Rarity of tagSNPs across transferred mtDNA inserts in human genome

The study deals with the single nucleotide polymorphism (SNPs, HapMap data) around the mtDNA insertions in human genome. The results obtained from this study suggest that application of tagSNP approach for large scale genotyping targeting NUMT integration sites may be difficult due to lack of informative mutations around these loci. This warrants development of new approaches to tag mtDNA inserts in genome-wide association studies.     

Rarity of TagSNPs across transferred mtDNA inserts in human genome

The study deals with the single nucleotide polymorphism (SNPs, HapMap data) around the mtDNA insertions in human genome. The results obtained from this study suggest that application of tagSNP approach for large scale genotyping targeting NUMT integration sites may be difficult due to lack of informative mutations around these loci. This warrants development of new approaches to tag mtDNA inserts in genome-wide association studies.     

香菇三个功能基因部分序列的单核苷酸多态性分析

以15个香菇栽培品种为材料,对尿嘧啶核苷酸-胞嘧啶核苷酸激酶基因(UMP-CMP kinase gene,uck1)、分裂原活化蛋白激酶基因(mitogen-activated protein kinase gene,mapk)和外切β-1,3葡聚糖酶基因(exo-β-1,3-glucanase-encoding gene,exg1)进行了部分序列的单核苷酸多态性(single nucleotide polymorphism,SNP)分析。结果表明,测序中出现的双峰,是菌丝双核体细胞中两细胞核之间的差异造成的。在采用uck1、mapk和exg1的3,126bp中,共发现48处多态性位点,发生频率为1/65bp,其中36个属于转换、12个为颠换。从群体发生频率上,38个属于超过10%的常见SNP,10个属于罕见SNP。不同基因的SNP发生频率不同,uck1、mapk和exg1的SNP发生频率分别为1.40%、0.82%和2.41%。外显子区SNP数量高于内含子,3个基因在外显子区域分布28个,内含子分布20个。外显子的28个SNP位点中,11个为错义突变,17个为同义突变。错义突变引起了编码氨基酸的改变。对SNP位点的聚类分析表明,15个栽培品种间存在的多态性位点在1–36之间,15个品种的SNP类型不同。uck1,mapk,exg1的SNP可用于香菇栽培品种的鉴别。     

滇金丝猴线粒体D-loop 片段SNPs 位点的鉴别及初步分析

本文利用已测序的157 个滇金丝猴控制区(D-loop)片段,通过与参考序列比对,鉴别了线粒体D-loop 片段中的52 个SNP (Single Nucleotide Polymorphisms)位点,定义了30 种滇金丝猴单倍型,排除概率为0. 938。谱系及种群遗传结构分析结果与以前利用D-loop 片段的研究结果相似。同时表明基于粪便样品进行保护遗传学、谱系生物地理学、种群遗传学等研究时,与线粒体标记和微卫星标记相比,SNP 标记可能具有一定的优越性,并建议进一步分析滇金丝猴线粒体D-loop 全序列甚至线粒体全基因组上的SNPs 位点的信息,以促进滇金丝猴保护遗传学等研究的开展。     

Accumulation of mitochondrial DNA deletions in human oral tissues -- effects of betel quid chewing and oral cancer

Accumulation of mitochondrial DNA (mtDNA) mutations in human tissues has been associated with intrinsic aging and environmental insult. Recently, mtDNA mutations have been detected in various tumors, including head and neck tumors. However, the factors affecting the occurrence and accumulation of mtDNA deletions in tumor tissues are poorly understood. In Taiwan, betel quid chewing is a major risk factor for oral cancer. Using polymerase chain reaction (PCR) techniques, we examined large-scale deletions of mtDNA in 53 pairs of tumor and non-tumor oral tissues from the patients with or without betel quid chewing history. The results revealed that irrespective of the history of betel quid chewing, the incidences of the 4977bp deletion and other deletions of mtDNA were lower in the tumor portion as compared with the non-tumor portion. The average proportions of the 4977bp deleted mtDNA in the tumor tissues of the betel quid chewers and non-betel quid chewers were 13- and 5-fold, respectively, lower than those in the corresponding non-tumor tissues. Moreover, the average proportion of 4977bp deleted mtDNA was significantly higher (P<0.05) in the non-tumor oral tissues of the patients with betel quid chewing history than that of the patients without the history of betel quid chewing. These results suggest that betel quid chewing may increase mtDNA mutation in human oral tissues and that accumulation of mtDNA deletions and subsequent cytoplasmic segregation of these mutations during cell division could be an important contributor to the early phase of oral carcinogenesis.     

