2. Materials and methods

2.1. Subjects

A total of 556 unrelated longevous individuals (202 males and 354 females aged 90–108 years) and 403 unrelated controls (204 males and 199 females aged 22–73 years) were recruited from Dujiangyan city and self-identified as being Han Chinese (Table 1). The ages of elderly subjects were authenticated by both official certification and the accounts of their offspring and other elderly neighbors. Written informed consents were obtained from all participants, and a clear explanation of the possible risks of the study had been provided. This research was approved by the Ethics Committee on Human Experimentation of the Kunming Institute of Zoology, Chinese Academy of Sciences, and the relevant bodies in Dujiangyan city. The study was performed in accordance with the tenets of the Declaration of Helsinki for research involving human subjects.

3. Results

In this study, mtDNA C150T mutation frequency in the longevous group 124/556 (22.30%) was not significantly different compared to control group 89/403 (22.08%; p=0.936) (Table 2). In the East Asian mtDNA phylogeny tree, haplogroups B4c1b, B4c1c, B6, D4b2b2, D5, G1a1, G1a2, G3a1, M7b and N9a have C150T as one of their definition motifs (Kong et al., 2006). In addition, three of the ten haplogroups: D5, M7b and N9a have comparatively higher frequencies than the remaining seven haplogroups in Han Chinese populations. Therefore, we divided all the samples bearing C150T mutations into four subgroups: the D5, M7b, N9a haplogroups mentioned above and “other”. No significant difference between longevous group and control group was discovered in any of these four subgroups. Additionally, of the entire 64 samples that fell under the “other” category, more than half could be attributed into the remaining seven haplogroups, which meant that less than 15% of the C150T mutations were not regarded as mtDNA haplogroup definition motifs. Namely, these C150T carriers didn't belong to any of the ten haplogroups mentioned above (data not shown). Meanwhile, when stratified by gender, the results are the same as haplogroup results and no significance was found as well.

2.2. Genotyping

DNA extraction from white blood cells in whole blood was done following the standard phenol/chloroform method (Sambrook et al., 1989). Fragments of the mtDNA control region from 16000-408 in the Cambridge sequence (Anderson et al., 1981) were amplified and sequenced on an ABI 3700 (AppliedBiosystems, Foster City, CA) using the methods described by Yao et al. (2003). Haplogroups were defined on the basis of coding region RFLPs (as described by Yao et al. (2002a)) and control region polymorphisms in terms of the updated East Asian mtDNA phylogeny tree (Kong et al., 2006).

2.3. Statistical analysis

Table 2 also highlights the distribution of the mtDNA C150T mutation, taking both haplogroup and gender into account. Neither male nor female samples showed significant higher prevalence of

Statistical analyses were carried out using SPSS version 17.0. Pearson's chi-square was used in order to evaluate overall haplogroup

Table 1

Demographic parameters of the subjects in this study.

Group N (male/female) Age (mean±SD, years) Ethnicity Range of age (years) Longevous 556 (202/354) 94.59±3.34 Han 90-108 Control 403 (204/199) 56.24±7.81 Han 22-73

Table 2

Frequencies of subjects with the mtDNA C150T mutation in longevous group and control group.

Longevous Control p-value Fisher's exact sig. (2-sided) n % n % All All haplogroups 124 22.30 89 22.08 0.936 1.000 Haplogroup D5 35 6.29 17 4.22 0.161 0.194 Haplogroup M7b 34 6.12 31 7.69 0.338 0.364 Haplogroup N9a 18 3.24 14 3.47 0.840 0.857 Other haplogroups 37 6.65 27 6.70 0.978 1.000 Males All haplogroups 36 17.82 46 22.55 0.236 0.266 Haplogroup D5 11 5.45 8 3.92 0.467 0.491 Haplogroup M7b 11 5.45 17 8.33 0.251 0.328 Haplogroup N9a 2 0.99 10 4.90 0.020 0.036 Other haplogroups 12 5.94 11 5.39 0.811 0.833 Females All haplogroups 88 24.86 43 21.61 0.388 0.406 Haplogroup D5 24 6.78 9 4.52 0.282 0.351 Haplogroup M7b 23 6.50 14 7.03 0.808 0.860 Haplogroup N9a 16 4.52 4 2.01 0.129 0.158 Other haplogroups 25 7.06 16 8.04 0.674 0.736

All p-values are unadjusted for multiple comparisons and p-values b0.05 are indicated in bold. No significant contrasts have been found at Bonferroni adjusted alpha=0.003.

Table 3

Distribution of mtDNA C150T mutation in Japanese population and in present study.

All haplogroups Haplogroup Haplogroup Haplogroup Other D5 M7b N9a haplogroups n % n % n % n % n % Chinese Han Longevous (n=556) 124 22.30 35 6.29 34 6.12 18 3.24 37 6.65 Control (n=403) 89 22.08 17 4.22 31 7.69 14 2.47 27 6.70 Japanesea Longevous (n=96) 19 19.79 7 7.29 7 7.29 2 2.08 3 3.12 Control (n=96) 9 9.37 1 1.04 1 1.04 4 4.17 3 3.12

a Data from Niemi et al. (2005).

C150T mutation in longevous group according to the D5, M7b, N9a haplogroups and “other”, whereas the frequency of haplogroup N9a in male samples decreased from 4.90% in control group to 0.99% in longevous group (Crosstab's chi-square value of p=0.020; Fisher's exact test value of p=0.036). But when this p-value was corrected for multiple comparisons using post hoc Bonferroni analysis, leading to a significance level much lower than 0.05, this difference turned out to be non-significant.