Experimental Gerontology 39 (2004) 629-635 www.elsevier.com/locate/expgero polymorphisms PON1 192 and 55 and longevity in Italian centenarians and Irish nonagenarians. A pooled analysis, a,* b b b c Irene M. Rea, Pascal P. McKeown, Dorothy McMaster, Ian S. Young, Chris Patterson, d b e e Maurice J. Savage, Christine Belton, Francesca Marchegiani, Fabiola Olivieri, f f Massimiliano Bonafe, Claudio Franceschi a Department of Geriatric Medicine, Queens University of Belfast, Whitla Medical Building, 97 Lisburn Road, Belfast BT7 9AB b Department of Medicine, Queens University of Belfast, Belfast, United Kingdom c Epidemiology, Queens University of Belfast, Belfast, United Kingdom d Child Health, Queens University of Belfast, Belfast, United Kingdom e Italian National Research Centres on Aging, Ancona, Italy Received 5 August 2003; received in revised form 8 October 2003; accepted 21 November 2003 Abstract Background: PON1 is an arylesterase associated with high-density lipoprotein (HDL) that protects low-density lipoprotein (LDL) against oxidative modification. Common polymorphisms PON1 55 (L/M) and 192 (Q/R) in the PON1 gene are associated with atherosclerosis and heart disease. Because long-lived people seem protected from premature vascular death, we conducted a pooled statistical analysis to assess any association between these polymorphisms and longevity in a large combined group of Italian centenarians and octo/nonagenarians from two large cohorts of very elderly subjects - centenarians in Italy and octo/nonagenarians from the Belfast Elderly Longitudinal Free-living Ageing Study (BELFAST) in Northern Ireland.

Materials and methods: Separated DNA was available from 1479 subjects from Italy and Northern Ireland (NI). In Italy, 308 (males 67, females 241, mean age 100.8, SD 2.1 years) and 579 young controls (males 347, females 232, mean age 40.7, SD 12.7 years) included in the study. In NI, 296 octo/nonagenarians (males 92, females 204, mean age (mean age 13.0, SD 1.4 years) had available DNA. PON1 55 (L/M) and 192 (Q/R) polymorphisms were studied using a PCR-RFLP approach.

Results: There was a significant difference in PON1 192 genotypes in Italian centenarians compared to younger controls (X2 = 6.8; df = 2; p = 0.03) and a similar but non-significant trend between octo/nonagenarian and young subjects in NI (X2 = 4.0; df = 2; p = 0.14). Using logistic regression analysis on the combined Italian and Irish datasets, there was a small survival advantage for centenarian and octo/nonagenarian subjects who were heterozygous for PON1 192 R allele (OR 1.3, CI 1-1.6; p = 0.04) with a stepwise increase for RR homozygous subjects (OR 1.7, CI 1.1-2.6; p = 0.02), compared to QQ subjects. Comparing R and Q alleles, there was a survival advantage for octo/nonagenarian/centenarian subjects who carried the R allele (OR1.3, CI1.1-1.5; p = 0.007), but there was no sex-specific effect (p = 0.77). LL, LM, and MM genotypes of PON 55 polymorphisms showed similar frequencies in Italy (39.9, 47.0, 13.1%) and Ireland (39.5, 48.6, 11.9%) with no age or sex-related differences. The PON1 192R/Q and PON55L/M loci were in strong linkage disequilibrium with a Lewontins D0 coefficient -0.928 (elderly) and -0.965 (young). There was a significant difference in haplotype frequency of these linked loci in older compared to younger subjects (Likelihood Ratio X2 = 9.60; df = 3; p = 0.02).

Conclusion: These data suggest a modest association between the 192R allele and longevity in two very elderly populations in two European countries. Being homozygous for 192 RR further enhances survival advantage but this effect was not found to be sex-specific.analysis of I.M. Rea et al. / Experimental Gerontology 39 (2004) 629-635 

3. Results 

DNA samples were digested with 10 U Hsp92II (Promega, Southampton, UK), and the PON1 192 product (99 bp) was digested with 8 U Alw1 (New England Biolabs, Hitchin, UK) at 37°C for 16 h. The samples were then resolved on a 3% agarose gel and visualized by UV after ethidium bromide staining. Alleles L (leucine) and M (methionine) for the PON1 55 position were assigned based on the presence of a 170 bp (undigested) fragment and 126 and 44 bp (digested) fragments, respectively. Alleles Q (glutamine) and R (arginine) for the PON1 192 position were assigned based on the presence of a 99 bp (undigested) fragment and 69 and 30 bp (digested) fragments, respectively. Control DNA samples with known genotypes were included within each run to ensure accurate genotype assignation. There was no significant deviation of PON1 192 and 55 genotype frequencies from Hardy-Weinberg equilibrium within age groups and countries.

3.1. Genotype-based analysis 

Table 1 shows a comparison of PON1 192 and 55 genotype distributions for centenarians from Italy and octo/nonagenarians from Northern Ireland versus their young controls. There was a significant difference between PON1 192 genotypes in Italy for centenarian and young subjects, with a greater proportion of QR heterozygotes among the older Italians.

Polymerase chain reactions were performed using primer sequences derived from published data specific for the amplification of regions surrounding codon 192 and codon 55 sites (Humbert et al., 1993). The amplification reactions and methodologies have been previously described (Bonafe et al., 2002). Some genotypes at both loci were confirmed by direct sequencing of amplified DNA fragments with the automatic sequencer (ABI PRISM 310, Perkins Elmer Biosystems).

Table 1: Comparison of PON1 192 and 55 genotype and allele numbers and frequencies for centenarians in Italy and octo/nonagenarians from Northern Ireland versus young controls 

3.2. Allele-based analysis