SciELO - Scientific Electronic Library Online

vol.52 issue3Psychometric properties of the Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire (PISQ-12) on Chilean womenPerception of the healthcare accreditation system in Colombia author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand




Related links

  • On index processCited by Google
  • Have no similar articlesSimilars in SciELO
  • On index processSimilars in Google


Colombia Médica

On-line version ISSN 1657-9534

Colomb. Med. vol.52 no.3 Cali July/Sept. 2021  Epub Sep 30, 2021 

Original article

Susceptibility to thiopurine toxicity by TPMT and NUDT15 variants in Colombian children with acute lymphoblastic leukemia

Oscar Correa-Jimenez1

Juan José Yunis2

Adriana Linares-Ballesteros1  3

Isabel Sarmiento-Urbina1  4

1 Universidad Nacional de Colombia, Facultad de Medicina, Departamento de Pediatría, Bogotá, Colombia.

2 Universidad Nacional de Colombia, Facultad de Medicina, Instituto de Genética, Departamento de Patología, Bogotá, Colombia.

3 HOMI Fundación Hospital Pediátrico la Misericordia, Division of Pediatric Hematology and Oncology. Bogotá, Colombia.

4 Clínica del Country, Division of Pediatric Hematology and Oncology, Bogota, Colombia.



This study aimed to correlate the genetic profile of the NUDT15 and TPMT genes with the side effects of the treatment of pediatric patients with acute lymphoid leukemia who were undergoing maintenance therapy at a tertiary care hospital in 2017.


This was an analytical, longitudinal, observational study in which the genotypes of the genes of interest were determined by PCR allelic discrimination with TaqMan® probes in patients receiving chemotherapy during the maintenance phase in the Pediatric Hematology and Oncology Unit in 2017. Sociodemographic and clinical data corresponding to the first six months of their maintenance chemotherapy were collected, and the correlation between the genotypes obtained and the development of side effects during the maintenance phase of chemotherapy in these patients was evaluated.


Seventy pediatric patients were included in the study. Genetic analyses were carried out of these for NUDT15 and TPMT (rs1800462 and rs1800460) on 68 patients, while for the rs1142345 polymorphism, typing was achieved in 42 patients. 4/68 patients were heterozygous for NUDT15, and the same number of patients were heterozygous for rs1800462 and rs1142345, while for rs1800460, 6 heterozygous patients were identified. No statistically significant association was identified between the genetic variants and the outcomes of interest.


Studies with a larger population size are needed and the evaluation of other genetic variants that may influence the development of side effects during maintenance chemotherapy.

Keywords: Acute lymphoid leukemia; drug-related side effects and adverse reactions; pharmacogenetics; children; mercaptopurine; pharmacogenomic testing



la finalidad de este estudio fue evaluar las asociaciones entre los perfiles de los genes NUDT15 y TPMT con los efectos adversos del tratamiento de mantenimiento en pacientes pediátricos con Leucemia Linfoblástica Aguda atendidos en un hospital de referencia durante el 2017.


Este fue un estudio observacional analítico, de corte longitudinal en el que los genotipos de los genes de interés fueron determinados mediante PCR de discriminación alélica con sondas TaqMan® en pacientes que estaban recibiendo quimioterapia de mantenimiento en la Unidad de Oncohematología Pediátrica durante el 2017. Los datos clínicos y sociodemográficos correspondientes a los primeros 6 meses de sus tratamientos de mantenimiento fueron colectados, y se evaluó la correlación entre los genotipos identificados y el desarrollo de efectos secundarios en estos pacientes.


setenta pacientes fueron incluidos en el estudio, de estos, los análisis genéticos para NUDT15 y TPMT (rs1800462 and rs1800460) fueron realizados en 68 pacientes, en tanto que para el polimorfismo rs1142345 se logró la tipificación en 42 pacientes. 4/68 pacientes fueron heterocigotos para NUDT15 y el mismo número de pacientes fueron heterocigotos para rs1800462 and rs1142345, mientras que para rs1800460, 6 pacientes heterocigotos fueron identificados. No se identificaron asociaciones estadísticamente significantes entre las variants genéticas y los resultados clínicos de interés.


