线粒体DNA的异质性与检测方法

线粒体DNA的异质性在生物学、医学、法医、生物技术等领域有重要的应用。本文从自然发生(包括体细胞突变、父系渗入和杂交)和人工产生(包括转基因、细胞融合、核移植和转线粒体)两大方面系统地介绍了线粒体DNA异质性的产生机制及其遗传。并介绍了线粒体DNA异质性的检测方法,已知突变位点mtDNA异质性的检测方法有原位PCR技术、PCR-RFLP和实时荧光定量PCR技术,未知突变位点mtDNA异质性的检测方法有长PCR技术、时相温度梯度凝胶电泳(TTGE)和变性高效液相色谱(DHPLC)等。最后对克隆生物中线粒体异质性检测的应用实例作了介绍。     

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.     

基于新一代测序技术的mtDNA突变检测方法学的建立

目的:大量研究证实线粒体DNA(mtDNA)突变与肿瘤发生及进展密切相关,但使用传统测序方法难以高通量、高精确度的检测mtDNA突变,为此本研究建立了基于新一代测序技术的mtDNA突变检测方法.方法:提取肝癌患者癌、癌旁组织以及外周血细胞总DNA,利用PCR技术对线粒体基因组进行富集并对PCR产物进行平末端、粘性末端连接或对PCR引物进行氨基修饰,构建mtDNA测序文库.经Illumina HiSeq 2000平台测序后利用生物信息学方法与人类mtDNA参考序列进行比对,并进行测序数据分析.结果:通过对不同质量基因组DNA进行评估后,发现三对引物法适用于大部分DNA样本的mtDNA富集.进一步我们发现PCR引物的氨基修饰可显著提高测序数据覆盖均一性,降低测序成本.结论:本研究利用新一代测序技术通过对线粒体DNA富集方法以及测序覆盖度均一性进行优化,建立了一套灵敏、特异、高通量的mtDNA突变检测策略,为mtDNA突变与疾病研究提供了新方法.     

人类线粒体DNA突变与癌症

细胞中的线粒体在细胞的能量代谢等功能中发挥了重要的作用。 人类的肿瘤形成与线粒体DNA (mtDNA)存在着复杂的关联, 在多种癌细胞中均检测到mtDNA的突变。文章综述了癌细胞中的mtDNA突变与癌症发 生的相关性, 并讨论了部分突变产生的原因及在癌症中进行mtDNA突变检测的应用前景。     

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。     

线粒体DNA突变与肿瘤发生的关系

线粒体DNA（mitochondrial DNA,mtDNA）是真核细胞内的核外遗传物质。事实证明,由于线粒体特殊的生物学结构与功能,和核基因（nDNA）相比,mtDNA更容易发生突变和氧化损伤。目前研究已经发现许多肿瘤组织中mtDNA结构和拷贝量发生了变化。文章主要对mtDNA突变、整合和不稳定性与肿瘤发生的关系以及可能的机理进行了综述。     

线粒体疾病与核基因－线粒体基因的表达调控

线粒体与疾病是当前生物医学领域最前沿之一。本文简单介绍线粒体生物医学的基础知识、线粒体疾病的遗传模式,综述了近年来在线粒体DNA（mtDNA）突变和疾病、核基因突变和疾病等领域的研究进展,着重阐明核基因（特别是核修饰基因）调控mtDNA突变致病表达的分子机制。     

人工耳蜗植入聋儿术前基因检测及家系分析

摘要: 国内外研究表明GJB2、SLC26A4(PDS)和线粒体DNA(Mitochondrial DNA, mtDNA)的病理性突变导致了大部分的遗传性聋。 文章收集了2006年4月~2007年9月接受人工耳蜗(Cochlear implant, CI)植入的14 例患儿及其父母的外周血, 应用基因诊断方法进行 GJB2、SLC26A4(PDS)和mtDNA 1555位点突变检测。结果显示, 35.7%的患儿检测到致病突变, 其中28.6%为GJB2基因突变, 类型均为235delC纯和突变, 其父母为携带GJB2 235delC的杂和子; 7.1%为mtDNA A1555G突变, 其母亲亦携带mtDNA A1555G突变。这表明CI 植入聋儿最常见的基因突变是GJB2 235delC突变, 其次是mtDNA A1555G突变, 通过对耳聋家系常见致病基因的检测和家系分析, 可以对优生优育及减少耳聋发病率提供科学准确的遗传信息。     

