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Background & Aims: Myelosuppression in patients with Crohn's disease (CD) treated with azathioprine has been attributed to low activity of thiopurine S-methyltransferase (TPMT). Allelic variants of the TPMT gene responsible for changes in the enzyme activity have been characterized. We investigated the distribution of mutant alleles associated with TPMT deficiency in patients with CD and myelosuppression during azathioprine/6-mercaptopurine therapy. Methods: Forty-one patients with CD were included. They developed leukopenia or thrombocytopenia during azathioprine or 6-mercaptopurine treatment. Polymerase chain reaction-based methods were used to search for mutations associated with TPMT deficiency. Results: Four patients (10%) had 2 mutant alleles associated with TPMT deficiency, 7 (17%) had 1 mutant allele, and 30 (73%) had no known TPMT mutation. The delay between administration of the drug and occurrence of bone marrow toxicity was less than 1.5 months in the 4 patients with 2 mutant alleles, and ranged from 1 to 18 months in patients with 1 mutant allele and from 0.5 to 87 months in patients with normal genotype. Conclusions: Twenty-seven percent of patients with CD and myelosuppression during azathioprine therapy had mutant alleles of the TPMT gene associated with enzyme deficiency. Myelosuppression is more often caused by other factors. Continued monitoring of blood cell counts remains mandatory in patients treated with azathioprine. GASTROENTEROLOGY 2000;118:1025-1030

Abstract: Objective: Among Caucasians, a lack of cytochrome P 450 enzyme CYP2D6 is observed in 5–10% of individuals, named poor metabolizers (PMs). A consequence may be an impaired metabolism of many drugs such as most of the psychotropic drugs with an increased risk of drug side effects. This enzyme is also involved in the metabolism of endogenous compounds, including neurotransmitters such as dopamine and dopamine-related neurotransmitters which play a role in the mechanism of action of extrapyramidal drug side effects. The present study investigates whether patients who have developed and those who have not developed extrapyramidal drug side effects differ in their CYP2D6 genotypes and phenotypes. Methods: The CP2D6 genotype (method involving restriction length fragment polymorphism and polymerase chain reaction-single strand conformation polymorphism) was determined in 65 drug-treated in-patients, and the CYP2D6 phenotype (with dextromethorphan probe) in 62 of them. Two groups were constituted, one with 22 patients who had developed extrapyramidal drug side effects, and the second with 43 patients without such side effects. Results: In the whole population, there was an over-representation of PM phenotypes – more marked in the first group than the second (45% vs 14%). Concerning the genotypes, we observed that the percentage of functional alleles (with extensive metabolic capacity) was higher in group 2, whereas the percentage of non-functional alleles (without metabolic activity) was higher in group 1; this frequency difference was only marginally significant (χ2 5.95; P < 0.0509; degrees of freedom=2). Consequently, there was a higher percentage of genotypes with no (extensive) functional alleles in the group of patients suffering from extrapyramidal side effects than in the other group (P < 0.00001). Conclusion: CYP2D6-impaired metabolic capacity may be a contributory factor in extrapyramidal drug side effects.

Previous reports of an association between the polymorphic cytochrome P450 CYP2D6 and systemic lupus erythematosus are conflicting. Following the elucidation of the molecular basis of the CYP2D6 genetic polymorphism, we re-examined the hypothesis of an association of this gene with a susceptibility to systemic lupus erythematosus by analysing the complete CYP2D6coding sequence. For this purpose, we studied the occurrence of 16 mutations in genomic DNA from 69 systemic lupus erythematosus patients and a large control group using a previously described polymerase chain reaction-single strand confirmation polymorphism analysis. In addition, we studied the occurrence of 11 alleles and 21 genotypes in the same individuals by the combined use of restriction fragment length polymorphism and allele-specific polymerase chain reaction followed by polymerase chain reaction-single strand confirmation polymorphism analysis. No significant differences in the distribution of overall genotypes and predicted phenotypes were observed between systemic lupus erythematosus patients and controls. The only new finding of our study is the higher frequency of one non functional allele, namely the CYP2D64A, in systemic lupus erythematosus versus control individuals (P= 0.007). This increased frequency was not statistically significant in multiple comparison analysis and was not related to any specific clinical features of systemic lupus erythematosus. These results suggest that CYP2D6genotype as well as CYP2D6 phenotype are not determinant of susceptibility to systemic lupus erythematosus but the presence of the inactive CYP2D64Aallele may be a contributory factor.

