Enhanced proteasomal degradation of mutant human thiopurine S-methyltransferase (TPMT) in mammalian cells: mechanism for TPMT protein deficiency inherited by TPMT*2, TPMT*3A, TPMT*3B or TPMT*3C.

Inheritance of the TPMT*2, TPMT*3A and TPMT*3C mutant alleles is associated with deficiency of thiopurine S-methyltransferase (TPMT) activity in humans. However, unlike TPMT*2 and TPMT*3A, the catalytically active protein coded by TPMT*3C does not undergo enhanced proteolysis when heterologously expressed in yeast, making it unclear why this common mutant allele should be associated with inheritance of TPMT-deficiency. To further elucidate the mechanism for TPMT deficiency associated with these alleles, we characterized TPMT proteolysis following heterologous expression of wild-type and mutant proteins in mammalian cells. When expressed in COS-1 cells, proteins encoded by TPMT*2, TPMT*3A, and TPMT*3C cDNAs had significantly reduced steady-state levels and shorter degradation half-lives compared with the wild-type protein. Similarly, in rabbit reticulocyte lysate (RRL), these mutant TPMT proteins were degraded significantly faster than the wild-type protein. Thus, enhanced proteolysis of TPMT*3C protein in mammalian cells is in contrast to its stability in yeast, but consistent with TPMT-deficiency in humans. Proteolysis was ATP-dependent and sensitive to proteasomal inhibitors MG115, MG132 and lactacystin, but not to calpain inhibitor II. We conclude that all of these mutant TPMT proteins undergo enhanced proteolysis in mammalian cells, through an ATP-dependent proteasomal pathway, leading to low or undetectable levels of TPMT protein in humans who inherit these mutant alleles.

Polymorphism of the thiopurine S-methyltransferase gene in African-Americans.

The molecular basis for the genetic polymorphism of thiopurine S -methyltransferase (TPMT) has been estab-lished for Caucasians, but it remains to be elucidated in African populations. In the current study, we determined TPMT genotypes in a population of 248 African-Americans and compared it with allele frequencies in 282 Caucasian Americans. TPMT genotype was determined in all individuals with TPMT activity indicative of a heterozygous genotype (10.2 U/ml pRBC, n = 23 African-Americans, n = 21 Caucasians). No mutant alleles were found in the high activity control groups. The overall mutant allele frequencies were similar in African-Americans and Caucasians (4.6 and 3.7% of alleles, respectively). However, while TPMT*3C was the most prevalent mutant allele in African-Americans (52.2% of mutant alleles), it represented only 4.8% of mutant alleles in Caucasians ( P < 0.001). In contrast, TPMT*3A and TPMT*2 were less common in African-Americans (17.4 and 8.7% of mutant alleles), whereas TPMT*3A was the most prevalent mutant allele in Caucasians (85.7% of mutant alleles). A novel allele ( TPMT*8 ), containing a single nucleotide transition (G644A), leading to an amino acid change at codon 215 (Arg-->His), was found in one African-American with intermediate activity. These data indicate that the same TPMT mutant alleles are found in American black and white populations, but that the predominant mutant alleles differ in these two ethnic groups.

Functional characterization of the human thiopurine S-methyltransferase (TPMT) gene promoter.

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes S-methylation of aromatic and heterocyclic sulfhydryl compounds, including anticancer and immunosuppressive thiopurines. We recently isolated the human TPMT promoter, which does not contain TATA box or CCAAT element consensus sequences, but is GC rich with multiple GC boxes and other putative cis-regulatory elements. Here, we report the functional characterization of the TPMT promoter, revealing several positive regulatory elements and identifying stimulating protein 1 (Sp1) as an important trans-activator essential for constitutive activity in cell culture. One major and two closely located minor transcription start points were identified in HepG2 cells. Deletion analysis revealed positive cis-regulatory elements located in the regions -85 to -75, -68 to -58, -58 to -51 and +34 to +60 relative to the transcription start site. DNaseI footprinting analysis and cotransfection in Drosophila Schneider SL2 cells documented that Sp1 binds to the TPMT promoter and is important for constitutive activity. We conclude that constitutive transcription of the TPMT gene involves a limited upstream GC-rich DNA sequence, containing multiple GC boxes, and that transcription factor Sp1 [or related protein(s)] is an important trans-activator of this TATA-less promoter.

Isolation of a human thiopurine S-methyltransferase (TPMT) complementary DNA with a single nucleotide transition A719G (TPMT*3C) and its association with loss of TPMT protein and catalytic activity in humans.

