whose frequency in the 145 wild mice examined is low, about 0.04. Also notable is the apparent homogeneity of mtDNA in domesticus races that have fixed six or more fused chromosomes and the close relationship of some of these mtDNAs to those of karyotypically normal mice.—In addition, this paper discusses fossil and other evidence for the view that in mice, as in many other mammals, the average rate of point mutational divergence in mtDNA is 2–4% per million years. From this, it is estimated that the commensal association between mice and our ancestors began more than a million years ago, i.e., at an early stage in the evolution of Homo erectus.     

Mitochondrial DNA Length Variation and Heteroplasmy in Populations of White Sturgeon (Acipenser Transmontanus)

Southern blot analysis was used to quantify the extent of mtDNA D-loop length variation in two populations of white sturgeon, Acipenser transmontanus. Over 42% of individuals were heteroplasmic for up to six different mtDNA length variants attributable to varying copy numbers of an 82-bp repeat sequence. Chi-square analyses revealed that the frequencies of length genotypes and the incidence of heteroplasmy were significantly different between Fraser and Columbia River sturgeon populations but not between restriction site haplotypes. Heteroplasmic fish have, on average, higher copy number than homoplasmic fish. Forty-five of 101 homoplasmic individuals carry only a single copy of the repeat, while none of the 73 heteroplasmic fish has the single repeat as the predominant variant. On the basis of differences in frequency distributions of copy number within and between fish, we suggest that (1) heteroplasmy is maintained by high recurrent mutation of multiple copy genomes, favoring increased copy number and (2) the mutation pressure toward higher copy number heteroplasmy is partially offset by selection to reduced genome size and segregation to the homoplasmic condition.     

Polymorphism, Heteroplasmy, Mitochondrial Fusion and Diabetes

Mitochondrial DNA (mtDNA) is highly susceptible to mutations that result in polymorphisms and diseases including diabetes. We analyzed heteroplasmy, polymorphisms related to diabetes, and complementation by fusogenic proteins. Cytoplast fusion and microinjection allow, defects in mutated mtDNA inside a heteroplasmic cell to be complemented by fusing two mitochondria via human fusogenic proteins. We characterized three hfzos as well as two OPAls that prevent apoptosis. Two coiled coil domains and GTPase domains in these fusogenic proteins regulate membrane fusion. The hfzo genes were expressed mainly in the brain and in muscle that are postmitotic, but not in the pancreas. Under the influence of polymorphisms of mtDNA and nDNA, the vicious circle of reactive oxygen species and mutations in cell can be alleviated by mitochondrial fusion.     

Genetic Analysis of Systematic Mitochondrial Heteroplasmy in Rabbits

One unusual property of rabbit mitochondrial DNA (mtDNA) is the existence of repeated 153-bp motifs in the vicinity of the replication origin of its H strand. Furthermore, every individual is heteroplasmic: it carries mtDNA molecules with a variable number of repeats. A systematic study of 8 females and their progeny has been devised to analyze mtDNA transmission through generations. The results suggest that three mechanisms are acting simultaneously. (1) Genetic drift in the germ line is revealed by the evolution of heteroplasmy when two major molecular forms are present in a female. (2) A high mutation rate (around 10(-2) per animal generation) generating molecular diversity, by deletion and addition of repeated units, is required to explain the observation of heteroplasmy in every individual. Moreover, the rates of mutation from the most frequent type to the other types are unequal. The deletion of one unit is more frequent than a deletion of two units, which is in turn more frequent than a deletion of three. (3) Selection for shorter molecules in somatic cells is probable. The frequency distribution of mtDNA types depends on the organ analyzed (kidney-spleen and liver vs. gonads).     

Heteroplasmy of Short Tandem Repeats in Mitochondrial DNA of Atlantic Cod, Gadus Morhua

The mitochondrial DNA of the Atlantic cod (Gadus morhua) contains a tandem array of 40-bp repeats in the D-loop region of the molecule. Variation among molecules in the copy number of these repeats results in mtDNA length variation and heteroplasmy (the presence of more than one form of mtDNA in an individual). In a sample of fish collected from different localities around Iceland and off George's Bank, each individual was heteroplasmic for two or more mtDNAs ranging in repeat copy number from two (common) to six (rare). An earlier report on mtDNA heteroplasmy in sturgeon (Acipenser transmontanus) presented a competitive displacement model for length mutations in mtDNAs containing tandem arrays and the cod data deviate from this model. Depending on the nature of putative secondary structures and the location of D-loop strand termination, additional mechanisms of length mutation may be needed to explain the range of mtDNA length variants maintained in these populations. The balance between genetic drift and mutation in maintaining this length polymorphism is estimated through a hierarchical analysis of diversity of mtDNA length variation in the Iceland samples. Eighty percent of the diversity lies within individuals, 8% among individuals and 12% among localities. An estimate of theta = 2N(eo) mu greater than 1 indicates that this system is characterized by a high mutation rate and is governed primarily by deterministic dynamics. The sequences of repeat arrays from fish collected in Norway, Iceland and George's Bank show no nucleotide variation suggesting that there is very little substructuring to the North Atlantic cod population.     

Frequency and Pattern of Heteroplasmy in the Complete Human Mitochondrial Genome

Determining the levels of human mitochondrial heteroplasmy is of utmost importance in several fields. In spite of this, there are currently few published works that have focused on this issue. In order to increase the knowledge of mitochondrial DNA (mtDNA) heteroplasmy, the main goal of this work is to investigate the frequency and the mutational spectrum of heteroplasmy in the human mtDNA genome. To address this, a set of nine primer pairs designed to avoid co-amplification of nuclear DNA (nDNA) sequences of mitochondrial origin (NUMTs) was used to amplify the mitochondrial genome in 101 individuals. The analysed individuals represent a collection with a balanced representation of genders and mtDNA haplogroup distribution, similar to that of a Western European population. The results show that the frequency of heteroplasmic individuals exceeds 61%. The frequency of point heteroplasmy is 28.7%, with a widespread distribution across the entire mtDNA. In addition, an excess of transitions in heteroplasmy were detected, suggesting that genetic drift and/or selection may be acting to reduce its frequency at population level. In fact, heteroplasmy at highly stable positions might have a greater impact on the viability of mitochondria, suggesting that purifying selection must be operating to prevent their fixation within individuals. This study analyses the frequency of heteroplasmy in a healthy population, carrying out an evolutionary analysis of the detected changes and providing a new perspective with important consequences in medical, evolutionary and forensic fields.