There are two common DNA polymorphisms in the PON1 gene. One located at amino acid position 55 is due to a leucine (L allele) and methionine (M allele) interchange. The second at amino acid position 192 is due to a glutamine (Q allele) and arginine (R allele) interchange. Humbert et al., 1993, reported that arginine at position 192(R) conferred higher PON activity, using paraoxon as the substrate, compared to glutamine at position 192(Q), though activity was reversed with other exogenous substrates such as diazoxon, soman, and sarin, or not altered (Davies et al., 1996; Mackness et al., 1997a). Mackness et al. (1997b, 1998), showed that the ability of HDL to protect LDL against oxidative modification may be determined by these polymorphisms, with the paraoxonase 55L and 192R homozygotes having a low ability of HDL to prevent lipid peroxidation of LDL, when compared to 55M and 192Q

recruited as part of an ongoing study of ageing BELFAST, (Rea et al., 2000). Blood was collected from 296 elderly subjects (92 male and 204 female, mean age 89.8 SD 5.7 years) who were apparently well, mobile and living in the community. Young subjects were part of the Young Hearts Study (Boreham et al., 2002) described previously, and comprised a sample of 296 subjects (92 males, 204 females, age 13.0 SD 1.4 years) with DNA samples randomly selected and sex-matched to the BELFAST octo/nonagen- arian group. The study was approved by the Research Ethics Committee of the Queen’s University of Belfast and all patients and controls gave informed written consent.

2.2. Genotyping

Several groups have conducted association studies between paraoxonase gene polymorphisms and coronary heart disease (CHD). The 192R allele has been found to be associated with an increased risk of CHD in some but not all populations (Ruiz et al., 1995; Serrato and Marian, 1995; Sanghera et al., 1997, 1998a; Antikainen et al., 1996; Herrmann et al., 1996; Jarvik et al., 2000; Mackness et al., 2001). The situation regarding cardio- vascular risk and the PON1 55 polymorphism is confusing; Salonen et al. (1999) reported an odds ratio of 3.4 for those with the MM genotype relative to those who carried the LL genotype, Blatter Garin et al. (1994) reported an odds ratio of 1.98 for those NIDDM patients homozygous for the L allele, while Sanghera et al. (1998b), reported no association.

Longitudinal Free-living Aging STudy and Young Hearts blood samples using the ‘salting-out’ method (Miller et al., 1988). The PON1 genotypes were determined by PCR amplification of the region containing the poly- morphism followed by restriction enzyme digestion (Humbert et al., 1993), previously described by Hasselwander et al. (1999). Briefly, the DNA fragment containing the codon 192 polymorphism was amplified 0 0 using forward 5 TATTGTTGCTGTGGGACCTGAG3 0 0 and reverse 5 CACGCTAAACCCAAATACATCTC3 primers. The DNA fragment containing the codon 55 polymorphism was amplified using forward 0 0 5 GAAGAGTGATGTATAGCCCCAG3 and reverse 0 0 5 TTTAATCCAGAGCTAATGAAAGCC3 primers. The following PCR conditions were used: 95 8C for 9 min; 35 cycles consisting of 95 8C for 1 min, 60 8C (PON1 55 fragment) or 58 8C (PON1 192 fragment) for 1 min, and 72 8C for 1 min; followed by a final extension step of

In view of the role of PON1 in metabolism of toxic chemicals and its proposed role modulating LDL oxidation we have investigated whether the PON1 55 and 192 polymorphisms influence longevity in a pooled analysis of

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

3. Results

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 8C for 16 h and then resolved on a 3% agarose gel, visualised 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) fragment, respectively. Control DNA samples with known genotypes were included within each run to ensure accurate genotype assignation.

There was no significant deviation of PON 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 NI 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

Italy. Polymerase chain reactions were performed using primer sequences derived from published data and 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 octolnonagerarians from Northern Ireland versus young controls