Experimental Gerontology 46 (2011) 511–515

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Experimental Gerontology

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Short report

Absence of association between mitochondrial DNA C150T polymorphism andlongevity in a Han Chinese population

Hui Pan a,b,c, Qing-peng Kong a,b, Yao-ting Cheng a,b,c, Shi-gang Lian d, Juan Yang d, Shou-jun Gao e,Liang-you Xu e, Ya-ping Zhang a,b,⁎a State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Chinab Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091, Chinac Graduate University of the Chinese Academy of Sciences, Beijing 100049, Chinad People’s Hospital of Dujiangyan City, Dujiangyan 611830, Chinae Dujiangyan Longevity Research Centre, Dujiangyan 611830, China

⁎ Corresponding author at: State Key Laboratory of GeKunming Institute of Zoology, Chinese Academy of SciencChina. Tel.: +86 871 519 9030; fax: +86 871 519 5430

E-mail addresses: zhangyp@mail.kiz.ac.cn, zhangyp1

0531-5565/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.exger.2011.01.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 May 2010Received in revised form 1 November 2010Accepted 14 January 2011Available online 22 January 2011

Section Editor: R. Westendorp

Keywords:LongevityMitochondrial DNAC150TAssociation studyHan Chinese population

Human longevity has been associated with mitochondrial DNA (mtDNA) coding region polymorphisms, aswell as the C150T polymorphism in the non-coding region in previous studies especially in Europeans. Thisstudy investigated the potential association between the mtDNA C150T polymorphism and longevity in a HanChinese population. Leukocyte mtDNAs from two groups of a Han Chinese population living in Dujiangyancity of Sichuan province, including 556 longevous individuals (90–108 years-old) and 403 unrelated controls,were analyzed and mtDNA haplogroups were determined by sequencing control regions and restrictionfragment length polymorphisms (RFLPs) in coding regions. Our results did not show a universal associationbetween the mitochondrial C150T polymorphism and longevity in this population. Even when mtDNAhaplogroups defined by C150T and gender were taken into account, there was no significant association withlongevity. In conclusion, the mtDNA C150T polymorphism could not present an accumulation in an elderlyHan Chinese population. Previous association studies might have been influenced by nuclear DNA and/orenvironment factors.

netic Resources and Evolution,es, Kunming, Yunnan, 650223,.@263.net.cn (Y. Zhang).

l rights reserved.

© 2011 Elsevier Inc. All rights reserved.

1. Introduction

The relationship between the human ageing process and mito-chondrial DNA (mtDNA) changes, including germ-line point muta-tions, somatic point mutations, and fragment deletions, has beenintensely investigated over the last decade. One of the mostcontroversial associations reported is that of human longevity witha C150T mutation on mtDNA. An early report of mtDNA haplogroup Jenrichment in elderly people focused on male centenarians ofnorthern Italy (De Benedictis et al., 1999). Subsequently, the C150Tmutation defining haplogroup J2 was revealed to be the true factorassociated with longevity (Niemi et al., 2003). At the same time, ananalysis of nascent heavy mtDNA strands revealed a new origin ofreplication at position 149, substituting for that at position 151, onlyin cell samples carrying the C150T mutation (Zhang et al., 2003).Indeed, a number of studies with positive or negative results have

been published in this area based on different populations. Theseparadoxical findings have motivated broader investigations for thepurpose of clarifying whether the C150T mutation does contribute tohuman longevity and whether the association is universal to allpopulations or unique to particular ethnic groups. Most studies so farhave focused on European populations or their derivatives, includingItalian (Dato et al., 2004; De Benedictis et al., 1999; Rose et al., 2007),Finnish (Niemi et al., 2003, 2005), Irish (Ross et al., 2001), Spanish(Dominguez-Garrido et al., 2009)and Ashkenazi Jewish populations(Iwata et al., 2007). In addition, populations from Japan (Niemi et al.,2005) and from Xinjiang province in China (Ren et al., 2008) havebeen investigated, however, the former study was limited by its smallsample size and the latter was limited to Uygur subjects. To ourknowledge, no previous study has investigated the putative associ-ation between the C150T mutation and longevity in a Han Chinesepopulation, which is the largest ethnic group in the world.

