Human aging and somatic point mutations in mtDNA: A comparative study of generational differences (grandparents and grandchildren)

The accumulation of somatic mutations in mtDNA is correlated with aging. In this work, we sought to identify somatic mutations in the HVS-1 region (D-loop) of mtDNA that might be associated with aging. For this, we compared 31 grandmothers (mean age: 63 ± 2.3 years) and their 62 grandchildren (mean age: 15 ± 4.1 years), the offspring of their daughters. Direct DNA sequencing showed that mutations absent in the grandchildren were detected in a presumably homoplasmic state in three grandmothers and in a heteroplasmic state in an additional 13 grandmothers; no mutations were detected in the remaining 15 grandmothers. However, cloning followed by DNA sequencing in 12 grandmothers confirmed homoplasia in only one of the three mutations previously considered to be homoplasmic and did not confirm heteroplasmy in three out of nine grandmothers found to be heteroplasmic by direct sequencing. Thus, of 12 grandmothers in whom mtDNA was analyzed by cloning, eight were heteroplasmic for mutations not detected in their grandchildren. In this study, the use of genetically related subjects allowed us to demonstrate the occurrence of age-related (> 60 years old) mutations (homoplasia and heteroplasmy). It is possible that both of these situations (homoplasia and heteroplasmy) were a long-term consequence of mitochondrial oxidative phosphorylation that can lead to the accumulation of mtDNA mutations throughout life.     

Analysis of heteroplasmy in the major noncoding region of mitochondrial DNA in the blood and atherosclerotic plaques of carotid arteries

For identification of somatic mitochondrial DNA (mtDNA) mutations, the mtDNA major noncoding region (D-loop) sequence in blood samples and carotid atherosclerosis plaques from patients with atherosclerosis was analyzed. Five point heteroplasmic positions were observed in 4 of 23 individuals (17%). Only in two cases could heteroplasmy have resulted from somatic mutation, whereas three heteroplasmic positions were found in both vascular tissue and blood. In addition, length heteroplasmy in a polycytosine stretches was registered at nucleotide positions 303–315 in 16 individuals, and also in the 16184–16193 region in four patients. The results suggest that somatic mtDNA mutations can occur during atherosclerosis, but some heteroplasmic mutations may appear in all tissues, possibly being inherited.     

Mitochondrial Mutations in Subjects with Psychiatric Disorders

A considerable body of evidence supports the role of mitochondrial dysfunction in psychiatric disorders and mitochondrial DNA (mtDNA) mutations are known to alter brain energy metabolism, neurotransmission, and cause neurodegenerative disorders. Genetic studies focusing on common nuclear genome variants associated with these disorders have produced genome wide significant results but those studies have not directly studied mtDNA variants. The purpose of this study is to investigate, using next generation sequencing, the involvement of mtDNA variation in bipolar disorder, schizophrenia, major depressive disorder, and methamphetamine use. MtDNA extracted from multiple brain regions and blood were sequenced (121 mtDNA samples with an average of 8,800x coverage) and compared to an electronic database containing 26,850 mtDNA genomes. We confirmed novel and rare variants, and confirmed next generation sequencing error hotspots by traditional sequencing and genotyping methods. We observed a significant increase of non-synonymous mutations found in individuals with schizophrenia. Novel and rare non-synonymous mutations were found in psychiatric cases in mtDNA genes: ND6, ATP6, CYTB, and ND2. We also observed mtDNA heteroplasmy in brain at a locus previously associated with schizophrenia (T16519C). Large differences in heteroplasmy levels across brain regions within subjects suggest that somatic mutations accumulate differentially in brain regions. Finally, multiplasmy, a heteroplasmic measure of repeat length, was observed in brain from selective cases at a higher frequency than controls. These results offer support for increased rates of mtDNA substitutions in schizophrenia shown in our prior results. The variable levels of heteroplasmic/multiplasmic somatic mutations that occur in brain may be indicators of genetic instability in mtDNA.     