线粒体基因突变与糖尿病的相关性研究进展

线粒体DNA(mtDNA)突变可引起多种遗传性疾病,其中包括糖尿病.与mtDNA突变相关的糖尿病中最常见的变异是tRNALeu(UUR)] 3243 A→G.文章描述了mtDNA线粒体突变与糖尿病的相关性,介绍了线粒体糖尿病的临床特点和发病机制,概括了线粒体糖尿病相关变异基因位点,重点介绍了m.3243A→G、3310C→T、16189T→C基因突变与线粒体糖尿病病理生理的联系.文章认为mtDNA突变位点的研究为糖尿病的发生机制提供新的视角,也为糖尿病的治疗提供了新方向.     

高血压相关的线粒体DNA突变

线粒体DNA(mtDNA)突变是高血压发病的分子机制之一。已经报道的与原发性高血压相关的mtDNA突变包括:tRNAMet A4435G,tRNAMet/tRNAGln A4401G,tRNAIle A4263G,T4291C和A4295G突变。这些高血压相关的mtDNA突变改变了相应的线粒体tRNA的结构,导致线粒体tRNA的代谢障碍。而线粒体tRNAs的代谢缺陷则影响蛋白质合成,造成氧化磷酸化缺陷,降低ATP的合成,增加活性氧的产生。因此,线粒体的功能缺陷可能在高血压的发生发展中起一定的作用。mtDNA突变发病的组织特异性则可能与线粒体tRNAs的代谢以及核修饰基因相关。目前发现的这些高血压相关的mtDNA突变则应该作为今后高血压诊断的遗传风险因子。高血压相关的线粒体功能缺陷的深入研究也将进一步诠释母系遗传高血压的分子致病机制,为高血压的预防、控制和治疗提供依据。文章对高血压相关的mtDNA突变进行了综述。     

Classification of European Mtdnas from an Analysis of Three European Populations

Mitochondrial DNA (mtDNA) sequence variation was examined in Finns, Swedes and Tuscans by PCR amplification and restriction analysis. About 99% of the mtDNAs were subsumed within 10 mtDNA haplogroups (H, I, J, K, M, T, U, V, W, and X) suggesting that the identified haplogroups could encompass virtually all European mtDNAs. Because both hypervariable segments of the mtDNA control region were previously sequenced in the Tuscan samples, the mtDNA haplogroups and control region sequences could be compared. Using a combination of haplogroup-specific restriction site changes and control region nucleotide substitutions, the distribution of the haplogroups was surveyed through the published restriction site polymorphism and control region sequence data of Caucasoids. This supported the conclusion that most haplogroups observed in Europe are Caucasoid-specific, and that at least some of them occur at varying frequencies in different Caucasoid populations. The classification of almost all European mtDNA variation in a number of well defined haplogroups could provide additional insights about the origin and relationships of Caucasoid populations and the process of human colonization of Europe, and is valuable for the definition of the role played by mtDNA backgrounds in the expression of pathological mtDNA mutations     

The A1555G Mutation in the 12S rRNA Gene of Human mtDNA: Recurrent Origins and Founder Events in Families Affected by Sensorineural Deafness

The mtDNA variation of 50 Spanish and 4 Cuban families affected by nonsyndromic sensorineural deafness due to the A1555G mutation in the 12S rRNA gene was studied by high-resolution RFLP analysis and sequencing of the control region. Phylogenetic analyses of haplotypes and detailed survey of population controls revealed that the A1555G mutation can be attributed to >/=30 independent mutational events among the 50 Spanish families and that it occurs on mtDNA haplogroups that are common in all European populations. This indicates that the relatively high detection rate of this mutation in Spain is not due to sampling biases or to a single major founder event. Moreover, the distribution of these mutational events on different haplogroups is compatible with a random occurrence of the A1555G mutation and tends to support the conclusion that mtDNA backgrounds do not play a significant role in the expression of the mutation. Overall, these findings appear to indicate that the rare detection of this mutation in other populations is most likely due to inadequacy in patient ascertainment and molecular screening. This probable lack of identification of the A1555G mutation in subjects affected by sensorineural hearing loss implies that their maternally related relatives are not benefiting from presymptomatic detection and information concerning their increased risk of ototoxicity due to aminoglycoside treatments.     