We set out to explore the possible association between the mtDNAC150T polymorphism and longevity in Han Chinese subjects living inDujiangyan city in Sichuan province, China. Dujiangyan city isrenowned for having a longevous population in China, with afrequency of centenarians of more than 0.01% as indexed in the 5thNational Census in China in 2000. Such a population is ideal to detectpossible relations between mtDNA mutations and longevity.

512 H. Pan et al. / Experimental Gerontology 46 (2011) 511–515

2. Materials and methods

2.1. Subjects

A total of 556 unrelated longevous individuals (202 males and 354females aged 90–108 years) and 403 unrelated controls (204 malesand 199 females aged 22–73 years) were recruited from Dujiangyancity and self-identified as being Han Chinese (Table 1). The ages ofelderly subjects were authenticated by both official certification andthe accounts of their offspring and other elderly neighbors. Writteninformed consents were obtained from all participants, and a clearexplanation of the possible risks of the study had been provided. Thisresearch was approved by the Ethics Committee on HumanExperimentation of the Kunming Institute of Zoology, ChineseAcademy of Sciences, and the relevant bodies in Dujiangyan city.The study was performed in accordance with the tenets of theDeclaration of Helsinki for research involving human subjects.

2.2. Genotyping

DNA extraction from white blood cells in whole blood was donefollowing the standard phenol/chloroform method (Sambrook et al.,1989). Fragments of the mtDNA control region from 16000-408 in theCambridge sequence (Anderson et al., 1981) were amplified andsequenced on an ABI 3700 (AppliedBiosystems, Foster City, CA) usingthe methods described by Yao et al. (2003). Haplogroups weredefined on the basis of coding region RFLPs (as described by Yao et al.(2002a)) and control region polymorphisms in terms of the updatedEast Asian mtDNA phylogeny tree (Kong et al., 2006).

2.3. Statistical analysis

Statistical analyses were carried out using SPSS version 17.0.Pearson's chi-square was used in order to evaluate overall haplogroup

Table 1Demographic parameters of the subjects in this study.

Group N (male/female) Age (mean±SD

Longevous 556 (202/354) 94.59±3.34Control 403 (204/199) 56.24±7.81

Table 2Frequencies of subjects with the mtDNA C150T mutation in longevous group and control g

Longevous Co

n % n

AllAll haplogroups 124 22.30 89Haplogroup D5 35 6.29 17Haplogroup M7b 34 6.12 31Haplogroup N9a 18 3.24 14Other haplogroups 37 6.65 27

MalesAll haplogroups 36 17.82 46Haplogroup D5 11 5.45 8Haplogroup M7b 11 5.45 17Haplogroup N9a 2 0.99 10Other haplogroups 12 5.94 11

FemalesAll haplogroups 88 24.86 43Haplogroup D5 24 6.78 9Haplogroup M7b 23 6.50 14Haplogroup N9a 16 4.52 4Other haplogroups 25 7.06 16

All p-values are unadjusted for multiple comparisons and p-values b0.05 are indicated in b

frequency differences between the two populations studied. Cross-tab's chi-square test as well as Fisher's exact test (the p-value was twotailed) were also performed in comparisons of total C150T mutationfrequencies and dipartite rates in terms of specific haplogroups andgender. P-values were corrected for multiple comparison by Bonfer-roni analysis (required significance level=0.05/number of compar-isons), leading to a new required significance level of b0.003(numberof comparisons=15).

