Accuracy of genotyping using the TaqMan PCR assay for single nucleotide polymorphisms responsible for thiopurine sensitivity in Japanese patients with inflammatory bowel disease

Osaki,Rie; Imaeda,Hirotsugu; Ban,Hiromitsu; Aomatsu,Tomoki; Bamba,Shigeki; Tsujikawa,Tomoyuki; Sasaki,Masaya; Fujiyama,Yoshihide; Andoh,Akira

doi:10.3892/etm.2011.287

Accuracy of genotyping using the TaqMan PCR assay for single nucleotide polymorphisms responsible for thiopurine sensitivity in Japanese patients with inflammatory bowel disease

Authors:
- Rie Osaki
- Hirotsugu Imaeda
- Hiromitsu Ban
- Tomoki Aomatsu
- Shigeki Bamba
- Tomoyuki Tsujikawa
- Masaya Sasaki
- Yoshihide Fujiyama
- Akira Andoh
View Affiliations

Affiliations: Department of Medicine, Graduate School of Medicine, Shiga University of Medical Science, Seta Tukinowa, Otsu 520-2192, Japan, Division of Mucosal Immunology, Graduate School of Medicine, Shiga University of Medical Science, Seta Tukinowa, Otsu 520-2192, Japan
Published online on: June 16, 2011 https://doi.org/10.3892/etm.2011.287
Pages: 783-786

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Thiopurine drugs are the most common drugs used to maintain clinical remission in inflammatory bowel disease (IBD). Three single-nucleotide polymorphisms (SNPs), TPMT A719G (rs1142345), inosine triphosphate pyrophosphatase (ITPase) C94A (rs1127354) and multidrug resistance protein 4 MRP4 G2269A (rs3765534), have been reported to account for heightened sensitivity to thiopurine drugs in the Japanese population. We investigated the usefulness of the TaqMan® PCR assay (Applied Biosystems) for the rapid detection of these SNPs to improve the safety of thiopurine therapy. We enrolled 44 healthy volunteers and 235 IBD patients. Genotyping of the SNPs was performed using Custom TaqMan SNP genotyping assays, direct sequencing and PCR-RFLP. Genotyping for MRP4 G2269A by the TaqMan PCR assay was successfully achieved in all samples. Comparison with our previous data using direct sequencing indicated one discordant result, and re-sequencing showed that the TaqMan PCR assay was correct. The overall accuracy of the TaqMan assay for MRP4 G2269A was 100%. The TaqMan PCR genotyping for TPMT A719G and ITPase C94A was successfully performed in all samples. The results of TPMT A719G by the TaqMan assay were identical with those of PCR-RFLP. In ITPase C94A, a comparison of the TaqMan assay and PCR-RFLP yielded 12 discordant results, and direct sequencing showed that the TaqMan PCR assay was correct. The allelic frequency determined by the TaqMan assay was 0.145 for MRP4 G2269A, 0.009 for TPMT A719G and 0.121 for ITPase C94A, respectively. In conclusion, the TaqMan® PCR assay is useful for genotyping of SNPs responsible for thiopurine sensitivity in Japanese IBD patients.

