Iranian Journal of Pathology

Thiopurine S-methyltransferase and Pemphigus Vulgaris: A Phenotype-Genotype Study

Document Type : Original Research

Authors

Maral Mokhtari 1
Farzaneh Mostanbet 1
Saideh Nekooee Fard 1
Golsa Shekarkhar 1
Mozhdeh Sepaskhah 2
Maryam Sadat Sadati 2

1 Pathology Department, Shahid Faghihi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran

2 Dermatology Department, Shahid Faghihi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran

https://doi.org/10.30699/ijp.2020.121365.2320
Abstract
Background & Objective:  Thiopurine drugs are considered as a treatment modality in various autoimmune disorders including pemphigus vulgaris (PV). These drugs are metabolized by an enzyme “Thiopurine S-methyl transferase” (TPMT). Various variants of this enzyme may have decreased activity leading to serious drug side effects. To investigate the phenotype and genotype of TPMT in PV patients receiving thiopurine drugs.
Methods:  50 patients (29 women and 21 men) with pemphigus vulgaris treating with standard dose of Thiopurine drugs were selected. Sex, age, result of liver function test and complete blood count were recorded. Genotyping of two common non-functional allele (TPMT*2 and TPMT*3C) by Allele-specific and RFLP-PCR was performed. TPMT enzymatic level was determined by an ELISA based method.
Results:  Of patients, 36 (72%) were found to have normal TPMT level; and 12, (24%) had higher level of enzyme and 2, 4% had low TPMT enzyme, but none of the patients showed mutant TPMT*2 and TPMT*3C alleles. None of the patients showed hepatotoxicity and bone marrow suppression.
Conclusion:  The phenotypic assay based on ELISA method may have false positive and misleading results but genotyping using PCR-RFLP and allele specific PCR is accurate, simple and cost-effective and can be used in patients decided to undergo thiopurine treatment.

Keywords

Allele specific PCR
PCR-RFLP
Thiopurine S- methyl transferase
Thiopurine drugs
ELISA test