3. Results

In this study, mtDNA C150T mutation frequency in the longevousgroup 124/556 (22.30%) was not significantly different compared tocontrol group 89/403 (22.08%; p=0.936) (Table 2). In the East AsianmtDNA phylogeny tree, haplogroups B4c1b, B4c1c, B6, D4b2b2, D5,G1a1, G1a2, G3a1, M7b and N9a have C150T as one of their definitionmotifs (Kong et al., 2006). In addition, three of the ten haplogroups:D5, M7b and N9a have comparatively higher frequencies than theremaining seven haplogroups in Han Chinese populations. Therefore,we divided all the samples bearing C150T mutations into foursubgroups: the D5, M7b, N9a haplogroups mentioned above and“other”. No significant difference between longevous group andcontrol group was discovered in any of these four subgroups.Additionally, of the entire 64 samples that fell under the “other”category, more than half could be attributed into the remaining sevenhaplogroups, which meant that less than 15% of the C150T mutationswere not regarded as mtDNA haplogroup definition motifs. Namely,these C150T carriers didn't belong to any of the ten haplogroupsmentioned above (data not shown). Meanwhile, when stratified bygender, the results are the same as haplogroup results and nosignificance was found as well.

Table 2 also highlights the distribution of the mtDNA C150Tmutation, taking both haplogroup and gender into account. Neithermale nor female samples showed significant higher prevalence of

, years) Ethnicity Range of age (years)

Han 90-108Han 22-73

roup.

ntrol p-value Fisher's exactsig. (2-sided)

%

22.08 0.936 1.0004.22 0.161 0.1947.69 0.338 0.3643.47 0.840 0.8576.70 0.978 1.000

22.55 0.236 0.2663.92 0.467 0.4918.33 0.251 0.3284.90 0.020 0.0365.39 0.811 0.833

21.61 0.388 0.4064.52 0.282 0.3517.03 0.808 0.8602.01 0.129 0.1588.04 0.674 0.736

old. No significant contrasts have been found at Bonferroni adjusted alpha=0.003.

Table 3Distribution of mtDNA C150T mutation in Japanese population and in present study.

All haplogroups HaplogroupD5

HaplogroupM7b

HaplogroupN9a

Otherhaplogroups

n % n % n % n % n %

Chinese Han Longevous (n=556) 124 22.30 35 6.29 34 6.12 18 3.24 37 6.65Control (n=403) 89 22.08 17 4.22 31 7.69 14 2.47 27 6.70

Japanesea Longevous (n=96) 19 19.79 7 7.29 7 7.29 2 2.08 3 3.12Control (n=96) 9 9.37 1 1.04 1 1.04 4 4.17 3 3.12

a Data from Niemi et al. (2005).

513H. Pan et al. / Experimental Gerontology 46 (2011) 511–515

C150T mutation in longevous group according to the D5, M7b, N9ahaplogroups and “other”, whereas the frequency of haplogroup N9a inmale samples decreased from 4.90% in control group to 0.99% inlongevous group (Crosstab's chi-square value of p=0.020; Fisher'sexact test value of p=0.036). But when this p-value was corrected formultiple comparisons using post hoc Bonferroni analysis, leading to asignificance level much lower than 0.05, this difference turned out tobe non-significant.

4. Discussion

The present results indicate that the C150T polymorphism is notrobustly associated with longevity in Han Chinese populations wehave investigated. Pearson Chi-Square analysis revealed that the twopopulations in this study had similar mtDNA genetic backgrounds andshowed no differences in the overall haplogroup frequencies (data notshown). A similar situation occurred in another study on longevity ina Chinese population (Cai et al., 2009). However, we still cannot ruleout that other mtDNA variants might be associated with humanlongevity potentially, despite the negative results found presently.