View Figures

View References

1.	Adler DJ and Korelitz BI: The therapeutic efficacy of 6-mercaptopurine in refractory ulcerative colitis. Am J Gastroenterol. 85:717–722. 1990.PubMed/NCBI
2.	Sandborn W, Sutherland L, Pearson D, May G, Modigliani R and Prantera C: Azathioprine or 6-mercaptopurine for inducing remission of Crohn’s disease. Cochrane Database Syst Rev. 6:CD0005452000.
3.	Hibi T and Ogata H: Novel pathophysiological concepts of inflammatory bowel disease. J Gastroenterol. 41:10–16. 2006. View Article : Google Scholar
4.	Gearry RB, Barclay ML, Burt MJ, Collett JA and Chapman BA: Thiopurine drug adverse effects in a population of New Zealand patients with inflammatory bowel disease. Pharmacoepidemiol Drug Saf. 13:563–567. 2004. View Article : Google Scholar : PubMed/NCBI
5.	Van Dieren JM, Hansen BE, Kuipers EJ, Nieuwenhuis EE and Van der Woude CJ: Meta-analysis: inosine triphosphate pyrophosphatase polymorphisms and thiopurine toxicity in the treatment of inflammatory bowel disease. Aliment Pharmacol Ther. 26:643–652. 2007.
6.	Gisbert JP and Gomollon F: Thiopurine-induced myelotoxicity in patients with inflammatory bowel disease: a review. Am J Gastroenterol. 103:1783–1800. 2008. View Article : Google Scholar : PubMed/NCBI
7.	Takatsu N, Matsui T, Murakami Y, et al: Adverse reactions to azathioprine cannot be predicted by thiopurine S-methyltransferase genotype in Japanese patients with inflammatory bowel disease. J Gastroenterol Hepatol. 24:1258–1264. 2009. View Article : Google Scholar : PubMed/NCBI
8.	Lees CW, Maan AK, Hansoti B, Satsangi J and Arnott ID: Tolerability and safety of mercaptopurine in azathioprine-intolerant patients with inflammatory bowel disease. Aliment Pharmacol Ther. 27:220–227. 2008. View Article : Google Scholar : PubMed/NCBI
9.	Winter JW, Gaffney D, Shapiro D, et al: Assessment of thiopurine methyltransferase enzyme activity is superior to genotype in predicting myelosuppression following azathioprine therapy in patients with inflammatory bowel disease. Aliment Pharmacol Ther. 25:1069–1077. 2007. View Article : Google Scholar
10.	Lichtenstein GR, Sbreu MT, Cohen R and Tremaine W: American Gastroenterological Association Institute technical review on corticosteroids, immunomodulators, and infliximab in inflammatory bowel disease. Rev Gastroenterol Mex. 71:351–401. 2006.(In Spanish).
11.	Hibi T, Naganuma M, Kitahora T, Kinjyo F and Shimoyama T: Low-dose azathioprine is effective and safe for maintenance of remission in patients with ulcerative colitis. J Gastroenterol. 38:740–746. 2003. View Article : Google Scholar : PubMed/NCBI
12.	Derijks LJ, Gilissen LP, Engels LG, et al: Pharmacokinetics of 6-thioguanine in patients with inflammatory bowel disease. Ther Drug Monit. 28:45–50. 2006. View Article : Google Scholar : PubMed/NCBI
13.	Haglund S, Taipalensuu J, Peterson C and Almer S: IMPDH activity in thiopurine-treated patients with inflammatory bowel disease – relation to TPMT activity and metabolite concentrations. Br J Clin Pharmacol. 65:69–77. 2007.PubMed/NCBI
14.	Ban H, Andoh A, Tanaka A, et al: Analysis of thiopurine S-methyltransferase genotypes in Japanese patients with inflammatory bowel disease. Intern Med. 47:1645–1648. 2008. View Article : Google Scholar : PubMed/NCBI
15.	Hiratsuka M, Inoue T, Omori F, Agatsuma Y and Mizugaki M: Genetic analysis of thiopurine methyltransferase polymorphism in a Japanese population. Mutat Res. 448:91–95. 2000. View Article : Google Scholar : PubMed/NCBI
16.	Uchiyama K, Nakamura M, Kubota T, Yamane T, Fujise K and Tajiri H: Thiopurine S-methyltransferase and inosine triphosphate pyrophosphohydrolase genes in Japanese patients with inflammatory bowel disease in whom adverse drug reactions were induced by azathioprine/6-mercaptopurine treatment. J Gastroenterol. 44:197–203. 2009. View Article : Google Scholar
17.	Kurzawski M, Dziewanowski K, Lener A and Drozdzik M: TPMT but not ITPA gene polymorphism influences the risk of azathioprine intolerance in renal transplant recipients. Eur J Clin Pharmacol. 65:533–540. 2009. View Article : Google Scholar : PubMed/NCBI
18.	Allorge D, Hamdan R, Broly F, Libersa C and Colombel JF: ITPA genotyping test does not improve detection of Crohn’s disease patients at risk of azathioprine/6-mercaptopurine induced myelosuppression. Gut. 54:5652005.
19.	Zelinkova Z, Derijks LJ, Stokkers PC, et al: Inosine triphosphate pyrophosphatase and thiopurine s-methyltransferase genotypes relationship to azathioprine-induced myelosuppression. Clin Gastroenterol Hepatol. 4:44–49. 2006. View Article : Google Scholar
20.	Wielinga PR, Reid G, Challa EE, et al: Thiopurine metabolism and identification of the thiopurine metabolites transported by MRP4 and MRP5 overexpressed in human embryonic kidney cells. Mol Pharmacol. 62:1321–1331. 2002. View Article : Google Scholar
21.	Krishnamurthy P, Schwab M, Takenaka K, et al: Transporter-mediated protection against thiopurine-induced hematopoietic toxicity. Cancer Res. 68:4983–4989. 2008. View Article : Google Scholar : PubMed/NCBI
22.	Peng XX, Shi Z, Damaraju VL, et al: Up-regulation of MRP4 and down-regulation of influx transporters in human leukemic cells with acquired resistance to 6-mercaptopurine. Leuk Res. 32:799–809. 2008. View Article : Google Scholar : PubMed/NCBI
23.	Borst P, Evers R, Kool M and Wijnholds J: A family of drug transporters: the multidrug resistance-associated proteins. J Natl Cancer Inst. 92:1295–1302. 2000. View Article : Google Scholar : PubMed/NCBI
24.	Ban H, Andoh A, Imaeda H, et al: The multidrug-resistance protein 4 polymorphism is a new factor accounting for thiopurine sensitivity in Japanese patients with inflammatory bowel disease. J Gastroenterol. 45:1014–1021. 2010. View Article : Google Scholar : PubMed/NCBI
25.	Yates CR, Krynetski EY, Loennechen T, et al: Molecular diagnosis of thiopurine S-methyltransferase deficiency: genetic basis for azathioprine and mercaptopurine intolerance. Ann Intern Med. 126:608–614. 1997. View Article : Google Scholar : PubMed/NCBI
26.	Marinaki AM, Ansari A, Duley JA, et al: Adverse drug reactions to azathioprine therapy are associated with polymorphism in the gene encoding inosine triphosphate pyrophosphatase (ITPase). Pharmacogenetics. 14:181–187. 2004. View Article : Google Scholar : PubMed/NCBI
27.	Hui L, DelMonte T and Ranade K: Genotyping using the TaqMan assay. Current Protocols Human Genetics. Haines JL: John Wiley; New York: 2008, View Article : Google Scholar
28.	Mitani Y, Lezhava A, Sakurai A, et al: Rapid and cost-effective SNP detection method: application of SmartAmp2 to pharmaco-genomics research. Pharmacogenomics. 10:1187–1197. 2009. View Article : Google Scholar : PubMed/NCBI
29.	Morita A, Nakayama T, Doba N, Hinohara S, Mizutani T and Soma M: Genotyping of triallelic SNPs using TaqMan PCR. Mol Cell Probes. 21:171–176. 2007. View Article : Google Scholar : PubMed/NCBI
30.	Livak KJ: Allelic discrimination using fluorogenic probes and the 5′ nuclease assay. Genet Anal. 14:143–149. 1999.