Dataset Information

Validation of new allele-specific real-time PCR system for thiopurine methyltransferase genotyping in Korean population.

ABSTRACT: INTRODUCTION: Thiopurine drugs are metabolized via S-methylation and catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low TPMT activity are at high risk of fatal bone marrow toxicity when standard doses of thiopurine drugs are administered. TPMT genotyping can predict TPMT activity and is not affected by transfusion or red blood cell defects. Here, we report a new allele-specific real-time polymerase chain reaction (PCR) system for thiopurine methyltransferase genotyping that is validated in Korean population. MATERIALS AND METHODS: Three major TPMT single-nucleotide polymorphisms (TPMT 2, 3B, and 3C) were genotyped using real-time PCR with the allele-specific primers and probes. Internal positive controls were included in each well, and an automatic interpretative algorithm was applied. This system was validated using 244 clinical samples and 2 commercial DNA samples that had been previously genotyped using PCR-direct sequencing. Results. All of the obtained results are concordant with those of the reference method. All of the internal positive control reactions were successful. The allele frequency of TPMT 3C was 2.05% (10 of 488 alleles). All of the patients with variant alleles were heterozygotes, and no homozygotes were detected. No TPMT 2, 3A, or 3B alleles were observed in this Korean population. CONCLUSION: This rapid, accurate, and user-friendly genotyping system can be readily used to improve the efficacy and safety of thiopurine treatments in clinical practice.

SUBMITTER: Kim S

PROVIDER: S-EPMC3600258 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Validation of new allele-specific real-time PCR system for thiopurine methyltransferase genotyping in Korean population.

Kim Sollip S Lee Hye Won HW Lee Woochang W Chun Sail S Min Won-Ki WK

BioMed research international 20130228

<h4>Introduction</h4>Thiopurine drugs are metabolized via S-methylation and catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low TPMT activity are at high risk of fatal bone marrow toxicity when standard doses of thiopurine drugs are administered. TPMT genotyping can predict TPMT activity and is not affected by transfusion or red blood cell defects. Here, we report a new allele-specific real-time polymerase chain reaction (PCR) system for thiopurine methyltransferase genoty ...[more]

PMID: 23555080

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Project description:Pediatric Crohn's disease (CD) carries a higher genetic susceptibility and an increased risk of a more aggressive disease course than adult CD. Treatment of CD is based on immunomodulatory drugs, such as thiopurines. The enzyme mainly involved in drug metabolism is thiopurine S-methyltransferase (TPMT). An increased concentration of drug metabolites can cause adverse drug effects, such as myelosuppression and hepatotoxicity; therefore, assessing the activity of TPMT is essential both before and during treatment. TPMT genotyping result is not affected by previous thiopurine dose and currently is the primary component of TPMT activity and disease monitoring. Until now, more than 40 allelic variants of the TPMT gene have been reported, with most of them having an uncertain or no enzyme function. In this article, we report the first case of a novel TPMT allele, TPMT*45, that was identified in a Korean girl with CD whose findings suggested decreased TPMT activity. This newly observed variant is caused by a single nucleotide polymorphism resulting in nonsense mutation (c.676C>T, p.R226*) and the partial loss of amino acids in the TPMT protein. Initially, the patient began azathioprine at a standard dosage (1.5 mg/kg/day), and her laboratory results, including red blood cell (RBC) TPMT activity (6-methylmercaptopurine 2.68 nmol/mL/h and 6-methylmercaptopurine riboside 4.82 nmol/mL/h) along with thiopurine metabolite levels (6-thioguanine nucleotides 479.3 pmol/8×108 RBC), suggested an enzyme deficiency. The thiopurine dose was reduced to half (0.7 mg/kg/day), and the follow-up metabolite results as well as the associated inflammatory markers were continuously within reference ranges. Along with an improvement in the patient's subjective reports and clinical symptoms, the patient demonstrated a good treatment response to the adjusted dose. The results of our report illustrate the importance of TPMT genotyping and pharmacogenetic-based thiopurine dose adjustment. Further research should focus on the functional characterization and impact on this novel allele's treatment effect.

Project description:BackgroundWhile improvements in genotyping technology have allowed for increased throughput and reduced time and expense, protocols remain hindered by the slow upstream steps of isolating, purifying, and normalizing DNA. Various methods exist for genotyping samples directly through blood, without having to purify the DNA first. These procedures were designed to be used on smaller throughput systems, however, and have not yet been tested for use on current high-throughput real-time (q)PCR based genotyping platforms. In this paper, a method of quantitative qPCR-based genotyping on blood without DNA purification was developed using a high-throughput qPCR platform.FindingsThe performances of either DNA purified from blood or the same blood samples without DNA purification were evaluated through qPCR-based genotyping. First, 60 different mutations prevalent in the Ashkenazi Jewish population were genotyped in 12 Ashkenazi Jewish individuals using the QuantStudio™12K Flex Real-Time PCR System. Genotyping directly from blood gave a call rate of 99.21%, and an accuracy of 100%, while the purified DNA gave a call rate of 92.49%, and an accuracy of 99.74%. Although no statistical difference was found for these parameters, an F test comparing the standard deviations of the wild type clusters for the two different methods indicated significantly less variation when genotyping directly from blood instead of after DNA purification. To further establish the ability to perform high-throughput qPCR based genotyping directly from blood, 96 individuals of Ashkenazi Jewish decent were genotyped for the same 60 mutations (5,760 genotypes in 5 hours) and resulted in a call rate of 98.38% and a diagnostic accuracy of 99.77%.ConclusionThis study shows that accurate qPCR-based high-throughput genotyping can be performed without DNA purification. The direct use of blood may further expedite the entire genotyping process, reduce costs, and avoid tracking errors which can occur during sample DNA purification.

Dataset Information

Validation of new allele-specific real-time PCR system for thiopurine methyltransferase genotyping in Korean population.

Publications

Validation of new allele-specific real-time PCR system for thiopurine methyltransferase genotyping in Korean population.

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