hydroxylase

(CYP2D6) has a role in the metabolism of a wide range of drugs, particularly cardiovascular agents and psychotropic agents (Cholerton et al. 1992). Its polymorphism is one of the most thoroughly studied among human enzymes. Ap- proximately 5–10% of Caucasians from Europe and North America lack CYP2D6 activity and are referred to as poor metabolizers (PM). Several studies indicate that the exten- sive-metabolizer (EM) phenotype, for which liver metabo- lism is normal, is associated with increased risk of various cancers, especially tobacco smoke-induced lung cancer (Ayesh et al. 1984), while other studies failed to detect such an association (Wolf et al. 1992). Phenotyping stud- ies suggested an association of the CYP2D6 polymor- phism with Parkinson’s disease (Poirier et al. 1987). This was confirmed later by genotyping studies (Smith et al. 1992; Agundez et al. 1995; Lucotte et al. 1996), in which mutant alleles of CYP2D6 were about twice as frequent among Parkinson’s disease patients than in controls.

In order to test the working hypothesis that the highly variable, genetically determined xenobiotic-metabolizing capacity of humans may represent one genetic component of human longevity, we compared in the present study the frequencies of polymorphic alleles of the CYP2D6, GSTM1, and NAT2 genes in centenarians and controls.

Materials and methods

Subjects and DNA samples

A total of 817 DNA samples was studied (CYP2D6*3: 243 controls and 552 centenarians; CYP2D6*4: 252 and 523; NAT2: 88 and 118; GSTM1: 229 and 565). Centenarians had been recruited from all over France at their home or in health-care institutions, with personal informed consent, through the Chronos project at the Fondation Jean Dausset-CEPH. They were Caucasians, at least 99 years old on the day of blood collection, and had their residence in France (regional distributions of the samples tested: greater Paris, 33.5%; north-west, 20.1%; south-west, 11.7%; north-east, 11.3%; south-east, 23.4%). The mean age of the centenarians studied here was 101 years on the day of blood collection, and 90% of them were women. Controls aged 20–70 years were chosen among unrelated normal individuals from the French CEPH cell bank (Dausset et al. 1990). DNA was ex- tracted from EBV-immortalized cell lines by standard methods (Sambrook et al. 1989), resuspended in TE buffer (10 mM TRIS, 1 mM EDTA pH 7.6), and kept frozen until used as described below.

The human population can be divided in two main sub- groups, associated with the N-acetylation pathway of detoxification, termed slow and rapid acetylators. Two highly homologous human NAT genes (NAT1 and NAT2) were shown to encode proteins with overlapping substrate specificities, and the NAT2 gene was first identified as the polymorphic one. NATs are able to metabolize a number of carcinogens and other xenobiotics such as aromatic amines, present in dyes, antioxidants, pesticides or other environmental and occupational pollutants. Three main DNA variants lacking in vitro NAT activity have been characterized in several studies from different populations (Hickman and Sim 1991; Bell et al. 1993; Lin et al. 1994; Cascorbi et al. 1995) and at least 95% of slow acetylators can now be identified by PCR assays. A number of phenotyping analyses suggested association between the slow-acetylator phenotype and individual susceptibility for diseases such as breast and bladder cancer (Bulowvskaya et al. 1978; Cartwright et al. 1982), insulin-dependent (IDDM) and NIDDM (McLaren et al. 1977). However, re- cent genotyping studies failed to detect any association of NIDDM (Agundez et al. 1996) or IDDM (Mrozikiewicz et al. 1994) and NAT2 polymorphism.

PCR primers

PCR primers used for analysis of the NAT2, GSTM1, and CYP2D6 loci are summarized in Table 1.

Genotyping assay

CYP2D6 locus analysis

Subjects were genotyped for the CYP2D6*3 (A deletion) and 2637 CYP2D6*4 (G A transition) alleles according to the PCR-coupled 1934 RFLP method described by Smith et al. (1992). Briefly, primer E contains a 1-bp mismatch to the CYP2D6 sequence, which makes the 268-bp PCR fragment, produced in conjunction with primer F from individuals with the exon 5 mutation, sensitive to digestion with the restriction enzyme HpaII at the site of the A deletion. An addi- 2637 tional HpaII site is present in all individuals. Amplification with primers C and D generates a 334-bp fragment with a G to A transi- tion at the junction of intron3/exon 4 which, in affected individuals, is resistant to restriction with BstNI. PCR was carried out in a total volume of 50 m l containing 1 m g of genomic DNA, 62.4 m M of each dNTP, 0.4 m M of each primer, 1.5 mM MgCl , 10 mM TRIS-HCl pH 2

In addition to NATs, phase II reactions are also carried out by GSTs. GSTs are multifunctional enzymes that cata- lyze several reactions between glutathione (GSH) and electrophilic compounds, including carcinogens and cyto- toxic drugs, present for instance in the diet and in tobacco smoke. Several GST isoenzymes have been identified in humans. The GSTµ class comprises the enzymes

Table 1 Oligonucleotides for PCR and RFLP-coupled PCR analysis of the CYP2D6, NAT2, and GSTM1 loci according to Hickman and Sim (1991), Smith et al. (1992), Bell et al. (1993), and Zhong et al. (1993b). Annealing temperature for each primer couple and expected size of amplification products are indicated

Locus Primer Sequence 5’fi 3’ Annealing temperature (°C) Product size (bp) CYP2D6 E GATGAGCTGCTACATGAGCCC 60 268 F CCGAGAGCATACTCGGGAC C GCCTTCGCCAACCACTCCG 60 334 D AAATCCTGCTCTTCCGAGGC NAT2 Nat-HU7 GGCTATAAGAACTCTAGGAAC 60 840 Nat-HU8 AATAGTAAGGGATCCATCACC Nat-HU14 GACATTGAAGCATATTTTGAAAG 58 1000 Nat-HU16 GATGAAAGTATTTGATGTTTAGG N4 TCTAGCATGAATCACTCTGC 57 1093 N5 GGAACAAATTGGACTTGG GSTM1 P1 CGCCATCTTGTGCTACATTGCCCG 62 158 (P1/P2) P2 ATCTTCTCCTCTTCTGTCTC 231 (P1/P3) P3 TTCTGGATTGTAGCAGATCA