The mtDNA haplogroup structure in European populations isdistinct from that in East Asian populations because of their differentmain haplogroups. In the European human mtDNA phylogeny tree,the C150T mutation defines haplogroup J2 and some sub-branches ofhaplogroup U and T (van Oven and Kayser, 2009), while it also acts asa motif site in 10 haplogroups in the East Asian tree. Additionally, wenoticed that the C150T mutation frequencies in European populationsreported by previous longevity related case/control studies were 17%vs. 3.4% in Italians (Zhang et al., 2003), 20.4% vs. 14.5% in Finns (Niemiet al., 2005) and 1.6% vs. 3.4% in Ashkenazi Jews (Iwata et al., 2007),for cases and controls, respectively. In contrast, the Chinesepopulation in our study showed much higher frequencies (higherthan 20%) of the C150T mutation in both case and control groups. Asfor other East Asian population, a research based on Japanesepopulation found a significant higher frequency of C150T mutationin centenarians (19.79%) than in middle-aged controls (9.37%) (Niemiet al., 2005). Compared to the Han Chinese population, the C150Tmutation frequency in Japanese population was near in case butobviously lower in control (Table 3). More importantly, their p-valueswere unadjusted for multiple comparisons and the sample size wassmall. Besides, the frequencies of the D5, M7b and N9a haplogroups incurrent study were roughly analogous to those observed in alongevous Chinese population in Rugao (Cai et al., 2009). Thus, ifthere was a real function of the C150T mutation that could affecthuman lifespan, it might be easier to detect in Chinese populationsthan in Europeans; furthermore, the possibility of introducingstatistical bias would be smaller as well.

The putative contribution of mtDNA to human longevity and/orage-related diseases, such as Parkinson's disease and Alzheimer'sdisease has been the subject of much investigation. Apart from theC150T polymorphism and the haplogroups it defined, other mtDNApolymorphisms/haplogroups have been reported to relate to humanlongevity. For example, enrichment of mtDNA 5178CNA (Tanaka et al.,2000), haplogroups D4 (Bilal et al., 2008), D5, and two subgroups of

D4, D4b2b andD4a (Alexe et al., 2007) have been observed in Japanesecentenarians. In addition, an increased incidence of D4a, anddecreased incidences of M9, N9 and B4a haplogroups were observedin Chinese longevous subjects in Rugao (Cai et al., 2009). However,several of these associations were limited to specific populations andcould not be replicated in other studies. For instance, unlike thefindings of Tanaka et al. (2000) in a Japanese sample, an associationbetween longevity and mtDNA 5178CNA polymorphism was notfound in Chinese subjects (Yao et al., 2002b). Furthermore, compli-cated relationships between mtDNA haplogroups and longevity,Parkinson's disease, Alzheimer's disease or Type 2 diabetic havebeen demonstrated with several mtDNA haplogroups, including B5bandM7b2, found to be enriched in both longevous and disease groups(Takasaki, 2009). These seemingly discrepant findings may beattributed to a number of factors: sampling strategy limitations,such as inadequate matching of cases and controls, inadequateadjustment for multiple comparison testing, and cryptic populationstratification (Shlush et al., 2008); interactions between mtDNA andthe environment, for example, altitude may affect mtDNA haplogroupJ2 enrichment in elderly people (Dominguez-Garrido et al., 2009);finally, mtDNA and variants elsewhere in the genome may interact.Moreover, the latter two factors usually related to specific populationsand then create such paradoxes.

MtDNA is likely to play an important role in human ageing,althoughmost investigations thus far remainwithin the scope of case/control studies and the incidence difference was typically the onlyparameter that could be analyzed. However, there has been little focuson elucidating the mechanisms by which mtDNA variants couldimpact function at the cellular, tissue, or systems level, and therebyinfluence human lifespan. Functional studies on mtDNA variants arerequired that will help to further our understanding of the influence ofmitochondrial/nuclear genetic background and environmental factorson human longevity.

