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

Abstract

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.

Thiopurine S-methyltransferase pharmacogenetics: insights, challenges and future directions

“…The *14 SNP disrupts the translation initiation codon (Lindqvist et al, 2004) and prevents translation of the enzyme protein , while *4 and *15 involve alterations in canonical mRNA splice site sequences (Otterness et al, 1998;Lindqvist et al, 2004) and *3D contains a premature stop codon (Otterness et al, 1997). There is also a polymorphic GC-rich variable number of tandem repeats (VNTR) present in the 5 0 -flanking region of TPMT that can vary from 3 to 9 repeat elements (SpireVayron de la Moureyre et al, 1998Moureyre et al, , 1999Yan et al, 2000). This VNTR has been reported to modulate TPMT activity as a result of altered transcription.…”

“…This region contains a VNTR that consists of GC-rich highly homologous repeats that vary from 17 to 18 bp in length. Spire-Vayron de la Moureyre et al (1999) first described this VNTR in a study of 54 unrelated European individuals. This region contains three to nine repeats, with the most common number in Caucasians being four or five repeats (SpireVayron de la Moureyre et al, 1999;Yan et al, 2000).…”

“…Spire-Vayron de la Moureyre et al (1999) first described this VNTR in a study of 54 unrelated European individuals. This region contains three to nine repeats, with the most common number in Caucasians being four or five repeats (SpireVayron de la Moureyre et al, 1999;Yan et al, 2000). Reporter gene constructs created with different numbers of repeats showed a modest effect of repeat number on transcription (Spire-Vayron de la Moureyre et al, 1999).…”

“…This region contains three to nine repeats, with the most common number in Caucasians being four or five repeats (SpireVayron de la Moureyre et al, 1999;Yan et al, 2000). Reporter gene constructs created with different numbers of repeats showed a modest effect of repeat number on transcription (Spire-Vayron de la Moureyre et al, 1999). However, the in vivo implications of this VNTR remain controversial.…”

“…However, the in vivo implications of this VNTR remain controversial. The initial studies showed an inverse relationship between RBC TPMT activity and the sum of repeat element number on both alleles as a surrogate for VNTR genotype (Spire-Vayron de la Moureyre et al, 1999). A much larger study performed with over 2000 clinical samples for which RBC TPMT activity had been measured showed a similar, but less clearcut relationship (Yan et al, 2000).…”