Mitochondrial DNA somatic mutation burden and heteroplasmy are associated with chronological age,smoking, and HIV infection

The gradual accumulation of mitochondrial DNA (mtDNA) mutations is implicated in aging and may contribute to the accelerated aging phenotype seen with tobacco smoking and HIV infection. mtDNA mutations are thought to arise from oxidative damage; however, recent reports implicate polymerase γ errors during mtDNA replication. Investigations of somatic mtDNA mutations have been hampered by technical challenges in measuring low‐frequency mutations. We use primer ID‐based next‐generation sequencing to quantify both somatic and heteroplasmic blood mtDNA point mutations within the D‐loop, in 164 women and girls aged 2–72 years, of whom 35% were smokers and 56% were HIV‐positive. Somatic mutations and the occurrence of heteroplasmic mutations increased with age. While transitions are theorized to result from polymerase γ errors, transversions are believed to arise from DNA oxidative damage. In our study, both transition and transversion mutations were associated with age. However, transition somatic mutations were more prevalent than transversions, and no heteroplasmic transversions were observed. We also measured elevated somatic mutations, but not heteroplasmy, in association with high peak HIV viremia. Conversely, heteroplasmy was higher among smokers, but somatic mutations were not, suggesting that smoking promotes the expansion of preexisting mutations rather than de novo mutations. Taken together, our results are consistent with blood mtDNA mutations increasing with age, inferring a greater contribution of polymerase γ errors in mtDNA mutagenesis. We further suggest that smoking and HIV infection both contribute to the accumulation of mtDNA mutations, though in different ways.     

Point mutations of the mtDNA control region in normal and neurodegenerative human brains

Recent observations in cultured human fibroblasts suggest that the accumulation of point mutations in the noncoding control region of mtDNA may be important in human aging. We studied the mtDNA control region in brain tissue from 31 normal elderly individuals, from 35 individuals who had Alzheimer disease, and from 47 individuals who had dementia with Lewy bodies. We found no evidence that these somatic mtDNA point mutations accumulate either in the brains of normal elderly individuals or in the brains of individuals with neurodegenerative disease.     

Lack of Structural Variation but Extensive Length Polymorphisms and Heteroplasmic Length Variations in the Mitochondrial DNA Control Region of Highly Inbred Crested Ibis,Nipponia nippon

The animal mitochondrial DNA (mtDNA) length polymorphism and heteroplasmy are accepted to be universal. Here we report the lack of structural variation but the presence of length polymorphism as well as heteroplasmy in mtDNA control region of an endangered avian species – the Crested Ibis (Nipponia nippon). The complete control region was directly sequenced while the distribution pattern and inheritance of the length variations were examined using both direct sequencing and genotyping of the PCR fragments from captive birds with pedigrees, wild birds and a historical specimen. Our results demonstrated that there was no structural variation in the control region, however, different numbers of short tandem repeats with an identical motif of CA₃CA₂CA₃ at the 3′-end of the control region determined the length polymorphisms among and heteroplasmy within individual birds. There were one to three predominant fragments in every bird; nevertheless multiple minor fragments coexist in all birds. These extremely high polymorphisms were suggested to have derived from the ‘replication slippage’ of a perfect microsatellite evolution following the step-wise mutational model. The patterns of heteroplasmy were found to be shifted between generations and among siblings but rather stable between blood and feather samples. This study provides the first evidence of a very extensive mtDNA length polymorphism and heteroplasmy in the highly inbred Crested Ibis which carries an mtDNA genome lack of structural genetic diversity. The analysis of pedigreed samples also sheds light on the transmission of mtDNA length heteroplasmy in birds following the genetic bottleneck theory. Further research focusing on the generation and transmission of particular mtDNA heteroplasmy patterns in single germ line of Crested Ibis is encouraged by this study.     

