Table 1

Comparison of PON1 192 and 55 genotype and allele numbers and frequencies, for centenarians in Italy and octolnonagerarians from Northern Ireland versus young controls

Genotype based analysis PON1 192 QQ QR RR Italy Old (308) 142(46.1) 137(44.5) 29(9.4) P ¼ 0:03 Young (579) 320(55.3) 212(36.6) 47(8.1) N Ireland Old (296) 138(46.6) 130(43.9) 28(9.5) P ¼ 0:14 Young (296) 152(51.4) 128(43.2) 16(5.4) PON1 55 LL LM MM Italy Old (308) 121(39.3) 155(50.3) 32(10.4) P ¼ 0:15 Young (579) 233(40.2) 262(45.3) 84(14.5) N Ireland Old (294) 122(41.5) 135(45.9) 37(12.6) P ¼ 0:42 Young (296) 111(37.5) 152(51.4) 33(11.1) Allele based analysis PON1 192 Q R Italy Old (616) 421(68.3) 195(31.7) P ¼ 0:02 Young (1158) 852(73.6) 306(26.4) N Ireland Old (592) 406(68.6) 186(31.4) P ¼ 0:09 Young (592) 432(73.0) 160(27.0) PON1 55 L M Italy Old (616) 397(64.4) 219(35.6) P ¼ 0:51 Young (1158) 728(62.9) 430(37.1) N Ireland Old (588) 379(64.5) 209(35.5) P ¼ 0:65 Young (592) 374(63.2) 218(36.8)

2.3. Statistical analysis

increase the number of observations being examined, providing more vigorous testing of the orignal hypo- thesised relationships among the variables (Taioli and Bonassi, 2003). Gene polymorphisms for PON 1 192 and 55 were coded in both Italy and Ireland according to standard nomenclature (Humbert et al., 1993). All data received and included in the data-sets was anonymous. Deviation of genotype frequencies from the Hardy- Weinberg equilibrium was tested by a Chi square goodness of fit test. Genotype and allele frequencies for PON1 192 and PON 55 were compared by Chi square analysis between centenarian and nonagenarian subjects and their control populations separately and for the combined Italian and Irish dataset by logistic regression with country, sex, genotype or allele as coded binary 0 independent variables. Lewontin’s D coefficient was used to assess the strength of Linkage disequilibrium between the PON1 192 and 55 polymorphisms with values close to þ1 or 21 suggesting a high degree of linkage. EH was used to estimate haplotype frequencies and to derive likelihood ratio tests to compare haplotype combination frequencies in young and old (Terwilliger and Ott, 1994). Fast EH plus was used to check the validity of the asymptotic P value associated with the likelihood ratio test using 10,000 simulations (Zhao and Sham, 2002). :

I.M. Rea et al. / Experimental Gerontology 39 (2004) 629–635

subjects. In NI there was no significant difference between octo/nonagenarian versus young subjects but there was a trend for an increase in RR homozygotes in octo/ nonagenarian subjects. The PON 55, LL, LM and MM genotypes occurred with similar frequencies in Italy and Ireland with no significant differences between the age groups.

Table 3

Genotype combinations for PON1 192 and PON 55 for Italy and N Ireland pooled Old and Young groups

PON 55 LL LM MM OLD PON1 192 QQ 70 143 66 279 QR 119 144 3 266 RR 54 3 – 57 Total 243 290 69 602 YOUNG PON1 192 QQ 112 245 115 472 QR 171 167 2 340 RR 61 2 – 63 Total 344 414 117 875

3.2. Allele based analysis

Table 1 also shows a comparison of PON1 192 and 55 allelefrequencies for both Italy and NI 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 N Ireland. In keeping with the genotype based analysis, the R allele frequency increased by about 4–5% in old compared to young subjects. Table 1 shows similar frequencies for PON 55 L and M alleles in Italy and NI with no significant differences between age groups.

and (M versus L) for PON 55. The R allele of PON1 192 showed elevated odds ratio compared to the Q allele (1.26, CI 1.07–1.5; p ¼ 0: 007). PON 55 L and M alleles showed no differences between the age groups (0.94, CI 0.80–1.10, p ¼ 0: 44). There was no interaction between country for PON1 192 (X2 ¼ 0: 02; df ¼ 1; p ¼ 0: 88) nor for PON 55 (X2 ¼ 0: 03; df ¼ 1; p ¼ 0: 86). Tests for the interaction between sex and PON1 192 Q/R alleles showed no suggestion that the PON1 192 effects were sex specific (X2 ¼ 0: 08; df ¼ 1; p ¼ 0: 77).

3.3.1. Genotype analysis

Logistic regression was used to compare PON1 192 genotypes whilst 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 ¼ 0: 04) and the RR genotype 1.68 (CI, 1.08–2.62; p ¼ 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 ¼ 2: 04; df ¼ 2; p ¼ 0: 36) or PON 55 (X2 ¼ 3: 91; df ¼ 2; p ¼ 0: 14) genotypes, providing no evidence of heterogeneity between Italy and N 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 typing that linkage disequibilrium existed. The RRMM genotype combination was absent in our combined Italian and Irish young and old cohort of close to1500 subjects. The RR genotype of PON1 192 and LL genotype from PON55 were almost exclusively linked in the RRLL genotype combination with only 5 subjects out of 1500 showing the very rare RRLM combination. Formal assessment of linkage disequibilium was carried out using Lewontin’s D0 estimate which showed highly significant values ðp ,0: 0001Þ of close to þ1 or -1 in all subject groups: pooled old ðD0 ¼ 20: 928Þ; pooled young ðD0 ¼ 20: 965Þ;

3.3.2. Allele based analysis

Table 2 also shows the logistic regression used to compare alleles (R versus Q) for PON1 192

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