Phylogeographic differentiation of mitochondrial DNA in Han Chinese

To characterize the mitochondrial DNA (mtDNA) variation in Han Chinese from several provinces of China, we have sequenced the two hypervariable segments of the control region and the segment spanning nucleotide positions 10171-10659 of the coding region, and we have identified a number of specific coding-region mutations by direct sequencing or restriction-fragment-length-polymorphism tests. This allows us to define new haplogroups (clades of the mtDNA phylogeny) and to dissect the Han mtDNA pool on a phylogenetic basis, which is a prerequisite for any fine-grained phylogeographic analysis, the interpretation of ancient mtDNA, or future complete mtDNA sequencing efforts. Some of the haplogroups under study differ considerably in frequencies across different provinces. The southernmost provinces show more pronounced contrasts in their regional Han mtDNA pools than the central and northern provinces. These and other features of the geographical distribution of the mtDNA haplogroups observed in the Han Chinese make an initial Paleolithic colonization from south to north plausible but would suggest subsequent migration events in China that mainly proceeded from north to south and east to west. Lumping together all regional Han mtDNA pools into one fictive general mtDNA pool or choosing one or two regional Han populations to represent all Han Chinese is inappropriate for prehistoric considerations as well as for forensic purposes or medical disease studies.     

Mitochondrial DNA polymorphisms in Chilean aboriginal populations: implications for the peopling of the southern cone of the continent

The mitochondrial DNAs (mtDNAs) from individuals belonging to three Chilean tribes, the Mapuche, the Pehuenche, and the Yaghan, were studied both by RFLP analysis and D-loop (control region) sequencing. RFLP analysis showed that 3 individuals (1.3%) belonged to haplogroup A, 19 (8%) to haplogroup B, 102 (43%) to haplogroup C, and 113 (47.7%) to haplogroup D. Among the 73 individuals analyzed by D-loop sequencing, we observed 37 different haplotypes defined by 52 polymorphic sites. Joint analysis of data obtained by RFLP and sequencing methods demonstrated that, regardless of the method of analysis, the mtDNA haplotypes of these three contemporary South American aborigine groups clustered into four main haplogroups, in a way similar to those previously described for other Amerindians. These results further revealed the absence of haplogroup A in both the Mapuche and Yaghan as well as the absence of haplogroup B in the Yaghan. These results suggest that the people of Tierra del Fuego are related to tribes from south-central South America.     

The emerging limbs and twigs of the East Asian mtDNA tree

We determine the phylogenetic backbone of the East Asian mtDNA tree by using published complete mtDNA sequences and assessing both coding and control region variation in 69 Han individuals from southern China. This approach assists in the interpretation of published mtDNA data on East Asians based on either control region sequencing or restriction fragment length polymorphism (RFLP) typing. Our results confirm that the East Asian mtDNA pool is locally region-specific and completely covered by the two superhaplogroups M and N. The phylogenetic partitioning based on complete mtDNA sequences corroborates existing RFLP-based classification of Asian mtDNA types and supports the distinction between northern and southern populations. We describe new haplogroups M7, M8, M9, N9, and R9 and demonstrate by way of example that hierarchically subdividing the major branches of the mtDNA tree aids in recognizing the settlement processes of any particular region in appropriate time scale. This is illustrated by the characteristically southern distribution of haplogroup M7 in East Asia, whereas its daughter-groups, M7a and M7b2, specific for Japanese and Korean populations, testify to a presumably (pre-)Jomon contribution to the modern mtDNA pool of Japan.     

