|Year : 2014 | Volume
| Issue : 2 | Page : 86-90
TPMT gene polymorphism detection by conventional PCR in pediatric acute lymphoblastic leukemia and its toxic effect
Dalal M.N.E. El-Kaffash1, Hoda M.A.F. Hassab2, Abla A AbouZeid1, Rania S Swelem1, Mona M Tahoun1
1 Department of Clinical Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
2 Department of Pediatric, Faculty of Medicine, Alexandria University, Alexandria, Egypt
|Date of Submission||06-Jan-2014|
|Date of Acceptance||09-Feb-2014|
|Date of Web Publication||30-Aug-2014|
Rania S Swelem
Clinical Pathology Department, Faculty of Medicine, Alexandria University
Source of Support: None, Conflict of Interest: None
Introduction The purine analog mercaptopurine is a key medication for the successful treatment of childhood acute lymphoblastic leukemia, particularly for the consolidation and continuation therapies. Thiopurine S-methyltransferase (TPMT) catalyzes the inactivation of mercaptopurine. TPMT single-nucleotide polymorphisms can prospectively identify patients at higher risk for mercaptopurine toxicity.
Patients and methods The TPMT genotype was determined by in-house conventional PCR followed by digestion of the product with restriction enzymes, MwoI FastDigest and AccI FastDigest. The study was carried out on a total of 80 participants: 40 pediatric patients with standard risk B-cell acute lymphoblastic leukemia and 40 age-matched and sex-matched healthy controls. Mercaptopurine was given to the patients in consolidation phase with oral dose of 75 mg/m 2 daily for 4 weeks. Toxicity of the drug was assessed at the end of this phase by complete blood profile and liver function tests.
Results In the patients group, 97.5% were of the wild-type homozygous TPMT*1/*1 genotype and 2.5% were of the heterozygous TPMT*1/*3A genotype. In the control group, we identified 90% with the TPMT*1/*1 genotype, 7.5% with the TPMT*1/*3A genotype, and 2.5% with the TPMT*1/*3C genotype. Among the wild-type *1/*1 genotype patients in the patient group, 32.5% of patients suffered from either hepatoxicity and/or myelosuppression.
Conclusion The homozygous wild-type TPMT*1/*1 genotype was the most frequent genotype in both cases and controls. TPMT*1/*3A was the most prevalent mutant genotype in this study. Although some patients had wild-type allele genotyping, they developed signs of toxicity.
Keywords: B-cell acute lymphoblastic leukemia, mercaptopurine, polymorphism, thiopurine S-methyltransferase
|How to cite this article:|
El-Kaffash DM, Hassab HM, AbouZeid AA, Swelem RS, Tahoun MM. TPMT gene polymorphism detection by conventional PCR in pediatric acute lymphoblastic leukemia and its toxic effect
. Egypt J Haematol 2014;39:86-90
|How to cite this URL:|
El-Kaffash DM, Hassab HM, AbouZeid AA, Swelem RS, Tahoun MM. TPMT gene polymorphism detection by conventional PCR in pediatric acute lymphoblastic leukemia and its toxic effect
. Egypt J Haematol [serial online] 2014 [cited 2018 May 23];39:86-90. Available from: http://www.ehj.eg.net/text.asp?2014/39/2/86/139775
| Introduction|| |
Acute lymphoblastic leukemia (ALL) is a malignant disease that occurs because of accumulation of lymphoid precursor cells that have their origin in the marrow or thymus. The leukemic cells persistently accumulate in the intramedullary and extramedullary sites, constantly competing with normal hematopoietic cell production and function  .
Therapy for childhood ALL consists of protocols in which specific treatment approaches may differ but consistently comprise three major treatment phases: remission induction therapy followed by consolidation/intensification therapy, and then continuation/maintenance treatment to eliminate the residual leukemic cells, with cure rates now surpassing 80%  .
The purine analog mercaptopurine is a key medication for the successful treatment of childhood ALL, in particular for the consolidation and continuation therapies. Thiopurine S-methyltransferase (TPMT) is a cytoplasmic enzyme that preferentially catalyzes the S-methylation (inactivation) of aromatic and heterocyclic sulfhydryl compounds, which include anticancer thiopurine groups such as mercaptopurine. These drugs are transformed enzymatically by three competitive enzymatic pathways  .
