Direct nucleotide repeats can facilitate deletions of segments of mitochondrial genome1, leading to a wide range of neuromuscular disorders1,2 as well as aging2,3 in humans. We hypothesized that the number of the direct perfect repeats in human mitochondrial genomes influences longevity through the formation of harmful mtDNA deletions in the somatic cells. The analysis of the complete mitochondrial genomes of 762 unrelated Japanese individuals4-6 reveals a negative correlation between the abundance of the direct perfect repeats and the expected longevity. This association is largely due to the disruption of the common repeat (8470,13447) by a point mutation 8473C which occurred at the origin of the D4a haplogroup characterized by extreme longevity in Japan7. Our results provide the first evidence for correlation between the number of nucleotide repeats and the lifespan on intraspecific level

Georgii Bazykin

Konstantin Popadin

English

Nature Precedings

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Nucleotide repeats in mitochondrial genome determine human lifespan  
 
Konstantin Popadin1, Georgii Bazykin1. 
 
1 Institute for Information Transmission Problems of the Russian Academy of Sciences 
(Kharkevich Institute), Bolshoi Karetny pereulok 19, Moscow 127994, Russia. 
 
Direct nucleotide repeats can facilitate deletions of segments of mitochondrial genome1, leading to a 
wide range of neuromuscular disorders1,2 as well as aging2,3 in humans. We hypothesized that the 
number of the direct perfect repeats in human mitochondrial genomes influences longevity through 
the formation of harmful mtDNA deletions in the somatic cells. The analysis of the complete 
mitochondrial genomes of 762 unrelated Japanese individuals4-6 reveals a negative correlation 
between the abundance of the direct perfect repeats and the expected longevity. This association is 
largely due to the disruption of the common repeat (8470,13447) by a point mutation 8473C which 
occurred at the origin of the D4a haplogroup characterized by extreme longevity in Japan7. Our 
results provide the first evidence for correlation between the number of nucleotide repeats and the 
lifespan on intraspecific level.  
 
 
 
 
 
 
 
