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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 de®ned 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'-¯anking 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 identi®ed. VNTR*V4 and *V5a which harbour four or ®ve repeats of a 17 ± 18 bp unit, were the most frequent (55% and 34%, respectively). The other VNTR alleles, having from ®ve 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.

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. Hum Mutat 12:177–185, 1998. © 1998 Wiley‐Liss, Inc.

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