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 additional HpaII site is present in all individuals. Amplification with primers C and D generates a 334-bp fragment with a G to A transition 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 ml containing 1 µg of genomic DNA, 62.4 mM of each dNTP, 0.4 mM of each primer, 1.5 mM MgCl2, 10 mM TRIS-HCl pH 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).

In addition to NATs, phase II reactions are also carried out by GSTs. GSTs are multifunctional enzymes that catalyze several reactions between glutathione (GSH) and electrophilic compounds, including carcinogens and cytotoxic drugs, present for instance in the diet and in tobacco smoke. Several GST isoenzymes have been identified in humans. The GST 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' 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 (P1Thus, an efficient gene or separately on the two chromosomes. Genotype and phenotype data observed for the NAT2 polymorphisms detected among a population of 88 controls and 118 centenarians are summarized in Table 3. As described in detail in Materials and Methods, two complementary 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 the NAT2 genotypes and the predicted phenotype in centenarian and control populations.

Genotype definition: WT/WT, homozygote wild-type; WT/M1M4, heterozygote; M1M4/M1M4, homozygote mutant. Only the observed genotypes are reported. Phenotype definitions deduced from combinations of NAT2 genotypes: slow acetylator, homozygote mutants; fast acetylator, genotype with one or two wild-type alleles.

NAT2 genotypes Controls Centenarians n Frequency n Frequency 

NAT2*4/NAT2*4 0 0 3 0.025 
NAT2*4/NAT2*5A 2 0.023 5 0.042 
NAT2*4/NAT2*6A 8 0.091 5 0.042 
NAT2*4/NAT2*7A 1 0.011 0 0 
NAT2*5A/NAT2*5A 24 0.273 39 0.331 
NAT2*5A/NAT2*6A 41 0.466 36 0.305 
NAT2*5A/NAT2*7A 2 0.023 6 0.051 
NAT2*6A/NAT2*6A 9 0.102 19 0.161 
NAT2*6A/NAT2*7A 1 0.011 5 0.042 

NAT2 predicted phenotypes Controls Centenarians 

Slow acetylator 77 (0.875) 105 (0.890) 
Fast acetylator 11 (0.125) 13 (0.110) 
Total 88 (1) 118 (1) 

The proportion of the GSTM1*0/GSTM1*0 genotype in the centenarian population (52.7%; n = 565) was not significantly different from that observed in controls (49.3%; n = 229). Table 4 shows the distribution of the GSTM1 genotypes in centenarian and control populations (GSTM1 gene absent, + GSTM1 gene detected).

GSTM1 gene Controls Centenarians n Frequency n Frequency 

- 113 0.493 298 0.527 
+ 116 0.507 267 0.473 
Total 229 (1) 565 (1) 

When the study populations were analyzed according to the combined predicted phenotype based on the knowledge of the genotypes (Table 5), 60 and 110 combinations were available for the control and the centenarian populations, respectively. Of the controls, 50% were characterized by the following combinations: CYP2D6/EM; NAT2/slow; GSTM1/present. The second major haplotype was identical except for the fact that it included the GSTM1 null version.

By comparing the distribution of haplotypes observed at the CYP2D6, NAT2, and GSTM1 loci for the polymorphisms tested, we found no significant difference between the two populations. The two most frequent NAT2 combinations were NAT2*5A/*5A (27.3% and 33.1%, respectively, for control and centenarian populations) and NAT2*6A/*6A (46.6% and 30.5%). However, no significant difference between the two populations was found as far as allele and genotype distributions were concerned. Mutant alleles are known to be associated with the slow phenotype when present without the wild-type allele, while the fast phenotype includes homozygotes for the wild-type allele or heterozygotes with one wild-type and one of the mutant alleles. As shown in Table 3, results of 88 genotypes from control subjects and 118 from centenarians predicted a frequency of slow phenotypes of 87.5% and 89%, respectively. These frequencies are clearly higher than those described in previous studies on other Caucasian populations in which the frequency of the slow acetylator phenotype is roughly 50% in Caucasians (Hickman and Sim 1991There is ethnic and geographical variation in the slow acetylation phenotype, with the percentage spanning from about 10% in the Japanese population to more than 90% in some Mediterranean populations (Hirvonen 1995).

References: