MATERIALS AND METHODS

Nonagenarians and centenarians in good health, i.e., belonging to category A and B according to Franceschi et al. (1) were identified in Northern Italy (Milan, Modena, Parma, Genoa), as part of an ongo- ing multicentric study in Italy with the aim of studying the mecha- nisms responsible for successful aging (1). A total of 94 subjects were included in the present study. The mean age of the group was 100.2 6 2.1 years (range 95–105 years; median 100 years), 22% of whom were males and 88% females. The male to female ratio was in agreement with the recent data on the nation-wide study on Italian centenarians (1). None was a current smoker. In the rest of the present paper, for simplicity, these very old people are collectively indicated as “centenarians”.

c “Null” refers to homozygotes for deletion allele, while “Present” refers to both homozygotes and heterozygotes which cannot be ex- perimentally distinguished.

Statistical Analysis

Chi-square was used to compare the frequency of each polymor- phism and of the combined genotype, in centenarians and in control subjects. Monte Carlo simulation was used (19), to take into account for cells with small numbers. Data were adjusted for sex, in order to control for a possible confounding factor, by multiple logistic regres- sion, using the SAS package version 6.12. Linear regression analysis was used to test the association between age and each genotype.

Control subjects were a sample of 418 healthy volunteers of the same ethnic group as the nonagenarians and centenarians, recruited in Northern Italy to study the geographic distribution of drug- metabolizing enzymes polymorphisms. They were recruited among blood donors (n 5 98), or health screening programs (n 5 320). All the subjects gave informed consent to participate in the study. The mean age of the control subjects was 46.0 6 11.3 years (range 4–82 years; median 47 years), 46% males and 54% females. As by study

RESULTS

The frequency of the polymorphisms analyzed in this is reported in Table 1.

Genomic DNA was extracted from peripheral blood lymphocytes, and polymorphisms were assessed by PCR method, as previously described (17, 18). A multiplex PCR method was used to detect the presence or absence of the GSTM1 and GSTT1 genes in genomic DNA samples. This method uses both GST primer sets (GSTM1, 59 GAACTCCCTGAAAAGCTAAAGC 59 GTTGGGCTCAAATATACG- GTGG; and GSTT1, 59 TCCTTACTG GTCCTCACATCTC, 59 TCAC- CGGATCATGGCCAGCA) in the same PCR and includes a third primer set for albumin (59 GCCCTCTGCTAACAAGTCCTAC, 59 GC- CCTAAAAAGAAAATCGCCAATC), and uses 30 cycles with denatur- ing at 94°C for 1 min, annealing at 64°C for 1 min, and extension at 72°C for 1 min. For analysis of CYP1A1 the following primers were used: 59 TTAGGAGTCTTGTCTCATGCCT and 39 CAGTGAAGAG- GTGTAGCCGCT for analysis of the Msp1 RFLP using PCR condi- tions as previously described (17).

A significant difference in the proportion of centenar- ians homozygotes for GSTT1 deletion was observed, in comparison to younger subjects (28% vs 19%: x2 5 5 4.18; P 0.03). Linear regression analysis confirmed the independent association between age and GSTT1 5 5 deletion (F 4.61, P 0.032). When the analysis was restricted to control subjects below the age of 60 5 years (n 386), the results did not change (data not shown). The distribution of the other gene polymor- phisms did not differ in the two groups (CYP1A1*2A: x2 5 0.94, P 5 0.68; GSTM1: x2 5 0.10, P 5 0.74). When the multiloci genotypes for CYP1A1, GSTM1, and GSTT1 were examined (Table 2), a statistical bor- derline difference was observed between centenarians and control subjects (x2 5 15.67; P 5 0.079). The difference was mainly due to the CYP1A1-GSTM1- GSTT1 combinations 1-0-2 and 1-2-2 (Table 2), both containing the combination of CYP1A1 heterozygotes and GSTT1 deletion genotypes (8% in centenarians vs

Nomenclature used: for CYP1A1, the term “wild type” refers to the homozygotes for the common allele (absence of the restriction site), “heterozygous” refers to the presence of the polymorphism on one allele, “homozygous” refers to the presence of the restriction site on both alleles. For GSTM1 and GSTT1, “null” refers to homozygotes for deletion allele, while “present” refers to both homozygotes and het- erozygotes (which cannot be distinguished by the experimental procedure).