Fig. 1. The WRN gene and proteinstructurewith a graphic representation of the i1-C/T, L1074Fand C1367R SNP localization. The positionsof the SNPs in the WRN gene and protein are indicated with arrows. The WRN gene is located in chromosome 8 and it spans a genomic region of 140 kb, containing 35 exons. The WRN protein is 1432 amino acids long and has 5 functional domains: exonuclease domain, helicase domain, RecQ helicase domain (RQC), helicase and RNaseD-C- terminal (HRDC) domain and C-terminal nuclear localization signal (NLS). The WRN protein has in addition acidic residues with unknown function.

and resulting allelic frequencies of the SNPs were similar to these found in Caucasians (Bendixen et al., 2004; Castro et al., 1999, 2000). In this study, the calculated allele and genotype

frequencies did not differ between the elderly and young subjects, showing no enrichment for any of the alleles (Table 1).

The prevalence of cardiovascular pathologies dependent on the i1-C/T, L1074F and C1367R polymorphisms, and the influence of these SNPs on cognitive functioning were assessed only in cohort ’97. At baseline the risks of myocardial infarction, myocardial ischemia, intermittent claudication, arterial surgery and stroke dependent on the i1-C/T, L1074F and C1367R polymorphisms did not vary between the different genotypes (Table 2).

Table 1

Characteristics of the study subjects

Young control Cohort ’87 Cohort ’97 Number 247 682 563 Age 19–40 84–100 85 Female 137 (56) 491 (72) 375 (67) i1-C/T CC 223 (92) 586 (91) 489 (91) CT 18 (8) 56 (9) 47 (9) TT – – – Total 241 642 536 MAF 0.04 0.04 0.04 HWE 0.83 0.51 0.57 L1074F LL 66 (27) 202 (32) 175 (32) LF 126 (51) 316 (51) 260 (47) FF 55 (22) 105 (17) 117 (21) Total 247 623 552 MAF 0.48 0.42 0.45 HWE 0.94 0.61 0.54 C1367R CC 139 (58) 329 (49) 316 (57) CR 81 (34) 285 (42) 192 (34) RR 21 (8) 58 (9) 50 (9) Total 241 672 558 MAF 0.26 0.30 0.26 HWE 0.20 0.95 0.04

annually during a mean follow-up of 4.2 years. Global cognition, as measured with MMSE, was similar between the different genotype carriers of the i1-C/T, L1074F and C1367R polymorphisms (Table 3). From the specific domains of cognitive functioning, only differences in attention were observed for the C1367R polymorphism. Homozygous carriers of the 1367R allele had worse attention compared to the 1367C homozygotes (94.2 (4.35) versus 84.8 (1.84), p = 0.04). The same trend was observed for the heterozygous 1367R allele carriers. The results remained unaltered after the adjustment for depressive feelings. No associations with processing speed, immediate and delayed memory were observed with the different genotypes (Table 3).

The influence of the i1-C/T, L1074F and C1367R polymorphisms on mortality risks was assessed separately for cohort ’87 and ’97, and also in the combined cohort of 1245 participants (Supplementary Table 1). Since in some instances the mortality risk estimates found in cohort ’87 were not replicated in cohort ’97, the results for the combined cohort are presented. The higher number of participants in the combined cohort provides better statistical power and enables to calculate more accurate estimates. During the total follow-up of 5164

MAF: minor allele frequency, HWE: Hardy–Weinberg equilibrium. Values in parenthesis are in percentage.

Table 2

Risk of cardiovascular pathologies at baseline dependent on i1-C/T, L1074F and C1367R

i1-C/T L1074F C1367R CC CTa LL LFa FFa CC CRa RRa Myocardial infarction (n = 103) 1 1.17 (0.56–2.46) 1 1.47 (0.89–2.43) 1.04 (0.55–1.97) 1 0.86 (0.54–1.39) 1.41 (0.69–2.89) Myocardial ischemia (n = 234) 1 0.51 (0.26–1.00) 1 1.22 (0.83–1.82) 1.53 (0.95–2.46) 1 1.02 (0.71–1.47) 0.60 (0.32–1.15) Intermittent claudication (n = 36) 1 2.26 (0.87–5.83) 1 0.91 (0.41–1.98) 0.90 (0.34–2.38) 1 1.64 (0.79–3.39) 1.57 (0.50–4.97) Arterial surgery (n = 37) 1 1.72 (0.62–4.75) 1 0.83 (0.38–1.78) 0.84 (0.32–2.20) 1 1.25 (0.60–2.59) 1.40 (0.45–4.40) Stroke (n = 57) 1 0.85 (0.29–2.47) 1 0.99 (0.52–1.90) 1.17 (0.54–2.51) 1 0.83 (0.45–1.51) 0.69 (0.24–2.04)

adjusted odds ratios (OR) with 95% confidence intervals (CI), estimated in the cohort ’97.

Sex adjusted odds ratios (OR) with 95% confidence intervals (CI), estimated in the cohort ’97. a OR (95% CI).

Differences in various domains of cognitive function dependent on the i1-C/T, L1074F and C1367R genotypes during follow-up

i1-C/T L1074F C1367R CCa CTa LLa LFa FFa CCa CRa RRa Global cognitive function (points) 22.9 (0.31) 23.1 (0.93) 22.6 (0.49) 23.4 (0.40) 22.2 (0.60) 22.8 (0.38) 22.8 (0.47) 23.1 (0.90) Attention (seconds) 87.3 (1.53) 83.3 (4.61) 88.4 (2.39) 87.1 (1.97) 84.6 (3.04) 84.8 (1.84) 88.9 (2.27) 94.2 (4.35) Processing speed (digits) 15.5 (0.33) 16.7 (0.99) 15.3 (0.52) 15.7 (0.43) 16.3 (0.66) 15.8 (0.40) 15.6 (0.50) 15.3 (0.95) Immediate memory (pictures) 20.3 (0.31) 21.2 (0.96) 20.5 (0.49) 20.5 (0.41) 20.1 (0.62) 20.4 (0.38) 20.3 (0.47) 20.2 (0.91) Delayed memory (pictures) 7.00 (0.15) 7.47 (0.45) 7.05 (0.23) 7.05 (0.19) 6.97 (0.29) 6.99 (0.18) 7.00 (0.22) 7.14 (0.43)

Estimates represent the overall mean difference in cognitive function during the mean 4.2-year follow-up, dependent on genotypes in the cohort ’97. The common allele homozygotes were taken as the referent group, and a significant difference (p < 0.05) is indicated in bold. Analyses were adjusted for sex and education. S.E.: standard error. a