Statistical Analysis

Descriptive analyses of the subject characteristics were per- formed using x2 tests for categorical variables and Mann-Whitney U test for continuous variables (i.e. packyears in ever smokers and age). The genotype frequencies were tested for Hardy-Weinberg Equilibrium (HWE) by x2 analysis. Differences in genotype distribution between dead and alive subjects were tested using x2 tests. SNP rs10823108 was tested in a general genetic model. Due to the low frequency of individuals being homozygous for the minor allele for rs12778366 (n= 14) and rs2273773 (n= 9) heterozygotes and homozygotes for the minor allele were analyzed in a one group. Cox proportional hazards regression models adjusted for gender, age and packyears of smoking (all at the survey in 1989/1990) were used to evaluate the association between SNPs and all-cause mortality. Time was defined from the examination in 1989/1990 until death, end of follow-up in 2008 or last registration if subjects were lost to follow-up. Survival curves are depicted based on these Cox models. Stratified analyses according to gender, smoking habits (never smokers vs ever smokers), BMI and age (dichotomized based on a median age at visit in 1989/1990, i.e. 52 yrs) were performed. Subjects with BMI $25 kg/m2 were categorized into the overweight/obese group according to World Health Organization (WHO) criteria.

The 3 tested SNPs (rs12778366, rs7069102 and rs2273773) were in Hardy-Weinberg equilibrium. Among subjects who died, 83% had the wild type genotype of rs12778366 which is significantly higher (p =0.015) than the 76% in the subjects who were still alive.

Cox regression showed that carriers of the minor allele of rs12778366 had a significantly reduced risk of mortality compared to wild types: HR 0.69 (95% CI: 0.50 to 0.96; p =0.025; see Table 2). Survival curves according to genotypes of rs12778366 clearly show the difference in mortality risk (Figure 1a). The same directions of the effect of rs12778366 were observed within groups that have different mortality risks, i.e. females (HR= 0.82 (0.5021.35)) and males (HR= 0.63 (0.4120.96)), never smokers (HR =0.53 (0.2521.11)) and ever smokers (HR= 0.75 (0.5221.08)), younger subjects (age # 52 yrs) (HR= 0.68 (0.3021.54)) and older subjects (age . 52 yrs) (HR= 0.69 (0.4820.98)), (Table 3 and Figure 1 b–c, e). Remarkably, the protective effect of rs12778366 was observed in overweight/obese subjects (HR= 0.62 (0.4320.91)) but not in subjects with normal weight (HR= 0.95 (0.5021.80). The survival curves clearly show that overweight/obese minor allele carriers of rs12778366 had survival comparable to subjects with normal weight while overweight/obese subjects with the wildtype genotype had an increased mortality risk (Figure 1d). The 2 other SNPs did not show significant associations between genotypes and mortality risk (Table 2).

A linear regression model adjusted for age at the measurement was used to evaluate the associations between SNPs in SIRT1 and glucose tolerance.

P values ,0.05 were considered statistically significant (tested 2- sided). All statistical analyses were performed using SPSS version 18.0 for Windows.

Table 3. HR for all-cause mortality for rs12778366 TC+CC genotypes in stratified analysis.

We analyzed the available data from 535 male subjects from the current study (aged at the measurement 18–61 years) who underwent the glucose tolerance test (OGTT). We found that

Stratification HR (95% CI)** p value a) gender Females, n*=653 0.82 (0.5021.35) 0.424 Males, n=680 0.63 (0.4120.96) 0.032 b) smoking habits Never smokers, n=424 0.53 (0.2521.11) 0.092 Ever smokers, n=909 0.75 (0.5221.08) 0.125 c) BMI Normal weight, n=344 0.95 (0.5021.80) 0.870 Overweight and obese, n=987 0.62 (0.4320.91) 0.014 d) age # 52 yrs, n=670 0.68 (0.3021.54) 0.350 . 52 yrs, n=663 0.69 (0.4820.98) 0.040

Table 4. Glucose levels (mmol/l) measured in males after the oral glucose tolerance test (OGGT).

SNP Genotype n Mean (SD) B* (mmol/l) SE p value rs12778366 TT 414 6.33 (1.55) TC+CC 119 5.99 (1.22) 20.34 0.16 0.030 rs7069102 GG 229 6.33 (1.59) GC 232 6.02 (1.43) 20.12 0.14 0.401 CC 59 6.32 (1.44) 0.02 0.22 0.931 rs2273773 TT 446 6.27 (1.51) TC+CC 89 6.22 (1.44) 20.04 0.17 0.830

*n=number of all subjects included in the analysis (excluding those who died due to external causes) **rs12778366 TT genotype as a reference doi:10.1371/journal.pone.0058636.t003

*Regression coefficient (B), its standard error (SE) and p value obtained with linear regression analysis adjusted for age at the measurement. doi:10.1371/journal.pone.0058636.t004

effect remained the same, but was not significant. In this light the better glucose tolerance in overweight/obese minor allele carriers of SNP rs12778366 could be considered a condition leading to better survival in this group. This additional result emphasizes the relevance of rs12778366 and indicates its possible use as a screening tool for clinical purposes.

the minor allele carriers of SNP rs12778366 had better glucose tolerance, since they had a 0.34 mmol/l lower glucose levels compared to wild type subjects (p = 0.03, see Table 4). When this association was further investigated in subjects with normal weight (n =249) and overweight/obese subjects (n =284) separately, we found significantly better glucose tolerance in overweight/obese carriers of the minor allele of rs12778366 (i.e. 0.60 mmol/l lower glucose levels (p= 0.01)) compared to overweight/obese wild type subjects. In subjects with normal weight the same direction was observed (0.18 mmol/l lower glucose levels), but the effect was not significant (p =0.50).