Subjects

  1. Diaz LA, Giudice GJ. End of the century overview of skin blisters. Arch Dermatol. 2000;136(1):106-12. [DOI:10.1001/archderm.136.1.106] [PMID]
  2. Broussard KC, Leung TG, Moradi A, Thorne JE, Fine JD. Autoimmune bullous diseases with skin and eye involvement: Cicatricial pemphigoid, pemphigus vulgaris, and pemphigus paraneoplastica. Clin Dermatol. 2016;34(2):205-13. [DOI:10.1016/j.clindermatol.2015.11.006] [PMID]
  3. Gregoriou S, Efthymiou O, Stefanaki C, Rigopoulos D. Management of pemphigus vulgaris: challenges and solutions. Clin Cosmet Investig Dermatol. 2015;8(1):521-7. [DOI:10.2147/CCID.S75908] [PMID] [PMCID]
  4. Chande N, Patton PH, Tsoulis DJ, Thomas BS, MacDonald JK. Azathioprine or 6-mercaptopurine for maintenance of remission in Crohn's disease. Cochrane Database Syst Rev. 2015(10):Cd000067. [DOI:10.1002/14651858.CD000067.pub3] [PMID]
  5. Gordon M, Taylor K, Akobeng AK, Thomas AG. Azathioprine and 6-mercaptopurine for maintenance of surgically-induced remission in Crohn's disease. Cochrane Database Syst Rev. 2014(8):Cd010233. [DOI:10.1002/14651858.CD010233.pub2] [PMCID]
  6. Ujiie S, Sasaki T, Mizugaki M, Ishikawa M, Hiratsuka M. Functional characterization of 23 allelic variants of thiopurine S-methyltransferase gene (TPMT*2 - *24). Pharmacogenet Genomics. 2008;18(10):887-93. [DOI:10.1097/FPC.0b013e3283097328] [PMID]
  7. McLeod HL, Pritchard SC, Githang'a J, Indalo A, Ameyaw MM, Powrie RH, et al. Ethnic differences in thiopurine methyltransferase pharmacogenetics: evidence for allele specificity in Caucasian and Kenyan individuals. Pharmacogenetics. 1999;9(6):773-6. [DOI:10.1097/00008571-199912000-00012] [PMID]
  8. Oselin K, Anier K, Tamm R, Kallassalu K, Maeorg U. Determination of thiopurine S-methyltransferase (TPMT) activity by comparing various normalization factors: reference values for Estonian population using HPLC-UV assay. J Chromatogr B Analyt Technol Biomed Life Sci. 2006;834(1-2):77-83. [DOI:10.1016/j.jchromb.2006.02.031] [PMID]
  9. Zhang B, Xu XW, Zeng XJ, Li DK. Correlation of thiopurine methyltransferase activity and 6-thioguanine nucleotide concentration in Han Chinese patients treated with azathioprine 25 to 100 mg: A 1-year, single-center, prospective study. Curr Ther Res Clin Exp. 2006;67(4):270-82. [DOI:10.1016/j.curtheres.2006.07.002] [PMID] [PMCID]
  10. Zhang LR, Song DK, Zhang W, Zhao J, Jia LJ, Xing DL. Efficient screening method of the thiopurine methyltransferase polymorphisms for patients considering taking thiopurine drugs in a Chinese Han population in Henan Province (central China). Clin Chim Acta. 2007;376(1-2):45-51. [DOI:10.1016/j.cca.2006.07.010] [PMID]
  11. Keizer-Garritsen JJ, Brouwer C, Lambooy LH, Ter Riet P, Bokkerink JP, Trijbels FJ, et al. Measurement of thiopurine S-methyltransferase activity in human blood samples based on high-performance liquid chromatography: reference values in erythrocytes from children. Ann Clin Biochem. 2003;40(Pt 1):86-93. [DOI:10.1258/000456303321016222] [PMID]
  12. Booth RA, Ansari MT, Loit E, Tricco AC, Weeks L, Doucette S, et al. Assessment of thiopurine S-methyltransferase activity in patients prescribed thiopurines: a systematic review. Ann Intern Med. 2011;154(12):814-23, w-295-8. [DOI:10.7326/0003-4819-154-12-201106210-00009] [PMID]
  13. Nguyen CM, Mendes MA, Ma JD. Thiopurine methyltransferase (TPMT) genotyping to predict myelosuppression risk. PLoS Curr. 2011;3(1):Rrn1236. [DOI:10.1371/currents.RRN1236] [PMID] [PMCID]
  14. Azimi F, Jafariyan M, Khatami S, Mortazavi Y, Azad M. Assessment of Thiopurine-based drugs according to Thiopurine S-methyltransferase genotype in patients with Acute Lymphoblastic Leukemia. Iran J Ped Hematol Oncol. 2014;4(1):32-8.
  15. DiPiero J, Teng K, Hicks JK. Should thiopurine methyltransferase (TPMT) activity be determined before prescribing azathioprine, mercaptopurine, or thioguanine?. Cleve Clin J Med. 2015;82(7):409-13. [DOI:10.3949/ccjm.82a.14106] [PMID]
