Additionally, circumstantial evidence suggests that mtDNA varia- tions could influence aging and longevity either by bearing mutations that can modulate the mitochondrial function or by relying upon more complex relationships between mitochondrial and nuclear ge- nomes (Santoro et al., 2006). It has been observed in cybrid cell models that mtDNA haplogroup H or J differently modulates the ex- pression of some nuclear genes related to the stress response, such as interleukin-1β, tumor necrosis factor receptor 2 and interleukin- 6, not only under basal conditions but also under oxidative stress con- ditions (Bellizzi et al., 2006; Santoro et al., 2006).

dard demographic indicators of quality of age reporting, in the Chuang population in China were 100.1–104.3 and 2.3–2.9 respec- tively, based on the 1982, 1990 and 2000 census data (Zeng, 2009). According to the United Nations' criteria, a Whipple's Index b105 and a Myer's Index b10 indicate good quality of age reporting. Thus, we are confident that the age information of the subjects who belong to Chuang ethnic group recruited in this study is reliable. In the con- trol group of this study, we excluded individuals with one or two par- ents who survived to 90 years old or above, and in fact, they were not able to be randomly chosen. Twelve baseline and metabolic indexes, such as, age, gender, weight, height, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting blood glucose (FBG), serum total triglyceride (TG), serum total cholesterol (TC), HDL-cholesterol (HDL-c) and LDL-cholesterol (LDL-c) are mea- sured in all selected ‘longevity’ and control subjects.

However, the effect of mtDNA haplogroups on longevity is popula- tion specific because haplogroups are defined by common mtDNA single nucleotide polymorphisms (mtSNP) in each ethnic group and the distribution of mtSNP allele frequency varies considerably depending on the population studied. For example, the mtDNA hap- logroup D (characteristic of m.5178CNA) in the Japanese population was associated with longevity. However the association between haplogroup D and longevity has not been found in southern Chinese populations (Yao et al., 2002b). Similarly, the association between haplogroup J and longevity was found in the population of northern Italy but not of southern Italy (Dato et al., 2004).

The research ethics committee of Beijing Hospital approved this study. Informed consent was obtained from all enrolled subjects, and the study was carried out in full compliance with all principles of the Helsinki Accord.

The contribution of mtDNA variations to longevity and longevity- related phenotypes in the Chuang people in China has not been established. Based on previous studies, we hypothesized that mtDNA variations could be associated with exceptional longevity in the Chuang Chinese population, and we speculated that the support- ing evidence for this association might be gained by further studying the effect of mtDNA variations on longevity-related phenotypes such as serum lipid or blood glucose levels. Previous studies have shown that subjects with exceptional longevity have favorable lipid profiles (Arai et al., 2001; Atzmon et al., 2005; Barzilai et al., 2003; Paolisso et al., 1997) and that these phenotypes are associated with several ge- netic variants located in the nuclear genome (Barzilai et al., 2003; Koropatnick et al., 2008; Pablos-Mendez et al., 1997; Rontu et al., 2006). However, conclusive evidence linking mtDNA variations to metabolic phenotypes such as serum lipid levels or blood glucose levels is still not clear among individuals with successful aging.

2.2. Study strategy

To control for population stratification, we restricted our analysis to a singleethnicgroup(Chuang)inageneticallyisolated,homogeneouslocal population from a rural county (Bama) in the southern China. We chose two samples of ‘longevity’ and younger control subjects which have not been matched by age to identify the association between mtDNA varia- tions and longevity. The strategy was as follows: 1) we identified 25 hap- logroups/subhaplogroups that include 225 mtDNA variants in long-lived individuals by sequencing twenty complete mitochondrial genomes and constructing a phylogenetic tree in preliminary study; 2) we determined which mtDNA haplogroups were associated with longevity in the Bama Chuangpopulationbythecase–controlassociationanalysiswithinadata- set of 738 subjects consisting of 367 centenarians and 371 controls; 3) we examined the effect of gender on longevity-associated mtDNA variations by stratification analysis for women and men separately; and 4) we gath- ered further supporting evidence of the association of mtDNA variations with longevity by analyzing the relationship of mtDNA variations with longevity-related phenotypes.

To pursue this hypothesis, we selected genetically isolated, homo- geneous, long-lived Chuang individuals from Bama as our study pop- ulation. Bama is a rural county in southern China with a population of 250,000 residents and an area of 1971 km2. The population lives in a valley surrounded by many steep mountains, serving as a closed geo- graphical environment in which the population has historically had little communication with outside populations. Therefore, the elders, with exceptional longevity selectively, maintained genetic homoge- neity because there was very little migration. Based on our prelimi- nary study, mtDNA haplogroups/subhaplogroups (M7/M7b′c/M7b1′ 2, F/F3, M8/C and D/D4/D4a) were selected from 25 haplogroups/sub- haplogroups in the population as our work loci used to examine the association with longevity.

2.3. Sequencing complete mitochondrial genome and phylogeny construction