Acknowledgements

We are greatly indebted to the persons who participated in thisresearch. In addition, we thank Dr. Hui Li and Dr. Yuanying Gong fortheir help in sample collection, and Dr. Wendy Adams's help inpreparing the paper. This work was supported by the National BasicResearch Program of China (No. 2007CB507405), “Light in WesternChina” of the Chinese Academy of Sciences, the National NaturalScience Foundation of China (No. 30621092), and the Bureau ofScience and Technology of Yunnan Province.

References

Alexe, G., Fuku, N., Bilal, E., Ueno, H., Nishigaki, Y., Fujita, Y., Ito, M., Arai, Y., Hirose, N.,Bhanot, G., Tanaka, M., 2007. Enrichment of longevity phenotype in mtDNAhaplogroups D4b2b, D4a, and D5 in the Japanese population. Hum. Genet. 121,347–356.

Anderson, S., Bankier, A.T., Barrell, B.G., deBruijn,M.H., Coulson,A.R., Drouin, J., Eperon, I.C.,Nierlich,D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith,A.J., Staden,R., Young, I.G., 1981.Sequence and organization of the human mitochondrial genome. Nature 290,457–465.

514 H. Pan et al. / Experimental Gerontology 46 (2011) 511–515

Bilal, E., Rabadan, R., Alexe, G., Fuku, N., Ueno, H., Nishigaki, Y., Fujita, Y., Ito, M., Arai, Y.,Hirose, N., Ruckenstein, A., Bhanot, G., Tanaka, M., 2008. Mitochondrial DNAhaplogroup D4a is a marker for extreme longevity in Japan. PLoS ONE 3, e2421.

Cai, X.Y., Wang, X.F., Li, S.L., Qian, J., Qian, D.G., Chen, F., Yang, Y.J., Yuan, Z.Y., Xu, J., Bai,Y., Yu, S.Z., Jin, L., 2009. Association of mitochondrial DNA haplogroups withexceptional longevity in a Chinese population. PLoS ONE 4, e6423.

Dato, S., Passarino, G., Rose, G., Altomare, K., Bellizzi, D., Mari, V., Feraco, E., Franceschi,C., De Benedictis, G., 2004. Association of the mitochondrial DNA haplogroup J withlongevity is population specific. Eur. J. Hum. Genet. 12, 1080–1082.

De Benedictis, G., Rose, G., Carrieri, G., De Luca, M., Falcone, E., Passarino, G., Bonafe, M.,Monti, D., Baggio, G., Bertolini, S., Mari, D., Mattace, R., Franceschi, C., 1999.Mitochondrial DNA inherited variants are associated with successful aging andlongevity in humans. FASEB J. 13, 1532–1536.

Dominguez-Garrido, E., Martinez-Redondo, D., Martin-Ruiz, C., Gomez-Duran, A., Ruiz-Pesini, E., Madero, P., Tamparillas, M., Montoya, J., von Zglinicki, T., Diez-Sanchez, C.,Lopez-Perez, M.J., 2009. Association of mitochondrial haplogroup J and mtDNAoxidative damage in two different North Spain elderly populations. Biogerontology10, 435–442.

Iwata, N., Zhang, J., Atzmon, G., Leanza, S., Cho, J., Chomyn, A., Burk, R.D., Barzilai, N.,Attardi, G., 2007. Aging-related occurrence in Ashkenazi Jews of leukocyteheteroplasmic mtDNA mutation adjacent to replication origin frequently remo-deled in Italian centenarians. Mitochondrion 7, 267–272.

Kong, Q.P., Bandelt, H.J., Sun, C., Yao, Y.G., Salas, A., Achilli, A., Wang, C.Y., Zhong, L., Zhu,C.L., Wu, S.F., Torroni, A., Zhang, Y.P., 2006. Updating the East Asian mtDNAphylogeny: a prerequisite for the identification of pathogenic mutations. Hum. Mol.Genet. 15, 2076–2086.

Niemi, A.K., Hervonen, A., Hurme, M., Karhunen, P.J., Jylha, M., Majamaa, K., 2003.Mitochondrial DNA polymorphisms associated with longevity in a Finnishpopulation. Hum. Genet. 112, 29–33.

