2.3. Cognitive function tests in cohort ’97

Overall cognitive function was measured with the Mini-Mental State Examination (MMSE). Individuals who scored equal to or above 19 points also performed tests measuring attention (Stroop Test) (Klein et al., 1997), processing speed (Letter Digit Coding Test) (Houx et al., 2002), immediate recall memory (Word Learning Test Immediate Recall) and delayed recall memory (Word Learning Test Delayed Recall) (Brand and Jolles, 1985). All participants were visited annually for re-measurement of cognitive functioning during a mean follow-up of 4.2 years. Parallel versions of the tests were used. Details of cognitive testing are described elsewhere (Houx et al., 2002).

2.4. Genotyping

The aim of this study was to assess the impact of the i1-C/T, L1074F and C1367R polymorphisms in the WRN gene on the occurrence of cardiovascular pathologies, on cognitive performance and on the risks of all-cause and cause-specific mortalities in the population at large. Since these polymorph- isms are potentially functional (Bohr et al., 2004; Kamath- Loeb et al., 2004), univariate analyses were performed. The study was carried out in elderly aged 85 years and older, using cross-sectional and prospective study designs. The use of elderly participants provides the best opportunities for determining the impact of genetic variations on aging trajectories, since at that age the effects of truly functional variations should be most pronounced.

The i1-C/T (rs2725335) and C1367R (rs1346044) variations were geno- typed using an Assay-by-Design (Applied Biosystems), consisting of PCR primers and TaqMan MGB probes. Amplification reactions were made at standard conditions except for the following modifications. A qPCR core kit was used (Eurogentec) and a half of the amount of primers and probes. Real time PCR was performed on ABI 7900 HT (Applied Biosystems). The L1074F (rs2725362) polymorphism was genotyped by matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), using the Sequenom MassARRAYtm (Sequenom Inc.) methodology. Amplification reac- tions and parameters were based on the manufacturer’s instructions.

2.5. Statistical analysis

Allele frequencies were calculated and analyzed for deviation from the Hardy–Weinberg equilibrium using the x2-test. Differences in the prevalence of cardiovascular pathologies between genotypes were tested using the binary logistic regression model adjusted for sex. Associations between genotypes and cognitive functioning were analyzed using a linear mixed model, estimating the overall mean difference in cognition during the follow-up, adjusted for sex and level of education. Mortality was first estimated using the Kaplan–Meier method, followed by the calculation of sex adjusted mortality risks and 95% confidence intervals (CI) for all-cause, cardiovascular and cancer mortality with the Cox proportional hazard model, using left censoring to correct for the delayed entry into the risk set according to age. For each polymorphism hazard ratios (HR) were calculated using common allele homozygotes as the referent group.Allanalyseswere performedwithSPSS statistical software,Version12.0 (Chicago, IL, USA), with the exception of the mortality analyses, which were performed with STATA software, Version 9.0 (TX, USA).

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 1 April 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 grand- parents 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 the 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

M. Kuningas et al./Mechanisms of Ageing and Development 127 (2006) 307–313

WAN gene5 140499 bp 3i1-C/T L1074F C1367R¥v ¥ vExon: 1 2 8 17 19 2 25 32 35Ae tta5'UTR Coding region 3'UTRWRN proteinAmino acid: 1 L1074F C1367R 4432‘7       Exonuclease Acidic Helicase° RQC  HRDC NLSresidues