Mitochondrial mutational spectra in human cells, tissues and derived tumors for bp 10,030–10,130 ... more Mitochondrial mutational spectra in human cells, tissues and derived tumors for bp 10,030–10,130 are essentially identical, suggesting a predominant mutagenic role for endogenous processes. We hypothesized that errors mediated by mitochondrial DNA polymerase γ were the primary sources of mutations. Point mutations created in this sequence by human DNA pol γ in vitro were thus compared to the eighteen mutational hotspots, all single base substitutions, previously found in human tissues. The set of concordant hotspots accounted for 83% of these in vivo mutational events. About half of these mutations are insensitive to prolonged heating of DNA during PCR and half increase proportionally with heating time at 98 °C. Primary misincorporation errors and miscopying errors past thermal denaturing products such as deaminated cytosines (uracils) thus appear to be of approximately equal importance. For the sequence studied, these data support the conclusion that, endogenous error mediated by DNA pol γ constitutes the primary source of mitochondrial point mutations in human tissues.
A-to-I editing is an adenosine-to-inosine modification of mRNA particularly widespread in the hum... more A-to-I editing is an adenosine-to-inosine modification of mRNA particularly widespread in the human brain, where it affects thousands of genes. A growing body of evidence suggests that A-to-I RNA editing is necessary for normal development and maintenance in mammals and that its deficiencies contribute to a number of pathological states. In this study, we examined whether mRNA editing levels of two mRNA species, CYFIP2 and GABRA3, change with aging. CYFIP2 has been implicated in synaptic maintenance, while GABRA3 is a GABA receptor subunit, a part of the major inhibitory neurotransmitter system in the CNS. The levels of mRNA editing were assessed in cortex samples of 20 subjects 22–102 years old. The data show an age-dependent statistically significant decrease in editing in CYFIP2. GABRA3 editing remained much more stable with age, implying that age-related decline of RNA editing is gene-specific. This is the first report of age-dependent decline in A-to-I editing. Further examination of these and other vulnerable genes may reveal specific RNA editing mechanisms that contribute to the aging phenotype.
It has been proposed that age-dependent accumulation of somatic mutations in mtDNA is responsible... more It has been proposed that age-dependent accumulation of somatic mutations in mtDNA is responsible for some aspects of the aging process. However, most cells contain hundreds to thousands of mtDNA molecules. Any nascent somatic mutant therefore appears as a single copy among a majority of wild-type species. A single mutant molecule is unlikely to influence the physiology of the cell and thus cannot play a role in the aging process. To affect cellular physiology, the nascent somatic mutants must somehow accumulate clonally in the cell to significant levels. The evidence supporting the view that, indeed, clonal expansion of mtDNA mutations is a widespread process in various human tissues, and the mechanisms by which clonal expansions may affect the aging process, are reviewed. Originally, clonal expansion was demonstrated for mtDNA with large deletions in muscle. Cell-by-cell analysis of human cardiomyocytes and buccal epithelial cells revealed that clonal expansion affects point mtDNA mutations as well as deletions. Expansions are not limited to muscle, but likely are present in most tissues, and almost every cell of an aged tissue is likely to be affected by an expansion. While the very existence of clonal expansion is beyond doubt, the mechanisms driving this process are largely controversial. The hypotheses explaining expansion includes random or various selective mechanisms, or both. We show that the spectra of expanded point mutations are drastically different in cardiomyocytes and epithelial cells. This suggests that the mechanisms of expansion in these tissues are different. In particular, we propose random segregation and positive selection models for epithelial and muscle cells, respectively.
Researchers in several laboratories have reported a high frequency of homoplasmic mitochondrial D... more Researchers in several laboratories have reported a high frequency of homoplasmic mitochondrial DNA (mtDNA) mutations in human tumors. This observation has been interpreted to reflect a replicative advantage for mutated mtDNA copies, a growth advantage for a cell containing certain mtDNA mutations, and/or tumorigenic properties of mtDNA mutations. We consider another possibility-that the observed homoplasmy arose entirely by chance in tumor progenitor cells, without any physiological advantage or tumorigenic requirement. Through extensive computer modeling, we demonstrate that there is sufficient opportunity for a tumor progenitor cell to achieve homoplasmy through unbiased mtDNA replication and sorting during cell division. To test our model in vivo, we analyzed mtDNA homoplasmy in healthy human epithelial tissues and discovered that the model correctly predicts the considerable observed frequency of homoplasmic cells. Based on the available data on mitochondrial mutant fractions and cell division kinetics, we show that the predicted frequency of homoplasmy in tumor progenitor cells in the absence of selection is similar to the reported frequency of homoplasmic mutations in tumors. Although a role for other mechanisms is not excluded, random processes are sufficient to explain the incidence of homoplasmic mtDNA mutations in human tumors.
Mitochondrial mutational spectra in human cells, tissues and derived tumors for bp 10,030–10,130 ... more Mitochondrial mutational spectra in human cells, tissues and derived tumors for bp 10,030–10,130 are essentially identical, suggesting a predominant mutagenic role for endogenous processes. We hypothesized that errors mediated by mitochondrial DNA polymerase γ were the primary sources of mutations. Point mutations created in this sequence by human DNA pol γ in vitro were thus compared to the eighteen mutational hotspots, all single base substitutions, previously found in human tissues. The set of concordant hotspots accounted for 83% of these in vivo mutational events. About half of these mutations are insensitive to prolonged heating of DNA during PCR and half increase proportionally with heating time at 98 °C. Primary misincorporation errors and miscopying errors past thermal denaturing products such as deaminated cytosines (uracils) thus appear to be of approximately equal importance. For the sequence studied, these data support the conclusion that, endogenous error mediated by DNA pol γ constitutes the primary source of mitochondrial point mutations in human tissues.