Niemi, A.K., Moilanen, J.S., Tanaka, M., Hervonen, A., Hurme, M., Lehtimaki, T., Arai, Y.,Hirose, N., Majamaa, K., 2005. A combination of three common inheritedmitochondrial DNA polymorphisms promotes longevity in Finnish and Japanesesubjects. Eur. J. Hum. Genet. 13, 166–170.

Ren, W.H., Li, X.H., Zhang, H.G., Deng, F.M., Liao, W.Q., Pang, Y., Liu, Y.H., Qiu, M.J., Zhang,G.Y., Zhang, Y.G., 2008. Mitochondrial DNA haplogroups in a Chinese Uygurpopulation and their potential association with longevity. Clin. Exp. Pharmacol.Physiol. 35, 1477–1481.

Rose, G., Passarino, G., Scornaienchi, V., Romeo, G., Dato, S., Bellizzi, D., Mari, V., Feraco,E., Maletta, R., Bruni, A., Franceschi, C., De Benedictis, G., 2007. The mitochondrialDNA control region shows genetically correlated levels of heteroplasmy inleukocytes of centenarians and their offspring. BMC Genomics 8, 293.

Ross, O.A., McCormack, R., Curran, M.D., Duguid, R.A., Barnett, Y.A., Rea, I.M., Middleton,D., 2001. Mitochondrial DNA polymorphism: its role in longevity of the Irishpopulation. Exp. Gerontol. 36, 1161–1178.

Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual,2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Shlush, L.I., Atzmon, G., Weisshof, R., Behar, D., Yudkovsky, G., Barzilai, N., Skorecki, K.,2008. Ashkenazi Jewish centenarians do not demonstrate enrichment in mito-chondrial haplogroup J. PLoS ONE 3, e3425.

Takasaki, S., 2009. Mitochondrial haplogroups associated with Japanese centenarians,Alzheimer's patients, Parkinson's patients, type 2 diabetic patients and healthynon-obese young males. J. Genet. Genomics 36, 425–434.

Tanaka, M., Gong, J., Zhang, J., Yamada, Y., Borgeld, H.J., Yagi, K., 2000. Mitochondrialgenotype associated with longevity and its inhibitory effect on mutagenesis. Mech.Ageing Dev. 116, 65–76.

van Oven, M., Kayser, M., 2009. Updated comprehensive phylogenetic tree of globalhuman mitochondrial DNA variation. Hum. Mutat. 30, E386–E394.

Yao, Y.G., Kong, Q.P., Bandelt, H.J., Kivisild, T., Zhang, Y.P., 2002a. Phylogeographicdifferentiation of mitochondrial DNA in Han Chinese. Am. J. Hum. Genet. 70,635–651.

Yao, Y.G., Kong, Q.P., Zhang, Y.P., 2002b. Mitochondrial DNA 5178A polymorphism andlongevity. Hum. Genet. 111, 462–463.

Yao, Y.G., Kong, Q.P., Man, X.Y., Bandelt, H.J., Zhang, Y.P., 2003. Reconstructing theevolutionary history of China: a caveat about inferences drawn from ancient DNA.Mol. Biol. Evol. 20, 214–219.

Zhang, J., Asin-Cayuela, J., Fish, J., Michikawa, Y., Bonafe, M., Olivieri, F., Passarino, G., DeBenedictis, G., Franceschi, C., Attardi, G., 2003. Strikingly higher frequency incentenarians and twins of mtDNA mutation causing remodeling of replicationorigin in leukocytes. Proc. Natl Acad. Sci. USA 100, 1116–1121.