A mutant allele of the CYP2D6 gene (CYP2D6C) characterized by a 3-base-pair deletion in exon 5 (mutation D6-C) and carried by a XbaI 29 kb restriction fragment (haplotype 29-C) was previously presumed to be associated with the debrisoquine poor metabolizer phenotype on the basis of in vitroenzymatic criteria. In order to determine whether D6-C was related to a deficient CYP2D6 activity in in vivo, we first analysed the CYP2D6 gene in the family of a carrier of the haplotype 29-C by combining XbaI-restriction-fragment-length polymorphism analysis and specific CYP2D 6mutation detection by polymerase chain reaction assays. Moreover, each member of the family was phenotyped using debrisoquine as probe drug. Secondly, we used polymerase chain reaction assays to test for the CYP2D6Cmutation DNA samples from 146 unrelated healthy volunteers with the extensive metabolizer phenotype and previously identified as heterozygous carrier of one of the haplotypes known to be associated with the poor metabolizer phenotype. All family members were extensive metabolizers and three were compound heterozygotes for the haplotype 29-C and a 11.5 kb haplotype that has been shown to lack the entire CYP2D6 gene. In addition, two extensive metabolizer individuals among the 146 tested for were compound hétérozygotes for the haplotype 29-C and a 29 kb haplotype carrying the defective CYP2D6Ballele (haplotype 29-B). These data demonstrate that in vivothe mutation D6-C does not result in an enzyme deficiency severe enough to cause the poor metabolizer phenotype, in contrast to previous expectations resulting from in in vitrodata. However, the possibility of a deficiency of the mutant CYP2D6C protein towards other CYP2D6 substrates cannot be ruled out.

A novel loss-of function allele of the CYP2D6 gene was characterized in a PM individual using exon-by-exon PCR-SSCP analysis. This allele, we termed CYP2D6(F), harbours four mutations including a new mutation (D6-F) which abolishes the splice acceptor site of the 1st intron and results in a premature stop codon. DNA samples from a large population of healthy unrelated volunteers were tested for D6-F using a PCR-assay we developed for the specific identification of the mutation in genomic DNA. The prevalence of D6-F was very low. However, its identification combined with that of the previously reported gene inactivating mutations would further increase the phenotype prediction rate by genotyping.

To detect mutations in the cytochrome P450 CYP2D6 gene (CYP2D6), we developed a strategy based on single-strand conformation polymorphism (SSCP) analysis of the gene amplified by polymerase chain reaction (PCR). The efficiency of the method was evaluated by analysing DNA samples from extensive metabolizers (EM) and poor metabolizers (PM) of debrisoquine. Haplotypes, alleles and mutations of CYP2D6 had previously been characterized in each individual using PCR assays, Xba I restriction fragment length polymorphism (RFLP) and sequencing. PCR-SSCP results were in complete agreement with those obtained using established methods. All previously characterized mutations were associated with particular shifts in the electrophoretic mobility of DNA fragments allowing their identification. We further tested the efficiency of PCR-SSCP for detecting new CYP2D6 mutations. DNA from a PM subject presumed to carry an unknown non-functional mutant allele of CYP2D6 was amplified and bands with aberrant migration patterns were observed on SSCP gels. Sequence analysis of the corresponding DNA fragments revealed the causative mutations. In this way, a novel non-functional allele of the gene, carrying three previously reported mutations and a new mutation in the third exon which results in a premature termination codon, was characterized. Finally, CYP2D6 SSCP analysis was performed on DNA amplified with fluorescent primers and an automated DNA sequencer was used for SSCP analysis of products. We conclude that the PCR-SSCP approach is a powerful method of identifying simultaneously known and new mutations of the CYP2D6 gene.