The TPMT gene is localized on chromosome 6p22.3 and is ˜34 kb in length and encodes a 245-amino acid peptide with a molecular mass of ˜35 kDa  . TPMT activity measurement and genotyping methods can be used to diagnose TPMT deficiency; however, TPMT polymorphism detection is more frequently used  .
It was stated that the variant alleles TPMT*2 (238G > C), TPMT*3A (460G > A and 719A > G), and TPMT*3C (only 719A > G) account for at least 95% of low or intermediate TPMT enzyme activity. About 90% of individuals have two wild-type TPMT alleles (TPMT*1) and high enzyme activity, about 10% of individuals inherit one wild-type TPMT allele and one nonfunctional variant allele and have intermediate activity, and the rare (one in 300) individuals who inherit two nonfunctional variant alleles are completely TPMT deficient , .
Thiopurine toxicity was evaluated according to complete blood count (CBC) findings and liver function tests  . Myelotoxicity in ALL patients has been defined according to the following criteria: leukopenia less than 3.0 × 10 9 /l, neutropenia less than 1.5 × 10 9 /l, and thrombocytopenia less than 75 × 10 9 /l. Hepatic toxicity was defined as a greater than two-fold increase in serum liver transaminases (AST and ALT)  .
| Patients and methods|| |
This study was carried out on a total of 80 pediatric patients divided into two groups. The first group included 40 patients with B-cell acute lymphoblastic leukemia (B-ALL), 23 boys and 17 girls, and 21 had ages equal to or less than 5 years, whereas the remaining 19 were above the age of 5 years. The second group included 40 age-matched and sex-matched healthy controls. Informed consent was obtained from the parents of both patients and controls before initiation of the study, and the study was approved by the Ethics Committee of the Faculty of Medicine, Alexandria University. Mercaptopurine was given to ALL patients in the consolidation phase with an oral dose of 75 mg/m 2 daily for 4 weeks. Toxicity of the drug was assessed at the end of this phase.
All the patients in the present study were subjected to full history taking, thorough clinical examination, and laboratory investigations including CBC performed on Sysmex XT-1800 (Siemens Healthcare Diagnostics Inc., USA)  , liver function tests (ALT and AST) performed on RXL Max (Siemens Healthcare Diagnostics Inc.)  , bone marrow aspirate for patients at diagnosis and for follow-up after therapy  , immunophenotyping for patients at diagnosis only using Becton Dickinson FACS Calibur Flow Cytometer (Becton Dickinson, BD Biosciences, San Jose, California, USA) equipped with CellQuest Software (BD Biosciences, San Jose, California, USA)  , and in-house conventional PCR for detection of TPMT gene polymorphism using thermal cycler Biocycler TC-S (HVD Life Science, Vienna, Austria), and finally digestion of PCR-amplified DNA product by restriction endonuclease enzymes, Mwol FastDigest and AccI FastDigest.
In brief, genomic DNA was extracted from EDTA whole blood samples by the column method using Qiagen DNA Extraction Kit (QIAamp Genomic Blood DNA Purification Kit; Qiagen, Hilden city, Germany). Detection of TPMT gene polymorphism was performed by in-house conventional PCR technique using Maxima Hot Start PCR Master Mix (Fermentas, Burlington city, Ontaria, Canada) and the following TPMT-specific primers (Fermentas): for TPMT exon 7 - TPMT7F 5?-ACGCAGACGTGAGATCCTAAT-3? and TPMT7R 5?-TGATTGAGCCACAAGCCTTA-3? -and for TPMT exon 10 - TPMT10F 5?-AATCCCTGATGTCATTCTTCATAG-3? and TPMT10R 5?-CACATCATAATCTCCTCTCC-3?.
