Thiopurine Methyltransferase Pharmacogenetics: Human Gene Cloning and Characterization of a Common Polymorphism

Abstract

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs. Individual variation in the toxicity and therapeutic efficacy of these drugs is associated with a common genetic polymorphism that controls levels of TPMT activity and immunoreactive protein in human tissues. Because of the clinical significance of the "pharmacogenetic" regulation of this enzyme, it would be important to clone the gene for TPMT in humans and to study the molecular basis for the genetic polymorphism. As a first step toward cloning the gene for TPMT, we used the rapid amplification of genomic DNA ends to obtain a TPMT-specific intron sequence. That DNA sequence was used to design primers for the polymerase chain reaction (PCR), which made it possible to determine that the active gene for TPMT is located on human chromosome 6. A TPMT-positive cosmid clone was then isolated from a human chromosome 6-specific genomic DNA library, and the gene was sublocalized to chromosome band 6p22.3 by fluorescence in situ hybridization. The gene for TPMT was found to be approximately 34 kb in length and consisted of 10 exons and 9 introns. On the basis of the results of 5'-rapid amplification of cDNA ends, transcription initiation occurred at or near a point 89 nucleotides upstream from the translation initiation codon of previously reported TPMT cDNAs. Once the structure of the TPMT gene had been determined, it was possible to perform the PCR with primers complementary to the sequences of introns flanking each exon that encodes enzyme protein with template DNA obtained from subjects with known phenotypes for the TPMT genetic polymorphism. This DNA was isolated from blood samples from 4 unrelated subjects with genetically low TPMT activity and 4 unrelated subjects with high TPMT activity. All subjects with low TPMT activity were homozygous for two point mutations--a G-->A transition at nucleotide 460 in exon 7 and an A-->G transition at nucleotide 719 in exon 10. Both mutations resulted in alterations in amino acid sequence, with Ala-154-->Thr and Tyr-240-->Cys, respectively. All DNA samples isolated from the blood of subjects with high TPMT activity contained "wild-type" sequence. Results obtained with these blood samples were confirmed when DNA from four human liver samples with high TPMT activity were found to have wild-type sequence at nucleotides 460 and 719, while three liver samples with intermediate enzyme activity (i.e., samples presumed to be heterozygous for the polymorphism) were heterozygous for the exon 7 and exon 10 mutations present in the blood samples of homozygous low subjects. Transient expression in COS-1 cells of TPMT expression constructs that contained both of the mutations in exons 7 and 10, as well as each independently, demonstrated that each mutation, as well as both together, resulted in decreased expression of TPMT enzymatic activity and immunoreactive protein. Molecular cloning and structural characterization of the TPMT gene as well as elucidation of the molecular basis for a common TPMT genetic poly...

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

“…Furthermore, as pointed out previously, there is a strong correlation between relative levels of TPMT activity in the RBC and in other human tissues (Woodson et al, 1982;Szumlanski et al, 1992). Following the cloning of the TPMT gene and characterization of its most important polymorphisms (Szumlanski et al, 1996;Tai et al, 1996), determination of TPMT genotype was also shown to be useful for individualizing thiopurine drug therapy. However, both genotyping and phenotyping have their limitations.…”

“…Of the allozymes studied by Salavaggione et al (2005), TPMT*3A, *3B, *3C and *2 displayed the most striking effects. This phenomenon was first observed a decade ago when TPMT*3A, *3B and *3C were initially described (Szumlanski et al, 1996). In the course of those early experiments, these three allozymes, as well as WT TPMT, were expressed in COS-1 cells, and Western blot analysis was performed.…”

“…Levels of TPMT activity in the RBC reflect relative levels of that enzyme in the kidney, liver and lymphocyte (Van Loon and Weinshilboum, 1982;Woodson et al, 1982;Szumlanski et al, 1992;Coulthard et al, 1998). After the human TPMT cDNA and gene had been cloned and characterized (Honchel et al, 1993;Szumlanski et al, 1996;Tai et al, 1996), these phenotypic variations were shown to result primarily from variation in the sequence of the TPMT gene itself. A total of 21 TPMT genetic polymorphisms have been identified which are, or may be associated with decreased levels of TPMT enzyme activity and/or thiopurine drug-induced toxicity .…”

“…A total of 21 TPMT genetic polymorphisms have been identified which are, or may be associated with decreased levels of TPMT enzyme activity and/or thiopurine drug-induced toxicity . Eighteen of those polymorphisms (*2, *3A, *3B, *3C, *5-*14 and *16-*19) involve nonsynonymous coding single-nucleotide polymorphisms (cSNPs) (Krynetski et al, 1995;Szumlanski et al, 1996;Otterness et al, 1997;Lindqvist et al, 2004;Schaeffeler et al, 2004;Hamdan-Khalil et al, 2005), that is, alterations in single DNA nucleotides that alter the encoded amino acid. 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).…”

“…TPMT *3C, the most common functionally significant variant allele in East Asia (frequency approximately 2%), includes only the codon 240 SNP, and the rare TPMT *3B allele has only the codon 154 SNP (Figure 2) (Szumlanski et al, 1996). The presence of TPMT*3A and *3B result in a virtual lack of TPMT enzyme activity and protein in the tissues of subjects who carry these alleles and, as a result, patients homozygous for these alleles can suffer severe, life-threatening myelosuppression when treated with standard doses of thiopurines, that is, they are 'overdosed' on standard doses (Lennard et al, 1989(Lennard et al, , 1990Evans et al, 1991).…”