Random intracellular drift explains the clonal expansion of mitochondrial DNA mutations with age

Human tissues acquire somatic mitochondrial DNA (mtDNA) mutations with age. Very high levels of specific mtDNA mutations accumulate within individual cells, causing a defect of mitochondrial oxidative metabolism. This is a fundamental property of nondividing tissues, but it is not known how it comes about. To explore this problem, we developed a model of mtDNA replication within single human cells. Using this model, we show that relaxed replication of mtDNA alone can lead, through random genetic drift, to the clonal expansion of single mutant events during human life. Significant expansions primarily develop from mutations acquired during a critical period in childhood or early adult life.     

The low abundance of clonally expanded mitochondrial DNA point mutations in aged substantia nigra neurons

Clonally expanded mitochondrial DNA (mtDNA) deletions accumulate with age in human substantia nigra (SN) and high levels cause respiratory chain deficiency. In other human tissues, mtDNA point mutations clonally expand with age. Here, the abundance of mtDNA point mutations within single SN neurons from aged controls was investigated. From 31 single cytochrome c oxidase normal SN neurons, only one clonally expanded mtDNA point mutation was identified, suggesting in these neurons mtDNA point mutations occur rarely, whereas mtDNA deletions are frequently observed. This contrasts observations in mitotic tissues and suggests that different forms of mtDNA maintenance may exist in these two cell types.     

Sequencing and alignment of mitochondrial genomes of Tibetan chicken and two lowland chicken breeds

Tibetan chicken lives in high-altitude area and has adapted well to hypoxia genetically. Shouguang chicken and Silky chicken are both lowland chicken breeds. In the present study, the complete mitochondrial genome sequences of the three chicken breeds were all sequenced. The results showed that the mitochondrial DNAs (mtDNAs) of Shouguang chicken and Silky chicken consist of 16784 bp and 16785 bp respectively, and Tibetan chicken mitochondrial genome varies from 16784 bp to 16786 bp. After sequence analysis, 120 mutations, including 4 single nucleotide polymorphisms (SNPs) in tRNA genes, 9 SNPs and 1 insertion in rRNA genes, 38 SNPs and 1 deletion in D-LOOP, 66 SNPs in protein-coding genes, were found. This work will provide clues for the future study on the association between mitochondrial genes and the adaptation to hypoxia.     

Clonal Expansion of Early to Mid-Life Mitochondrial DNA Point Mutations Drives Mitochondrial Dysfunction during Human Ageing

Age-related decline in the integrity of mitochondria is an important contributor to the human ageing process. In a number of ageing stem cell populations, this decline in mitochondrial function is due to clonal expansion of individual mitochondrial DNA (mtDNA) point mutations within single cells. However the dynamics of this process and when these mtDNA mutations occur initially are poorly understood. Using human colorectal epithelium as an exemplar tissue with a well-defined stem cell population, we analysed samples from 207 healthy participants aged 17–78 years using a combination of techniques (Random Mutation Capture, Next Generation Sequencing and mitochondrial enzyme histochemistry), and show that: 1) non-pathogenic mtDNA mutations are present from early embryogenesis or may be transmitted through the germline, whereas pathogenic mtDNA mutations are detected in the somatic cells, providing evidence for purifying selection in humans, 2) pathogenic mtDNA mutations are present from early adulthood (<20 years of age), at both low levels and as clonal expansions, 3) low level mtDNA mutation frequency does not change significantly with age, suggesting that mtDNA mutation rate does not increase significantly with age, and 4) clonally expanded mtDNA mutations increase dramatically with age. These data confirm that clonal expansion of mtDNA mutations, some of which are generated very early in life, is the major driving force behind the mitochondrial dysfunction associated with ageing of the human colorectal epithelium.     

Sequencing and alignment of mitochondrial genomes of Tibetan chicken and two lowland chicken breeds

Tibetan chicken lives in high-altitude area and has adapted well to hypoxia genetically. Shouguang chicken and Silky chicken are both lowland chicken breeds. In the present study, the complete mito-chondrial genome sequences of the three chicken breeds were all sequenced. The results showed that the mitochondrial DNAs (mtDNAs) of Shouguang chicken and Silky chicken consist of 16784 bp and 16785 bp respectively, and Tibetan chicken mitochondrial genome varies from 16784 bp to 16786 bp. After sequence analysis, 120 mutations, including 4 single nucleotide polymorphisms (SNPs) in tRNA genes, 9 SNPs and 1 insertion in rRNA genes, 38 SNPs and 1 deletion in D-LOOP, 66 SNPs in pro-tein-coding genes, were found. This work will provide clues for the future study on the association between mitochondrial genes and the adaptation to hypoxia.Tibetan chicken, lowland chicken, mitochondrial genome, hypoxia.     

Age-dependent 6kb deletion in human liver mitochondrial DNA.