Determination of human caucasian mitochondrial DNA haplogroups by means of a hierarchical approach

In this paper we propose a hierarchical approach that allows the screening of mitochondrial DNA (mtDNA) haplogroups in populations that have essentially West Eurasian mtDNA backgrounds but that could have some non-West Eurasian contributions. To develop and validate this scheme, we used data on 18 coding region polymorphisms (17 analyzed by RFLP analysis and 1 by sequencing) and sequences of hypervariable segment I (HVSI) of the mtDNA control region from the Azores Islands (Portugal) population. The proposed scheme allows the characterization of almost all West Eurasian and African major clusters by means of RFLPs. Furthermore, the scheme includes information on situations in which sequencing is pertinent to defining a particular haplogroup. The validity of the scheme is ensured by (1) using relatively stable polymorphic positions, (2) screening more than one position to define a specific haplogroup, and (3) typing confirmatory positions. Dubious samples can be resolved by sequencing. The robustness of this approach was assessed by sequencing all samples for HVSI, taking advantage of the previously established relationships between RFLPs and control region sequence polymorphisms. The use of this hierarchical approach avoids the screening of unnecessary control region polymorphisms and therefore results in a more rapid and cost-efficient screening than one in which all polymorphic positions are analyzed. Even if this approach leads to a lower level of phylogeographic resolution than the sequencing of all samples, it allows us to define population movements on a continental level and can be applied, unlike sequencing all samples, with a low cost in any laboratory.     

Phylogenetic network for European mtDNA

The sequence in the first hypervariable segment (HVS-I) of the control region has been used as a source of evolutionary information in most phylogenetic analyses of mtDNA. Population genetic inference would benefit from a better understanding of the variation in the mtDNA coding region, but, thus far, complete mtDNA sequences have been rare. We determined the nucleotide sequence in the coding region of mtDNA from 121 Finns, by conformation-sensitive gel electrophoresis and subsequent sequencing and by direct sequencing of the D loop. Furthermore, 71 sequences from our previous reports were included, so that the samples represented all the mtDNA haplogroups present in the Finnish population. We found a total of 297 variable sites in the coding region, which allowed the compilation of unambiguous phylogenetic networks. The D loop harbored 104 variable sites, and, in most cases, these could be localized within the coding-region networks, without discrepancies. Interestingly, many homoplasies were detected in the coding region. Nucleotide variation in the rRNA and tRNA genes was 6%, and that in the third nucleotide positions of structural genes amounted to 22% of that in the HVS-I. The complete networks enabled the relationships between the mtDNA haplogroups to be analyzed. Phylogenetic networks based on the entire coding-region sequence in mtDNA provide a rich source for further population genetic studies, and complete sequences make it easier to differentiate between disease-causing mutations and rare polymorphisms.     

Mitochondrial sequence diversity within a subspecies of savanna monkeys (Cercopithecus aethiops) is similar to that between subspecies

Cercopithecus aethiops can be classified into four subspecies by morphology and by geographic distribution. However, the phylogenetic relationship between these subspecies is unclear. We previously found five distinct haplogroups of mitochondrial DNA (mtDNA) in the subspecies C. aethiops aethiops at the restriction fragment length polymorphism (RFLP) level, and found that those haplogroups are parapatrically distributed in their habitat. To determine the relationship between subspeciation and haplogroup formation in a subspecies, we compared mtDNA control region and 12S rRNA gene sequences (approximately 700 bp) in C. a. aethiops, two other subspecies of C. aethiops, and two species of Cercopithecus: The diversity between haplogroups in C. a. aethiops was almost the same as that between subspecies. This similar level of diversification between and within haplogroups may explain why a previously obtained mtDNA tree did not show monophyletic branching according to subspecies.     

Polymorphism of human mitochondrial DNA

To date, a large data set on the mitochondrial DNA (mtDNA) sequence variation in human populations has been accumulated. The use of direct sequencing of the main noncoding region of mtDNA along with the RFLP analysis provide performance of complex analysis of mtDNA polymorphism in human populations. This approach proved to be effective for obtaining molecular genetic portraits of the world populations, as well as for the elucidation of the human evolutionary history and past migrations.     

Polymorphism of Human Mitochondrial DNA

To date, a large data set on the mitochondrial DNA (mtDNA) sequence variation in human populations has been accumulated. The use of direct sequencing of the main noncoding region of mtDNA along with the RFLP analysis provide performance of complex analysis of mtDNA polymorphism in human populations. This approach proved to be effective for obtaining molecular genetic portraits of the world populations, as well as for the elucidation of the human evolutionary history and past migrations.