DNA extraction and phenotype measurements. Blood

samples, anticoagulated with 2% ethylenediaminetetra- acetic acid, were drawn for DNA extraction and bio- chemical examination. Genomic DNA was extracted from white blood cells using the proteinase K/phenol/ chloroform purification, followed by ethanol precipita- tion, and stored in 10 mmol/L Tris-HCl, 1 mmol/L Na -EDTA, pH 8.0. Body weight and height, as well 2 as blood pressure levels, were calibrated according to the last measurement at the time of survey. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters. Blood pressure was measured at the right arm with a conventional mer- cury sphygmomanometer after the subjects rested at least 5 min. Plasma triglyceride, total cholesterol, high- density lipoprotein cholesterol, apolipoprotein A, and apolipoprotein B concentrations were determined enzy- matically using available kits and an autoanalyzer in Xinjiang Medical University. Plasma low-density lipo- protein cholesterol was estimated using Friedewald’s formula. Fasting plasma glucose was detected by the Glucorder analyzer (Roche, Basel, Switzerland).

Genotyping. The genotypes of the 3 studied variants were determined by the polymerase chain reaction (PCR) and restriction fragment length polymorphism techniques. With respect to the HSPA1L 2437 T/C vari- ant, the sequences of the forward and reverse primers were GTC CCT GGG GCT GGA GAC GG and GTG ATG ATA GGG TTA CAC ATC TGC T, respectively. PCR amplification was carried out in a PTC-200 MJ Re- search Peltier Thermal Cycler (Bio-Rad Laboratories, Mass) for 35 cycles with denaturation at 94(cid:2)C for 45 s, annealing at 60(cid:2)C for 45 s, and extension at 72(cid:2)C for 45 s. All amplifications were performed with 50-100 ng of DNA in a total volume of 25 mL containing 10 pmol of each primer, 200 mmol/L dNTPs, 2.0 mmol/L

Statistical analysis. Database management and simple statistical calculations were conducted by SAS software version 9.1.3 (Institute Inc., Cary, NC). The means of continuous variables were compared by unpaired t-test. The c2 test was used to assess the goodness of fit between the observed allele frequencies and the expected counterparts by Hardy-Weinberg equilibrium to evaluate the differences in genotype and allele distributions between the 2 groups. A trend analysis was performed using a 1-way analysis of variance. A 2-tailed P . 0.05 was used to determine statistical significance.

Table I. The basic characteristics of the study population

Characteristics Long-lived people (n5191) Short-lived people (n553) P* Age, years 97 6 3 67 6 3 . 0.001 Gender, male/female 131/60 29/24 NS† BMI, kg/m2 20.64 6 2.34 21.29 6 2.49 NS Systolic BP, mm Hg 116.23 6 9.65 116.82 6 8.31 NS Diastolic BP, mm Hg 77.48 6 11.13 70.62 6 5.36 NS Triglyceride, mmol/L 1.66 6 0.72 1.81 6 0.75 NS Total cholesterol, mmol/L 4.81 6 1.02 4.76 6 0.97 NS HDL cholesterol, mmol/L 1.07 6 0.25 1.10 6 0.27 NS LDL cholesterol, mmol/L 3.22 6 0.90 2.95 6 0.95 NS Apolipoprotein A, g/L 1.28 6 0.19 1.32 6 0.17 NS Apolipoprotein B, g/L 1.01 6 0.27 1.15 6 0.52 NS Fasting glucose, mmol/L 5.92 6 2.13 5.71 6 1.17 NS

Abbreviations: BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SD, standard deviation. Continuous data are expressed as mean6SD and categorical data as number. *P was calculated using an unpaired t test. †P was calculated using a c2 test.

II. The genotype and allele distributions of 3 studied Hsp70 family variants between long-lived and short- people

Long-lived Short-lived Long-lived Short-lived Variants Genotypes people (n5191) people (n553) Alleles people (n5191) people (n553) AA 65 (34.03) 14 (26.42) AG 215 (56.28) 50 (47.17) A1267 G AG 85 (44.50) 22 (41.51) 167 (43.72) 56 (52.83) GG 41 (21.47) 17 (32.08) P 5 0.225/0.310 c2 5 2.79 P 5 0.246* c2 5 2.78 P 5 0.096* H-W GG 58 (30.37) 15 (28.30) GC 213 (55.76) 58 (54.72) G190C GC 97 (50.79) 28 (52.83) 169 (44.24) 48 (45.28) CC 36 (18.85) 10 (18.87) P 5 0.824/0.893 c2 5 0.09 P 5 0.955* c2 5 0.04 P 5 0.849* H-W TT 130 (68.06) 41 (77.36) TC 314 (82.20) 93 (87.74) T2437C TC 54 (28.27) 11 (20.75) 68 (17.80) 13 (12.26) CC 7 (3.66) 1 (1.89) P 5 0.771/1.000 c2 5 1.79 P 5 0.408* c2 5 1.84 P 5 0.175* H-W

P was the goodness-of-fitness value for the Hardy-Weinberg (H-W) test of long-lived people/short-lived people. H-W *P values were calculated using a c2 test from a 332 contingency table for genotype data and a 232 contingency table

people (mean age, 67 years). Except for age, no statisti- cal significance was observed in terms of gender, BMI, blood pressure, fasting glucose, and plasma lipid-related biomarkers between long-lived people and short-lived people (P . 0.05).

The EH/EH1 program was employed to estimate and the haplotype frequencies between longevity control groups by c2 test from a series of 2 3 2 con- tables by combining other haplotypes. To in- study power, only haplotypes with a frequency than 3% were considered for analysis. An inter- analysis was implemented in the open-source software package (v.1.0.0), which is available www.epistasis.org. Using the MDR constructive function, we constructed all possible combina- of 1 to 3 variants. The genetic data were collapsed 2 categories with high and low risk in each cell. An dendrogram was applied to visualize the of the dependencies.

For the genotype/allele distributions of 3 studied var- iants, no evidence was found for Hardy-Weinberg dis- equilibrium in both groups (P . 0.05). As presented in Table II, no significant differences were apparent between the 2 groups in the genotype and allele distribu- tions of all the studied variants by single-locus analysis (P . 0.05). After stratifing the study popualtion by gender, we still failed to observed any differences for these distributions (data not shown).