Explaining TPMT genotype/phenotype discrepancy by haplotyping of TPMT*3A and identification of a novel sequence variant, TPMT*23.

Thiopurine methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism of thiopurine drugs. Owing to polymorphisms in the TPMT gene (TPMT*2-*22), the enzyme activity varies interindividually. Patients with reduced TPMT activity may develop adverse reactions when treated with standard doses of thiopurines. This work focuses on a TPMT genotype/phenotype discrepancy found in a patient during routine testing. The patient displayed very low TPMT enzyme activity and she was genotyped by pyrosequencing as being heterozygous for the 460G>A and 719A>G polymorphisms (TPMT*3A). Complete sequencing in combination with haplotyping of the TPMT gene revealed a novel sequence variant, 500C>G, on one allele and TPMT*3A on the other allele, giving rise to the novel genotype TPMT*3A/*23. When investigating the patient's relatives, they too had the TPMT*3A/*23 genotype in combination with low enzyme activity. We conclude that this novel variant allele affects enzyme activity, as the individuals carrying it had almost undetectable TPMT activity.

Involvement of the concentrative nucleoside transporter 3 and equilibrative nucleoside transporter 2 in the resistance of T-lymphoblastic cell lines to thiopurines.

Mechanisms of resistance to thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) were investigated in human leukemia cell lines. We developed two 6-MP- and 6-TG-resistant cell lines from the human T-lymphoblastic cell line (MOLT-4) by prolonged exposure to these drugs. The resistant cells were highly cross resistant to 6-MP and 6-TG, and exhibited marked reduction in cellular uptake of 6-MP (70% and 80%, respectively). No significant modification of the activities of hypoxanthine-guanine phosphoribosyl transferase, thiopurine methyltransferase or inosine monophosphate dehydrogenase was observed. Real-time PCR of concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2) of resistant cells showed substantial reductions in expression of messenger RNAs. Small interfering RNA designed to silence the CNT3 and ENT2 genes down-regulated the expression of these genes in leukemia cells. These decreases were accompanied by reduction of transport of 6-MP (47% and 21%, respectively) as well as its cytocidal effect (30% and 21%, respectively). Taken together these results show that CNT3 and ENT2 play a key role in the transport of 6-MP and 6-TG by leukemia cells. From a clinical point of view determination of CNT3 and ENT2 levels in leukemia cells may be useful in predicting the efficacy of thiopurine treatment.

Failure of the PAXgene Blood RNA System to maintain mRNA stability in whole blood.

In multicentre studies of malignant and inflammatory diseases, whole blood, cell or tissue samples are often collected for analyses of gene expression to predict or monitor treatment effects. For correct analysis, sample stability during handling and transport is crucial. In developing the logistics for multicentre studies in malignant melanoma and inflammatory bowel disease, we found poor stability of a number of transcripts using the PAXgene Blood RNA System, which was advertised to maintain RNA stability for several days at room temperature. The results indicate that general statements on sample stability are not reliable and have to be verified for the specific transcripts of interest.

Identification of two novel sequence variants affecting thiopurine methyltransferase enzyme activity.

The polymorphic enzyme thiopurine methyltransferase (TPMT) is involved in the methylation of thiopurines. On comparing the phenotype with the genotype in Swedish patients with inflammatory bowel disease and healthy individuals, we found two discordant cases with low TPMT enzyme activity (0.3 and 0.4 U/ml packed red blood cells (pRBC). Genotyping by pyrosequencing revealed that they carried the nucleotide substitutions 460G>A and 719A>G, giving two possible genotypes (TPMT*1/*3A or TPMT*3B/*3C). DNA sequencing of exon III to X was performed in the patients and their parents. We identified an A>G transition in the start codon (exon III, 1A>G, Met>Val, TPMT*14) in one of the patients and her father (6.3 U/ml pRBC). The mother in this family carried the 460G>A and 719A>G nucleotide substitutions (TPMT*1/*3A; 5.0 U/ml pRBC). In the second family, sequencing revealed a G>A transition in the acceptor splice site in intron VII/exon VIII (IVS7 -1G>A, TPMT*15) in the patient and his mother (6.9 U/ml pRBC). His father was genotyped as TPMT*1/*3A (6.0 U/ml pRBC). Hence, we report the identification of two novel sequence variants, present in highly conserved nucleotide positions of the human TPMT gene, resulting in a loss of enzyme activity.

