Regression Analysis of PON1 192 and PON 55 genotypes and after adjustment for possible confounding by country and gender

Comparison of haplotype frequencies in young and old subjects

Odds ratio (95% CI) P Genotype analysis PON1 192 RQ vs QQ 1.29 (1.01, 1.65) 0.04 RR vs QQ 1.68 (1.08, 2.62) 0.02 PON1 55 LM vs LL 1.10 (0.86, 1.41) 0.46 MM vs LL 1.07 (0.72, 1.58) 0.73 Allele analysis PON1 192 R vs Q 1.26 (1.07, 1.49) 0.007 PON1 55 M vs L 0.94 (0.80, 1.10) 0.44

Italy and N Ireland (pooled) Allele at PON1 192 Allele at PON1 55 Haplotype frequency Old Young Q L 0.337 0.367 Q M 0.348 0.367 R L 0.308 0.263 R M 0.008 0.004

Likelihood ratio chi square ¼ 9.60, df ¼ 3; P ¼ 0:02:

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frequency of 8.5% for RR homozygotes in the oldest cohort but showed no enrichment for older age groups compared to their younger controls. As in the present study they show a tight linkage disequilibrium between the PON1 192 and PON 55 loci, in this study linking R and L alleles.

Italian centenarians ðD0 ¼ 20: 880Þ and Italian young ðD0 ¼20: 975Þ; Irish octo/nonagenarians ðD0 ¼ 20: 975Þ and Irish young ðD0 ¼ 20: 936Þ: A similar linkage has been documented by Heijmans et al. (2000) in Netherlands, 0 with a similar estimated linkage D value.

Table 4 shows the estimated haplotype frequencies and the Likelihood Ratio (X2 ¼ 9: 6; df ¼; p ¼ 0: 02) obtained from EM which suggested that there was a small but significant difference in PON1 192 and PON55 genotype combinations in octo/nonagenarians and centenarians com- pared to the younger subject groups across Italy and Ireland. Fast EH plus confirmed the validity of the asymptotic P value given by the likelihood ratio test by using 10,000 simulations (Zhao and Sham, 2002).

The mechanism underlying such a possible survival effect from carriage of the R allele in nonagenarian and centenarian subjects is not clear particularly since carriers of the R allele have appeared to be at risk of increased cardiovascular disease in earlier life in many though not all studies (Mackness et al., 2001). In younger people the mechanism has been related to the reduced ability of R allele carriers to protect their LDL cholesterol from damaging oxidation (Aviram et al., 1999; Mackness et al., 1997a). Interestingly, several studies have noted a decrease in the effect of PON1 192 with age in cardiovascular-related studies which is so far unexplained (Senti et al., 2001; Leviev et al., 2002), though Mackness et al. (2003) in the Caerphilly study suggest that risk factors for a multifactorial disease like atherosclerosis may become diluted with age. A further argument against an important cardiovascular mechanism for PON1 in survival comes from the separate analysis by sex which showed no evidence that sex differentially enhanced longevity between male and female subjects.

4. Discussion

Pooled analysis of Italian and Irish data in this study allowed us to increase the number of subjects being examined and provided for more vigorous testing of the original hypothesis that there might be attrition of the PON1 gene variants in the oldest age groups. In addition it allowed us to perform an analysis of the effect of sex which had not been specifically evaluated in either study (Taioli and Bonassi, 2003). Contra-intuitively, we demonstrated a modest increase in the frequency of the carriage of the R allele of the PON1 192 gene, between centenarian and octo/ nonagenarian subjects and their young controls, in the pooled data from Italy and Northern Ireland. This finding was independent of the country of the subjects’ origin and of their sex. The oldest subjects in both Italy and Ireland, in this study, demonstrate an increased frequency of the PON1 192 R allele and this effect is increased stepwise when the oldest subjects were homozygous for the R allele. When compared to younger subjects in either Ireland or Italy, surviving octo/nonagenarians in Ireland and centenarians in Italy, had an increased chance of carrying either one copy of the PON1 192 R allele (odds ratio 1.3) or two copies of the R allele (odds ratio 1.7). In some previous studies in the oldest subject groups, there has been a documented attrition in the frequency of genes which have been associated with earlier mortality, though this has not always been the case. In the most consistent finding, several groups have demonstrated that ApoE4, associated with hypercholesterolaemia, pre- mature atherosclerosis and late-onset Alzheimer’s disease (Katzman, 1994) is significantly less frequent in their populations of centenarians or nonagenarians compared to control groups, whereas ApoE 2 frequency is increased (Schachter et al., 1994; Kervinen et al., 1994; Jian-Gang et al., 1998; Frisioni et al., 1991; Rea et al., 2001). The findings in this study would seem to suggest that carriage of the PON1 192R allele is associated with a modest increased chance of survival into very old age. In the only other study to date of PON1 192 polymorphisms in longevity, Heijmans et al. (2000) in the Leiden .85 study also report a similar