Subjects

conducted in 2001, the population of Rugao was 1,362.5 thousand, and the life expectancy of Rugao is 75.58 years [18]. Until Dec, 2007, the registered number of individuals with exceptional longevity (EL, survival in good health to aged $95 yr) in the Longevity Institute of Rugao is 755, including 112 centenarians. The field investigation of EL subjects were conducted between Dec 24, 2007 and Feb 29, 2008. We verified the age of EL subjects by the following steps: (1) verification of dates of birth (DOB) – linkage to the 2000 population Census Office of Rugao (a part of the fifth national censuses of China) and statistic annual of Rugao; (2) verification of dates of death – linkage to the death record of local police station and Civil Administration Bureau; (3) internal consistency checks of dates by using generation of the family and the relation of the year of one’s birth to one of The Twelve Animal Years which is a kind of Chinese zodiac; (4) internal consistency checks of dates in the fieldtrip. After verification, the number of persons aged $95 years is 705 (149 male, and 556 female), including 102 centenarians (18 male and 84 female) in Rugao. The people who had already deceased but did not deleted from the register list were excluded. Among the EL subjects, 463 subjects (360 women and 103 men; mean [SD] age, 101.23 [1.37] years; range, 95–107 years) were recruited in this study, with a response rate of 71.6%. About 94% of the EL subjects living with their offspring of three or four generations. The gender ratio of unresponded subjects did not show significant difference from that of

We introduced the SNaPshot (Applied Biosystems, Foster City, CA) minisequencing reaction assay to genotype mitochondrial deoxyribonucleic acid single nucleotide polymorphism (SNPs) [24] with minor modifications. We genotyped the aforementioned 32 mtDNA SNPs in two independent multiplex amplifications and one SNaPshot reaction. Briefly, the PCR multiplex reaction was carried out in a total volume of 15 mL using 1.5 mL of 10X FastStart Taq DNA Polymerase PCR Buffer(Roche Diagnostics, Mannheim, Germany), 8 ng of DNA template, 2.25 mL of premixed dNTPs(Promega, Shanghai, China), 0.9 mL of 25 mM MgCl (Qiagen, Hilden, Germany), 0.12 mL of FastStart Taq 2 DNA Polymerase(Roche Diagnostics, Mannheim, Germany) and 5.12 mL and 2.6 mL premixed primers for each multiplex amplification with the multiplex PCR cycling conditions:

Table 1. Decision mtSNPs for haplogroup assignment.

Haplogroup Key multiplex SNP variants M* 10400T D* 5178A 10400T D4 3010A 5178A 10400T D4a 14979C 3010A 5178A 10400T D4b 8020A 3010A 5178A 10400T D4b2 8964T 8020A 3010A 5178A 10400T D4b2b 9296T 8964T 8020A 3010A 5178A 10400T D5 1107C 5178A 10400T M12 14569A 10400T G* 4833G 14569A 10400T G2 7600A 4833G 14569A 10400T M7* 9824C 10400T M7b 12811C 9824C 10400T M8* 7196A 10400T M8a 8684T 7196A 10400T C 13263G 7196A 10400T M9 4491A 3705G 10400T M10 10646A 10400T N* 10873T N9* 5417A 10873T N9a 12358G 5417A 10873T A 1736G 10873T F* 3970T 12705C 10873T F1 12406A 3970T 12705C 10873T B* 9bp- deletion 10873T B5 8584A 9bp- deletion 10873T B5a 15235G 8584A 9bp-deletion 10873T B5b 15662G 8584A 9bp- deletion 10873T B4a 5465C 9bp- deletion 10873T B4b 13590A 9bp- deletion 10873T

Overall 926 subjects aged 60–69 years (elderly group) were randomly recruited from the resident registry at the local government offices of Rugao. For both genders of the case subjects, two-fold elderly subjects were chosen. To test the consistency of original case-control observations, another cohort of middle-aged control subjects including 463 individuals aged 40– 49 years (middle-aged group) was randomly recruited from the resident registry at the local government offices of Rugao, matched for gender with the extreme longevity group.

Protocol and Measurements

All subjects were contacted at home and examined by physicians previously trained to administer a structured question- naire. The questionnaire include socioeconomic status, demo- graphic characteristics, personal habits, dietary habits, family disease histories, personal disease histories, surgery histories, menstrual and reproductive histories, hormone use histories (for women only), daily activity histories, and cognitive function appraisals, etc. Physical examinations were accomplished and fasting blood specimens were drawn for laboratory examination (e.g. lipid, glucose, liver function). For the purpose of the quality control of the investigation, all the field staff involved in data collection and data handling was trained. All interviews were tape recorded, and 5 percent of the recorded interviews were evaluated for interviewing quality. About 3–5 percent of subjects were re- contacted by phone to evaluate the interviewers’ work. Written informed consent was obtained from each participant or a member of his/her immediate family. The Human Ethnics Committee of Fudan University School of Life Sciences approved the research.

Experimental procedures

Genomic DNA was extracted by using a method described by Boom et al, with minor modifications [19]. In order to examined the correlations between the haplogroups and longevity, we

doi:10.1371/journal.pone.0006423.t001

although a decreasing trend of N9 haplogroups was still observed from middle-aged subjects (8.6%), elderly (7.1%) and EL subjects (5.3%) in female samples, the statistical significance originally observed in all subjects diminished, which may be due to lack of statistic power (Table 3).