The impact of mitochondrial DNA on human lifespan: a view from studies on centenarians

The role of inherited and somatic mutations of mitochondrial DNA (mtDNA) in aging and longevity is complex and highly controversial, owing to its peculiar genetics, including the phenomenon of heteroplasmy. Most of the data on mtDNA and longevity have been obtained on humans and particularly on centenarians, i. e., people who escaped or delayed the major age-related pathologies and reached the extreme limit of human lifespan. In this review we summarize the most recent advances in this field that suggest a consistent role in human longevity of both germ-line inherited and somatically acquired mutations. The particular case of the association with longevity of the somatic C150T mutation is extensively discussed, challenging the tenet that mtDNA mutations are basically detrimental. We also stress several limitations of our present knowledge, regarding the difficulty in extrapolating to humans the results obtained in animal models, owing to a variety of biological differences, including the very limited genetic variability of mtDNA in the strains used in laboratory experiments. The use of high-throughput technologies and the extensive analysis, possibly at the single cell level, of different tissues and cell types derived from the same individual will help in disentangling the complexity of mtDNA in aging and longevity.     

The use of next generation sequencing technology to study the effect of radiation therapy on mitochondrial DNA mutation

The human mitochondrial genome has an exclusively maternal mode of inheritance. Mitochondrial DNA (mtDNA) is particularly vulnerable to environmental insults due in part to an underdeveloped DNA repair system, limited to base excision and homologous recombination repair. Radiation exposure to the ovaries may cause mtDNA mutations in oocytes, which may in turn be transmitted to offspring. We hypothesized that the children of female cancer survivors who received radiation therapy may have an increased rate of mtDNA heteroplasmy mutations, which conceivably could increase their risk of developing cancer and other diseases. We evaluated 44 DNA blood samples from 17 Danish and 1 Finnish families (18 mothers and 26 children). All mothers had been treated for cancer as children and radiation doses to their ovaries were determined based on medical records and computational models. DNA samples were sequenced for the entire mitochondrial genome using the Illumina GAII system. Mother's age at sample collection was positively correlated with mtDNA heteroplasmy mutations. There was evidence of heteroplasmy inheritance in that 9 of the 18 families had at least one child who inherited at least one heteroplasmy site from his or her mother. No significant difference in single nucleotide polymorphisms between mother and offspring, however, was observed. Radiation therapy dose to ovaries also was not significantly associated with the heteroplasmy mutation rate among mothers and children. No evidence was found that radiotherapy for pediatric cancer is associated with the mitochondrial genome mutation rate in female cancer survivors and their children.     

Length Variation, Heteroplasmy and Sequence Divergence in the Mitochondrial DNA of Four Species of Sturgeon (Acipenser)

The extent of mtDNA length variation and heteroplasmy as well as DNA sequences of the control region and two tRNA genes were determined for four North American sturgeon species: Acipenser transmontanus, A. medirostris, A. fulvescens and A. oxyrhnychus. Across the Continental Divide, a division in the occurrence of length variation and heteroplasmy was observed that was concordant with species biogeography as well as with phylogenies inferred from restriction fragment length polymorphisms (RFLP) of whole mtDNA and pairwise comparisons of unique sequences of the control region. In all species, mtDNA length variation was due to repeated arrays of 78-82-bp sequences each containing a D-loop strand synthesis termination associated sequence (TAS). Individual repeats showed greater sequence conservation within individuals and species rather than between species, which is suggestive of concerted evolution. Differences in the frequencies of multiple copy genomes and heteroplasmy among the four species may be ascribed to differences in the rates of recurrent mutation. A mechanism that may offset the high rate of mutation for increased copy number is suggested on the basis that an increase in the number of functional TAS motifs might reduce the frequency of successfully initiated H-strand replications.     

