A Simple Method for TPMT and ITPA Genotyping Using Multiplex HRMA for Patients Treated with Thiopurine Drugs.
Thiopurine methyltransferase (TPMT) and inosine triphosphatase (ITPA) are crucial enzymes involved in the metabolism of thiopurine drugs: azathioprine and 6-mercaptopurine, used in the treatment of leukemia or inflammatory bowel diseases (IBD). The activity in these enzymes correlates with the genetic polymorphism of the TPMT and ITPA genes, respectively, which determines an individual reaction and dosing of thiopurines. Three main TPMT alleles: TPMT*2 (c.238G>C), TPMT*3A (c.460G>A, c.719A>G) and TPMT*3C (c.719A>G) account for 80-95 % of inherited TPMT deficiency in different populations in the world. In the ITPA gene, a c.94C>A mutation is significantly associated with an adverse thiopurine reaction. The aim of this study was to develop a quick and highly sensitive method for determining major TPMT and ITPA alleles. Here we present the molecular test for genotyping c.238G>C, c.460G>A, c.719A>G and c.94C>A changes based on multiplex high resolution melting analysis (HRMA). We analyzed DNA samples from 100 clinically diagnosed IBD patients treated with thiopurine drugs, and a known genotype in the positions 238, 460 and 719 of the TPMT gene as well as in position 94 of the ITPA gene. Our results obtained with multiplex HRMA indicated 100 % accuracy in comparison with data from restriction fragments length polymorphism (RFLP) and standard DNA sequencing. We conclude, that multiplex HRMA can be used as a quick, sensitive and efficient alternative diagnostic method compared to conventional techniques for the determination of TPMT*2, TPMT*3A and TPMT*3C alleles and c.94C>A change in the ITPA gene.
Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszynska 32, 60-479, Poznan, Poland. mskrzypczakzielinska@gmail.com.
Marzena
M
Skrzypczak-Zielinska
Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszynska 32, 60-479, Poznan, Poland.
Pawel
P
Borun
Laboratory of Cell Biophysics, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
Anna
A
Bartkowiak-Kaczmarek
Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszynska 32, 60-479, Poznan, Poland.
Oliwia
O
Zakerska-Banaszak
Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszynska 32, 60-479, Poznan, Poland.
Michal
M
Walczak
Department of Gastroenterology, Human Nutrition and Internal Diseases, University of Medical Sciences, Poznan, Poland.
Agnieszka
A
Dobrowolska
Department of Experimental and Clinical Pharmacology, Pomeranian Medical University, Szczecin, Poland.
Mateusz
M
Kurzawski
Department of Functional Anatomy, University School of Physical Education, Poznan, Poland.
Malgorzata
M
Waszak
Department of Biochemistry and Biotechnology, University of Life Sciences, Poznan, Poland.
Daniel
D
Lipinski
Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszynska 32, 60-479, Poznan, Poland.
Andrzej
A
Plawski
Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszynska 32, 60-479, Poznan, Poland.|Department of Biochemistry and Biotechnology, University of Life Sciences, Poznan, Poland.
Ryszard
R
Slomski
D000483:Alleles; D001379:Azathioprine / Q000494:pharmacology / Q000627:therapeutic use; D005787:Gene Frequency; D005838:Genotype; D060005:Genotyping Techniques; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000188:drug therapy / Q000235:genetics; D015122:Mercaptopurine / Q000494:pharmacology / Q000627:therapeutic use; D008780:Methyltransferases / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D000071185:Pharmacogenomic Testing; D011755:Pyrophosphatases / Q000235:genetics*; D000097630:Inosine Triphosphatase
D001379:Azathioprine; D015122:Mercaptopurine; D008780:Methyltransferases; D011755:Pyrophosphatases; D000097630:Inosine Triphosphatase; C022745:thiopurine methyltransferase