Characterization of a variable number tandem repeat region in the thiopurine S-methyltransferase gene promoter.

Characterization of the genetic polymorphism of thiopurine S-methyltransferase enzyme (TPMT; EC 2.1.1.67) is required because of its clinical importance for patients exposed to thiopurine drugs. A number of point mutations have already been characterized in exons and introns of the TPMT gene. Here we report the identification of a polymorphic locus within the promoter region of the gene. This polymorphism was detected by polymerase chain reaction - single strand conformation polymorphism analysis of DNA samples from 54 unrelated European individuals. A total of five alleles with length variations were distinguished through the 5'-flanking region involved in the TPMT gene expression. Sequence analysis revealed that these variations were due to a variable number of tandem repeats (VNTR), ranging from four to eight repeats. Each repeat consists of 17 or 18 bp units and contains putative binding sites for transcription factors. The most frequent alleles harbour four or five tandem repeats, a heterozygosity rate of 0.44 was calculated, and a stable Mendelian inheritance of alleles was demonstrated. Analysis of the effect of each VNTR allele on promoter activity of a reporter gene was further performed in various cell lines by transient transfection assay. A modulatory effect of VNTR alleles was observed in vitro, but the repeat polymorphism did not display a significative role in TPMT gene regulation in vivo. Further studies need to be carried out to support the hypothesis that VNTR may contribute to the large interindividual variations of TPMT activity.

CYP2D6 polymorphism and Parkinson's disease susceptibility.

Following the recent identification of multiple novel mutations and alleles of the cytochrome P450 CYP2D6 gene which cause decreased, increased, or absent enzyme activity, we re-examined the controversial hypothesis of a role of the CYP2D6 polymorphism in Parkinson's disease (PD) susceptibility. For this purpose, a strategy based on PCR-SSCP and RFLP analyses allowing the detection of all known CYP2D6 alleles was performed in DNA from 109 patients with sporadic PD. This strategy was also applied to DNA from 68 members of PD families including 18 affected and 50 unaffected members. Seventeen mutations occurring alone or in various combination on 14 alleles of CYP2D6 have been identified in patients with sporadic PD. Moreover, 12 mutations and nine alleles of the gene have been characterized in members of PD families. No significant difference was observed when the distribution of mutations and alleles of CYP2D6 was compared between the PD patients and 514 control subjects previously analyzed using the same strategy. There was also no difference in the distribution of phenotypes predicted from genotypes between both groups. In addition, when the distribution of CYP2D6 genotypes was compared, no difference between affected and unaffected members of PD families was observed. These data indicate that CYP2D6 polymorphism is not a susceptibility factor to PD.

Genotypic and phenotypic analysis of the polymorphic thiopurine S-methyltransferase gene (TPMT) in a European population.

1. Characterization of allelic variants of the TPMT gene (TPMT) responsible for changes in TPMT activity, and elucidation of the mechanism by which these alleles act, are required because of the clinical importance of this polymorphism for patients receiving thiopurine drugs. 2. We defined the mutational and allelic spectrum of TPMT in a group of 191 Europeans. Using PCR-SSCP, we screened for mutation the entire coding sequence, the exon-intron boundaries, the promoter region and the 3'-flanking region of the gene. Six mutations were detected throughout the ten exons and seven TPMT alleles were characterized. Four of them, TPMT*2, *3A, *3C and *7, harbouring the known mutations, G238C, G460A, A719G or T681G, were nonfunctional and accounted for 0.5, 5.7, 0.8 and 0.3% of the allele totality, respectively. 3. Within the promoter region, six alleles corresponding to a variable number of tandem repeats (VNTR), were identified. VNTR*V4 and *V5a which harbour four or five repeats of a 17-18 bp unit, were the most frequent (55% and 34%, respectively). The other VNTR alleles, having from five to eight repeats, were rarer. 4. The TPMT phenotype was correctly predicted by genotyping for 87% of individuals. A clear negative correlation between the total number of repeats from both alleles and the TPMT activity level was observed, indicating that VNTRs contribute to interindividual variations of TPMT activity. Therefore, additional analysis of the promoter region of TPMT can improve the phenotype prediction rate by genotyping.

Detection of known and new mutations in the thiopurine S-methyltransferase gene by single-strand conformation polymorphism analysis.