Pyrosequencing of TPMT alleles in a general Swedish population and in patients with inflammatory bowel disease.

BACKGROUND: Interindividual differences in therapeutic efficacy in patients treated with thiopurines might be explained by the presence of thiopurine S-methyltransferase (TPMT) alleles that encode for reduced TPMT enzymatic activity. It is therefore of value to know an individual's inherent capacity to express TPMT. METHOD: We developed a pyrosequencing method to detect 10 single-nucleotide polymorphisms (SNPs) in TPMT. A Swedish population (n = 800) was examined for TPMT*3A, TPMT*3B, TPMT*3C, and TPMT*2. Patients with inflammatory bowel disease (n = 24) and healthy volunteers (n = 6), selected on the basis of TPMT enzymatic activity, were investigated for all 10 SNPs to determine the relationship between TPMT genotype and phenotype. RESULTS: In the general population we identified the following genotypes with nonfunctional alleles: TPMT*1/*3A (*3A allelic frequency, 3.75%), TPMT*1/*3C (*3C allelic frequency, 0.44%), TPMT*1/*3B (*3B allelic frequency, 0.13%), and TPMT*1/*2 (*2 allelic frequency, 0.06%). All nine individuals with normal enzymatic activity were wild-type TPMT*1/*1. Thirteen individuals with intermediate activity were either TPMT*1/*3A (n = 12) or TPMT*1/*2 (n = 1). Eight individuals with low enzymatic activity were TPMT*3A/*3A (n = 4), TPMT*3A/*3C (n = 2), or TPMT*1/*3A (n = 2). CONCLUSION: Next to wild type, the most frequent alleles in Sweden are TPMT*3A and TPMT*3C. A previously established phenotypic cutoff for distinguishing normal from intermediate metabolizers was confirmed. To identify the majority of cases (90%) with low or intermediate TPMT activity, it was sufficient to analyze individuals for only 3 of the 10 SNPs investigated. Nevertheless, this investigation indicates that other mutations might be of relevance for decreased enzymatic activity.

Real-time RT-PCR methodology for quantification of thiopurine methyltransferase gene expression.

OBJECTIVE: The aim of the present study was to develop a real-time reverse-transcription polymerase chain reaction (RT-PCR) methodology for the quantification of thiopurine methyltransferase (TPMT) gene expression in whole blood and compare it with the TPMT enzyme activity measured in red blood cells. METHODS: TPMT gene expression was quantified relative to the housekeeping gene cyclophilin (huCYC) and expressed as a TPMT/huCYC ratio. TPMT activity in red blood cells was determined by measuring the formation rate of 6-(14)C-methylmercaptopurine from 6-MP using S-adenosyl-L-((14)C-methyl)-methionine as methyl donor. Thirty-nine individuals were included in the study. A cut-off value of 9 U/ml pRBC was used to distinguish intermediate TPMT enzyme activity from high TPMT enzyme activity. RESULTS: Sequencing of the real-time RT-PCR amplicon proved that the method was specific for the TPMT cDNA, without co-amplification of the highly similar TPMT processed pseudogene. The intra-assay coefficients of variation (CVs), as determined by the threshold cycle, were 0.7% for TPMT and 0.5% for huCYC. The interassay CVs were 1.5% for TPMT and 4.0% for huCYC. The intra- and interassay CVs, as determined by the TPMT/huCYC ratio, were 8.6% and 25%, respectively. There was a statistically significant correlation between TPMT enzyme activity and mRNA level in blood cells from individuals with an enzyme activity above 9 U/ml pRBC (r(s)=0.66, P=0.0001). However, we did not find any statistically significant correlation in individuals with lower enzyme activity or when analysing the whole population. CONCLUSION: We present a specific and robust real-time RT-PCR method for quantifying TPMT gene expression. The method may be used for studies on TPMT gene regulation.