Understanding differences between phylogenetic and pedigree-derived mtDNA mutation rate: a model using families from the Azores Islands (Portugal)

We analyzed the control region of the mitochondrial DNA (mtDNA)from maternally related individuals originating from the AzoresIslands (Portugal) in order to estimate the mutation rate ofmtDNA and to gain insights into the process by which a new mutationarises and segregates into heteroplasmy. Length and/or pointheteroplasmies were found at least in one individual of 72%of the studied families. Eleven new point substitutions werefound, all of them in heteroplasmy, from which five appear tobe somatic mutations and six can be considered germinal, evidencingthe high frequency of somatic mutations in mtDNA in healthyyoung individuals. Different values of the mutation rate accordingto different assumptions were estimated. When considering allthe germinal mutations, the value of the mutation rate obtainedis one of the highest reported so far in family studies. However,when corrected for gender (assuming that the mutations presentin men have the same evolutionary weight of somatic mutationsbecause they will inevitably be lost) and for the probabilityof intraindividual fixation, the value for the mutation rateobtained for HVRI and HVRII (0.2415 mutations/site/Myr) wasin the upper end of the values provided by phylogenetic estimations.These results indicate that the discrepancy, that has been reportedpreviously, between the human mtDNA mutation rates observedalong evolutionary timescales and the estimations obtained usingfamily pedigrees can be minimized when corrections for genderproportions in newborn individuals and for the probability ofintraindividual fixation are introduced. The analyses performedsupport the hypothesis that (1) in a constant, tight bottleneckgenetic drift alone can explain different patterns of heteroplasmysegregation and (2) in neutral conditions, the destiny of anew mutation is strictly related to the initial proportion ofthe new variant. Another important point arising from the dataobtained is that, even in the absence of a paternal contributionof mtDNA, recombination may occur between mtDNA molecules presentin an individual, which is only observable if it occurs betweenmtDNA types that differ at two or more positions.     

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。     

The distribution of mitochondrial DNA heteroplasmy due to random genetic drift

Cells containing pathogenic mutations in mitochondrial DNA (mtDNA) generally also contain the wild-type mtDNA, a condition called heteroplasmy. The amount of mutant mtDNA in a cell, called the heteroplasmy level, is an important factor in determining the amount of mitochondrial dysfunction and therefore the disease severity. mtDNA is inherited maternally, and there are large random shifts in heteroplasmy level between mother and offspring. Understanding the distribution in heteroplasmy levels across a group of offspring is an important step in understanding the inheritance of diseases caused by mtDNA mutations. Previously, our understanding of the heteroplasmy distribution has been limited to just the mean and variance of the distribution. Here we give equations, adapted from the work of Kimura on random genetic drift, for the full mtDNA heteroplasmy distribution. We describe how to use the Kimura distribution in mitochondrial genetics, and we test the Kimura distribution against human, mouse, and Drosophila data sets.     

Heteroplasmy of the human mtDNA control region remains constant during life

In a longitudinal, retrospective study, we monitored the level of heteroplasmy at nucleotide position (nt) 309 and nt 16189 of the control region of human mtDNA. As a unique source of DNA, we analyzed multiple cervical-cell samples collected, during 1 or 2 decades, from four women with heteroplasmy at either nt 309 or nt 16189. According to accurate, quantitative analysis by solid-phase minisequencing, the level of heteroplasmy remained stable in the cervical-cell samples from all four women during the time studied. We also analyzed autopsy samples from several different tissues, all containing nt 309 in heteroplasmic form, of one of the women, who was deceased. On the basis of our results, heteroplasmy in the control region of mtDNA seems to be inherited and is not the result of somatic age-related accumulation.     

Low mutational burden of individual acquired mitochondrial DNA mutations in brain

Neurons may be particularly susceptible to oxidative damage, which has been proposed to induce somatic mutations, particularly in mitochondrial DNA (mtDNA). Therefore, acquired mtDNA mutations might preferentially accumulate in the brain and could play a role in aging and neurodegenerative disorders. Recently, a somatic T to G mtDNA mutation at noncoding nucleotide position 414 was reported in fibroblasts specifically from elderly subjects, with mutational burdens of up to 50%. We screened for this mutation in brain-derived mtDNA from 8 Alzheimer's disease patients, 27 Parkinson's disease patients, 4 multiple system atrophy patients, and 44 controls using up to three RFLP analyses. A total of 73 of these subjects were over the age of 65. The 414 mutation was absent in all cases. Next, individual mtDNA fragments from 6 elderly subjects were cloned, and a total of 70 clones were sequenced. The 414 mutation was absent in all clones, though occasional sequence variations were identified at other sites in single clones. The 414 mutation also was absent in blood (n = 6) and fibroblasts (n = 11) from elderly subjects. Our data suggest that it is rare for any one particular acquired mtDNA mutation to reach levels in the brain that are functionally significant. This does not exclude the possibility that the cumulative burden of multiple, individually rare, acquired mutations impairs mitochondrial function.     