OBJECTIVE: Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes the S-methylation of mercaptopurine, azathioprine, thioguanine and most of their nucleotide metabolites. TPMT exhibits genetic polymorphism, with about 10% of individuals having intermediate TPMT activity because of heterozygosity at the TPMT locus and about 1 in 300 inheriting TPMT deficiency as an autosomal recessive trait. Although several mutant alleles have now been associated with inheritance of TPMT deficiency in humans, the expression of only TPMT*2 and TPMT*3A has been established by isolation and characterization of complementary DNA (cDNA) from individuals with low TPMT activity. METHODS: Radiochemical assay, Western blot analysis, polymerase chain reaction (PCR) genotyping, and cDNA sequencing were used to analyze TPMT activity and protein levels in erythrocytes and to determine TPMT genotype. RESULTS: We established expression of another common mutant allele, TPMT*3C (containing only the A719G mutation), by sequence analysis of cDNA isolated from an individual with a heterozygous TPMT phenotype (7 units/ml packed erythrocytes). The TPMT*3C allele was also confirmed in an unrelated individual by sequencing TPMT coding exons after PCR amplification of genomic DNA. Moreover, Western blot analysis of erythrocytes obtained from five heterozygous individuals with the TPMT*3C allele (i.e., TPMT*1/TPMT*3C) exhibited about 50% less immunodetectable TPMT protein compared with homozygous wild-type individuals, and a TPMT-deficient individual with a TPMT*3A/TPMT*3C genotype had no immunodetectable TPMT protein. CONCLUSION: These data establish that the TPMT*3C allele is expressed in humans and is associated with lower immunodetectable TPMT protein and catalytic activity.

Molecular cloning and functional characterization of the cDNA encoding the murine thiopurine S-methyltransferase (TPMT).

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyzes S-methylation of aromatic and heterocyclic sulfhydryl compounds, including anticancer and immunosuppressive thiopurines. Here we report the isolation and functional characterization of the murine TPMT cDNA. The screening of expressed sequence tags database led to isolation of a murine cDNA clone containing an uninterrupted ORF encoding the protein with an amino acid sequence that is 82% similar and 78% identical to the human TPMT. The expression product of the murine cDNA in rabbit reticulocyte and wheat germ lysate coupled transcription-translation systems showed TPMT enzymatic activity. We conclude that the isolated cDNA clone represents the murine TPMT cDNA.

Molecular diagnosis of thiopurine S-methyltransferase deficiency: genetic basis for azathioprine and mercaptopurine intolerance.

BACKGROUND: Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation (that is, inactivation) of mercaptopurine, azathioprine, and thioguanine and exhibits genetic polymorphism. About 10% of patients have intermediate TPMT activity because of heterozygosity, and about 1 in 300 inherit TPMT deficiency as an autosomal recessive trait. If they receive standard doses of thiopurine medications (for example, 75 mg/m2 body surface area per day), TPMT-deficient patients accumulate excessive thioguanine nucleotides in hematopoietic tissues, which leads to severe and possibly fatal myelosuppression. OBJECTIVE: To elucidate the genetic basis and develop molecular methods for the diagnosis of TPMT deficiency and heterozygosity. DESIGN: Diagnostic test evaluation. SETTING: Research hospital. PATIENTS: The TPMT phenotype was determined in 282 unrelated white persons, and TPMT genotype was determined in all persons who had intermediate TPMT activity (heterozygotes) and a randomly selected, equal number of persons who had high activity. In addition, genotype was determined in 6 TPMT-deficient patients. MEASUREMENTS: Polymerase chain reaction (PCR) assays were developed to detect the G238C transversion in TPMT*2 and the G460A and A719G transitions in TPMT*3 alleles. Radiochemical assay was used to measure TPMT activity. Mutations of TPMT were identified in genomic DNA, and the concordance of TPMT genotype and phenotype was determined. RESULTS: 21 patients who had a heterozygous phenotype were identified (7.4% of sample [95% CI, 4.7% to 11.2%]). TPMT*3A was the most prevalent mutant allele (18 of 21 mutant alleles in heterozygotes; 85%); TPMT*2 and TPMT*3C were more rare (about 5% each). All 6 patients who had TPMT deficiency had two mutant alleles, 20 of 21 patients (95% [CI, 76% to 99.9%]) who had intermediate TPMT activity had one mutant allele, and 21 of 21 patients (100% [CI, 83% to 100%]) who had high activity had no known TPMT mutation. Detection of TPMT mutations in genomic DNA by PCR coincided perfectly with genotypes detected by complementary DNA sequencing. CONCLUSIONS: The major inactivating mutations at the human TPMT locus have been identified and can be reliably detected by PCR-based methods, which show an excellent concordance between genotype and phenotype. The detection of TPMT mutations provides a molecular diagnostic method for prospectively identifying TPMT-deficient and heterozygous patients.

Promoter and intronic sequences of the human thiopurine S-methyltransferase (TPMT) gene isolated from a human PAC1 genomic library.

