Jose M. Ladero, MD, PhD, and Julio Benitez, MD, PhD Badajoz and Madrid, Spain. The genetic polymorphisms of drug-metabolizing enzymes are a common ...
Genetic basis for differences in debrisoquin polymorphism between a Spanish and other white populations 1
The debrisoquin hydroxylation polymorphism is an autosomic recessive trait of the cytochrome P450IID6, an enzyme involved in drug metabolism, that affects 5% to 10% of white subjects. The genetic basis of this polymorphism was studied in 258 unrelated Spanish white subjects. The results revealed that about 5% of the subjects were homozygous for mutant alleles and that about 1% of the subjects carried alleles that suggested C T P W 6 gene duplication. The extensive metabolizers who were homozygous for the wild-type allele had higher metabolic ratio than the heterozygous extensive metabolizers, indicating a gene-dose effect for the wild type allele. The C T P W 6 allele frequencies indicate a reduced frequency for the CTPW6(B) allele and a higher frequency for the wild-type allele compared with other white populations. This is also reflected in an increased frequency of the subjects who were homozygous for the wild-type allele among extensive metabolizers. We conclude that the same C T P W 6 mutations are present in Spaniards and other white subjects. Nevertheless, the frequencies of such mutations are different in our population. This implies that a high number of Spanish subjects may behave differently than other white subjects in the effect of drugs metabolized by the CYP2D6 enzyme. (CLIN PHAFUUCOL THER1994;55:412-7.)
Jose A. G. Agindez, MD, PhD, Carmen Martinez, MD, PhD, Maria C. Ledesma, BSc, Margarita G. Ladona, MD, PhD, Jose M. Ladero, MD, PhD, and Julio Benitez, MD, PhD Badajoz and Madrid, Spain
The genetic polymorphisms of drug-metabolizing enzymes are a common determinant of interindividual differences in the therapeutic effect and toxicity for many drugs.' Such polymorphisms lead to an extremely high efficiency of drug metabolism in very extensive metabolizers or to impaired drug biotransformation in individuals who are poor metabolizers From the Department of Pharmacology, Medical School, University of Extremadura Badajoz, and the Gastroenterology Unit, San Carlos University Hospital, University Complutense, Madrid. Supported in part by grants CYCIT-SAF92-0333 and FISss 9310632 from Comision Interministerial de Ciencia y Tecnologia (Madrid) and Fondo de Investigaciones Sanitarias de la Seguridad Social (Madrid), respectively. This work has been done within a European collaborative group of the Center for Cooperation in the Field of Scientific and Technical Research (COST) of the European Communities, project B l , phase I1 (Brussels, Belgium). Received for publication Aug. 6, 1993; accepted Oct. 25, 1993. Reprint requests: Julio Benitez, MD, PhD, Departamento de Farmacologia, Facultad de Medicina, Universidad de Extremadura, Avda. de Elvas sln, 06071 Badajoz, Spain. Copyright O 1994 by Mosby-Yearbook, Inc. 0009-9236/94/$3.00 + 0 13/1/52410
compared with normal extensive metabolizers. Poor metabolizers are a high-risk group with tendency to develop adverse drug effect^,"^ whereas very extensive metabolizers may need higher doses of certain drugs to achieve a therapeutic e f f e ~ t . ~ The frequency of poor metabolizers among Spaniards is in the range of the frequency described for Europeans, but it is close to the minimum 95% confidence interval Moreover, it has been shown that Spanish extensive metabolizers tend to have lower metabolic ratios than other white extensive metabolizThe cause for these differences is still unknown. It may be attributable either to a different environment or to a different genetic background. Nevertheless, although several white populations have been studied for CYP2D6 there is no available CYP2D6 genotype data on a Spanish population. We have analyzed genomic deoxyribonucleic acid (DNA) from 258 unrelated Spanish individuals to study the CYP2D6 allele frequency in a Spanish population. In addition, we have checked the agreement between oxidative phenotype and genotype to assess
Amundez e t al. 413 the efficiency of phenotype prediction in a Spanish population. Because an unexpected high frequency of subjects who were homozygous for the wild-type allele was found among Spaniards, we compared the metabolic ratios of homozygous and heterozygous (carriers of mutant alleles) extensive metabolizers to determine the influence of homozygosity for the wildtype allele on the oxidative phenotype.
METHODS The 258 participants were included in the study after giving informed written consent. The protocol was approved by the Ethics Committee of the University Hospital Infanta Cristina, Badajoz, Spain. All the subjects participating in this study were healthy Spanish volunteers. Most of them were medical students; 107 were men and 151 were women. All subjects were white, and the age range was from 18 to 83 years (mean age, 25). Determination of the oxidative phenotype. Phenotype was determined by the urinary metabolic ratio of debrisoquin: Individuals were given oral doses of 10 mg debrisoquin (Declinax, Roche). Urine was collected until 6 hours after administration. Debrisoquin and its metabolite 4-hydroxydebrisoquin were assayed by gas chromatography as described e ~ s e w h e r e The .~ metabolic ratio of the parent drug and its metabolite was established, and poor metabolizers were defined as those subjects with a debrisoquinl4-hydroxydebrisoquin ratio higher than 12.6 according to Evans et al.'" Determination of the CYP2D6 genotype. 'Venous blood (about 20 ml) was collected in heparinized (sodium heparin, 143 USP units) sterile glass tubes (Vacutainer, Becton Dickinson Systems Europe, Meylan, France). Total genomic DNA was prepared from peripheral leukocyte^,'^ kept at 4" C in sterile plastic vials, and subjected to a polymerase chain reaction (PCR)-based allele-specific amplification'5~'6 and Xba I restriction fragment length polymorphism (RFLP) analysis as d e s ~ r i b e d . ' ~The * ' ~ CYP2D6 complementary DNA probe used for the analysis of RFLP was provided by Urs A. Meyer, MD (Base1 University, Basel, Switzerland). The probe was labeled by random priming with digoxigenin- 1 1-2'-deoxyuridine5'-triphosphate by use of a digoxigenin DNA labeling and detection kit (Boehringer Mannheim, Barcelona, Spain). Statistical analysis. The intergroup comparison values were calculated by use of the X 2 test. The 95% confidence intervals were calculated according to Bulpitt.
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Table I. Frequencies of allelic combinations of the CYP2D6 gene determined by Xba I RFLP analysis of genomic deoxyribonucleic acid of 258 unrelated Spanish individuals Genoype (kb)
29129 2911 1.5 29/44 29116+9 29116+9+ 11.5 44144 44111.5 16+911 1.5 16+9116+9 Other (291>44)*
Number of subjecrs
173 2 57 3 1
13 3 1
2 3
Frequenq
0.67 [0.612-0.7271