In a total volume of 25 μl, the reaction mixture was incubated at 95°C for 5 min for initial denaturation, followed by 40 cycles of PCR, each of which consisted of denaturation at 94°C for 30 s, annealing at 55°C for 35 s, and extension at 72°C for 40 s, and then a final extension step at 72°C for 7 min. Next, PCR amplicon was separated by electrophoresis on 2% agarose gel at 100 V for 30 min, and the bands were visualized by ultraviolet transillumination after staining with ethidium bromide, and photographed using Wealtec Dolphin-Doc Imaging System (Wealtec Corp., USA). After amplification, the PCR products were digested by AccI FastDigest restriction enzyme for 719A > G at exon 10 and by MwoI FastDigest restriction enzyme for 460G > A at exon 7. The digestion products were separated by agarose gel electrophoresis and were detected under ultraviolet light after staining with ethidium bromide. The expected pattern after digestion of PCR product at exon 7 using restriction endonuclease MwoI was one of the following [Figure 1]: two fragments of 172 and 246 bp for the homozygous wild-type allele 460G > A, three fragments of 172, 246, and 418 bp for the heterozygous allele 460G > A, or one uncut fragment of 418 bp for mutant homozygous allele 460G > A. The expected pattern after digestion of the amplified product at exon 10 using restriction endonuclease AccI was one of the following [Figure 2]: one undigested fragment of 401 bp for wild-type allele 719A > G, three fragments of 151, 250, and 401 bp for the heterozygous allele 719A > G, or two fragments of 151 and 250 bp for mutant homozygous allele 719A > G , .
|Figure 1: Gel electrophoresis results after digestion of PCR product at exon 7 by MwoI for detection of 460G > A mutation. C, cut (after digestion); U, uncut (before digestion).|
Click here to view
|Figure 2: Gel electrophoresis results after digestion of PCR product at exon 10 by AccI for detection of 719A > G mutation. C, cut (after digestion); U, uncut (before digestion).|
Click here to view
TPMT*3A was detected if both exons showed mutations. TPMT*3B would be detected if 460G > A mutation only was present. TPMT*3C was detected if there was mutation 719A > G only  .
Data were fed into the computer using IBM SPSS Software Package (version 20.1; IBM, USA) for statistical analysis.
| Results|| |
The results of the study showed that the only TPMT alleles detected were TPMT*1/*1 wide-type, *1/*3A, and *1/*3C mutant genotypes [Figure 3] and [Figure 4]. The frequency of TPMT alleles of the patients were 39 (97.5%) TPMT*1/*1 wild-type and one (2.5%) heterozygous mutant *1/*3A. In the control group, 36 (90%) had the wild-type *1/*1 genotype, three (7.5%) had the *1/*3A genotype, and one (2.5%) had the *1/*3C genotype [Table 1]. Regarding Hardy-Weinberg equilibrium, there was no statistical significance between the observed and the expected results of the genotypes (P = 0.9938).
|Figure 3: Gel electrophoresis showed digestion of PCR product at exon 7 by MwoI restriction endonuclease. Lanes 1, 2, and 4– 6 showed wild TPMT*1/*1. Lanes 3 and 7 showed heterozygous mutation of 460G > A at exon 7. Lane 8 showed 100 bp DNA ladder. TPMT, thiopurine|
Click here to view
|Figure 4: Gel electrophoresis shows digestion of PCR product at exon 10 by AccI restriction endonuclease. Lanes 1– 5, 7, and 8 showed wild TPMT*1/*1. Lane 6 showed heterozygous mutation of 719A > G at exon 10. Lane 9 showed 100 bp DNA ladder. TPMT, thiopurine S-methyltransferase.|
Click here to view
No homozygous mutant genotypes were detected in any group and no statistically significant difference was detected between both groups regarding the different genotypes.
On clinical basis, among the wild-type TPMT*1/*1 genotype, pallor (79.5%) was the most common presenting symptom followed by fever (74.4%), hepatomegaly (69.2%), splenomegaly (61.5%), lymphadenopathy (57.5%), and bone pain (35.9%), whereas purpura and ecchymosis were the least common (28.2%). The only patient with the TPMT*1/*3A genotype presented with pallor, bone pain, hepatomegaly, and splenomegaly.