Web references

Alexe, G., Fuku, N., Bilal, E., Ueno, H., Nishigaki, Y., Fujita, Y., Ito, M., Arai, Y., Hirose, N.,Bhanot, G., Tanaka, M., 2007. Enrichment of longevity phenotype in mtDNAhaplogroups D4b2b, D4a, and D5 in the Japanese population. Hum. Genet. 121,347–56. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17308896.

Anderson, S., Bankier,A.T., Barrell, B.G., deBruijn,M.H., Coulson,A.R., Drouin, J., Eperon, I.C.,Nierlich, D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith, A.J., Staden, R., Young, I.G.,1981. Sequence and organization of the human mitochondrial genome. Nature 290,457–65. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7219534.

Bilal, E., Rabadan, R., Alexe, G., Fuku, N., Ueno, H., Nishigaki, Y., Fujita, Y., Ito, M., Arai, Y.,Hirose, N., Ruckenstein, A., Bhanot, G., Tanaka, M., 2008. Mitochondrial DNA

haplogroup D4a is a marker for extreme longevity in Japan. PLoS One 3, e2421.URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18545700.

Cai, X.Y., Wang, X.F., Li, S.L., Qian, J., Qian, D.G., Chen, F., Yang, Y.J., Yuan, Z.Y., Xu, J., Bai, Y.,Yu, S.Z., Jin, L., 2009. Association of mitochondrial DNA haplogroups with exceptionallongevity in a Chinese population. PLoS One 4, e6423. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19641616.

Dato, S., Passarino, G., Rose, G., Altomare, K., Bellizzi, D., Mari, V., Feraco, E., Franceschi, C.,De Benedictis, G., 2004. Association of the mitochondrial DNA haplogroup J withlongevity ispopulation specific. Eur. J.Hum.Genet. 12, 1080–2.URL:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15470367.

De Benedictis, G., Rose, G., Carrieri, G., De Luca, M., Falcone, E., Passarino, G., Bonafe, M.,Monti, D., Baggio, G., Bertolini, S., Mari, D., Mattace, R., Franceschi, C., 1999.Mitochondrial DNA inherited variants are associated with successful aging andlongevity in humans. Faseb. J. 13, 1532–6. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10463944.

Dominguez-Garrido, E., Martinez-Redondo, D., Martin-Ruiz, C., Gomez-Duran, A., Ruiz-Pesini, E., Madero, P., Tamparillas, M., Montoya, J., von Zglinicki, T., Diez-Sanchez,C., Lopez-Perez, M.J., 2009. Association of mitochondrial haplogroup J and mtDNAoxidative damage in two different North Spain elderly populations. Biogerontology10, 435–42. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18931934.

Iwata,N., Zhang, J., Atzmon,G., Leanza, S., Cho, J., Chomyn, A., Burk, R.D., Barzilai, N., Attardi,G., 2007. Aging-related occurrence in Ashkenazi Jews of leukocyte heteroplasmicmtDNA mutation adjacent to replication origin frequently remodeled in Italiancentenarians. Mitochondrion 7, 267–72. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17452024.

Kong, Q.P., Bandelt, H.J., Sun, C., Yao, Y.G., Salas, A., Achilli, A., Wang, C.Y., Zhong, L., Zhu,C.L., Wu, S.F., Torroni, A., Zhang, Y.P., 2006. Updating the East Asian mtDNAphylogeny: a prerequisite for the identification of pathogenic mutations. Hum. Mol.Genet. 15, 2076–86. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16714301.

Niemi, A.K., Hervonen, A., Hurme, M., Karhunen, P.J., Jylha, M., Majamaa, K., 2003.Mitochondrial DNA polymorphisms associated with longevity in a Finnish popula-tion. Hum. Genet. 112, 29–33. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12483296.

Niemi, A.K., Moilanen, J.S., Tanaka, M., Hervonen, A., Hurme, M., Lehtimaki, T., Arai, Y.,Hirose, N., Majamaa, K., 2005. A combination of three common inheritedmitochondrial DNA polymorphisms promotes longevity in Finnish and Japanesesubjects. Eur. J. Hum. Genet. 13, 166–70. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15483642.

