8.3, 50 mM KCl, 2.5% DMSO, and 2.5 U of Taq polymerase (Perkin Elmer). Samples were subjected to an initial denaturation of 5 min at 94°C and 25 cycles consisting of 1 min at 94°C, 1 min at 60°C, and 2 min at 72°C. Restriction fragments were electrophoresed in 4% Nusieve gels (Tebu).

(initial denaturation 2 min at 94°C, 30 cycles of 94°C for 1 min, 62°C for 1.5 min, 72°C for 30 s) was carried out in a total volume of 50 m l in the presence of 500 ng of genomic DNA, 0.2 m M of each primer, 1.5 mM MgCl , 10 mM TRIS-HCl pH 8.3, 50 mM KCl, 62.4 2 m M of each dNTP, and 2.5 U of Taq polymerase. PCR products were electrophoresed in 2% agarose gels.

NAT2 locus analysis

Statistical method

were used to discriminate NAT2 alleles. The first one (Hickman and Sim 1991) allows discrimination of NAT2*5A (C T), NAT2*6A 481 (G A), and NAT2*7A (G A) alleles, while the second one identi- 590 857 fied these three alleles and a fourth, NAT2*14A (G A) (Bell et al. 191 1993). Briefly, three separate amplification reactions were carried out on genomic DNA in the presence of three sets of oligonucle- otides, Nat-Hu7/Nat-Hu8, Nat-Hu14/Nat-Hu16, and N4/N5. PCR was carried out in a total volume of 50 m l (except for the N4/N5 set where the total volume was 100 m l), comprising 80–120 ng of ge- nomic DNA, 31.2 m M of each dNTP, 40 pmol of each primer (100 pmol for N4/N5), 1–3 mM MgCl , 10 mM TRIS-HCl pH 8.3, 50 2 mM KCl, and 2 U of Taq polymerase. Samples were subjected to an initial denaturation of 2 min at 94°C and 35 cycles consisting of 30 s at 94°C, 1 min at adequate annealing temperature, 2 min at 72°C, and a final elongation step of 5 min at 72°C. The predicted sizes of the amplified products are 840 bp (Nat-Hu7/Nat-Hu8), 1000 bp (Nat-Hu14/Nat-Hu16), and 1093 bp (N4/N5). PCR products (7 m l and 20 m l, respectively, for Nat-Hu7/8/14/16- and N4/5-amplified products) were subjected to restriction digestion with KpnI (NAT2*5A), TaqI (NAT2*6A), BamHI (NAT2*7A), and MspI/AluI (NAT2*14A). The restriction digests were electrophoresed in 2% agarose (NAT2*5A and NAT2*7A) or 4% NuSieve (NAT2*6A and NAT2*14A) gels.

Statistical analysis of results was done by comparing allele, geno- type, and predicted phenotype distributions using the c 2 test.

Results

CYP2D6 locus analysis

We analyzed allelic variants of the CYP2D6 gene in a pop- ulation of 243 controls versus 552 centenarians for CYP2D6*3 (1-bp deletion), and 252 controls compared to 523 centenarians for CYP2D6*4 (G to A transition). The frequencies of the CYP2D6*3 and CYP2D6*4 alleles in control subjects (1.9% and 18.1%, respectively) were sim- ilar to those reported previously in two British, one ger- man, and two french Caucasian populations (Smith et al. 1992; Bordet et al. 1996; Lucotte et al. 1996; Pirmohamed et al. 1996; Sachse et al. 1997). No significant difference was found between centenarians and control subjects, ei- ther in allelic variant or CYP2D6 genotypes (Table 2). In- dividuals for whom both mutation loci were analyzed al- lowed us to discriminate haplotypes for 238 controls and 513 centenarians (Table 2), thus predicting the CYP2D6 phenotype for these individuals. Approximately 5% of each population was identified as PM. Five double het- erozygotes (one control, four centenarians) did not allow phenotype prediction since the two mutations could both

GSTM1 locus analysis

DNA samples were analyzed by a PCR-based method described pre- viously (Zhong et al. 1993b). Briefly, two of the three primers used could also anneal to another m class gene, GSTM4 (Zhong et al. 1993a), whereas the third primer was specific for the GSTM1 gene. Amplification with P1 and P3 primers gave a 231-bp GSTM1 gene- specific fragment, while the P1 and P2 set gave a 158-bp product in all samples, serving as an internal control. The amplification reaction

CYP2D6 genotypes Controls Centenarians n Frequency n Frequency CYP2D6*3 Wt/Wt 235 0.967 542 0.982 Wt/D6*3 7 0.029 8 0.014 D6*3/D6*3 1 0.004 2 0.004 Total 243 1 552 1 CYP2D6*4 Wt/Wt 171 0.678 323 0.618 Wt/D6*4 71 0.282 178 0.340 D6*4/D6*4 10 0.040 22 0.042 Total 252 1 523 1 CYP2D6 predicted phenotypes Extensive W/W 157 0.660 311 0.606 metabolizer Het/W 69 0.290 174 0.339 Poor metabolizer PM 11 0.046 24 0.047 Undefined Het/Het 1 0.004 4 0.008 Total 238 1 513 1

NAT2*14A. So far this mutation has only been detected in black African or black American populations (Bell et al. 1993; Lin et al. 1994), therefore, the failure to detect the *14A allele in our French Caucasian population was antic- ipated. NAT2*5A and *6A mutant alleles were the most prevalent in our control group, with frequencies of 52.8% and 38.6%, respectively. The wild-type allele (NAT2*4) was present only in 6.3% of our Caucasian control popula- tion, although a higher percentage might have been ex- pected according to several previous studies (Hickman and Sim 1991; Bell et al. 1993; Mrozikiewicz et al. 1994; Cascorbi et al. 1995). Nine genotype combinations were observed, and only three of them reached a frequency above 10% (namely, NAT2*5A/*6A, NAT2*5A/*5A, and NAT2*6A/*6A), while the others remained rare (see Table 3). The two most common genotypes were NAT2*5A/*5A

be located on the same chromosome (leading to two wild- type loci on the second chromosome, thus an efficient gene) or separately on the two chromosomes.

Genotype and phenotype data observed for the NAT2 poly- morphisms detected among a population of 88 controls and 118 centenarians are summarized in Table 3. As de- scribed in detail in Materials and methods, two comple- mentary PCR–RFLP approaches were used to discriminate NAT2 alleles. Both allowed discrimination of the three main mutations of the NAT2 gene, i.e., the NAT2*5A, *6A, and *7A alleles, but only one was also aimed at revealing