2.3. Sequencing complete mitochondrial genome and phylogeny construction

GenomicDNA wasextractedfromperipheralblood leukocytesusing a Wizard® Genomic DNA Purification Kit (Cat.No.A1120, Promega, U.S.A.). The complete mtDNA of 20 individuals (selected from 367 ‘lon- gevity subjects’) (age range 90–107 years, mean [SD] age, 96.4[5.31] years) who were of normal physical, psychological and cognitive func- tioning was 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 In- dustry Park, Building 204 BDA, Beijing 100176, P.R. China). The detailed condition is listed in Supplementary Table 1. For the complete mito- chondrial 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, in- cluding 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 ated by the neighbor-joining program (1000× bootstrapped)

2.5. Statistical methods

MEGA version 3.1 (Kumar et al., 2004).Then, using sequence alignment and sequence comparisons, the positions of the individual nucleotide changes were incorporated into nodes of the tree. Each mtDNA was assigned to its respective haplogroup as described in the literature (Kivisild et al., 2002; Kong et al., 2006; Kong et al., 2003; Tanaka et al., 2004; Yao et al., 2002a). Coding-region mutation motifs were mainly employed for the definition of the haplogroup (Bandelt et al., 2000). Each mtDNA sequence was analyzed for possible sequencing errors, identified by base changes inconsistent with the most likely hap- logroup. Sequences with multiple potential errors were excluded from the dataset and subsequently re-sequenced. On the basis of our phy- logeny (Fig. 1) and East Asian haplogroups as previously described (Kivisild et al., 2002; Kong et al., 2003, 2006; Tanaka et al., 2004; Yao et al., 2002a), 25 haplogroups/subhaplogroups in long-lived Bama individuals were identified, as shown in Fig. 1. And the frequen- cies of all observed haplogroups/subhaplogroups are presented in Sup- plementary Table 2.

All statistical analyses were conducted using the SPSS 11.5 soft- ware package. An unpaired Student's t-test and a chi-squared test were used to compare quantitative and qualitative phenotypes and demographic data between long-lived subjects and controls. The chi-squared and Fisher's exact tests were carried out to explore the association of each mtDNA variant or haplogroup with longevity. We performed gender stratification analysis by Mantel–Haenszel chi-squared test. The p-value, odds ratios (ORs) and their 95% confi- dence intervals (CIs) were calculated. For haplogroup analyses, we compared each haplogroup with all other haplogroups pooled into one group. Because there are no recombination mechanisms in mito- chondria, some of the mtSNPs in the present study are tightly linked to each other. Since some of the markers are in linkage disequilibrium and the haplogroup comparisons are not independent, p values reported for mtSNPs and haplogroups are not adjusted for multiple testing (Rosa et al., 2008). A significant threshold was set at pb0.05.

2.4. Selection of mtSNPs for haplogroups classification and genotyping of mtSNPs

3. Results

3.1. The characteristics of the study subjects

10 candidate haplogroups/subhaplogroups that included 15 com- mon mtDNA variants were selected as our working loci for the asso- ciation study and further genotyping (details in Supplementary Table 3). Genotypes of 15 mtDNA variants were analyzed by PCR or restriction site-generating PCR (RG-PCR) followed by restriction di- gestion analysis. The mtDNA variants 10,400, 15,487, 6455, 4071, 12,811, 3010, 3970 and 10,310 were genotyped by RG-PCR-RFLP using a base change on the 3′ terminal of the forward or reverse prim- er to form a restriction site. The mitochondrial DNA variants 13,263, 4048, 5178 (Castri et al., 2009), 14,979, 13,928, 10,320 and 5585 were typed by PCR-RFLP. The detailed condition and restriction en- zyme are listed in Supplementary Table 4. We subjected DNA samples to direct sequencing to confirm the accuracy of genotyping by the two methods.

The baseline characteristics of the 738 Chuang individuals are pre- sented in Table 1. In terms of biological characteristics, BMI and serum concentration of TG were lower in long-lived individuals than in the controls (pb0.001). The SBP, DBP and FBG levels, serum concentration of TC and LDL-c were higher in long-lived individuals than in the controls (pb0.001).

3.2. mtDNA variation associated with longevity

Ten mtDNA haplogroups/subhaplogroups were investigated by the association analysis. Haplogroup F was found to be significantly more frequent in longevity subjects (21.0%) than in local controls

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Fig. 1. The phylogenetic tree of complete mtDNA sequence MtDNA phylogenetic tree includes 20 complete mtDNA sequences (SEQA1-10 and SEQB1-10). The tree is rooted in macrohaplogroup M and N. Suffixes A and T refer to transversions, ‘d’ means deletion. B5* represents an undefined subhaplogroup of B5.

3.3. The effect of gender on the association of mtDNA variation with longevity

Table 1

The characteristics in 738 Chuang subjects in China.