The polymorphic cytochrome P450 CYP2D6 is involved in the metabolism of various drugs of wide therapeutic use and is a presumed susceptibility factor for certain environmentally-induced diseases. Our aim was to define the mutations and alleles of the CYP2D6 gene and to evaluate their frequencies in the European population. Using polymerase chain reaction-single strand conformation polymorphism analysis, 672 unrelated subjects were screened for mutations in the 9 exons of the gene and their exon-intron boundaries. A total of 48 point mutations were identified, of which 29 were novel. Mutations 1749 G-C, 2938 C-T and 4268 G-C represented 52.6%, 34.3% and 52.9% of the mutations in the total population, respectively. Of the eight detrimental mutations detected, the 1934 G-A, the 1795 Tdel and the 2637 Adel accounted for 65.8%, 6.2% and 4.8% respectively, within the poor metabolizer subgroup. Fifty-three different alleles were characterized from the mutation pattern and by allele-specific sequencing. They are derived from three major alleles, namely the wild-type CYP2D61A, the functional CYP2D62 and the null CYP2D64A. Five allelic variants (CYP2D61A, 2, 2B, 4A and 5) account for about 87% of all alleles, while the remaining alleles occur with a frequency of 0.1%-2.7%. These data provide a solid basis for future epidemiological, clinical as well as interethnic studies of the CYP2D6 polymorphism and highlight that the described single strand conformation polymorphism method can be successfully used in designing such studies.

To standardize CYP2D6 allele nomenclature, and to conform with international human gene nomenclature guidelines, an alternative to the current arbitrary system is described. Based on recommendations for human genome nomenclature, we propose that alleles be designated by CYP2D6 followed by an asterisk and a combination of roman letters and arabic numerals distinct for each allele with the number specifying the key mutation and, where appropriate, a letter specifying additional mutations. Criteria for classification as a separate allele and protein nomenclature are also presented.

Many studies have been performed in an attempt to establish a link between the polymorphism of the cytochrome P45O CYP2D6 gene and the incidence of lung cancer. Nevertheless, whether or not this genetic polymorphism has a role in the development of the disease remains unclear. Recently, new advances in our knowledge of the CYP2D6 gene and its locus (CYP2D) have been achieved. In particular, CYP2D6 was found to be highly polymorphic and multiple novel mutations and allelic variants of the gene have been identified. In addition, a number of CYP2D rearrangements, including those with amplification of the gene, have been demonstrated. Taking this new information into account, we have reconsidered the potential influence of CYP2D6 polymorphism in lung cancer susceptibility by performing a comparative analysis of the overall mutational spectrum of CYP2D6 and of the rearrangements of CYP2D in 249 patients with lung cancer and in 265 control individuals matched on age, sex, hospital and residence area. For this purpose, a strategy based on SSCP analysis of the entire coding sequence of CYP2D6 and on RFLP analysis of the gene locus was carried out in DNA samples from each individual. Forty mutations occuring in various combinations on 42 alleles of the gene and 82 different genotypes were identified. No significant difference in the distribution of the mutations, alleles or genotypes was observed between the two groups, except a particular genotype (CYP2D6lA/2), which was more common in the sub-group of moderate smokers (< 30 pack-years) suffering from small cell carcinoma (Odds Ratio (OR) 3.6, 95 CI 1.1-11.9). When the phenotype was predicted according to genotype, only a trend toward a higher frequency of ultrarapid metabolizers in patients was obtained. In spite of a complete analysis of the CYP2D6 gene and its locus, this case-control study provides elements against an influence of the CYP2D6 polymorphism on lung cancer Susceptibility.

