Few studies, however, have investigated the impact of PON1 polymorphisms on survival. Heijmans et al10 found no significant effect of the Q192R and M55L polymorph- isms on mortality risk in 666 subjects age 85þ years in a 10-year follow-up study, whereas Bonafe´ et al11 reported an increase in the frequency of 55M/192R carriers in 308 centenarians as compared to 579 younger individuals. In this study, we examined the association between the (cid:1)107C/T, M55L and Q192R polymorphisms in a sample of Danish subjects aged 47–93 years drawn among participants of three populations-based studies. We used the sample both in a cross-sectional design and in a longitudinal follow-up study 5 years in average. All three polymorphism were investigated in a total of 1932 individuals and to confirm positive findings in the initial cohorts, a further 541 subjects 93 years of age were examined.

DNA was amplified in a total volume of 10ml containing 5ml Taqman Universal Master Mix (Applied Biosystems), 900nM of each primer (DNA Technology), 200nM of each B probe (MWG Biotech) and 10ng template DNA. PCR was performed in the ABI Prism 7700 using the conditions recommended by the manufacturer (Applied Biosystems) and analyzed using the Sequence Detection System soft- ware.

Materials and methods

The subjects included in this study were drawn from three major population-based surveys; the Danish 1905 Cohort, which includes all Danes born in 1905;12 the Longitudinal Study of Ageing Danish Twins (LSADT), including all Danish twins age 70 years or older;13,14 the Study of Middle-aged Danish Twins (MADT), including Danish twins born between 1931 and 1952.14,15 Initially, we genotyped a total of 1932 unrelated individuals (sample 1) previously used for gene-longevity studies.16,17 Of these, 724 originated from the 1905 cohort (93 years of age), 626 participants originated from LSADT (age 70–90 years) and 582 participants originated from MADT (age 47–68 years). For twins only one twin from each pair was included. We then extended the study by replicating the initial positive findings in an independent sample of 541 individuals available from the 1905 survey (sample 2), which is the survey with the most events (highest mortality).

Statistical analysis

Genotype distribution frequencies were compared with the w2 test. Haplotype frequencies were estimated using an expectation maximization algorithm and distribution differences tested using CLUMP.18 Linkage disequilibrium coefficients were estimated according to the method described by Chakravarti et al.19

Mortality risk was estimated using Cox’s proportional- hazard model, adjusting for age and sex. Statistic proce- dures were performed with the STATA 8.0 statistical program (Stata Corporation).

Results

Baseline characteristics and genotype frequencies for each of the three polymorphisms are summarized in Table 1. Genotype frequencies were not different between men and women and combined frequencies are thus reported. A cross-sectional analysis indicated that there was no differ- ence in genotype distribution in the three cohorts. Dividing the entire population into groups based on 10- year intervals revealed no difference in genotype distribu- tion either (results not shown). The estimated haplotype frequencies using all three polymorphisms were not significantly different in any groups and examination of haplotypes for two polymorphisms at a time did not

All participants were followed from the date of inclusion (November, 1996–May, 1999) to the date of either death or January 8, 2004, whichever came first, resulting in a mean follow-up time of 5 years for survivors (range 4.6–7.2 years). Information on survival status was retrieved from the Danish Central Population Register, which is continu- ously updated.

Self-reported information with regard to health issues was available from all surveys. As a part of the original survey, the participants were asked whether they did now or had previously suffered from various diseases. Only

1 Characteristics and PON1 genotype frequencies

MADT LSADT 1905 cohort 1905 cohort N¼582 (sample 1) N¼626 (sample 1) N¼724 (sample 1) N¼541 (sample 2) Agea 53.67 5.7 77.87 4.4 937 0 937 0 Male/female 272/310 309/317 282/442 111/430 Events (deaths) Male/female 10/6 135/120 232/319 85/319 PON1 (cid:1)107C/T CC 175 (30.1%) 173 (27.6%) 192 (26.5%) CT 250 (42.9%) 298 (47.6%) 347 (47.9%) TT 157 (27.0%) 155 (24.8%) 185 (25.6%) PON1 55M/L MM 74 (12.7%) 87 (13.9%) 96 (13.3%) 69 (13.3%) ML 265 (45.5%) 304 (48.6%) 334 (46.1%) 242 (46.5%) LL 243 (41.8%) 235 (37.5%) 294 (40.6%) 209 (40.2%)b PON1 192Q/R QQ 307 (52.8%) 352 (56.2%) 381 (52.6%) 284 (52.5%) QR 230 (39.5%) 225 (36.0%) 290 (40.1%) 215 (39.7%) RR 45 (7.7%) 49 (7.8%) 53 (7.3%) 42 (7.8%)

MADT¼Study of Middle-Aged Danish Twins; LSADT¼Longitudinal study of Ageing Danish Twins; 1905 cohort¼The Danish 1905 Cohort.

Table 2 Mortality risk according to PON1 genotypes in a 5-year follow-upa in women

change this picture (results not shown). All three morphisms showed linkage disequilibrium to some with D¼(cid:1)0.43 for 108C/T and 55M/L, D¼(cid:1)0.23 for T and 192Q/R and D ¼0.43 for 55M/L and respectively.

Hazard rateb 95% CI P-value PON1 genotypes Sample 1 (N¼1069) (cid:1)107CC 1 (cid:1)107CT 0.99 0.79–1.23 0.90 (cid:1)107TT 1.08 0.83–1.40 0.56 55MM 1 55ML 0.69 0.52–0.90 0.007 55LL 0.83 0.63–1.09 0.17 192QQ 1 192QR 0.86 0.70–1.05 0.13 192RR 1.38 1.01–1.87 0.04 Sample 2 (N¼430) 192QQ 1 192QR 1.05 0.83–1.33 0.69 192RR 1.38 0.95–2.00 0.09 55MM 1 55ML 1.17 0.82–1.69 0.37 55LL 1.38 0.96–1.99 0.08 Sample 1+2 (N¼1499) 192QQ 1 192QR 0.93 0.80–1.09 0.37 192RR 1.38 1.09–1.75 0.008