Using PCR technique, restriction mapping and DNA sequencing, we analyzed liver mitochondrial DNA (mtDNA) of 2 stillborn babies and 62 Chinese subjects with non-liver disease from 27 to 86 years old. The results showed an age-dependent 6,063 bp deletion in the liver mtDNA of older subjects. We found a TAACAGAC sequence flanking the 5'-end breakpoint at 7,842 nucleotide position and an imperfect repeat sequence CAACATAC flanking the 3'-end breakpoint at 13,905 nucleotide position. The incidence of the deleted mtDNA was found to increase with age. The deleted mtDNA was not detected in the liver of the stillbirth or blood cells of all the subjects. This is the first account that an age-related 6,063 bp deletion occurs in the liver mtDNA of old humans. The occurrence of this and previously reported 4,977 bp deletions is consistent with our recent finding that liver mitochondrial respiratory functions decline with age and support the hypothesis that continuous accumulation of mtDNA mutations is an important contributor to ageing process in the human.     

The complete nucleotide sequence of the mitochondrial genome of Tetraodon nigroviridis.

The fresh water pufferfish Tetraodon nigroviridis is a model organism for studying evolution of genome and gene functions, but its mitochondrial genome (mtDNA) sequence is still not available. We determined the complete nucleotide sequence of its mtDNA using shotgun sequencing. The T. nigroviridis mtDNA was 16,462 bp, and contained 13 protein coding genes, 22 tRNAs, 2 rRNAs and a major non-coding region. The gene order was identical to the common type of vertebrate mtDNA, whereas the G + C content in the sense strand was 46.9%, much higher than most other fish species. One hundred and three SNPs were detected in the control region of the mtDNA of 35 individuals, a majority of SNPs were detected in the 5' end of the control region. A phylogenetic study including 21 fish species was performed on concatenated amino acid sequences of 12 protein coding genes, and revealed that the T. nigroviridis was clustered with Fugu rubripes into a group. The complete mtDNA sequence and SNPs in its control region will be useful in studying fish evolution, in differentiating different Tetraodon species and in analyzing genetic diversity within T. nigroviridis.     

线粒体DNA突变与相关人类疾病

在过去的20年里, 人们发现线粒体DNA(mitochondrial DNA, mtDNA)突变与多种人类疾病相关, 其致病范围从单器官组织损害到多系统受累。文章目的在于探讨mtDNA突变与人类疾病的关系。文章重点论述: (1)线粒体遗传学特征; (2) mtDNA突变与人类遗传性疾病; (3)体细胞mtDNA突变在衰老和肿瘤中的作用; (4)mtDNA疾病的诊断和治疗。     

Comparing phylogeny and the predicted pathogenicity of protein variations reveals equal purifying selection across the global human mtDNA diversity

We used detailed phylogenetic trees for human mtDNA, combined with pathogenicity predictions for each amino acid change, to evaluate selection on mtDNA-encoded protein variants. Protein variants with high pathogenicity scores were significantly rarer in the older branches of the tree. Variants that have formed and survived multiple times in the human phylogenetics tree had significantly lower pathogenicity scores than those that only appear once in the tree. We compared the distribution of pathogenicity scores observed on the human phylogenetic tree to the distribution of all possible protein variations to define a measure of the effect of selection on these protein variations. The measured effect of selection increased exponentially with increasing pathogenicity score. We found no measurable difference in this measure of purifying selection in mtDNA across the global population, represented by the macrohaplogroups L, M, and N. We provide a list of all possible single amino acid variations for the human mtDNA-encoded proteins with their predicted pathogenicity scores and our measured selection effect as a tool for assessing novel protein variations that are often reported in patients with mitochondrial disease of unknown origin or for assessing somatic mutations acquired through aging or detected in tumors.     

Base composition analysis of human mitochondrial DNA using electrospray ionization mass spectrometry: a novel tool for the identification and differentiation of humans

In traditional approaches, mitochondrial DNA (mtDNA) variation is exploited for forensic identity testing by sequencing the two hypervariable regions of the human mtDNA control region. To reduce time and labor, single nucleotide polymorphism (SNP) assays are being sought to possibly replace sequencing. However, most SNP assays capture only a portion of the total variation within the desired regions, require a priori knowledge of the position of the SNP in the genome, and are generally not quantitative. Furthermore, with mtDNA, the clustering of SNPs complicates the design of SNP extension primers or hybridization probes. This article describes an automated electrospray ionization mass spectrometry method that can detect a number of clustered SNPs within an amplicon without a priori knowledge of specific SNP positions and can do so quantitatively. With this technique, the base composition of a PCR amplicon, less than 140 nucleotides in length, can be calculated. The difference in base composition between two samples indicates the presence of an SNP. Therefore, no post-PCR analytical construct needs to be developed to assess variation within a fragment. Of the 2754 different mtDNA sequences in the public forensic mtDNA database, nearly 90% could be resolved by the assay. The mass spectrometer is well suited to characterize and quantitate heteroplasmic samples or those containing mixtures. This makes possible the interpretation of mtDNA mixtures (as well as mixtures when assaying other SNPs). This assay can be expanded to assess genetic variation in the coding region of the mtDNA genome and can be automated to facilitate analysis of a large number of samples such as those encountered after a mass disaster.     

