The intronic polymorphisms p53 Msp1 and the p53 16 bp duplication are situated in a non-coding region of the gene and do not seem to be functionally involved in the carcinogenesis process, but they do show strong linkage disequili- bria with each other and with p53 codon 72 polymorphism (Sja¨lander et al. 1995); for this reason it is very useful to study the combined genotype distribution for these three polymorphisms when analysing differences between groups, or associations with any type of endpoint.

We studied the frequency of the simultaneous presence of polymorphisms in phase I and phase II genes, and of three p53 germline mutations, in a group of nonagenarians and a group of healthy young controls, to order to obtain insights into the genetic mechanisms of ageing.

Materials and methods

The study population comprised 66 nonagenarians and centenarians in good health, described in detail elsewhere (Franceschi et al. 2000), selected from a larger sample of a multicentre Italian study in Northern Italy on mechanisms responsible for successful ageing. The mean (9SD) age of the group was / 100.192.2 years (range 95(cid:1)105 years); 77% were females and 23% were males. / /

A sample of 150 young healthy volunteers of the same ethnic group as the centenarians were recruited in Northern Italy and used as a reference group. The mean (9SD) age of the control group was 42.59 / / 12.4 years (range 20(cid:1)65 years). Since the majority of the centenarians were females, we used a / population of female as controls.

Genotypinganalysis

Genomic DNA was extracted from peripheral blood lymphocytes and genotyping analyses were performed on all the subjects.

GSTM1 and GSTT1 genotypes were determined in a multiplex polymerase chain reaction (PCR) analysis using primer pairs specific for GSTM1 (5?-GAACTCCCTGAAAAGCTAAAGC-3? and 5?- GTTGGGCTCAAATAT ACGTGG-3?) and GSTT1 (5?-TTCCTTACTGGTCCTCACATCTC-3? and 5?-TCACCGGATCATGGCCAGCA-3?) together with a b-globin specific primer pair (5?- CAACTTCATCCACGTTCACC-3? and 5?-GAAGAGCCAAGGACAGGTAC-3?). After an initial step of denaturation, 35 cycles were performed with denaturation at 948C for 1 min, annealing at 648C for 1 min, and extension at 728C for 1 min. Briefly, the absence (deleted) or presence of the GSTM1- and GSTT1-specific PCR products determined the respective genotypes, whereas the b-globin

CYP1A1 Msp1 (CYP1A1*2) genotyping analyses were performed using PCR as previously described (Taioli et al. 1995) using the following primers: 5?-TTAGGAGTCTTGTCTCATGCCT-3? and 5?- CAGTGAAGAGGTGTAGCCGCT-3?.

The term ‘wild type’ refers to homozygotes for the common allele (absence of the Msp1 restriction site) and ‘heterozygotes’ refers to the presence of the polymorphism on one allele; when the polymorphism is present on both the alleles we used the term ‘homozygotes’.

p53 polymorphisms were determined using the method of Sja¨lander et al. (1995). A set of primers, 5?-GCAGAGACCTGTGGGAAGCGA-3? and 5?-ACCGTAGCTGCCCTGGTAGGT-3?, was used to analyse p53 codon 72 and p53 16 bp duplication polymorphisms: 33(cid:1)35 cycles of amplification were / performed with denaturation at 948C for 1 min, annealing at 658C for 1 min 30 sec and extension at 728C for 1.30 min. A fragment of 401 bp, or 417 bp if the 16 bp duplication is present, was amplified. To visualize the codon 72 polymorphism, 15 ml of PCR product was then digested with 8 U of BstU restriction enzyme and separated on 3% agarose gel stained with ethidium bromide. Two other primers, 5?-TATGAGCCGCCTGAGGTCTGG-3? and 5?-TACAGGCA TGAGCCACTGCGC-3? were used to study the Msp1 polymorphism; amplification conditions were similar to those previously described for the other p53 polymorphisms, with an annealing temperature of 688C for 1 min. PCR products were digested with 10 U of Msp1 and run on agarose gel.

The term ‘wild type’ refers to homozygotes for the common allele (presence of the BstU or Msp1 restriction site, and absence of the 16 bp duplication), ‘heterozygotes’ refers to the presence of the polymorphism on one allele, and ‘homozygotes’ referes to the presence of the polymorphism on both alleles.

Statisticalanalysis

Gene polymorphism frequencies are presented separately for centenarians and controls. Differences among gene frequencies were estimated using the x2 test or, when necessary, with the Monte Carlo simulation (Roff and Bentzen 1989). Values of p 50.05 were considered to be significant. / Analysis of the data was performed using SAS 8.2 software system.

We estimated the genotype frequencies of several genes involved in xenobiotic metabolism (GSTM1, GSTT1 and CYP1A1*2) and of various p53 polymorphisms (codon 72, Msp1 and a 16 bp duplication) for more than 95% of the study population (Table 1). No statistically significant differences in the genotype frequencies were found between the two groups analysed; GSTT1 deletion was

1. Frequencies of gene polymorphisms in centenarians and controls.

Centenarians (n(cid:4)66) Controls (n(cid:4)150) / / Gene polymorphism n % n % p value p53 Msp1 Wild type (G/G) 42 63.6 101 67.8 Heterozygous (G/A) 23 34.9 44 29.5 Homozygous (A/A) 1 1.5 4 2.7 0.65 p53 codon 72 Wild type (G/G) 33 50.0 81 54.3 Heterozygous (G/C) 28 42.4 57 38.3 Homozygous (C/C) 5 7.6 11 7.4 0.85 p53 16 bp duplication Wild type 34 60.7 98 65.8 Heterozygous 21 37.5 46 30.8 Homozygous 1 1.8 5 3.4 0.59 CYP1A1*2 Wild type (T/T) 46 73.0 124 82.7 Heterozygous (T/C) 16 25.4 23 15.3 Homozygous (C/C) 1 1.6 3 2.0 0.19 GSTM1 Present 27 43.6 70 47.0 Deleted 35 56.4 79 53.0 0.54 GSTT1 Present 45 72.6 119 79.9 Deleted 17 27.4 30 20.1 0.20