We also found that, among the wild-type TPMT*1/*1 genotype patients in the patient group, 32.5% suffered from either myelosuppression and/or hepatotoxicity. About 15% of the patients had leukopenia and absolute neutropenia, 12.5% had neutropenia alone, 2.5% had leukopenia, absolute neutropenia, and thrombocytopenia, and only 2.5% had leukopenia with hepatotoxicity.
| Discussion|| |
In the present study, genotyping of TPMT polymorphism using conventional PCR and digestion of PCR products by restriction enzymes, MwoI and AccI, in B-ALL pediatric patients and a control group to detect mutations at exon 7 and exon 10, respectively, was performed. Homozygous wild-type *1/*1 genotype was the most prevalent genotype in the two groups. Allele frequency obeyed the Hardy-Weinberg equation. On statistically analyzing the difference in genotype distribution among the two groups, these differences did not reach statistical significance.
In accordance to our results, in a study conducted by Ayesh et al.  , TPMT genotyping revealed that 98.2% of the patients had the wild-type TPMT*1/*1 genotype and only 1.8% had the heterozygous mutant genotype. Similar findings were proved by Oender et al.  , Tantawy et al.  , and Toft et al.  .
A study by Peregud-Pogorzelski et al.  showed that 92.12% of 203 ALL patients had the wild-type TPMT*1/*1 genotype, 7.39% had the heterozygous mutant genotype, and only 0.49% had the homozygous mutant genotype. Among 397 participants in the control group, 92.95% had wild-type, 6.8% had the heterozygous mutant genotype, and 0.25% had the homozygous mutant genotype. Similar results were found by Rossino et al.  and Azad et al.  .
In contrast to the present results, in a study conducted by Aboul Naga et al.  , TPMT genotyping showed that 76.9% had the wild-type and 23.1% had the mutant-type.
A study conducted by Chrzanowska et al.  detected that 88.8% were carrying the wild-type TPMT*1/*1 genotype and 11.2% had the mutant heterozygous genotype. Similar results were found by Hongeng et al.  , Toft et al.  , and Oliveira et al.  .
In the current study, TPMT*1/*3A was the predominant heterozygous mutant genotype in both cases and controls (2.5 and 7.5%, respectively), whereas *1/*3C was detected in 2.5% of controls only.
Similar to our findings, Oender et al.  showed that, among 8.82% heterozygous mutant genotypes, 8.6% carried the mutant TPMT*3A genotype and only 0.22% carried the heterozygous mutant TPMT*3C genotype. In accordance with these results were those concluded by Toft et al.  on the Danish population and those by Ayesh et al.  . In addition, Hongeng et al.  stated that no variant alleles, TPMT*2, TPMT*3A, TPMT*3B, or homozygous mutant TPMT genotypes, were found.
Aboul Naga et al.  study stated that, of the heterozygous mutant genotypes, TPMT*1/*3B was the most common (7.69%) followed by TPMT*1/*2 (5.77%) then TPMT*2/*2 and TPMT*3A, which had the same frequencies (3.85%). Only 1.92% showed the homozygous mutant genotype TPMT*3B/*3B. Chrzanowska et al.  showed that 11.2% had the mutant heterozygous genotypes, wherein 10.2% were TPMT*1/*3A and 1.0% was TPMT*1/*2.
The study conducted by Ayesh et al.  showed that 30.3% of patients developed side effects associated with 6-mercaptopurine (6-MP) therapy; all had the wild-type TPMT genotype except only 1.8% who had a heterozygous TPMT*3A genotype. Similar findings were detected by Ban et al.  .
In contrast, Hongeng et al.  found that patients who did not have genetic variation of this gene had no hematopoietic toxicity during treatment with full doses of mercaptopurine and thioguanine, whereas patients who had genetic variations developed neutropenia during full dose of either mercaptopurine or thioguanine administration. None of these patients developed severe hematopoietic toxicity complications after the doses of either mercaptopurine or thioguanine were reduced by 50-75% of the original dose.