PURPOSE: To isolate and characterize the polymorphic human thiopurine S-methyltransferase (TPMT) gene. METHODS: The human TPMT gene was isolated by PCR screening of a phage artificial chromosome (PAC) library, using exon- and intron-specific primers, then mapped and sequenced. RESULTS: Two separate PAC1 clones were isolated that contained the same 25 kb gene with 9 exons encompassing the entire TPMT open reading frame. Structural characterization revealed distinct differences when compared to a TPMT gene previously isolated from a chromosome 6-specific human genomic library; the 5'-flanking region (putative promoter) contains 17 additional nucleotides located at position-77 upstream from the transcription start site, in addition to several nucleotide sequence differences, and intron 8 is only 1.6 kb, 5 kb shorter than previously reported. Southern and PCR analyses of genomic DNA from 18 unrelated individuals revealed only the TPMT gene structure corresponding to the PAC clones we isolated. Analysis of the TPMT promoter activity using the 5'-terminal region confirmed transcriptional activity in human HepG2 and CCRF-CEM cells. The 5'-flank is 71% GC rich and does not contain consensus sequences for TATA box or CCAAT elements. FISH analysis demonstrated the presence of the TPMT-homologous sequence on the short arm of chromosome 6 (sublocalized to 6p22). CONCLUSIONS: These findings establish the genomic structure of the human TPMT gene, revealing differences in the promoter and intronic sequences compared to that previously reported and providing a basis for future studies to further elucidate its biological function and regulation.

Genetic polymorphism of thiopurine S-methyltransferase: clinical importance and molecular mechanisms.

Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurines such as mercaptopurine and thioguanine. TPMT activity exhibits genetic polymorphism, with about 1 in 300 inheriting TPMT-deficiency as an autosomal recessive trait. If treated with standard dosages of thiopurines. TPMT-deficient patients accumulate excessive thioguanine nucleotides (TGN) in hematopoietic tissues, leading to severe hematopoietic toxicity that can be fatal. However, TPMT-deficient patients can be successfully treated with a 10-15-fold lower dosage of these medications. The human gene encoding polymorphic TPMT has been cloned and characterized, and two mutant alleles have recently been isolated from TPMT-deficient and heterozygous patients (TPMT*2, TPMT*3), permitting development of PCR-based methods to identify TPMT-deficient and heterozygous patients prior to therapy. TPMT*3 is the predominant mutant allele in American whites, accounting for about 75% of mutations in this population. Ongoing studies aim to better define the influence of TPMT activity on thiopurine efficacy, to identify additional mutant alleles and determine their frequency in different ethnic groups, to elucidate the mechanism(s) for loss of function of mutant proteins, to identify potential endogenous substrates and to define the molecular mechanisms of TPMT regulation. Together, these advances bold the promise of improving the safety and efficacy of thiopurine therapy.

Thiopurine S-methyltransferase deficiency: two nucleotide transitions define the most prevalent mutant allele associated with loss of catalytic activity in Caucasians.

The autosomal recessive trait of thiopurine S-methytransferase (TPMT) deficiency is associated with severe hematopoietic toxicity when patients are treated with standard doses of mercaptopurine, azathioprine, or thioguanine. To define the molecular mechanism of this genetic polymorphism, we cloned and characterized the cDNA of a TPMT-deficient patient, which revealed a novel mutant allele (TPMT*3) containing two nucleotide transitions (G460-->A and A719-->G) producing amino acid changes at codons 154 (Ala-->Thr) and 240 (Tyr--> Cys), differing from the rare mutant TPMT allele we previously identified (i.e., TPMT*2 with only G238-->C). Site-directed mutagenesis and heterologous expression established that either TPMT*3 mutation alone leads to a reduction in catalytic activity (G460-->A, ninefold reduction; A719-->G, 1.4-fold reduction), while the presence of both mutations leads to complete loss of activity. Using mutation specific PCR-RFLP analysis, the TPMT*3 allele was detected in genomic DNA from approximately 75 percent of unrelated white subjects with heterozygous phenotypes, indicating that TPMT*3 is the most prevalent mutant allele associated with TPMT-deficiency in Caucasians.

Segment selection during 'phase variation'-type regulation of gene replacement mediated by FLP recombinase in the yeast Saccharomyces cerevisiae.

FLP recombinase has recently been used as a tool to direct the exchange between invertible DNA segments, called 'Phase variation'-type regulation of gene replacement in eukaryotic cells. Using an appropriate selective medium, positive segment selection was shown to be efficient during the regulation of gene replacement. The efficiency was determined from the copy number ratio of invertible segments with the use of the neomycinphosphotransferase II (NPTII) gene bearing invertible segments located on the episomal yeast plasmid, and the resident 2-microns circle. Without the selection the segments copy number ratio was retained in growing cells. The results obtained are an evidence for the efficiency of positive segment selection during the 'Phase variation'-type regulation of gene replacement in eukaryotic cells.