2.5. Statistical methods

MEGA version 3.1 (Kumar et al., 2004) was used to create the phylogenetic tree. 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 haplogroup. Sequences with multiple potential errors were excluded from the dataset and subsequently re-sequenced. Based on our phylogeny (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 frequencies of all observed haplogroups/subhaplogroups are presented in Supplementary Table 2. All statistical analyses were conducted using the SPSS 11.5 software 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% confidence 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 mitochondria, 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 p<0.05.

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

3. Results

3.1. The characteristics of the study subjects

Ten candidate haplogroups/subhaplogroups that included 15 common mtDNA variants were selected as our working loci for the association 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 digestion 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 primer to form a(N=367) (N=371)

Age (years) 95.34±4.75 46.75±4.79 p<0.001 
Gender (M/F) 98/269 107/264 
BMI (kg/m2) 18.69±3.21 24.03±2.69 p<0.001 
SBP (mm Hg) 146.31±21.94 117.21±13.71 p<0.001 
DBP (mm Hg) 80.13±11.38 75.27±9.38 p<0.001 
FBG (mmol/L) 5.34±1.54 4.83±0.59 p<0.001 
TC (mmol/L) 4.91±1.24 4.62±0.72 p=0.001 
HDL-c (mmol/L) 1.51±0.61 1.52±0.33 p=0.970 
LDL-c (mmol/L) 2.85±0.92 2.27±0.64 p<0.001 
TG (mmol/L) 1.37±0.93 1.51±0.44 p=0.022 

Sex differences exist in longevity, with women living longer than men.
When we stratified the Bama cohort by gender, we found a significant association of longevity in females for haplogroup F (p=0.003, OR: 2.01, 95% CI: 1.26-3.19) and the three mtSNPs (m.3970C>T, m13928G>C and m10310G>A) that defined haplogroup F (p=0.001, OR: 1.970, 95% CI: 1.30-2.98; p=0.004, OR: 1.799, 95% CI: 1.20-2.68 and p=0.003, OR: 2.010, 95% CI: 1.26-3.19). However, there was no such significant association found in males (Table 3). Additionally, subhaplogroup F3 and one mtDNA variant (m10320G>A) in subhaplogroup F3 were slightly associated with longevity.

Note: Values are given as means ± SDs.
BMI = body mass index, SBP = systolic blood pressure, DBP = diastolic blood pressure, FBG = fasting blood glucose, TG = serum total triglyceride, TC = serum total cholesterol, HDL-c = high density lipoprotein-cholesterol, LDL-c = low density lipoprotein-cholesterol.

3.4. mtDNA variation associated with longevity-related phenotype

To attain supporting evidence for the association of mtDNA variations with longevity, we tested the relationship of FBG, TG, TC, HDL-c and LDL-c levels with mtDNA haplogroup F among the Bama population. The results showed that mtDNA haplogroup F was associated with markedly higher HDL-c levels (p<0.05), but only in long-lived subjects (13.5%). There was a significant statistical difference for haplogroup F between the long-lived and control groups for the two genders combined (p=0.010, OR: 1.703, 95% CI: 1.153-2.514, see Table 2), and the power was 85.5% by the statistical power calculator (http://www.dssresearch.com/toolkit/sscalc/size_p2.asp). To further determine the influence of haplogroup F on longevity, every mtDNA variant (m.3970C>T, m.13928G>C or m.10310G>A) in haplogroup F was analyzed by the association study. The three mtDNA variants were found to be consistently associated with longevity for the two genders combined (p=0.009, OR: 1.602, 95% CI: 1.134-2.262; p=0.021, OR: 1.507, 95% CI: 1.075-2.114 and p=0.010, OR: 1.703, 95% CI: 1.153-2.514, respectively, see Table 2).

4. Discussion