The discrepancy between our results as a whole and those of other studies may be explained by the low proportion of eligible cases and controls included in the analysis and variations in genotype frequency among different ethnic groups. Such variations resulted in differences in expression of the toxic effect of 6-MP drug therapy. In addition, the absence of statistically significant differences between groups might be due to the still small sample sizes, particularly to the low number of heterozygous patients.
Commonly, wild-type is the predominant genotype with no evidence of 6-MP toxic side effects, and those with mutant genotypes sometimes showed signs of 6-MP drug toxicity. In our study, we found that some of the patients who had wild-type TPMT suffered from myelosuppression and/or hepatotoxicity. The only mutant patient with TPMT*1/*3A genotype showed leukopenia, absolute neutropenia, thrombocytopenia, and elevated ALT enzyme at diagnosis; however, after treatment all these results improved except for ALT. The presence of these abnormal results in the patients at diagnosis was mainly due to lymphoblasts infiltration of the bone marrow and/or extramedullary organs. However, after treatment, it was suggested that abnormal CBC findings and abnormal liver functions in those patients might be due to increased thioguanine nucleotides (TGNs) inside the cells and decreased activity of TPMT enzyme despite absence of polymorphism. In addition, infections or any other unknown environmental factors not related to 6-MP treatment might be responsible for these abnormal laboratory results.
Pretreatment determination of TPMT status (genotype and enzyme activity) and measuring TGN inside the erythrocytes using high-performance liquid chromatography were mandatory to predict 6-MP toxicity in patients who have wild-type TPMT*1/*1.
Similarly, detection of mutant genotypes in the control group confirmed that screening of TPMT genotype should be performed before initiating therapy with thiopurine drugs.
Thus, TPMT status and pharmacogenetically-guided thiopurine therapy should be performed to individually optimize 6-MP therapy and avoid adverse reactions to this drug, as TPMT represents a determinant of 6-MP response and ALL outcome.
| Acknowledgements|| |
| References|| |
|1.||Caldwell B. Acute leukemia. In: Ciesla B, editor. Haemtology in practice. 1 st ed. Philadelphia, Pennsylvania: F.A. Davis Company: FA Davis; 2007. 159-185. |
|2.||Campana D, Pui CH. Childhood acute lymphoblastic leukemia. In: Hoffbrand V, Catovsky D, Tuddenham EGD, Green AR, editors. Postgraduate heamatology 6 th ed. Oxford; Wiley-Blackwell Publishing; 2011. 448-462. |
|3.||Zhou SF, Chowbay B. Clinical significance of thiopurine S-methyltransferase gene polymorphism. Curr Pharmacogenomics 2007; 5 : 103-115. |
|4.||Coulthard SA, Rabello C, Robson J, Howell C, Minto L, Middleton PG, et al. A comparison of molecular and enzyme-based assays for the detection of thiopurine methyltransferase mutations. Br J Haematol 2000; 110 :599-604. |
|5.||Schaeffeler E, Fischer C, Brockmeier D, Wernet D, Moerike K, Eichelbaum M, et al. Comprehensive analysis of thiopurine S-methyltransferase phenotype-genotype correlation in a large population of German-Caucasians and identification of novel TPMT variants. Pharmacogenetics 2004; 14 :407-417. |
|6.||Cheng Q, Yang W, Raimondi SC, Pui SC, Relling MV, Evans WE. Karyotypic abnormalities create discordance of germline genotype and cancer cell phenotypes. Nature Genet 2005; 37 :878-882. |
|7.||Aboul Naga S, Ebid GT, Fahmi HM, Zamzam MF, Mahmoud S, Hafez HF, et al. Effects of thiopurine S-methyltransferase genetic polymorphism on mercaptopurine therapy in pediatric ALL. J Am Sci 2011; 7 :337-346. |
