Results

We typed 33 mtSNPs which defined 28 haplogroups in the exceptional longevity group, elderly group, and middle-aged group. The genotyping success rates ranged from 99.5% to 100%. The prevalence of the aforementioned 28 haplogroups ranged from 1.9% to 23% in the elderly control subjects (Table 2).

Significant reduction of M9 haplogroups was observed in EL subjects (0.2%) when compared with both elderly subjects (2.2%) and middle-aged subjects (1.7%) (p= 0.005 and p= 0.019, respectively). Linear-by-linear association test revealed a signifi- cant decreasing trend of N9 frequency from middle-aged subjects (8.6%), elderly subjects (7.2%) and EL subjects (4.8%) (p= 0.018) Table 2.

In subsequent analysis stratified by gender, linear-by-linear association test revealed a significant increasing trend of D4 frequency from middle-aged subjects (15.8%), elderly subjects (16.4%) and EL subjects (21.7%) in females (p= 0.025) (Table 3). Conversely, a significant decreasing trend of B4a frequency was observed from middle-aged subjects (4.2%), elderly subjects (3.8%) and longevity subjects (1.7%) in females (p =0.045). However,

Table 2. Distribution of mtDNA haplogroups in different age groups.

Haplogroup Longevity Elderly Middle-aged P(L vs. E) p(L vs. M) p-trend D 120(25.9%) 213(23.0%) 111(24.0%) 0.23 0.494 0.387 D4 88(19.0%) 141(15.2%) 74(16.0%) 0.074 0.226 0.141 D4a 14(3.0%) 24(2.6%) 13(2.8%) 0.642 0.845 0.781 D4b 20(4.3%) 33(3.6%) 17(3.7%) 0.488 0.615 0.549 D4b2 20(4.3%) 30(3.2%) 15(3.2%) 0.308 0.389 0.319 D4b2b 14(3.0%) 25(2.7%) 11(2.4%) 0.730 0.543 0.550 D5 30(6.5%) 69(7.5%) 34(7.3%) 0.507 0.604 0.559 M12 22(4.8%) 44(4.8%) 24(5.2%) 1.000 0.762 0.796 G 21(4.5%) 39(4.2%) 21(4.5%) 0.779 1.000 0.949 G2 13(2.8%) 18(1.9%) 7(1.5%) 0.304 0.175 0.153 M7 35(7.6%) 67(7.2%) 36(7.8%) 0.827 0.902 0.955 M7b 18(3.9%) 35(3.8%) 22(4.8%) 0.921 0.518 0.587 M8 47(10.2%) 112(12.1%) 42(9.1%) 0.283 0.577 0.847 M8a 10(2.2%) 37(4.0%) 12(2.6%) 0.074 0.666 0.457 C 22(4.8%) 43(4.6%) 17(3.7%) 0.928 0.413 0.485 M9 1(0.2%) 20(2.2%) 8(1.7%) 0.005 0.019 0.027 M10 9(1.9%) 15(1.6%) 8(1.7%) 0.662 0.807 0.753 N9 22(4.8%) 67(7.2%) 40(8.6%) 0.075 0.018 0.018 N9a 18(3.9%) 51(5.5%) 31(6.7%) 0.190 0.056 0.058 A 35(7.6%) 66(7.1%) 26(5.6%) 0.770 0.233 0.288 F 77(16.6%) 138(14.9%) 61(13.2%) 0.401 0.140 0.147 F1 45(9.7%) 81(8.7%) 36(7.8%) 0.552 0.295 0.304 B 77(16.8%) 152(16.5%) 91(19.7%) 0.898 0.251 0.311 B5 29(6.3%) 39(4.2%) 20(4.3%) 0.097 0.186 0.117 B5a 16(3.5%) 22(2.4%) 8(1.7%) 0.245 0.098 0.086 B5b 13(2.8%) 16(1.7%) 12(2.6%) 0.186 0.839 0.357 B4a 9(1.9%) 31(3.4%) 17(3.7%) 0.139 0.112 0.105 B4b 8(1.7%) 24(2.6%) 11(2.4%) 0.310 0.487 0.430

that B4a is negatively correlated with ages in Rugao population has never been reported elsewhere.

mtDNA haplogroups and longevity is consistent with the hypothesis that mtDNA variations have a higher impact on male than on female longevity [37]. This gender difference could be one of the consequences of the maternal inheritance of mtDNA, which in turn implies a higher relaxation and less purging of male- specific mitochondrial phenotype, being males’ evolutionary dead ends for mtDNA. Interestingly, the haplogroup D4 was not enriched in male Rugao subjects, but only in female subjects. Coincidently, longevity also shows maternal inheritance [38], and

In genetic association studies on longevity, the gender difference is common, where different polymorphisms of mtDNA were associated with longevity only in males [12,14,15]. The reason for such a gender difference is not clear. A possible reason for the observed gender difference could be that males are under stronger selection than females, and are thus less likely to reach the same age as women [36]. The male-specific association between

Table 3. Distribution of mtDNA haplogroups in female subjects of different age groups.

Haplogroups Longevity Elderly Middle-aged p(L vs. E) p(L vs. M) p-trend D4 78(21.7%) 118(16.4%) 57(15.8%) 0.034 0.045 0.025 B4a 6(1.7%) 27(3.8%) 15(4.2%) 0.060 0.046 0.045 M9 0(0%) 14(1.9%) 6(1.7%) 0.008 0.014 0.032 N9 19(5.3%) 51(7.1%) 31(8.6%) 0.256 0.079 0.082

doi:10.1371/journal.pone.0006423.t003

the female male ratio is 3.5:1 in Rugao long lived subjects. The fundamental mechanism of this phenomenon and the female correlation warrants further exploring.

exerted from the original population of case group and well matched for gender with the case group, thus minimized the population stratification and false positive results. The population was relatively homogeneous related to environmental exposures, which decreased the selection biases for case control matching. Finally, although our study included 463 cases and two control groups, unfortunately, interpreting the results is also limited by the relatively insufficient sample size. For example, our sample size had the power of 0.91, and 0.65 to detect the differences between longevity subjects and elderly subjects, between longevity subjects and middle-aged subjects, respectively, given the significant level of 0.05. However, when applying a conservative Bonferroni correction for multiple testing, the significant level will be set at 0.05/28 for 28 haplogroups we analyzed. Nevertheless, for D super-haplogroup, our aim was to validate the observations made by Bilal et al and Alexe et al [11,12], we still set the significant level at a = 0.05.