Ren,W.H., Li, X.H., Zhang,H.G., Deng, F.M., Liao,W.Q., Pang, Y., Liu, Y.H., Qiu,M.J., Zhang,G.Y.,Zhang, Y.G., 2008. Mitochondrial DNA haplogroups in a chinese Uygur population andtheir potential association with longevity. Clin. Exp. Pharmacol. Physiol. 35, 1477–81.URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18759861.

Rose, G., Passarino, G., Scornaienchi, V., Romeo, G., Dato, S., Bellizzi, D., Mari, V., Feraco, E.,Maletta, R., Bruni, A., Franceschi, C., De Benedictis, G., 2007. The mitochondrial DNAcontrol region shows genetically correlated levels of heteroplasmy in leukocytes ofcentenarians and their offspring. BMC Genomics 8, 293. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17727699.

Ross, O.A., McCormack, R., Curran,M.D., Duguid, R.A., Barnett, Y.A., Rea, I.M.,Middleton, D.,2001. Mitochondrial DNA polymorphism: its role in longevity of the Irish population.Exp. Gerontol. 36, 1161–78. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11404057.

Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular cloning : a laboratory manual, 2nded. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. City: Cold SpringHarbor, N.Y. Publisher: Cold Spring Harbor Laboratory Press. Number of Volumes: 3.ISBN: 9780879693091. URL: http://www.cshlpress.com/default.tpl?action=full&–eqskudatarq=144.

Shlush, L.I., Atzmon, G.,Weisshof, R., Behar, D., Yudkovsky, G., Barzilai, N., Skorecki, K., 2008.Ashkenazi Jewish centenarians do not demonstrate enrichment in mitochondrialhaplogroup J. PLoSOne3, e3425.URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18923645.

Takasaki, S., 2009. Mitochondrial haplogroups associated with Japanese centenarians,Alzheimer's patients, Parkinson's patients, type 2 diabetic patients and healthy non-obese young males. J. Genet. Genomics 36, 425–34. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19631917.

Tanaka, M., Gong, J., Zhang, J., Yamada, Y., Borgeld, H.J., Yagi, K., 2000. Mitochondrialgenotype associated with longevity and its inhibitory effect on mutagenesis. Mech.Ageing Dev. 116, 65–76. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10996007.

van Oven, M., Kayser, M., 2009. Updated comprehensive phylogenetic tree of globalhuman mitochondrial DNA variation. Hum. Mutat. 30, E386-94. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18853457.

Yao, Y.G., Kong, Q.P., Bandelt, H.J., Kivisild, T., Zhang, Y.P., 2002a. Phylogeographicdifferentiation of mitochondrial DNA in Han Chinese. Am. J. Hum. Genet. 70, 635–51.URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db= Pub-Med&dopt=Citation&list_uids=11836649Yao, Y.G., Kong, Q.P., Zhang, Y.P., 2002b.Mitochondrial DNA 5178A polymorphism and longevity. Hum. Genet. 111, 462–3.

515H. Pan et al. / Experimental Gerontology 46 (2011) 511–515

URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12384792.

Yao, Y.G., Kong, Q.P., Man, X.Y., Bandelt, H.J., Zhang, Y.P., 2003. Reconstructing theevolutionary history of China: a caveat about inferences drawn from ancientDNA. Mol. Biol. Evol. 20, 214–9. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12598688.

Zhang, J., Asin-Cayuela, J., Fish, J., Michikawa, Y., Bonafe, M., Olivieri, F., Passarino, G.,

De Benedictis, G., Franceschi, C., Attardi, G., 2003. Strikingly higher frequency incentenarians and twins of mtDNA mutation causing remodeling of replication originin leukocytes. Proc. Natl. Acad. Sci. U.S.A. 100, 1116–21. URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12538859.