Yiheyuan Road, Beijing

, China. Tel.: +

; fax: +

; e-mail: tianxiaoli@pku.edu.cn † These authors have contributed equally to the work. Accepted for publication

September

i

Xiao-Li Tian, Department of Human Population Genetics, Institute of Molecular Medicine, Peking University, 5 Yiheyuan Road, Beijing 100871, China. Tel.: +86 10 6275 5397; fax: +86 10 6275 6926; e-mail: tianxiaoli@pku.edu.cn

compared by the Pearson chi-square test. Of all SNPs analyzed, four from ADRB2 (rs1042718 and rs1042719), ADCY6 (rs2291727), and MAPK1 (rs2266968) exhibited significant differences (P < 0.05; Table S3, Sup- porting information).

Results

Characterization of populations

Chinese Longitudinal Healthy Longevity Survey (CLHLS) was a follow-up study initiated in 1998, investigating the effects of social–biological factors on healthy conditions of the oldest-old population (aged 80+ and mostly 90+). Of 9093 participants were interviewed and 4120 donated the blood samples in 1998. Of those who donated the blood samples, 1072 were centenarians (259 men and 813 women) from Han Chinese nationality and used in this study (Fig. S1, Supporting information). The reliability of age reports in this population has been assessed and validated on our cohort in previous study (Wang et al., 1998). In this study, population 1 (stage 1) included 384 centenarians and 384 gender- and geographically matched young individuals from northern China, while population 2 (stage 2) consisted of 579 cases with exceptional long life span (age   95 year for men and age   100 year for women) and 644 geographically matched young individuals from southern China (Table 1). All participants in this study were from Han Chinese. The matched design eliminated the potential effects of population stratifi- cation caused by gender and geographic region. However, as a consequence, the total number of individuals in this study was limited by a smaller number of male centenarians; thus, to include enough men with exceptional long lifespan in the population 2, the cutoff age was   95 years. The Han Chinese population in this study consisted of 963 long-lived (median age is 101 years) and 1028 geography-matched young individuals (median age is 44 years). The mean ages (mean ± SEM) of long-lived individuals in Population 1 and 2 were 102.1 ± 0.20 and 101.0 ± 0.16. The differences between case and control groups for age, gender, body mass index (BMI), systolic blood pressure (SBP), and diastolic blood pressure (DBP) in populations 1 and 2 are shown in Table 1.

We then genotyped the four SNPs in the remaining individuals of population 1 and found that the MAFs of SNPs rs1042718 and rs1042719 from ADRB2 increased in the centenarian group compared with controls (P = 0.014–0.028, Table 2). The SNPs, rs2291727 and rs2266968, from ADCY6 and MAPK1 did not differ in the distributions of their MAFs. Four SNPs, rs1042718, rs1042719, rs2291727, and rs2266968, were also genotyped by TaqMan method to validate the genotyping. The consistencies between two methods were greater than 99.3% (not shown).

The association of rs1042718 and rs1042719 from ADRB2 with longevity was replicated in population 2 (stage 2) (P = 0.003–0.011, Table 2). When two populations were combined, the P-values and statistical powers for rs1042718 and rs1042719 became (P/power) 0.00079/0.74 and 0.00011/0.85. The Bonferroni-corrected P-values were 0.012 and 0.0018, respectively.

Quantitative trait analysis demonstrated that SNPs rs1042718 and rs1042719 of ADRB2 did not exhibit any correlation with BMI, systolic, or diastolic blood pressures in either the oldest-old or their young-matched subjects (Fig. S3, Supporting information).

Gender effects on MAFs of SNPs

To eliminate gender effects on the distributions of MAFs, we compared the MAFs of SNPs between the oldest-old and control groups within gender using the chi-square test. We found that the MAFs of rs1042718 and rs1042719 from ADRB2 were only elevated in men (the Bonferroni- corrected P = 0.0005–0.010; statistical power = 0.92–0.93, Table 3) but not in female centenarians compared with their young counterparts (P = 0.091–0.102; Table S4, Supporting information). The interactions between genders and SNPs were then tested; the P-values for interaction were 0.07 and 0.019 for rs1042718 and rs1042719, respectively, as shown in Table S5 (Supporting information).

Genotyping and single SNP association study

Sixteen SNPs were selected from ADRB1, ADRB2, ADCY5, ADCY6, and MAPK1 from the b-adrenergic signaling pathway (Fig. S2, Supporting information). As a first step, the 16 SNPs were genotyped by directly sequencing the amplified genomic DNA from 96 centenarians and 96 young individuals from population 1 (Table S1, Supporting information) with the listed PCR primers (Table S2, Supporting information). None of the SNPs were found to depart from Hardy–Weinberg equilibrium expectations (Table S3, Supporting information). The distributions of minor allele frequencies (MAFs) between case and control groups were

Enrichment of MAF in groups with increased death ages

To test whether the MAFs of SNPs, rs1042718 and rs1042719 of ADRB2, were enriched with increased age of mortality, we calculated the MAFs of the two SNPs in four male groups: (i) young group (living subjects; life expectancy was reported as 71.3 years for men in China in 2009); (ii) death age = 95; (iii) death ages from 96 to 100; and (iv) death ages

Table 1 Characteristics of populations

Population 1 Population 2 Items Case (n = 384) Control (n = 384) P Case (n = 579) Control (n = 644) P Age (year) 102.1 ± 0.20 46.8 ± 0.55 < 0.05 101.0 ± 0.16 45.6 ± 0.50 < 0.05 Gender (m/f) 96/288 96/288 1.00 242/337 280/364 0.553 BMI (kg m(cid:3)2) 20.4 ± 0.3 24.2 ± 0.2 < 0.05 19.3 ± 0.2 23.1 ± 0.2 < 0.05 SBP (mmHg) 146 ± 1.3 123 ± 1.1 < 0.05 147 ± 1.0 120 ± 0.6 < 0.05 DBP (mmHg) 82 ± 0.7 78 ± 0.6 < 0.05 84 ± 0.6 76 ± 0.4 < 0.05