Estos hallazgos resaltan la importancia de realizar estudios de este tipo con un mayor número de sujetos de estudio, así como plantean la necesidad de evaluar otras variantes genéticas que podrían tener algún impacto en el desarrollo de efectos secundarios durante la quimioterapia de mantenimiento.

Palabras clave: leucemia linfoide aguda; efectos adversos; farmacogenética; niños; tiopurina; pruebas de farmacogenómica


1) Why was this study conducted?
Several studies carried out mainly in the United States and Asia have shown associations between the variants of the TPMT and NUDT15 genes with the toxicity outcomes in patients with acute lymphoid leukemia, the information referring to this topic is scarce in our country, so we decided to evaluate this association in our population which represents a sample with genetic admixture and in whom pharmacogenetic differences related to ancestry have been described.
2) What were the most relevant results of the study?
No statistically significant associations were identified between genetic variants in TPMT and NUDT15 with toxicity outcomes evaluated (myelotoxicity and liver toxicity, as well as the dose reduction or interruption of 6-MP).
3) What do these results contribute?
Our results point to the need to carry out national studies aimed at the search for “autochthonous biomarkers” that allow the identification of pediatric cancer patients who present a higher risk of developing toxicity outcomes during their treatments.


Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood, accounting for 25% of all malignancies in children under 15 years of age 1. It is classified into two large groups, according to the cell of origin. 85% of the cases correspond to ALL with B cell precursors and 15% with T cell 2,3. It occurs more frequently in children with Hispanic and Caucasian ancestry than in those of African ancestry 4,5. A few environmental risk factors have been associated with ALL in children (exposure to radiation and certain chemicals); however, these associations explain only a minority of cases 6.

One of the major milestones in the therapy of children with ALL was the development of an intensive regimen of 8 drugs, eight weeks of induction and consolidation, which formed the basis of the BFM (Berlin-Frankfurt-Münster) regimen, which is the backbone of current therapies for ALL. In addition, the chemotherapy strategy also includes intensification and maintenance therapy 6. Since the introduction of this therapeutic regimen, multiple collaborative clinical trials have been developed that have resulted in important advances on the survival of patients with ALL, reaching overall survival rates up to 90% 6,7.

Despite the improvements in the survival rate for most pediatric patients, relapses occur between 15% and 20% and are an important cause of morbidity and mortality in pediatric patients with ALL 8,9. At the same time, in the majority of multicenter trials of first-line treatment for ALL in children, up to 5% die due to the treatment toxic side effects10,11. Treatment-related deaths occur during recurrent and prolonged episodes of neutropenia and lymphopenia due to cytotoxic and immunosuppressive drugs or because leukemia itself could inhibit bone marrow recovery after induction therapy, especially in those patients classified as slow responders 10,12.

Technological progress has allowed the identification of genomic variations that determine characteristic pharmacogenomic patterns, responsible for the individual differences observed in response to treatment in terms of effectiveness or toxicity. Currently, the TPMT genotype is the main pharmacogenetic pattern with implications for treatment protocols for patients with ALL with treatment based on thiopurines 13. 6- mercaptopurine (6-MP) is specifically important in therapy maintenance, the longest phase during treatment. In patients with homozygous variants in TPMT, an 85%-90% dose reduction is required 14-17. Other genes involved in thiopurine metabolism have been evaluated for their behavior in ALL 15; Among these, the NUDT15 gene has recently emerged as an important prediction candidate for toxicity 18.

