Table 1. Characteristics of participants at visit 1989/1990 by vital status on Dec 31st,

Status on 31-12-2008 Alive Dead p value Number (%) 1087 (78.2) 270 (19.4) Males, n (%) 525 (48.3) 166 (61.5) 0.000 Age, median (range) 49.4 (36.0 to 72.6) 61.8 (37.3 to 79.1) 0.000 Ever smokers, n (%) 711 (65.4) 207 (76.7) 0.000 Packyears in ever smokers, median (range) 17.2 (0.1 to 117.1) 26.0 (0.6 to 262.2) 0.000 BMI Normal weight, n (%) 284 (26.2) 65 (24.2) Overweight, n (%) 540 (49.8) 139 (51.6) Obese, n (%) 261 (24.0) 65 (24.2) 0.781

doi:10.1371/journal.pone.0058636.t001

all participants in the study on December 31, 2008 was assessed. Causes of death were coded according to the International Classification of Diseases (ICD) and obtained from the Statistics Netherlands (The Hague). In order to avoid bias and provide true associations, the external causes of death (i.e. suicides, homicides, traffic accidents etc.) were excluded from the analyses, (ICD-9: codes$800 and in ICD-10: codes $S00).

involved in maintaining glucose homeostasis [27], we additionally investigated SIRT1 polymorphisms and glucose tolerance.

Methods

Ethics Statement

The study protocol was approved by the local university medical hospital ethics committee, University of Groningen, University Medical Center Groningen, The Netherlands and all participants gave their written informed consent. In 1984, the Committee on Human Subjects in Research of the University of Groningen reviewed the study and affirmed the safety of the protocol and study design.

Blood samples

In 1989/1990 neutrophil depots from peripheral blood samples were collected and stored at –20uC. In 2003–2004 DNA was extracted from these samples with a QIAamp DNA blood mini kit (Qiagen, Hilden, Germany) and checked for purity and concen- tration with a NanoDrop ND-1000 UV–Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE) [28].

Study population

We studied 1,390 subjects of the Vlagtwedde/Vlaardingen cohort participating in the last survey in 1989/1990 [28]. This general population-based cohort of white individuals of Dutch descent started in 1965 and has been followed for 25 years. The main focus of the study was on respiratory health. Surveys (median number of 7 per subject, range 1–8) were performed every 3 years, during which information was collected on smoking status, age, sex and respiratory symptoms by the Dutch version of the British Medical Council standardized questionnaire, BMI was deter- mined, spirometry was performed and and the number of eosinophils in peripheral blood was measured. The vital status of

SNP Selection and Genotyping

Four SNPs (rs12778366, rs10823108, rs7069102 and rs2273773), that tag all 21 SNPs in SIRT1 and its 5 kb up-/ downstream region with r2.0.8 and Minor Allele Frequency.5% (based on the HapMap release 23a/March 2008) were genotyped by K-Bioscience Ltd (UK) [29]. Since rs10823108 and rs2273773 were in complete linkage disequilibrium (r2 =1.0, Figure S1) in our study population, only rs2273773 was analyzed.

Table 2. Distribution of genotypes and hazard ratio (HR) for all-cause mortality.

All-cause mortality SNP Genotype Alive n=1,087 n=270 p value** HR (95% CI)*** p value rs12778366* TT 798 (76.0) 220 (83.0) 1 TC+CC 252 (24.0) 45 (17.0) 0.015 0.69 (0.5020.96) 0.025 rs7069102 GG 449 (43.6) 124 (48.8) 1 GC 458 (44.5) 103 (40.6) 0.85 (0.6521.11) 0.234 CC 123 (11.9) 27 (10.6) 0.323 0.90 (0.5921.37) 0.628 rs2273773* TT 897 (81.1) 164 (76.6) 1 TC+CC 209 (18.9) 50 (23.4) 0.132 1.26 (0.9221.73) 0.155

to the low frequency of individuals being homozygous for the minor allele heterozygotes and homozygotes variants were in genotype distribution between alive subjects and those who died (excluding external causes of death) tested with x2 regression adjusted for age, gender and packyears at visit in 1989/90

 Cum Survival1s12778366—TT--+ TO#CCI 1 1 1 10 5 10 15 20years of follow-up              ts12778366Cum Survival—— TT never smokers~--- TC+CC never smokers—— TT ever smokers- -+ TC+CC ever smokers0 5 10 15 20 Cum Survivalo7- 1812778366—— TTs52yrs---- TC+CC $52 yrs— TT>52yrsO06- --- TC+CC>52yrs1 1 1 1 I0 5 10 15 20Cum SurvivalCum Survival1.070.9-0.8-0.8-    1812778366—— TT females~--- TC+CC females— TT males~~ + TC+CC malesI 1 15 10 15years of follow-uprs12778366—— TT normal weight---- TC+CC normal weight— TT overweight and obese- - + TC+CC overweight and obeseT T T5 10 15years of follow-up20 20

0 5 10 15 20years of follow-up              ts12778366Cum Survival—— TT never smokers~--- TC+CC never smokers—— TT ever smokers- -+ TC+CC ever smokers Cum Survivalo7- 1812778366—— TTs52yrs---- TC+CC $82 yrs— TT>52yrsO6- --- TC+CC>52yrsI I I I r0 5 10 15 20 vears of follow-up

Figure 1. Survival curves for all-cause mortality according to SNP rs12778366. a. all subjects; b. stratified according to gender; c. stratified according to smoking habits; d. stratified according to BMI; e. stratified according to age (median age at visit in 1989/1900). *the Y axis scale in a figure 1e differs from Y axis scales in other figures. doi:10.1371/journal.pone.0058636.g001

Results

Oral glucose tolerance test

In 1970/1972, 1973 and 1976 male subjects underwent the oral glucose tolerance test (OGTT). They were given a drink of 100 g glucose solution, and blood glucose was measured two hours later.

Subjects with genetic data available and participating in the last survey in 1989/1990 were included in this study (n =1,390, see Table 1). After 18 years of follow-up, 78.2% (n= 1,087) of the cohort was still alive and 284 deaths (20.4%) were recorded. Out of all deaths, 14 (4.9%) occurred due to external causes and these were excluded form the analyses. Of the participants who had died 207 (76.7%) were ever smokers and these had significantly higher numbers of packyears compared to participants still alive. It is important to note that our study had an excellent follow-up rate, since only 19 subjects (1.4%) could not be traced back.