3.2. Allele-based analysis 

Table 1 also shows a comparison of PON1 192 and 55 allele frequencies for both Italy and Northern Ireland for very old and young groups. Comparing Q and R alleles of PON1 192, there were significant differences between the centenarian and young age groups from Italy with similar but non-significant changes in Northern Ireland. In keeping with the genotype-based analysis, the R allele frequency increased by about 45% in old compared to young subjects. Table 1 shows similar frequencies for PON1 55 L and M alleles in Italy and Northern Ireland with no significant differences between age groups.

3.3. Statistical analysis 

To increase the number of observations being examined, providing more vigorous testing of the original hypothesized relationships among the variables (Taioli and Bonassi, 2003), gene polymorphisms for PON1 192 and 55 were coded in both Italy and Ireland according to standard nomenclature (Humbert et al., 1993). All data received and included in the datasets were anonymous. Deviation of genotype frequencies from Hardy-Weinberg equilibrium was tested by a chi-square goodness of fit test.

3.3.1. Genotype analysis 

Logistic regression was used to compare PON1 192 genotypes while adjusting for any confounding influence of country or gender. In Table 2, the results show a significant stepwise elevation in odds ratio associated with the QR genotype 1.29 (CI, 1.01-1.65; p = 0.04) and the RR genotype 1.68 (CI, 1.08-2.62; p = 0.02), relative to the QQ genotype. When logistic regression was used to compare the PON 55 LL, LM, and MM genotypes between age groups, there was no significant effect. There was no interaction between country and PON1 192 (X2 = 2.04; df = 2; p = 0.36) or PON 55 (X2 = 3.91; df = 2; p = 0.14) genotypes, providing no evidence of heterogeneity between Italy and Northern Ireland in the association between PON1 192 and longevity.

Table 3 shows the possible genotype combinations for PON1 192 and PON 55 and subject numbers in each genotype combination since there was evidence from the typingThe effect of the carriage of the R allele in nonagenarian and centenarian subjects is unclear, particularly since carriers of the R allele have been shown to be at risk of increased cardiovascular disease earlier in life in many, but 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) 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.

Pooled analysis of Italian and Irish data in this study allowed us to increase the number of subjects being examined and provided more vigorous testing of the original hypothesis that there might be attrition of the PON1 gene variants in the oldest age groups. Additionally, 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). Contrary to intuition, we demonstrated a modest increase in the frequency of 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 origin and sex. The oldest subjects in both Italy and Ireland demonstrated an increased frequency of the PON1 192 R allele, and this effect 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 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, premature 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 ApoE2 frequency is increased (Schachter et al., 1994; Kervinen et al., 1994; Jian-Gang et al., 1998; Frisoni 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 reported a similar finding.