Given prior evidences on the associations of the Gln27Glu and Arg16Gly polymorphisms with various aging-related phenotypes including those studied in this paper (see Section 1) and very limited number of independent tests, Type I sampling error is unlikely to be an issue for our analyses. Consequently, correction for multiple comparisons was not carried out. We note that robustness of our findings is further justified by the pleiotropic effect of the Gln27Glu polymorphism (revealed in this study) as well as by a hypothesized role of the ADRB2 polymorphisms in cardiovascular development (see Section 2.3) and by the candidate-gene-focused genotyping technique that offsets potential problems relevant to genome-wide technologies (Ziegler et al., 2008).

2.3. ADRB2 polymorphisms in the FHSO

3. Results

DNA was collected for living participants of the FHSO in the late 1980s and through 1990s (Cupples et al., 2007). Genotyping of about 1900 offspring (mainly unrelated) subjects was performed under the Cardio-Genomics program (http:// cardiogenomics.med.harvard.edu/projects/p5/assoc-results) that focused on se- lected candidate genetic markers of cardiovascular development (a review of genotypedresourcesintheFHSandFHSOcanbefoundin(Govindarajuetal.,2008; Levy et al., 2006)). Two common Arg16Gly and the Gln27Glu polymorphisms of the ADRB2 gene were selected for this study because of their documented multiple physiological and health effects (see Section 1) that warrants exploration of their

3.1. The Gln27Glu polymorphism and health risks

Fig. 1A shows the highly significant protective role of the Glu27Glu homozygous genotype against cancer (all sites but skin) for men and women combined. To ascertain that this effect is not an artifact of biased attrition of the FHSO cohort before the blood

Probability of staying free of diseases°©a°©NS0.880.84M&W; cancer ‘Glu”Glu; N=279 (28) nyi Reference__. Gln?’Glu; N=733 (126)RR=1.79"; Cl=1.19-2.70N --M&W, Prostate or breastGlu7Glu; N=280 (15)be Reference_ Gin?”Glu; N=734 (61)RR=1.57; Cl=0.89-2.87         M&W, Other cancersGlu?’Glu; N=279 (15)Reference            M&W, CVD ‘ M&W, MIL Glu?”Glu; N=280 (20)             Time, monthsGlu?’Glu; N=279 (65) — RR=0.72; Cl=0.43-1.22Reference Gin"G_ . — Gin?"Glu; N=734 (158) 2 RReF RR=1.12; Cl=0.82-1.59 bt eee RR=0.64"; Cl=0.42-0.97_ Gin’Gin;N=477(109) NL eee | ecco Gin?7Gin; N=238 (45) yRR=1.15; Cl=0.83-1.59 % Reference r Reference(0), 14 | 11 YE) : 14 ! \ (F) ! . ! : ! . J100 200 300 0 100 200 300 400 100 200 300 400

Fig.1.ProbabilityofstayingfreeofselectedgeriatricdiseasesforcarriersofdifferentgenotypesoftheADRB2Gln27GlupolymorphismintheFHSOsample(seeinsets).M&W denotes combined sampleofmenandwomen.MI = myocardialinfarction.IC = intermittentclaudication.Theletter‘‘N’’inthe insetdenotes samplesizeforagivengenotype with non-missing information on all covariates. The numbers in parentheses are counts of individuals with onsets of respective condition within the follow-up period (months). The RR (CI) denotes relative risks (confidence intervals) calculated using the Cox regression model adjusted for age, sex (when necessary), systolic and diastolic blood pressures, total and high-density lipoprotein cholesterol, body-mass index, smoking, and diabetes. *0.01 < p   0.05; **0.0005 < p   0.01; ***p   0.0005.

3.2. The Arg16Gly and Gln27Glu compound genotypes and health risks

drawing (see Section 2.3), we evaluated survival patterns and the relative risks focusing on the 4th, 5th, and 6th examinations which were performed on or after the blood drawing. In all three cases, the results resemble those in Fig. 1A (e.g., the relative risk [RR] for the Gln27Gln homozygote was 2.03, 95% confidence interval [CI] = 1.29–3.21 compared to the Glu27Glu homozygote for the 5th FHSO examination). This protective effect holds for each sex, e.g., cancer risks are significantly higher for the Gln27Gln homozygote (RR = 2.14; CI = 1.23–3.72 for men and RR = 2.02; CI = 1.05–3.86 for women) compared to the Glu27Glu homozygote. Furthermore, the protective effect appears to be not specific to a cancer type; similar relationships were observed for breast and prostate cancers (Fig. 1B) as well as for the remaining cancer sites (Fig. 1C).

Table 1 shows that the Glu27Glu homozygote is tightly linked to the Gly16Gly homozygote. Consequently, the Gly16Gly/Glu27Glu compound genotype resembles the effect of the Glu27Glu genotype shown in Fig. 1. Because the LD between the Gln27Glu and Arg16Gly polymorphisms is modest, carriers of the other two genotypes of the Gln27Glu polymorphism can carry different genotypes of the Arg16Gly polymorphism, e.g., the Gln27Glu genotype can be linked either to the Arg16Gly or Gly16Gly genotype (Table 1). Conse- quently, the effects of the Gln27Gln and Gln27Glu genotypes can theoretically be mediated or modulated by the Arg16Gly polymor- phism.

Fig. 2 shows that the effects on cancer of the Arg16Gly/Gln27Glu and Gly16Gly/Gln27Glu compound genotypes (i.e., carrying the Gln27Glu genotype) as well as the Arg16Arg/Gln27Gln, Arg16Gly/ Gln27Gln, and Gly16Gly/Gln27Gln compound genotypes (i.e., carrying the Gln27Gln genotype) are the same (Fig. 2A), i.e., the Arg16Gly polymorphism neither modulates nor mediates the effect of the Gln27Glu polymorphism on cancer. Although the effect of the Gln27Glu polymorphism on compound phenotype of MI and IC can be more sensitive to the Arg16Gly polymorphism than in the case of cancer, this sensitivity is not significant as well (Fig. 2B). Therefore, these results provide no evidence on functional relationship between the Arg16Gly polymorphism and these aging-associated phenotypes. This result means that a protective effect is attributed to the Glu27 allele (that is equivalent to the Gly16Glu27 haplotype), whereas a detrimental effect is largely associated with the Gln27 allele of the Gln27Glu polymorphism.