|8.||US Department of Health and Human Services. National Institutes of Health. National Cancer Institute. Common Terminology Criteria for Adverse Events (CTCAE), version 4.0, 14 June 2010. Available at: http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_8.5x11.pdf. [Last accessed on 2014 April 3]. |
|9.||Briggs C, Bain BJ. Basic haematological techniques. In: Lewis SM, Bain BJ, Bates I, editors. Dacie and Lewis practical haematology. 11 th ed. Germany: Elselvier Ltd 2011. 23-53. |
|10.||Dufour DR. Liver diseases. In: Ashwood ER, Bruns DE, editors. Tietz textbook of clinical chemistry and molecular diagnostics. 4th ed. St. Louis: Elsevier Saunders 2006. 1777-1827. |
|11.||Bates I, Burthem J. Bone marrow biopsy. In: Lewis SM, Bain BJ, Bates I, editors. Dacie and Lewis practical haematology. 11 th ed. Germany: Elselvier Ltd 2011. 124-137. |
|12.||Matutes E, Morilla R, Morilla AM. Immunophenotyping. In: Bain BJ, Bates I, Laffan MA, Lewis SM, editors. Dacie and Lewis practical haematology 11th ed. London: Elsevier, Churchill Livingstone 2011. 353-371. |
|13.||Oender K, Lanschuetzer CM, Paulweber B, Laimer M, Klauseqqer A, 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 :396-400. |
|14.||Ronen O, Cohen SP, Rund D. Evaluating frequencies of thiopurine S-methyl transferase (TPMT) variant alleles in Israeli ethnic subpopulations using DNA analysis. Isr Med Assoc J 2010; 12 :721-725. |
|15.||Ayesh BM, Harb WM, Abed AA. Thiopurine methyltransferase genotyping in Palestinian childhood acute lymphoblastic leukemia patients. BMC Hematol 2013; 13 :1-4. |
|16.||Tantawy A, Adly A, AlGhoroury E, Abdel Maksoud M. Prevalence of thiopurine methyltransferase gene polymorphism in Egyptian children with acute lymphoblastic leukemia. Haematologica 2010; 95 :486. |
|17.||Toft N, Nygaard U, Gregers J, Schmiegelow K. Genetic analyses of thiopurine methyltransferase polymorphisms in Greenlandic and Danish populations. Acta Paediatr 2006; 95 :1665-1667. |
|18.||Peregud-Pogorzelski JA, Tetera-Rudnicka E, Kurzawski M, Brodkiewicz A, Adrianowska N, Mlynarski W, et al. Thiopurine S-methyltransferase (TPMT) polymorphisms in children with acute lymphoblastic leukemia, and the need for reduction or cessation of 6-mercaptopurine doses during maintenance therapy: the polish multicenter analysis. Pediatr Blood Cancer 2011; 57 :578-582. |
|19.||Rossino R, Vincis C, Alves S, Prata MJ, Macis MD, Nucaro AL, et al. Frequency of the thiopurine S-methyltransferase alleles in the ancient genetic population isolate of Sardinia. J Clin Pharm Ther 2006; 31 :283-287. |
|20.||Azad M, Kaviani S, Soleimani M, Noruzinia M, Hajfathali A. Common polymorphism's analysis of thiopurine S-methyltransferase (TPMT) in Iranian population. Yakhteh Med J 2009; 11 :311-316. |
|21.||Chrzanowska M, Kuehn M, Andowska DJL, Wski MK, Droedzik M. Thiopurine S-methyltransferase phenotype-genotype correlation in children with acute lymphoblastic leukemia. Acta Pol Pharm 2012; 69 :405-410. |
|22.||Hongeng S, Sasanakul W, Chuansumrit A, Pakakasama S, Chattananon A, Hathirat P. Frequency of thiopurine S-methyltransferase genetic variation in Thai children with acute leukemia. Med Pediatr Oncol 2000; 35 :410-414. |
|23.||Oliveira E, Alves S, Quental S, Ferreira F, Norton L, Costa V, et al. Outcome in acute lymphoblastic leukemia: influence of thiopurine methyltransferase genetic polymorphisms. Int Cong Ser 2006; 1288 :789-791. |
|24.||Ban H, Andoh A, Tanaka A, Tsujikawa T, Sasaki M, Saito Y, et al. Analysis of thiopurine S-methyltransferase genotypes in Japanese patients with inflammatory bowel disease. Intern Med 2008; 47 :1645-1648. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4]