Propafenone (Pf) is a class I antiarrhythmic drug that can be given both orally and intravenously. In order to examine whether its two major metabolites [5-hydroxypropafenone (5-OH-Pf) and N-depropylpropafenone (N-DP-Pf)] possess pharmacodynamical properties, we compared their electrophysiological effects to those of the parent drug on papillary muscle fibers from guinea pig ventricular myocardium. After baseline action potential and refractory period characteristics were measured at different pacing rates, the tissue preparations were superfused with either Pf, 5-OH-Pf, or N-DP-Pf at five different concentrations and electrophysiological characteristics were studied again. The maximal rate of depolarization (Vmax) was depressed by the three compounds only at the highest concentration, although the effect of N-DP-Pf was slightly less than the other two. Refractory periods were altered only by the highest concentration of 5-OH-Pf. Propafenone and N-DP-Pf exhibited equally slow on-set/off-set kinetics of the sodium channel block, whereas those of 5-OH-Pf were twice as long, which seems to suggest a slower rate of dissociation of the latter from the inactivated sodium channels.

Mexiletine, a class lb antiarrythmic drug, is used clinically as a racemic mixture of two enantiomers. Aromatic and aliphatic hydroxylation are the two major metabolic steps. In the liver, this metabolism is catalyzed by the cytochrome P450IID6, an isoenzyme of cytochrome P450 due to genetic polymorphism in humans. In the present study, the metabolism of the two stereoisomers was compared in vitro in human liver microsomes. Parahydroxylation (aromatic hydroxylation) is favored for S (+)-mexiletine with a mean intrinsic clearance higher than for R(-)-mexiletine. The R(-) enantiomer exhibits a threefold higher mean Vmaxvalue for aliphatic hydroxylation than S(+)-mexiletine. We showed that (i) the high-affinity component of dextrometorphan O-demethylation was competitively inhibited by R(-)-and S(+)-mexiletine and that (ii) hydroxylations of the two enantiomers were very strongly competitively inhibited by quinidine. Hydroxylation reactions of mexiletine exhibit stereoselectivity in vitro in human liver microsomes and are catalyzed by P450IID6.

Summary The debrisoquine/sparteine-type polymorphism of drug oxidation and the polymorphism for acetylation are two common inherited variations in human drug metabolism. The phenotypes for hydroxylation and acetylation can be predicted be newly developed methods based on mutation-specific amplification of DNA by the polymerase chain reaction (PCR), which also allow for identification of heterozygous carriers of one mutant allele.

Abstract: The identification of a novelCYP2D6 allele from a healthy Caucasian poor metabolizer was achieved by using a previously described polymerase chain reaction/single-strand conformation polymorphism strategy. Among the four point mutations that this allele carries, a missense mutation in exon 1 (212 G rarr A or D6-H) seems to be responsible for the loss of CYP2D6 function. Although the mutation D6-H has a low prevalence in a randomly selected population of healthy Caucasians, its identification should further increase the phenotype prediction rate by genotyping.

A novel mutation that generates a stop codon in the third exon of the gene encoding the cytochrome P-450 CYP2D6 was identified in a Caucasian having a deficiency of the isozyme, by means of single strand conformation polymorphism analysis of DNA fragments amplified by the polymerase chain reaction, followed by selective sequencing.

The possible association between lung cancer and the CYP2D6*9 mutant allele, which reduces the catalytic activity of cytochrome P450 CYP2D6, was examined by PCR-SSCP using peripheral blood DNA from 249 cases of lung cancer and 265 controls, with detailed data on smoking. The CYP2D6*9 mutant allele was present in 4.9% of controls and 6% of cases. Adjusted for age, hospital and smoking, the odds ratio (OR) of lung cancer associated with the presence of the CYP2D6*9 mutant allele was 1.2 [95% confidence interval (CI) 0.5-2.9]. According to histological type, adenocarcinoma and small cell carcinoma were not associated with the presence of the CYP2D6*9 mutant allele and a non-significant higher occurrence of the mutant allele was observed for squamous cell carcinoma (OR 1.74, 95% CI 0.6-4.8). Moreover, no associations were observed upon stratification by number of pack-years of cigarette smoking. These results do not confirm an earlier report that this CYP2D6*9 mutant allele may be an additional risk factor for the development of lung cancer.