2. Subjects and methods

2.1. Study population

The Leiden 85-plus Study is a prospective population-based study in which inhabitants of Leiden, The Netherlands, aged 85 years or over were invited to take part. There were no selection criteria related to health or demographic characteristics. The study population consists of two cohorts: cohort 87 and 97. Cohort 87 includes 977 participants aged 85 years and older, enrolled between 1987 and 1989 (Weverling-Rijnsburger et al., 1997). Cohort 97 consists of 599 subjects, all members of the 1912-1914 birth cohort who were enrolled in the month of their 85th birthday between 1997 and 1999 (Bootsma-van der Wiel et al., 2002). DNA was available for 682 participants from cohort 87 and for 563 people from cohort 97. All participants of the Leiden 85-plus Study were followed for mortality until April 1, 2004. Primary causes of death were obtained from the Dutch Central Bureau of Statistics and categorized according to the 10th International Classification of Diseases (ICD-10). The Medical Ethical Committee of the Leiden University Medical Center approved the study, and informed consent was obtained from all participants. In addition, 247 young blood donors (aged 19-40 years) from Leiden were included for a cross-sectional comparison of genotype frequencies. To avoid population stratification due to geographic differences between the elderly and young, we restricted the young control population to those with either two Leiden-born parents or one Leiden-born parent and the other born within a 12-km distance from Leiden. Information regarding the birthplace of their grandparents was obtained from a written questionnaire (Heijmans et al., 1999).

3. Results

All 1245 participants of the Leiden 85-plus Study and 247 young blood donors were genotyped for the i1-C/T, L1074F, and C1367R polymorphisms. The location of the SNPs in the WRN gene and protein are indicated in Fig. 1. The genotype and resulting allelic frequencies of the SNPs were in agreement with the Hardy-Weinberg equilibrium, except for the C1367R polymorphism in cohort 97 (Table 1). In that case, a deficit of heterozygotes and an excess of both homozygote allele carriers were observed. The overall genotype distributions and resulting allelic frequencies of the SNPs were similar to those 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 Total:642 Total:536 MAF:0.04 MAF:0.04 MAF:0.04 HWE:0.83 HWE:0.51 HWE:04.2-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 1.00 0.50 0.75
Cumulative mortality 25
1.00 0.00 0.0 0.50 0.75
Cumulative mortality 25
Cumulative mortality 0.50 0.75 1.00 0.00 0.0
25
All-cause mortality All-cause mortality All-cause mortality

it-cc3; L1074L8,1367 it-cT el lo7ar27 | C1367R3 FA074F o| R1367RR xgo go28 $8= aFR as6 oo8 885 90 95 100 105-985 90 95 100 105 
85 90 95 100 105Age (years) Age (years) Age (years)

Cardiovascular mortality @ Cardiovascular mortality Cardiovascular mortality

it-cc8 L1074L >8,1367CT zr L1074F2 | ci3e7Ro|_ Fr074F Ew R1367RBS Bo8 ZegO BS; i i2zu a3Os ogg g85 90 95 100 105 
85 90 95 100 105Age (years) Age (years) Age (years)

Cancer mortality Cancer mortality Cancer mortality

1 i1-CC8 L1074L8 C1367Cit-CT2 | wor2} ci367R_S|R1367R|# F1074F z eES go38 28ge B3 3Ew Ew6g = 6s2z S85 90 95 100 105 
85 90 95 100 105Age (years) Age (years) Age (years)

Fig. 2. Kaplan-Meier curves of cumulative all-cause, cardiovascular and cancer mortality, dependent on i1-C/T, L1074F and C1367R genotypes in the combined cohort of 1245 individuals from age 85 years onwards. 

Over the course of person-years, 957 (77%) participants had died, of which 365 (38%) died from cardiovascular disease and 143 (15%) due to cancer. The Kaplan-Meier estimates of cumulative mortality according to the genotypes of the different SNPs are presented in Fig. 2. According to the Kaplan-Meier mortality curves, it seems that carriers of the i1-CT genotype, and heterozygous and homozygous carriers of the 1367R allele have lower cancer mortality. Although not statistically significant, the mortality risk estimates of 0.56 (0.26-1.20) for i1-CT, 0.84 (0.59-1.20) for C1367R and 0.57 (0.27-1.17) for R1367R genotypes show lower cancer mortality. The same trend was observed in cohort 87 and 97 (Supplementary Table 1). The other all-cause and cause-specific mortality risks, calculated separately for all polymorphisms, were similar between the different genotype carriers of the i1-C/T, L1074F and C1367R polymorphisms, showing no evidence of altered survival.