aw2=3.754, df=2, P=0.155; bw2=0.077, df=1, P=0.829; cw2=0.433, df=1, P=0.535.

3 PON1 codon192/codon 55 haplotypes frequency distribution

Haplotype frequency+s.d. Young subjects Centenarians (1158 chromosomes) (616 chromosomes) Haplotypea Mle Exp Db Mle Exp Dc AL 0.367+0.014 0.462+0.015 70.0955 0.341+0.018 0.440+0.020 AM 0.368+0.016 0.273+0.013 +0.0955 0.342+0.019 0.243+0.017 +0.0996 BL 0.262+0.014 0.167+0.011 +0.0955 0.303+0.017 0.204+0.016 +0.0996 BM 0.003+0.002 0.098+0.009 70.0955 0.014+0.006 0.113+0.013

aw2=10.5, df=3, P=0.012; bD’=97.4%, w2=232.855, df=1, P50.001; cD’=88.5%, w2=123.1642, df=1, P50.001; Mle: Maximum likelihood estimation; Exp: expected.

4 Genotype combinations at PON1 codon 192 and codon 55

Young subjects Centenarians (n=579) n (%+s.e.) (n=308) n (%+s.e.) Genotype Observed genotype Observed genotype Combinationsa combinations combinations B+M+subjects 100 (0.173+0.016) 81 (0.263+0.025) (AB.LM+AB.MM+BB.LM) B+M7subjects 159 (0.275+0.018) 85 (0.276+0.025) (AB.LL+BB.LL) B7M+subjects 246 (0.424+0.021) 106 (0.344+0.027) (AA.LM+AA.MM) B7M7subjects 74 (0.128+0.014) 36 (0.117+0.018) (AA.LL)

(AB.LL+BB.LL) did not change appreciably (0.275 in controls vs 0.276 in centenarians).

192 23 On the other hand, carriers of B allele (B+ subjects) seem to be protected when other known risk factors for CVD are present, such as smoking,24 familiar hypercolesterolemia,25 low HDL-cholesterol levels.26 Anyway, even if the role of the B allele as risk factor were ascertained, the presence of a genetic risk factor in the gene pool of centenarians is not unusual. The above cited mathematical model19 is able to explain such ‘paradoxes in centenarians’ taking into account the biological and physiological role of risk alleles in survival, and the heterogeneity of the today population of centenar- ians. In any case, an increasing number of data indicate that long life expectancy is compatible with genetic risk factors,27–30 when the individual genetic background is able to cope with stress and consequent age-related accumulation of somatic damage.31

Discussion

Taking into account the physiological role of the enzyme Paraoxonase 1, which may play a central role in rate and quality of ageing,17 we investigated the possible association of PON1 genetic variability with longevity, i.e. the capacity of reaching the extreme limits of human life span escaping major age-related diseases.

192 55 separately (Tables 1 and 2), we found that PON1 variability at 192 codon was able to discriminate between young people and centenarians. In particular, the frequency of B allele, and consequently of B+ individuals, increased from young people to centenarians, thus indicating that this allele decreases mortality in carriers. When the two variants A/B and L/ 192 M were analysed together (Table 4) only B+M+ individuals 55 increased from young people to centenarians, thus indicating that this genotype combination decreases mortality in carriers.

PON1 activity is involved in the protection of LDL against oxidative/proinflammatory modifications which play a central role in the pathogenesis of arteriosclerosis and cardiovascular diseases.6 As the increase of B+M+ individuals among centenarians suggests a genetically determined Paraoxonase activity advantageous for longevity, it could be hypothesised that such individuals are provided with an elicited capacity to counteract the deleterious effects of the accumulation of pro-inflammatory molecules and the increase of the proinflammatory status which accompany human aging.31 Functional studies of correlation between PON1 genotype and PON1 activity in cells from centenarians may clarify this point.

observed gene/longevity association is not highly significant. This finding implies that the effect of PON1 variability on the overall population mortality is rather slight, and this finding is in line with the results obtained in several gene/longevity association studies.18 Moreover, the AB heterozygous 192 genotype appears to contribute rather largely to the observed association (Table 1). A recently developed mathematical model explains the variation of heterozygosity in an ageing cohort in terms of differential survival as affected by the biological role of the gene.19 According to the model, centenarians are expected to show increasing heterozygosity with respect to young subjects for stress-responder genes driving the individual adaptive capacity. Data in Table 1 suggest that PON1 is one of the genes affecting such adaptive capability, and therefore one of the genes affecting rate and quality of ageing.

Acknowledgements

This work was financed by Ministry of Health ‘Progetto finalizzato 1997: la prevenzione della morbilita` per malattie croniche nell’eta` avanzata. Il modello dei centenari’ and ‘Progetto finalizzato 1998: interazioni tra fattori di rischio cardiovascolari e fattori genetici dell’aterosclerosi e della trombosi: un approccio integrato genetico- funzionale in casistiche selezionate’ and by COFIN 2000–2002 Genetics Determinants of Human Longevity.

192 centenarians than in young people seems to be contradictory with literature data reporting the role of this allele as risk factor in artery and coronary diseases, carotid stenosis, cardiovascular diseases, vascular dementia.10,20–22 However, the data are still conflicting. What is more, these discrepan- cies may be related to different criteria of recruitment, different average age of the sample groups, different experimental design (for example the fact that in some studies A/B variants have been examined without taking 192 into account other variants of PON1 gene, such as L/M 55 variants). Probably, the population genetic history as well as the presence of other risk factor for CVD play a key role in determining association between A/B alleles and CVD. It is 192 of interest that studies carried out in the Mediterranean area do not support the existence of a significant association

References