  16. Tsuchiya A, Aomori T, Sakamoto M, Takeuchi A, Suzuki S, Jibiki A, et al. Effect of genetic polymorphisms of azathioprine-metabolizing enzymes on response to rheumatoid arthritis treatment. Pharmazie. 2017;72(1):22-8.
  17. Sanderson JD. TPMT Testing Before Starting Azathioprine or Mercaptopurine: Surely Just Do It?. Gastroenterology. 2015;149(4):850-3. [DOI:10.1053/j.gastro.2015.08.040] [PMID]
  18. Lennard L. Implementation of TPMT testing. B J Clin Pharmacol. 2014; 77(4):704-14. [DOI:10.1111/bcp.12226] [PMID] [PMCID]
  19. Dezhgir A, Talebzadeh T, Hosseini IA, Altafi D, Hojatian H, Mehrfard R, et al. The prevalence of polymorphisms of thiopurine s-methyltransferase gene In Iranian alopecia areata patients. Int Res J Med Med Sci. 2018;6(3):67-78. [DOI:10.30918/IRJMMS.63.18.034]
  20. Azad M, Kaviani S, Soleymani M, Nourouzinia M, Hajfathali A. Common polymorphism's analysis of thiopurine S-methyltransferase (TPMT) in Iranian population. Yakhteh Med J. 2009;11(3):311-6.
  21. Bahari A, Hashemi M, Bari Z, Moazeni-Roodi A, Kaykhaei MA, Narouie B. Frequency of thiopurine S-methyltransferase (TPMT) alleles in southeast Iranian population. Nucleosides Nucleotides Nucleic Acids. 2010;29(3):237-44. [DOI:10.1080/15257771003720418] [PMID]
  22. Relling MV, Gardner EE, Sandborn WJ, Schmiegelow K, Pui CH, Yee SW, et al. Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing. Clin Pharmacol Ther. 2011;89(3):387-91. [DOI:10.1038/clpt.2010.320] [PMID] [PMCID]
  23. Bahrehmand F, Vaisi-Raygani A, Kiani A, Bashiri H, Zobeiri M, Tanhapour M, et al. Whole-Blood Thiopurine S-Methyltransferase Genotype and Phenotype Concordance in Iranian Kurdish Ulcerative Colitis (UC) Patients. Clin Lab. 2017;63(5):947-54. [DOI:10.7754/Clin.Lab.2017.161201] [PMID]
  24. Oender K, Lanschuetzer CM, Laimer M, Klausegger A, Paulweber B, Kofler B, et al. Introducing a fast and simple PCR-RFLP analysis for the detection of mutant thiopurine S-methyltransferase alleles TPMT*3A and TPMT*3C. J Eur Acad Dermatol Venereol. 2006;20(4):396-400. [DOI:10.1111/j.1468-3083.2006.01459.x] [PMID]
  25. Thompson AJ, Newman WG, Elliott RA, Roberts SA, Tricker K, Payne K. The cost-effectiveness of a pharmacogenetic test: a trial-based evaluation of TPMT genotyping for azathioprine. Value Health. 2014;17(1):22-33. [DOI:10.1016/j.jval.2013.10.007] [PMID]
  26. Mozafari S, Einollahi N, Shahgholi E, Vardasbi S, Golestani A. Thiopurine S-Methyltransferase Assay by HPLC in Acute Lymphoblastic Leukemia Patients and a Healthy Iranian Population. Acta Med Iran. 2018;56(5):301-7.
  27. Fong WY, Ho CC, Poon WT. Comparison of direct sequencing, real-time PCR-high resolution melt (PCR-HRM) and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis for genotyping of common Thiopurine intolerant variant alleles NUDT15 c. 415C> T and TPMT c. 719A> G (TPMT* 3C). Diagnostics. 2017;7(2): E27. [DOI:10.3390/diagnostics7020027] [PMID] [PMCID]
  28. Ho CC, Fong WY, Lee YH, Poon WT. Novel Tetra-Primer ARMS-PCR Assays for Thiopurine Intolerance Susceptibility Mutations NUDT15 c. 415C> T and TPMT c. 719A> G (TPMT* 3C) in East Asians. Genes. 2017;8(10):E285. [DOI:10.3390/genes8100285] [PMID] [PMCID]
  29. Gisbert JP, Gomollon F. Thiopurine-induced myelotoxicity in patients with inflammatory bowel disease: a review. Am J Gastroenterol. 2008;103(7):1783-800. [DOI:10.1111/j.1572-0241.2008.01848.x] [PMID]
  30. Shipkova M, Niedmann PD, Armstrong VW, Oellerich M, Wieland E. Determination of thiopurine methyltransferase activity in isolated human erythrocytes does not reflect putative in vivo enzyme inhibition by sulfasalazine. Clin Chem. 2004;50(2):438-41. [DOI:10.1373/clinchem.2003.026096] [PMID]
  31. Brouwer C, De Abreu RA, Keizer-Garritsen JJ, Lambooy LH, Ament K, ter Riet PG, et al. Thiopurine methyltransferase in acute lymphoblastic leukaemia: biochemical and molecular biological aspects. Eur J Cancer. 2005;41(4):613-23. [DOI:10.1016/j.ejca.2004.10.027] [PMID]
Iranian Journal of Pathology
Volume 15, Issue 4
Autumn 2020
Pages 299-305

  • Receive Date 07 February 2020
  • Revise Date 09 March 2020
  • Accept Date 04 April 2020

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