Effects of haplotypes on translational efficiency of ADRB2 gene

Five common SNPs are found in the ADRB2 coding region: rs1042713, rs1042714, rs1042717, rs1042718, and rs1042719. Among these, two synonymous SNPs, rs1042718 (Arg175Arg) and rs1042719 (Gly351Gly), were associated with longevity in our study. It is common that an exonic SNP affects 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 measure 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.

Here, we assessed the genetic contribution of bAR signaling system to the human longevity trait in Han Chinese long-lived and younger individuals. The average lifespan in China was 71 and 74 years of ages for men and women, respectively. Thus, those who were at the ages of 95 or above were considered as the ‘long-lived,’ and middle-aged groups were taken as controls. The SNPs selected from ADRB1, ADRB2, ADCY5, ADCY6, and MAPK1 were genotyped and evaluated for their association with the longevity trait. We identified two synonymous SNPs of ADRB2 (rs1042718 and rs1042719) that were significantly associated with longevity in men through dominant and additive models. Despite the fact that no positive signals were observed in women in the allele association study, we noticed that these two SNP exhibited marginal P-values in genotypic association in women. This may suggest that the effect size in women is smaller than in men. The association with male longevity was replicated in two geographically isolated Han Chinese populations. The MAFs of the two SNPs were increased with the death ages, providing additional support for the association. In further studies, we demonstrated that the haplotype formed by these two SNPs, which increased the probability of becoming a centenarian, also led to decreased protein production of b2AR. In contrast, over-production of the b2AR is associated with decreased male lifespan. The other SNPs analyzed were not found to be associated with human longevity in the studied populations. To our knowledge, this is the first indication that ADRB2 is linked to human longevity and that a suppression of b2AR production may have beneficial effects on lifespan.

Discussion

Longevity is a complex trait that is affected by both genetic and environmental factors (Herskind et al., 1996; Li et al., 2009; Zeng et al., 2010). A great effort has been made to assess to what extent genetics contributes to the trait over the past decades. For instance, a Danish twin-based study indicated that the heritability of longevity was about 0.33 and 0.44 for men and women, respectively (Herskind et al., 1996). A recent meta-analysis of four GWAS identified 273 SNPs associated with longevity at low P-values (< 0.0001) (Newman et al., 2010).

A C G C G (A) (B) GG A A C 400 P = 0.002 ADRB2 350 3 4 7 8 9 300 1 1 1 1 1 7 7 7 7 7 2 2 2 2 2 4 4 4 4 4 250 0 0 0 0 0 1 1 1 1 1 s s s s s r r r r r 200 A C G C G 150 a CMV ADRB2 Flu C C 100 b CMV ADRB2 Flu 50 A C c CMV ADRB2 Flu 0 d CMV Flu Haplotype C-G C-C A-C e TK Rlu Frequency (%) 52.3/63.5 13.3/10.7 33.2/25.0 (case/control) Block (0 kb) 1 2 3 4 5 OR 0.63 1.29 1.49 (95% CI) P 0.000018 0.119 0.0007 Perm P 0.00005 0.323 0.0017 ulR/ulF fo oitaR 

Table 5 Haplotypic association of ADRB2 with male longevity in combined population

positively associated with longevity, while the haplotype C-G increased the translational efficiency of b2AR and was negatively associated with longevity. Notably, an existing study also demonstrated that the haplotype harboring A-C alleles decreases the translational level, providing strong evidence that A-C alleles are functional (Panebra et al., 2010). Thus, our data indicate that the b2AR-triggered signaling pathway contributes to human lifespan.

Haplotype Frequency (case/control) OR (95% CI) P Perm P C-G 0.523/0.635 0.63 (0.51–0.78) 1.86E-5 A-C 0.332/0.250 1.49 (1.17–1.88) 7.00E-4 C-C 0.133/0.107 1.29 (0.92–1.80) 0.119 0.323