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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 = 0.0005-0.01; 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 beta-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 >100. One-way ANOVA and post hoc multiple comparisons were used to test the differences between groups. It was found that the MAFs of the two SNPs were significantly increased with age of mortality (Fig. 1 and Table S6, Supporting information). Genotypic association analysis To determine through which inheritance models rs1042718 and rs1042719 of ADRB2 were associated with the longevity trait, a genotypic association test was performed under dominant, recessive, and additive models by logistic regression in the combined analysis. We found that rs1042718 and rs1042719 were strongly associated with male longevity in both dominant (P = 0.00001-0.003) and additive models (P = 0.00001-0.001; Table 4). Association signals were relatively weak in recessive model (P = 0.008-0.017). The marginal P-values for the associations were also observed in women (Table 4). Linkage disequilibrium and haplotypic association analysis Linkage disequilibrium (LD) and haplotype blocks were defined by r2 (> 0.6) and visualized by the solid spine of LD method (lower panel in Fig. 2). The common haplotypes (frequency <5%) were analyzed for an association with longevity. Three haplotypes, C-G, C-C, and A-C, formed by rs1042718 and rs1042719 of ADRB2 were thus selected and tested in a combined analysis (338 male cases and 460 male controls). The frequency of haplotype C-G was decreased (the frequency of CG is 0.523 and 0.635 in cases andExonic SNPs affect protein production by influencing RNA stability. To test whether the two SNPs were able to alter the translational efficiency of ADRB2, we respectively cloned the ADRB2 coding regions that harbored the three common haplotypes (C-G, C-C, and A-C) formed by rs1042718 and rs1042719 and fused each with the firefly luciferase reporter gene (Fig. 2). To exclude interferences from other SNPs on an expression assay, we uniformly used the major allele for rs1042713, rs1042714, and rs1042717 in each construct (Fig. 2). The expression vectors were independently transfected into HEK293A cells. Renilla luciferase was used to assess the transfection efficiency. As the only differences among the three fused genes are caused by haplotypic structures, the total translational activity is solely dependent on the respective haplotypes and measured by the ratio of firefly to Renilla luminescence. Therefore, this system accurately assesses the effects of haplotypes on translational efficiency (Tranque et al., 1998). We found that the haplotype A-C led to a reduction in translation efficiency compared with C-G (P = 0.002; Fig. 2). The resulting protein level from the fused gene that carried C-C was closer to that of C-G and marginally different from that of A-C (P = 0.059). The correlations between expression levels and ORs were shown in Fig. 2.