However, the effect of mtDNA haplogroups on longevity is population-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 haplogroup 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 supporting 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 these phenotypes are associated with several genetic 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 single ethnic group (Chuang) in a genetically isolated, homogeneous local population from a rural county (Bama) in southern China. We chose two samples of longevity and younger control subjects that have not been matched by age to identify the association between mtDNA variations and longevity. The strategy was as follows: 1) we identified 25 haplogroups/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 Chuang population by the case-control association analysis within a dataset 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 gathered further supporting evidence of the association of mtDNA variations with longevity by analyzing the relationship of mtDNA20 individuals (selected from 367 longevity subjects) (age range 90-107 years, mean [SD] age, 96.4 [5.31] years) who were of normal physical, psychological, and cognitive functioning were amplified by polymerase chain reaction in a total of 34 overlapping fragments of approximately 700 bp and sequenced by Invitrogen Corporation (No.7 Rong Chang East Street, Long Sheng Industry Park, Building 204 BDA, Beijing 100176, P.R. China). The detailed conditions are listed in Supplementary Table 1. For the complete mitochondrial genome sequencing, altogether 225 mtDNA variants were found in long-lived individuals using the revised Cambridge Reference Sequence (rCRS) (Andrews et al., 1999) as the reference.

2. Subjects and methods

2.1. Subjects

In this study, longevity subjects were classified as participants who had survived to age 90 years or more, with younger controls being less than 65 years of age. 738 individuals from Bama were recruited, including 367 long-lived individuals and 371 local and unrelated younger controls. The individuals with exceptional longevity included 269 women and 98 men (mean [SD] age: 95.4 [4.84] and 95.1 [4.51] years). The phylogenetic tree of 20 complete mtDNA sequences was created by the neighbor-joining program (1000 bootstrapped).

2.5. Statistical methods