A-to-I editing is an adenosine-to-inosine modification of mRNA particularly widespread in the hum... more A-to-I editing is an adenosine-to-inosine modification of mRNA particularly widespread in the human brain, where it affects thousands of genes. A growing body of evidence suggests that A-to-I RNA editing is necessary for normal development and maintenance in mammals and that its deficiencies contribute to a number of pathological states. In this study, we examined whether mRNA editing levels of two mRNA species, CYFIP2 and GABRA3, change with aging. CYFIP2 has been implicated in synaptic maintenance, while GABRA3 is a GABA receptor subunit, a part of the major inhibitory neurotransmitter system in the CNS. The levels of mRNA editing were assessed in cortex samples of 20 subjects 22–102 years old. The data show an age-dependent statistically significant decrease in editing in CYFIP2. GABRA3 editing remained much more stable with age, implying that age-related decline of RNA editing is gene-specific. This is the first report of age-dependent decline in A-to-I editing. Further examination of these and other vulnerable genes may reveal specific RNA editing mechanisms that contribute to the aging phenotype.
It has been proposed that age-dependent accumulation of somatic mutations in mtDNA is responsible... more It has been proposed that age-dependent accumulation of somatic mutations in mtDNA is responsible for some aspects of the aging process. However, most cells contain hundreds to thousands of mtDNA molecules. Any nascent somatic mutant therefore appears as a single copy among a majority of wild-type species. A single mutant molecule is unlikely to influence the physiology of the cell and thus cannot play a role in the aging process. To affect cellular physiology, the nascent somatic mutants must somehow accumulate clonally in the cell to significant levels. The evidence supporting the view that, indeed, clonal expansion of mtDNA mutations is a widespread process in various human tissues, and the mechanisms by which clonal expansions may affect the aging process, are reviewed. Originally, clonal expansion was demonstrated for mtDNA with large deletions in muscle. Cell-by-cell analysis of human cardiomyocytes and buccal epithelial cells revealed that clonal expansion affects point mtDNA mutations as well as deletions. Expansions are not limited to muscle, but likely are present in most tissues, and almost every cell of an aged tissue is likely to be affected by an expansion. While the very existence of clonal expansion is beyond doubt, the mechanisms driving this process are largely controversial. The hypotheses explaining expansion includes random or various selective mechanisms, or both. We show that the spectra of expanded point mutations are drastically different in cardiomyocytes and epithelial cells. This suggests that the mechanisms of expansion in these tissues are different. In particular, we propose random segregation and positive selection models for epithelial and muscle cells, respectively.
Researchers in several laboratories have reported a high frequency of homoplasmic mitochondrial D... more Researchers in several laboratories have reported a high frequency of homoplasmic mitochondrial DNA (mtDNA) mutations in human tumors. This observation has been interpreted to reflect a replicative advantage for mutated mtDNA copies, a growth advantage for a cell containing certain mtDNA mutations, and/or tumorigenic properties of mtDNA mutations. We consider another possibility-that the observed homoplasmy arose entirely by chance in tumor progenitor cells, without any physiological advantage or tumorigenic requirement. Through extensive computer modeling, we demonstrate that there is sufficient opportunity for a tumor progenitor cell to achieve homoplasmy through unbiased mtDNA replication and sorting during cell division. To test our model in vivo, we analyzed mtDNA homoplasmy in healthy human epithelial tissues and discovered that the model correctly predicts the considerable observed frequency of homoplasmic cells. Based on the available data on mitochondrial mutant fractions and cell division kinetics, we show that the predicted frequency of homoplasmy in tumor progenitor cells in the absence of selection is similar to the reported frequency of homoplasmic mutations in tumors. Although a role for other mechanisms is not excluded, random processes are sufficient to explain the incidence of homoplasmic mtDNA mutations in human tumors.
It has been shown recently that mitochondrial (mtDNA) somatic variants are numerous enough to tra... more It has been shown recently that mitochondrial (mtDNA) somatic variants are numerous enough to trace cellular lineages in our body. Here we extend this statement and demonstrate that mtDNA variants can be interpreted not only as neutral markers of cell divisions but the relative frequency of different mtDNA substitutions (i.e. mtDNA mutational spectrum) can inform us about important biological properties such as cell longevity. Analysing 7611 somatic mtDNA mutations from 37 types of human cancers and more than 2000 somatic mtDNA mutations from 25 healthy human tissues we observed that mtDNA mutational spectrum is associated with cell turnover rate: the ratio of T>C to G>A is increasing with cell longevity. To extend this logic we considered that, if universal, the discovered mutation bias may drive the differences in mtDNA mutational spectrum between mammalian species with short- (‘mice’) and long- (‘elephants’) lived oocytes. Based on presumably neutral polymorphisms in MT-CYB we reconstructed mutational spectra for 424 mammalian species and obtained that the fraction of T>C positively correlated with the species-specific generation length, which is a good proxy for oocyte longevity. Next, comparing complete mitochondrial genomes of 650 mammalian species we confirmed that exactly the same process shapes the nucleotide content of the most neutral sites in the whole mitochondrial genomes of short- (high T, low C) versus long- (low T, high C) lived mammals. Altogether analysing mtDNA mutations in time interval from dozens of years (somatic mutations) through the hundreds of thousands of years (within species polymorphisms) to millions of years (between species substitutions) we demonstrated that T>C/G>A positively correlates with cellular and organismal longevity. We hypothesize that the discovered mtDNA signature presents a chemical damage which is associated with the level of oxidative metabolism which, in turn, correlates with cellular and organismal longevity. The described properties of mtDNA mutational spectrum shed light on mtDNA replication, mtDNA evolution of mammals and can be used as a marker of cell longevity in single-cell analyses of heterogeneous samples.
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