Yang et al. (19, through a study of GWAS and its respective replication cohort, identified two loci related to the dose intensity of 6-MP: rs1142345 in the TPMT gene and rs116855232 in NUDT15. This last variant was common among Asians and Hispanics but infrequent among Europeans, and not observed in Africans, thus contributing to differences in mercaptopurine tolerance related to ancestry. Also, this study showed that heterozygous patients for TPMT or NUDT15 variants required a 50% decrease in the 6-MP dose compared to those with wild type genotype 19. Based on these results, the same group of researchers, subsequently replicated these results in cohorts in Guatemala, Singapore, and Japan, adding functional genomics studies where they concluded that the presence of SNPs resulted in a loss of nucleotide diphosphatase activity, and patients with these defective NUDT15 alleles exhibited high levels of active thiopurine metabolites, thus increasing their toxicity 20. Scientific interest in this gene has increased in recent years. It has led to its association with age related reductions in mercaptopurine dose21, association with myelosuppression 22, development of severe toxicity 23, and 6-MP intolerance in specific population groups (24, making it an important candidate for evaluation in pharmacogenetic studies related to 6-MP tolerance, although with less support in the literature than the TPMT.

To date, there are no published reports of pharmacogenomic approaches to the treatment response and chemotherapy toxicity for ALL in the pediatric population of Colombia. This becomes even more important when pharmacogenetic differences related to ancestry have been described 25-28. It has been suggested that pharmacogenetic studies should be carried out independently or collaboratively in different Latin American countries to better understand these relationships 29. This study aimed to evaluate the association between the genetic profile of the NUDT15 and TPMT genes and the side effects during maintenance phase of treatment in a sample of Colombian pediatric patients with acute lymphoid leukemia.

Materials and Methods


This was an observational, longitudinal study carried out at a university-affiliated pediatric cancer center in Bogota, Colombia, that receives patients from rural and urban areas, and it is a national referral hospital for childhood cancer care.

Patients and data collection

Patients from 1 to 18 years of age diagnosed with acute lymphoblastic leukemia who were admitted to the Pediatric Hematology and Oncology Unit and who were in the maintenance therapy of BFM ALLIC 2009 chemotherapy protocol during 2017 were enrolled in this study. Patients younger than 1-year-old or those with Down syndrome as comorbidity were excluded from this study. For this study, a convenience sampling was carried out, including consecutively all the patients who met the selection criteria and agreed to participate. The characteristics and clinical results obtained with this protocol in our setting have been previously published (30. The study was conducted in accordance with the national policy for clinical research and followed the Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. This research protocol was approved by the ethics committees of the participating university and hospital. (Act 015-184-16 of 08/25/16 and CEI-38-16 of 08/11/16). Patients' blood samples and clinical data were collected after obtaining parental consent. Additionally, for each patient from 14 years of age, their assent to participate in this study was obtained.

The surplus of the whole blood sample taken as part of the usual controls was used for DNA extraction. In addition, patients were followed up monthly for six months in order to identify the occurrence of adverse effects due to treatment (myelosuppression, liver toxicity, or toxic death). During this follow-up, information regarding laboratory (white blood cell (WBC) count and liver function) and clinical data related to adjustments in treatment and the development of complications were collected. For this study, follow-up was carried out in conjunction with follow-up visits during maintenance, which for these patients are carried out on a monthly basis.


The outcomes of interest were side effects as myelotoxicity and liver toxicity and the dose reduction or interruption of 6-MP. As an exploratory analysis, we evaluated other outcomes as febrile neutropenia, relapse and deaths.

DNA extraction

The DNA extraction was carried out using the QIamp DNA Blood Mini Kit™, following the manufacturer's specifications and as previously reported (31,32. Briefly, 200 μL of whole blood was subjected to the series of steps described in the manufacturer's guide, using the technique of column filtration by microcentrifugation. The DNA quantification was carried out by spectrophotometry in a Nanodrop2000™, and this same equipment was used to measure the 260/280 nm ratio to determine the purity of the genetic material. The genetic material obtained was stored in 1.5 mL Eppendorf™ tubes at a temperature of -20° C until its use in polymerase chain reaction (PCR) assays.