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.     

Mitochondrial DNA polymorphism in Finnish families with Leber's hereditary optic neuroretinopathy

Summary Leukocyte mitochondrial DNA (mtDNA) from 17 Finnish families iwth Leber's hereditary optic neuroretinopathy and 70 maternally unrelated controls as well as skeletal muscle mtDNA from four of the Leber families and three controls was analyzed with 30 restriction enzymes. By this means, over 10% of the nucleotides of mtDNA were screened. No major deletion or insertion was found in any of the mtDNAs studied. The restriction fragment patterns of mtDNA showed no evidence of mtDNA heteroplasmy (mixture of different mtDNA types) in either blood or muscle cells. In all, 24 mtDNA types were observed in the material. In the maternal lines of Leber families, 11 mtDNA types were found, indicating no recent common maternal ancestor for the Finnish Leber families. In spite of several previously unknown polymorphisms, no mutation of mtDNA could be found exclusively in families with Leber's disease. However, a couple of mutations leading to amino acid replacements of mitochondrially encoded proteins were observed in certain Leber families only. These mutations have occurred in genes coding for subunits of NADH dehydrogenase, suggesting that a defect of the respiratory chain complex I may cause Leber's disease.     

Increased variation in mtDNA in patients with familial sensorineural hearing impairment

Analyses of mitochondrial DNA (mtDNA) sequences have revealed non-neutral patterns, suggesting that many amino acid mutations in animal mtDNA may be mildly deleterious, but this has not been verified in human clinical series. Since sensorineural hearing impairment (SNHI) is a common manifestation in many of the syndromes caused by mutations in mtDNA, this may be regarded as the phenotype of choice in attempts to detect mutations that may have a mildly deleterious effect on mitochondrial function. We selected 32 subjects from among 117 unrelated SNHI patients with SNHI in maternal relatives by means of family history, determined the entire coding region sequence of mtDNA and compared the sequence variation with that in 32 haplogroup-matched controls taken at random from 192 Finnish sequences. The 32 control sequences differed from the remaining 160 sequences by 36±9 substitutions (mean ± SD), while the difference for the 32 patients was 58±4 substitutions (P=0.005 for difference; Wilcoxon signed rank test). Differences were also found in the number of new haplotypes and new non-synonymous mutations or mutations in tRNA or rRNA genes. A total of 12 rare mtDNA variants were detected in the patients, and only 3 of these were considered to be neutral in effect. It is proposed that increased sequence variation in mtDNA may be a genetic risk factor for SNHI, and the increased frequency of rare haplotypes in these patients points to the presence of mildly deleterious mutations in mtDNA.     

Accumulation of mtDNA variations in human single CD34+ cells from maternally related individuals: Effects of aging and family genetic background

Marked sequence variation in the mtDNA control region has been observed in human single CD34⁺ cells, which persist in vivo and are present also in differentiated hematopoietic cells. In this study, we analyzed 5071 single CD34⁺ cells from 49 individuals (including 31 maternally related members from four families and 18 unrelated donors) in order to determine the mutation spectrum within the mtDNA control region in single cells, as related to aging and family genetic background. Many highly mutated sites among family members were hypervariable sites in the mtDNA control region. Further, CD34⁺ cells from members of the same family also shared several unique mtDNA variants, suggesting pedigree-specific occurrence of these variants. Overall age-related accumulation of mtDNA mutations in CD34⁺ cells varied in different families, suggesting a specific accumulation pattern, which might be modulated by family genetic background. Our current findings have implications for the occurrence of mtDNA mutations in hematopoietic stem cells and progenitors.     

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.