To detect mutations in the thiopurine S-methyltransferase gene (TPMT), we have developed a strategy based on single-strand conformation polymorphism (SSCP) analysis of the gene amplified by polymerase chain reaction (PCR). The sensitivity of the method was first evaluated by analyzing DNA samples from five individuals, including two high methylators (HMs), two intermediate methylators (IMs), and one deficient methylator (DM). TPMT alleles and mutations in each of these individuals had previously been characterized by conventional PCR-based assays and direct sequencing analysis. All mutations were associated with particular shifts in the electrophoretic mobility of DNA fragments, allowing their identification. We further tested the efficiency of the strategy to detect new TPMT mutations. For this purpose, additional DNAs from 15 IMs and 15 HMs were submitted to PCR-SSCP analysis. A total of 7 alleles were characterized, including two new alleles. The first one, termed TPMT*1A, harbors a single mutation C-->T at nucleotide -178 in exon 1 and was detected in a HM subject. The second one, termed TPMT*7, was characterized by a T-->G transversion at nucleotide 681 in exon 10. This allele should be a nonfunctional allele of the TPMT gene since it was observed in combination with a wild-type allele in an intermediate methylator. We conclude that the PCR-SSCP strategy we developed could be advantageously used to fully characterize the extent of allelic variation at the TPMT gene locus in populations and thus to improve our understanding of the genetic polymorphism of TPMT activity, which has considerable consequences for the toxicity and efficacy of therapeutically important and widely used drugs.

Genetic analysis of the cytochrome P450 CYP2D6 polymorphism in patients with systemic lupus erythematosus.

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 system lupus erythematosus by analysing the complete CYP2D6 coding sequence. For this purpose, we studies 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 system lupus erythematosus patients and controls. The only new finding of our study is the higher frequency of one non functional allele, namely the CYP2D6*4A, 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 CYP2D6 genotype as well as CYP2D6 phenotype are not determinant of susceptibility to systemic lupus erythematosus but the presence of the inactive CYP2D6*4A allele may be a contributory factor.

Cytochrome P450 CYP2D6 gene polymorphism and lung cancer susceptibility in Caucasians.

Many studies have been performed in an attempt to establish a link between the polymorphism of the cytochrome P450 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 occurring 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 (CYP2D6*1A/*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.

Polymorphism of the cytochrome P450 CYP2D6 gene in a European population: characterization of 48 mutations and 53 alleles, their frequencies and evolution.

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 CYP2D6*1A, the functional CYP2D6*2 and the null CYP2D6*4A. Five allelic variants (CYP2D6*1A, *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.

NAT2 genotyping and efficacy of sulfasalazine in patients with chronic discoid lupus erythematosus.

Sulfasalazine is an effective agent for chronic discoid lupus erythematosus (CDLE) but the response to treatment is considerably variable between patients and is also unpredictable. The reason for this might relate to differences in metabolism of the drug which is extensively acetylated by the polymorphic enzyme N-acetyltransferase 2 (NAT2). To test this possibility, the N-acetylation phenotype of eleven patients with CDLE and treated by standard doses of sulfasalazine was retrospectively determined by genotyping. A clear-cut difference in the outcome of treatment was observed according to whether the patients were slow acetylators (SA) or rapid acetylators (RA). Eight out of 11 patients responded to treatment with a complete or marked remission of the disease. Seven of them were RA. The three other patients who did not respond at all to the drug were SA. In addition, SA seem to be more prone to toxic events. These findings strongly suggest that the genetic polymorphism of NAT2 is responsible for differences in the response to sulfasalazine in patients with CDLE. Therefore, candidates for sulfasalazine therapy should be genotyped to identify those patients who might benefit from the drug.

Evidence for CYP2D6 expression in human lung.

The cytochrome P450 CYP2D6 gene (CYP2D6) expression was examined in samples from human bronchial mucosa and lung parenchyma using reverse transcription-polymerase chain reaction (RT-PCR) and immunochemistry. Except specimen from a patient previously genotyped as homozygous for a complete deletion of the gene, all tissue samples were positive. When compared to that in the liver, the mean level of CYP2D6 mRNA was 3-fold lower in bronchial mucosa and 6-fold lower in lung parenchyma. To our knowledge, these data demonstrate for the first time the presence of CYP2D6 protein in human lung. They also indicate that the gene is nonuniformly distributed within this organ. The possibility that CYP2D6 has a role in lung carcinogenesis by locally activating inhaled chemicals should therefore be considered.

Influence of a mutation reducing the catalytic activity of the cytochrome P450 CYP2D6 on lung cancer susceptibility.

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.

An additional allelic variant of the CYP2D6 gene causing impaired metabolism of sparteine.

The identification of a novel CYP2D6 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-->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.

An efficient strategy for detection of known and new mutations of the CYP2D6 gene using single strand conformation polymorphism analysis.

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.

A nonsense mutation in the cytochrome P450 CYP2D6 gene identified in a Caucasian with an enzyme deficiency.

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.

A novel CYP2D6 allele with an abolished splice recognition site associated with the poor metabolizer phenotype.

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.