Despite the fact that our findings were strongly supported by several lines of evidence, the limitations need to be considered in this study. Firstly, only 96 centenarians and 96 controls were selected for primarily screen SNPs for the association. Clearly, this design may be restrictive to the identification of SNPs that were prominently associated with the longevity trait. Secondly, due to a limited number of SNPs genotyped in this study, the independence or structures of population 1 and 2 could not be evaluated by, for example, principal component analysis, which requires a large number of SNPs. Thirdly, the ages for control groups were relatively young. This design can maximize the contrast between cases and controls. On the other hand, it can also bring in other strata, for instance, reproductive age, early onset of diseases, and so on. Lastly,

Genotyping of SNPs and Validation

Human genomic DNA was isolated from a 5-mm-diameter punch-out from each blood spot or from the EDTA-anti-coagulated blood using the

Author contributions

proteinase K methods. Based on the HapMap (CHB+JPT), the 16 SNPs from the ADRB1, ADRB2, ADCY5, ADCY6, and MAPK1 genes were selected for genotyping by directly sequencing. The primers used to amplify the SNPs are presented (Table S2, Supporting information). (The detailed information on genotyping and validation was in ‘Sampling and genotyping’ of Data S1, Supporting information).

Xiao-Li Tian and Ling Zhao designed the study; Yi Zeng supervised the collection of blood samples and information from the oldest-old subjects; Hanbing Cui, Xiaomin Chen, Zhiming Zhu, Hongbo He, and Xianming Mo collected the blood samples and clinical information from geographically matched controls. Ling Zhao, Fan Yang, Ke Xu, Huiqing Cao, Gu-Yan Zheng, Yang Zhang, and Jianxin Li performed experiments; Xiao-Li Tian, Ling Zhao, Fan Yang, Brian K. Kennedy, and Yousin Suh analyzed and interpreted data; Xiao-Li Tian and Ling Zhao wrote the manuscript; Brian K. Kennedy, Fan Yang, Ke Xu, Yi Zeng, and Yousin Suh were involved in the final approval of the manuscript.

Constructs and cell transfection

The ADRB2 coding region was generated and cloned by PCR with primers 5′-CGCCCTCGAGATGGGGCAACCCGGGAAC-3′ and 5′- CTGCTAAGCTTTTACCAGCAGTGAGTCAT-3′. We constructed plasmids that carried a CMV promoter, the human full-length coding region of ADRB2, and firefly luciferase fused at its 3′-end. Three common haplotypes (frequencies > 5%) formed by rs1042718 (C/A) and rs1042719 (G/C) were generated by PCR-based mutagenesis and confirmed by sequencing. The major alleles of SNPs rs1042713, rs1042714, and rs1042717 were used to avoid any possible interference. The translational efficiency was measured by firefly luciferase activity. Transfection efficiency was standardized by reference to Renilla lucifer- ase activity resulting from the parallel introduction of this plasmid into the test cells.

References

Benovic JL (2002) Novel beta2-adrenergic receptor signaling pathways. J. Allergy Clin. Immunol. 110, S229–S235.

Clin. Immunol.

, S

–S

Bergman A, Atzmon G, Ye K, Ma

arthy T, Barzilai N (

) mechanisms in aging: a systems approach toward uncovering the component of aging. PL

Comput. Biol.

, e

Cahill L, Van Stegeren A (

) Sex-related impairment of memory for events with beta-adrenergic blockade. Neurobiol. Learn. Mem.

,

–

Chang L, Villacorta L, Zhang J, Garcia-Barrio MT, Yang K, Hamblin M, Whitesall D’Alecy LG, Chen YE (

) Vascular smooth muscle cell-selective proliferator-activated receptor-gamma deletion leads to hypotension.

,

–

Dhillon AS, Kolch W (

) Untying the regulation of the Raf-

kinase. Biochem. Biophys.

,

–

Enns LC, Morton JF, Treuting PR, Emond MJ, Wolf NS, Dai DF, M

night Rabinovitch PS, Ladiges WC (

) Disruption of protein kinase A healthy aging. PL

ONE

, e

Gao XM, Agrotis A, Autelitano DJ, Percy E, Woodcock EA, Jennings GL, Dart Du XJ (

) Sex hormones and cardiomyopathic phenotype induced by beta

-adrenergic receptor overexpression. Endocrinology

,

–

Ge D, Huang J, He J, Li B, Duan X, Chen R, Gu D (

) beta

-Adrenergic gene variations associated with stage-

hypertension in northern Han Ann. Hum. Genet.

,

–

Gendron CM, Minois N, Fabrizio P, Longo VD, Pletcher SD, Vaupel JW Biodemographic trajectories of age-specific reproliferation from stationary in the yeast Saccharomyces cerevisiae seem multiphasic. Mech. Ageing

,

–

Hara MR, Kovacs JJ, Whalen EJ, Rajagopal S, Strachan RT, Grant W, Towers Williams B, Lam CM, Xiao K, Shenoy SK, Gregory SG, Ahn S, Duckett Lefkowitz RJ (

) A stress response pathway regulates DNA damage beta

-adrenoreceptors and beta-arrestin-

Nature

,

–

Heckbert SR, Hindorff LA, Edwards KL, Psaty BM, Lumley T, Siscovick DS, Tang Durda JP, Kronmal RA, Tracy RP (

) Beta

-adrenergic receptor phisms and risk of incident cardiovascular events in the elderly. Circulation

–

Herskind AM, M

ue M, Iachine IA, Holm N, Sorensen TI, Harvald B, Vaupel (

) Untangling genetic influences on smoking, body mass index longevity: a multivariate study of

Danish twins followed for

Hum. Genet.

,

–

Holm S (

). A simple sequentially rejective Bonferroni test procedure. In Scand statisticsed eds, pp.

–

Krieg RJ Jr, Thorner MO, Evans WS (

) Sex differences in stimulation of growth hormone secretion in vitro. Endocrinology

,

Kulminski AM, Culminskaya I, Ukraintseva SV, Arbeev KG, Land KC, Yashin (

) Beta

-adrenergic receptor gene polymorphisms as systemic of healthy aging in an evolutionary context. Mech. Ageing Dev.

, Li Z, Duckles SP (

) Influence of gender on vascular reactivity in the rat. J. Pharmacol. Exp. Ther.

,

–

Li Y, Wang WJ, Cao H, Lu J, Wu C, Hu FY, Guo J, Zhao L, Yang F, Zhang YX, Li Zheng GY, Cui H, Chen X, Zhu Z, He H, Dong B, Mo X, Zeng Y, Tian XL Genetic association of FOXO

A and FOXO

A with longevity trait in Han populations. Hum. Mol. Genet.

,

–