The data are presented as mean SEM (standard error of the mean). BMI, body mass index; Case, long-lived individuals; Control, young individuals; DBP, diastolic blood pressure; m/f, male/female; P-values are calculated from t-test or chi-square test when comparing case and control within population; SBP, systolic blood pressure.

Table 2 Allelic association of ADRB2 with longevity

Population 1 Population 2 Combined Allele MAF MAF MAF (major/ (case/ OR (case/ OR (case/ OR SNPs minor) control) (95% CI) P/aP control) (95% CI) P/aP control) (95% CI) P/aP bP/PW rs1042718 C/A 0.350/0.298 1.27 0.028/0.028 0.345/0.297 1.25 0.011/ 0.347/0.297 1.25 7.92E-4/ 1.27E-2/0.74 (1.03–1.58) (1.05–1.48) 0.013 (1.10–1.43) 0.001 rs1042719 G/C 0.451/0.389 1.29 0.014/0.014 0.465/0.405 1.28 0.003/ 0.460/0.399 1.28 1.15E-4/ 1.84E-3/0.85 (1.05–1.58) (1.09–1.50) 0.003 (1.13–1.45) 1.27E-4

Combined, population 1 plus population 2; MAF, minor allele frequency; OR (95% CI), odds ratio with 95% confidence interval; P, P-value calculated from chi-square test; PW, power when alpha is set as 0.05; aP, P-value adjusted by gender; bP, P-value corrected by Bonferroni method.

Table 3 Allelic association of ADRB2 with longevity in males

Combined, population 1 plus population 2; MAF, minor allele frequency; OR (95% CI), odds ratio with 95% confidence interval; P, P-value calculated from chi-square test; PW, power when alpha is set as 0.05; bP, P-value corrected by Bonferroni method.

> 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).

0.5 * * 0.4 * * 0.3 0.2 rs1042719 rs1042718 0.1 0 1 2 3 4 Male groups FAM

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 C–G is 0.523 and 0.635 in cases and controls, respectively, OR = 0.63, CI 95% = 0.51–0.78, P = 0.000018), whereas that of A-C was elevated (the frequency of A–C is 0.332 and 0.250 in cases and controls, respectively, OR = 1.49, CI 95% = 1.17–1.88, P = 0.0007) in oldest- olds relative to their male controls (Table 5, Fig. 2); the permutation corrected P-values were 0.00005 and 0.0009, respectively. None of the three haplotypes showed statistical differences in their frequencies between case and control in women (Data not shown).

Fig. 1 Enrichment of MAF in groups with increased death ages. MAF, minor allele frequency; group 1, young group (living subjects, the life expectancy was reported as 71.3 year for men in China in 2009, n = 376); group 2 (n = 51), death age = 95; group 3 (n = 121), death ages from 96 to 100, and group 4 (n = 166), death ages > 100. The P-values for group 1 vs. 2, group 1 vs. 3, and group 1 vs. 4 were 0.480, 0.049, and 0.0003 for rs1042718 as well as 0.152, 0.002, and 0.0004 for rs1042719, respectively. *Statistically significant.

Table 4 Genotypic association of ADRB2 with longevity in males and females from the combined populations

Male Female Allele SNP (major/minor) Model Genotyping Case Control OR (95% CI) P Case Control OR (95% CI) P rs1042718 C/A Dom A/A + C/A 193 172 1.56 (1.16–2.09) 0.003 370 352 1.24 (0.99–1.54) C/C 145 201 255 300 Rec A/A 39 22 2.08 (1.21–3.59) 0.008 66 63 1.10 (0.77–1.59) C/A + C/C 299 351 559 589 Add A/A 39 22 1.50 (1.19–1.90) 0.001 66 63 1.16 (0.98–1.37) C/A 154 150 304 289 C/C 145 201 255 300 rs1042719 G/C Dom C/C + G/C 255 226 2.05 (1.48–2.83) 1.37E-5 459 434 1.41 (1.11–1.80) G/G 82 149 163 218 Rec C/C 59 42 1.68 (1.10–2.58) 0.017 109 118 0.96 (0.72–1.28) G/C + G/G 278 333 513 534 Add C/C 59 42 1.67 (1.32–2.10) 1.37E-5 109 118 1.16 (0.98–1.36) G/C 196 184 350 316 G/G 82 149 163 218

Add, additive model; Dom, dominant; OR (95% CI), odds ratio with 95% confidence interval; P, P-value calculated from chi-square test; Rec, recessive.

Although these studies may not directly indicate genetic contribution to longevity, these suggest there should be intrinsic relationship between certain genetic determinants and the trait. Interestingly, it was found that genetic factors have increasing effects, particularly after the certain age (60 years) (Von Hjelmborg et al., 2006). It is possible that genetic factors contribute to longevity directly by mediating the signaling pathways associated with aging process or indirectly by buffering the deleterious effects caused by other factors (Herskind et al., 1996; Bergman et al., 2007). It has been hypothesized that instituting beta- receptor blockage pharmacotherapy at an early age will increase longevity by countering the effects of sympathetically mediated stress (Milne & Hong, 2004). This, however, remains to be validated.

Effects of haplotypes on translational efficiency of ADRB2 gene