A Standard Reference Material to determine the sensitivity of techniques for detecting low-frequency mutations, SNPs, and heteroplasmies in mitochondrial DNA

Human mitochondrial DNA (mtDNA) mutations are important for forensic identifications and mitochondrial disease diagnostics. Low-frequency mutations, heteroplasmies, or SNPs scattered throughout the DNA in the presence of a majority of mtDNA with the Cambridge Reference Sequence (CRS) are almost impossible to detect. Therefore, the National Institute of Science and Technology has developed heteroplasmic human mtDNA Standard Reference Material (SRM) 2394 to allow scientists to determine their sensitivity in detecting such differences. SRM 2394 is composed of mixtures ranging from 1/99 to 50/50 of two 285-bp PCR products from two cell lines that differ at one nucleotide position. Twelve laboratories using various mutation detection methods participated in a blind interlaboratory evaluation of a prototype of SRM 2394. Most of these procedures were unable to detect the mutation when present below 20%, an indication that, in many real-life cases, low-frequency mutations remain undetected and that more sensitive mutation detection techniques are urgently needed.     

The Complete Mitochondrial Genome Sequence and Characterization of Single-Nucleotide Polymorphisms in the Control Region of the Asian Seabass (Lates calcarifer)

We determined the complete mtDNA nucleotide sequence of Lates calcarifer using the shotgun sequencing method. The mitochondrial DNA (mtDNA) was 16,535 base pairs (bp) in length, and contained 13 protein coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and one major noncoding control region (CR). The CR was unusually short at only 768 bp. A striking feature of the mitochondrial genome was the high G+C content (46.1%), which is among the highest in fish. The gene order was identical to that of a typical vertebrate. Phylogenetic analyses using concatenated amino acid sequences of 12 protein-coding genes of 30 fish species representing 14 suborders clearly showed Lates calcarifer was located in the cluster of fish species from the order Perciformes, supporting the traditional systematic classification. We characterized single-nucleotide polymorphisms (SNPs) in the CR by sequencing the complete CR of 25 individuals obtained from Australia and Singapore. A total of 68 SNPs were detected. Eighteen SNPs were fixed with alternative nucleotides in Australian and Singapore seabass, and these SNPs could be used for differentiating fish from the two countries.     

Detection and quantification of mitochondrial DNA deletions in individual cells by real-time PCR

Defects of mitochondrial DNA (mtDNA) are an important cause of disease and play a role in the ageing process. There are multiple copies of the mitochondrial genome in a single cell. In many patients with acquired or inherited mtDNA mutations, there exists a mixture of mutated and wild type genomes (termed heteroplasmy) within individual cells. As a biochemical and clinical defect is only observed when there are high levels of mutated mtDNA, a crucial investigation is to determine the level of heteroplasmic mutations within tissues and individual cells. We have developed an assay to determine the relative amount of deleted mtDNA using real-time fluorescence PCR. This assay detects the vast majority of deleted molecules, thus eliminating the need to develop specific probes. We have demonstrated an excellent correlation with other techniques (Southern blotting and three- primer competitive PCR), and have shown this technique to be sensitive to quantify the level of deleted mtDNA molecules in individual cells. Finally, we have used this assay to investigate patients with mitochondrial disease and shown in individual skeletal muscle fibres that there exist different patterns of abnormalities between patients with single or multiple mtDNA deletions. We believe that this technique has significant advantages over other methods to quantify deleted mtDNA and, employed alongside our method to sequence the mitochondrial genome from single cells, will further our understanding of the role of mtDNA mutations in human disease and ageing.     

Positive and negative selection on the human genome.

The distinction between deleterious, neutral, and adaptive mutations is a fundamental problem in the study of molecular evolution. Two significant quantities are the fraction of DNA variation in natural populations that is deleterious and destined to be eliminated and the fraction of fixed differences between species driven by positive Darwinian selection. We estimate these quantities using the large number of human genes for which there are polymorphism and divergence data. The fraction of amino acid mutations that is neutral is estimated to be 0.20 from the ratio of common amino acid (A) to synonymous (S) single nucleotide polymorphisms (SNPs) at frequencies of > or =15%. Among the 80% of amino acid mutations that are deleterious at least 20% of them are only slightly deleterious and often attain frequencies of 1-10%. We estimate that these slightly deleterious mutations comprise at least 3% of amino acid SNPs in the average individual or at least 300 per diploid genome. This estimate is not sensitive to human population history. The A/S ratio of fixed differences is greater than that of common SNPs and suggests that a large fraction of protein divergence is adaptive and driven by positive Darwinian selection.