95uC*4 min - [94uC*30 sec - 60uC (-0.5uC/cycle)*50 sec - 72uC*90 sec]*11cycles –[94uC*30 sec - 55uC*50 sec - 72uC*90 - sec]* 24cycles - 72uC*10 min - 4uC*‘, and the final concentra- tions of each forward and reverse primers for mtDNA SNPs are as follows: for Multiplex 1, 12705 and 12811 are 0.33 mM; 12406 and 12358 are 0.35 mM; 7600 and 10400 are 0.53 mM; 8281 is 0.6 mM; 14569 is 0.67 mM; 9296 is 0.7 mM. For Multiplex 2, 4491, 8020, 8584 and 8684 are 0.2 mM; 5417 and 5465 are 0.25 mM; 1107 is 0.27 mM; 3010, 3970, 4833, 5178, 7196, 9824, 10646, 10873, and 15662 are 0.3 mM; 15235 is 0.37 mM; 3705, 8964, 13263 and 13590 are 0.4 mM; 14979 is 0.47 mM; 1736 is 0.5 mM. After PCR amplification, 0.95 mL of the both two multiplexes amplification products are mixed and incubated with 0.1 mL Exonuclease I (New England Biolabs, Ipswich, MA) and 2 mL shrimp alkaline phosphatase (SAP) (Promega, Madison, WI) for 80 min at 37uC followed by 15 min at 75uC for enzyme inactivation. And then, multiplex primer extension reactions were carried out by SNaPshot Multiplex Ready eaction mix (Applied Biosystems, Foster City, CA), Sequencing Buffer 5 X (Applied Biosystems, Foster City, CA) and primers extension mix with the multiplex primer extension cycling conditions: 96uC*1 min - [96uC*10 sec - 52uC*5 sec-60uC*30 sec] *28cycles - 4uC*‘ and the final concentrations of each extension primers are also given: 1107, 1736, 3010, 3705, 3970, 4491, 4833, 5178, 7196, 7600, 8020, 8584, 8684, 8964, 9824, 10646, 10873, 12811, 13263, 13590, 15235 and 15662 are 0.16 mM; 5417 is 0.33 mM; 12406 is 0.37 mM; 5465, 12358, 12705 and 14979 are 0.41 mM; 14569 is 0.45 mM; 8281, 9296 and 10400 are 0.49 mM. After the SNaPshot reaction, 6.1 ml of the SNaPshot reaction was treated with 1 U of shrimp alkaline phosphatase (Promega, Madison, WI) to inactivate the excess ddNTPs. Electrophoresis of final minisequencing products was accomplished on the ABI 3130xl Genetic Analyzer (Applied Biosystems). Genemapper ver.4.0 software was used to

Discussion

In the present study, we conducted a population-based case- control study to explore the effects of mtDNA haplogroups on the phenomenon of extremely long life in a Chinese population. Crucial to our approach is the strong survival advantage of the extremely longevity people at Rugao (less than 1/10,000 Rugao people survive to 100 y, while the average lifespan in Rugao is 75.58 y). The enrichment in the frequency of certain genetic variants at this extremely old people probably reflects a selection effect that may enhance the likelihood of survival. We did observe the enrichment of certain haplogroups in Rugao population, such as D4 haplogroup. Interestingly, there is a reduction of M9, N9, and B4a in these long lived subjects.

Haplogroups D4 is one of the most characteristic mtDNA lineages found in Northeast Asian populations [25]. Historically, the population of Rugao was mainly admixed with the local residents and the immigrants from northern China in the last two millenniums [26]. It was reported that the 5178A of haplogroup D4 was enriched in Japanese centenarians [21]. Haplogroups D4a, D5, and D4b2 were significantly enriched in male centenarians compared with healthy young Japanese males. [12]. Further, D4a haplogroup were associated with certain beneficial patterns in Japanese centenarians and semi-supercentenarians [11]. In the present study, we also observed enrichment of D4a haplogroup in Rugao longevity subjects than two control groups, albeit significance only exists in females. Our results provided evidence that D4a contribute to longevity outside Japan.

Haplogroups M9 and N9, two major subclusters directly sprout from the basal M and N trunks, respectively, have originated from Southeast Asia conceivably and are prevailing over central or northern China [27–29]. Significantly higher prevalence of M9 was observed in Tibetan (20%) than in Chinese Han (2.0%) living in northern China. In Rugao subjects living in an area where admixture of southern and northern populations occurred, the prevalence of M9 (2.2%) is similar to those living in northern China. The specific characteristic mutation to M9 is G4491A from valine (V) to isoleucine (I), which is considered a potentially functional polymorphism. Nevertheless, although the presence of M9 haplogroup is negatively associated with the risk of longevity in Rugao population, the prevalence of M9 is only 2.2%. Therefore, the attributable risk of M9 to the longevity is not prominent at Rugao. In the present study, we presented the first report that N9 confers an increased risk against longevity. The specific charac- teristic mutation 5417 to N9 had silent amino acid change [30,31]. Thus, the potential mechanism for the involvement of N9 in longevity in female of Rugao calls for further investigation. Interestingly, it was reported recently that haplogroup N9a confers resistance against type 2 diabetes in Asians and haplogroup N9a protected against metabolic syndrome in Japanese women [32,33]. These may relate to N9a-specific mtSNPs or other potentially functional polymorphisms. The B4a haplogroups, defined as a subgroup of B4 by the presence of the 16261T polymorphism, are frequent along the southeastern coast of Taiwan, but also have a wide and ancient distribution all over East Asia and island Pacific [34,35]. To the best of our knowledge, there is no report linking B4a to the phenotype of complex trait. Therefore, our observation

Statistical Analysis

Mitochondrial DNA haplogroup frequencies were calculated by counting from the observed genotypes. The Pearson chi-square test and the chi-square test for linear-by-linear association were used to analyze the significances between the prevalence of haplogroups and different age groups. SPSS software (version 10.0) was used.

Results