 
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Phenotypic effects of aging are largely determined by the expression of hundreds of alleles 
which are deleterious in late life, and either neutral, slightly deleterious, or slightly beneficial in 
early life8. Germline mutations giving rise to these alleles can’t be eliminated effectively through 
purifying selection and accumulate due to genetic drift. Some of these alleles do not have a direct 
phenotypic effect of their own; instead, they facilitate the somatic mutations that do have phenotypic 
consequences. As the mitochondrial DNA replicates intensively during the whole life cycle, 
mitochondrial theory of aging9 postulates a vicious cycle whereby somatic mtDNA mutations 
generate excessive reactive oxygen species and these, in turn, further damage mtDNA. Since 
somatic mutations (predominantly deletions10) in mitochondrial genome are particularly likely to 
contribute to phenotypes associated with aging the germline mutations that increase the somatic 
mutability of the mtDNA are likely to have a strong effect late in life. For example, the defected 
nucleus-encoded mtDNA polymerase causes somatic accumulation of point mutations11  and 
deletions12 in mtDNA, which leads to reduced lifespan and premature onset of aging-specific 
phenotypes in mice.  
Similarly, the mtDNA repeats promote occurrence of harmful deletions in the mitochondrial 
genomes of somatic cells. Such mutated mitochondrial genomes are favored by intracellular 
selection, which amplifies the deleterious effect of the repeats for the organism. Since the 
probability of deletions relates to mtDNA replication events, and intracellular selection takes some 
time the deleterious effect of mtDNA repeats might be delayed until late in life2,3,13. Therefore, 
selection against direct repeats can be expected to be stronger in long-lived animals than in short-
lived ones. Indeed, a negative correlation between the number of the direct mitochondrial repeats 
and the maximal lifespan of mammalian species has been shown recently14,15. 
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Here we hypothesized that the number of the direct perfect repeats in human mitochondrial 
genomes influences longevity through the formation of harmful mtDNA deletions in the somatic 
cells. To test this hypothesis, we analyzed the fully sequenced mitochondrial genomes of 762 
unrelated Japanese individuals with known ages of sampling of blood for mtDNA analysis5,6.  
In each genome, we identified all direct perfect repeats of length 13 bp or more in which 
both copies of the repeat are located within the same (major or minor) arc of mtDNA16 and are at 
least 30 nucleotides from each other (Supplementary methods online). A total of 17 repeats match 
these constraints. Of these, only five repeats were frequent: cccataccccgaa (535, 4430), 
acctccctcacca (8470, 13447),  caacataaaaccc (6410, 12031), accaacaaactta (13858, 16278) and 
acccccctcccca (955, 3565), while all others were only observed once or twice in our dataset 
(Supplementary Table 1 online).  
The number of the direct repeats has a significant negative correlation with age (Kendall’s 
tau = -0.049,  P = 0.0225, N =762). To analyze this effect in more detail, we split the population into 
two parts depending on whether the patient was younger (“young”, N=366) or older (“seniors”, 
N=376) than the median age of 64 years in our population. The negative correlation between the age 
and the number of the repeats was highly significant in the seniors (Kendall tau = -0.08733382, p = 
0.005729) but not in the young (Kendall tau = -0.03316608, p = 0.1718), suggesting that the effect 
of the repeats is constrained to late in life.  
We determined the effect of each of the 17 individual repeats separately by multiple linear 
regression. Two repeats had a significant effect on age: acctccctcacca(8470,13447) (P = 0.0122) and 
atacttcctattc(11200,15562) (P = 0.0390), both on the major arc. The former is a frequent and well 
known common repeat1,8, while the latter is a rare repeat observed only in two individuals in our 
sample (Supplementary Table 1, Supplementary Figure 1 online) .  
Since these two repeats are the major determinants of age, we studied their effect on 
longevity in more detail. The subpopulation without either of these repeats reveals a significant 
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excess (P = 0.01, two-sided Fisher’s exact test) of centenarians and super centenarians (25 of 61, 
41.0%), compared to the rest of the population (178 of 701, 25.4%). The survival curves between 
the two subpopulations were significantly different (P < 0.01, Cochran-Mantel-Haenszel Chi-
Squared Test; Fig. 1), with the average expected lifespan of the individuals without these repeats 
(92.2 years) being 4 years longer than in individuals with either of these repeats (88.3 years).  
To understand the evolutionary dynamics of these two repeats, we reconstructed the 
phylogenetic tree (Supplementary Figure 2 online) and assessed their phylogenetic distribution. 
The common repeat acctccctcacca(8470,13447) was present in the last common ancestor of the 
sampled population and in most individual samples. Any single-nucleotide mutation in either copy 
of the repeated sequence is sufficient to destroy the repeat. Such substitutions occurred a total of 
four times independently on the phylogenetic tree (Supplementary Figure 2 online). All four 
substitutions were observed in the first arm of the repeat (i.e. the arm starting in position 8470), in 
the synonymous positions of the ATP8 gene; the second arm of the repeat remained invariant. The 
ancestral sequence of the repeat was disrupted by a 8473C mutation at the origin of a large 
monophyletic group consisting of 58 sampled individuals representing the D4a haplogroup. The 
other three mutations – 8479G before the origin of sequence NDsq0210 and 8470G before the origin 
of sequences HNsq0181 and PDsq0068 – occurred on the external branches. The second repeat 
atacttcctattc(11200,15562) arose by a single-nucleotide mutation only once, giving rise to two 
individuals from our sample (Supplementary Figure 2 online). 
The analyzed Japanese population was stratified by several disease-specific groups: 
individuals with Parkinson’s disease, individuals with Alzheimer’s disease, young obese males and 
type-2 diabetes individuals with or without severe vascular involvement. To assess the possible 
contribution of the diseases to our analysis, we performed a multiple logistic regression, using 
patient’s age, sex and disease group to predict the presence or absence of these two repeats. Again, 
 5
only age emerged as a significant factor (P = 0.0363), while neither sex nor belonging to any of the 
disease groups were associated with presence or absence of the repeats. 
D4a is a Japanese haplogroup well known for its unusual longevity. Several works4-7 
analyzed the mtSNPs characteristic of this haplogroup without accounting for the possible link 
between the repeat abundance and longevity. The most recent and extensive study7 of the association 
between the mtSNPs and longevity of the D4A haplogroup revealed four mtSNPs of strong effect, 
including the repeat-disrupting 8473C mtSNP, although the authors conservatively denied them 
function based on the strong phylogenetic clustering of the corresponding phenotypes evident of 
phylogenetic inertia. However, phylogenetic signal per se is not an evidence for the lack of 
functional significance. Indeed, all rare advantageous mutations in maternally inherited 
mitochondrial genome will have a strong phylogenetic signal. 
Instead, our observations of the association of the two mitochondrial repeats with longevity 
and the few independent events of loss of the common repeat, together with the known deleterious 
effect of the repeats1-3,13,16, indicate that the 8473C mtSNP caused by a mutation at the synonymous 
position of ATP8 gene is the main factor contributing to the longer lifespan of the D4a haplogroup, 
and point to the disruption of the nucleotide repeat as the likely mechanism of its effect.  
Most deletions in human mtDNA appear to be caused by the (8470,13447) repeat16. It has 
been suggested that this common repeat is the principal factor behind the formation of most 
deletions, irrespective of the precise deletion breakpoint, and that the disruption of this repeat will 
substantially reduce the chance of forming most mtDNA deletions16. The effect of the loss of the 
repeat in the human population is consistent with this hypothesis: even a single nucleotide 
substitution in the repeated segment is sufficient to significantly increase the lifespan, probably by 
reducing the total deletion score. The deleterious somatic effect of the nucleotide repeats makes it 
important to consider the effect of mtDNA mutations on abundance of nucleotide repeats in 
searching and explaining the associations between mtSNPs and phenotypes. 
 6
 
 
 
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Note: Supplementary information is available on the Nature Genetics website. 
 
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ACKNOWLEDGMENTS 
 
We thank Masashi Tanaka for discussion and providing the data of sex and age of each patient, 
Alexey Kondrashov, Mikhail Gelfand, Shamil Sunyaev, Anatolii Teriokhin and Leila Mamirova 
for comments and helpful discussions. This research was supported by the Molecular and 
Cellular Biology Program of the Russian Academy of Sciences, by Russian Foundation for 
Basic Research Grants 08-04-09680 and 08-04-01394, and by the Russian Science Support 
Foundation Grants to K.P. and G.B.  
 
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Figure 1. Survival curves for individuals with (dashed line) and without (solid line) either of 
the repeats acctccctcacca(8470,13447) and atacttcctattc(11200,15562).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 



and 27 coauthors.

Nucleotide repeats in mitochondrial genome determine human lifespan

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Nucleotide repeats in mitochondrial genome determine human lifespan

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