TaqMan® 5' nuclease real-time PCR

The determination of the genotypes of the SNPs (rs1800462, rs1800460, rs1142345, and rs116855232) in the TPMT and NUDT15 genes was carried out in real-time PCR for allelic discrimination by 5' nuclease using commercially available TaqMan® SNP Genotyping Assays (Assay ID: C__12091552_30, C__30634116_20, C_____19567_20 and C_154823200_10, Thermo Fisher Scientific, USA), following the recommendations described in the "TaqMan® Allelic Discrimination Guide". For this, we used a PCR mixture containing 15 ng DNA (3 μL), 1.25 μL of the corresponding TaqMan® probe (20X), 12.5 μL TaqMan® Gene Expression Master Mix and 8.25 μL of nuclease-free ultrapure water for a final volume per reaction of 25 μL. All the tests were carried out in duplicate. The tests were carried out using the CFX96 Touch ™ Real-Time PCR Detection System (Bio-Rad), following the operating conditions suggested by the manufacturer for each probe.

Data analysis

Data are presented as median plus interquartile ranges for the quantitative variables, while their respective frequencies are shown for the qualitative data. The allelic and genotypic frequencies, as well as the Hardy-Weinberg equilibrium, were evaluated using snpReady. TPMT typing was carried out following the standards of the Clinical Pharmacogenetic Implementation Consortium 13. Briefly, TPMT haplotypes were determined based on the combinations of SNPs rs1800462, rs1800460 and rs1142345 as follows: CCT = * 1, GCT = * 2, CTC = * 3A, CTT = * 3B and CCC = * 3C. To determine the association between the genetic variants and the outcome of interest (some type of toxicity), the Chi-square statistic was used. The statistical analyses were performed using SPSS for Mac, version 2.0, and a value of p <0.05 was considered statistically significant.


General characteristics of study subjects

A total of 73 patients with ALL diagnosis that were in maintenance phase during 2017 in our institution. Of these, 70 patients agreed to participate in the project. From these patients, in two cases, it was not possible to extract DNA for genetic studies, so the statistical analyzes were based on the 68 patients in whom genetic studies were performed. Figure 1 presents a flow diagram showing the selection of patients included in the analysis.

Figure 1 Patients' inclusion flowchart. This diagram shows how the inclusion of patients in the study was carried out and the details of the performance of the genetic studies. 

Table 1 summarizes the general characteristics of the study population. A total of 70 patients were included in the study, with a male-to-female ratio of 1:1. The median age was six years when entering the maintenance therapy (IQR: 7 years). More than 50% of the study population was from Bogota, close to 42% were from small towns and rural areas. Regarding the outcomes evaluated, myelotoxicity in 25% Liver toxicity in 27% and reduction of 6-MP during follow-up was documented in 32.9% of patients. It is important to highlight that in our 6-month follow-up, there were no loss of patients.

Table 1 General characteristics and clinical follow-up of study population. 

Characteristic Measure
n or median % or IQR
Gender Female 35 50%
Age (Years old) 6 7
Lineage B 65 92.9%
T 5 7.1%
Risk Standard 7 10.0%
Intermediate 34 48.6%
High 29 41.4%
6-MP Reduction 23 32.9%
6-MP Interruption 20 28.6%
Febrile Neutropenia 8 11.4%
Relapse 11 15.7%
Death 2 2.9%
Follow-up 1 ALT (IU/L) 48.7 91.3
AST (IU/L) 37.0 25.3
WBC/mm3 3,315 1,370
Neutrophils/mm3 1,820 1,137.5
Lymphocytes/mm3 780 690
Total Bilirubin (mg/dL) 0.5 0.3
Follow-up 2 ALT (IU/L) 42.5 97.5
AST (IU/L) 33.55 31.13
WBC/mm3 3,060 2,160
Neutrophils/mm3 1,721 1,682.5
Lymphocytes/mm3 630 675
Total Bilirubin (mg/dL) 0.5 0.3
Follow-up 3 ALT (IU/L) 47.00 98.48
AST (IU/L) 34 37
WBC/mm3 3,095 14,87.5
Neutrophils/mm3 1,615 1,196.5
Lymphocytes/mm3 742 546
Total Bilirubin (mg/dL) 0.5 0.4
Follow-up 4 ALT (IU/L) 50.35 76.43
AST (IU/L) 32.55 23.31
WBC/mm3 3,400 1,640
Neutrophils/mm3 1,940 1,276.75
Lymphocytes/mm3 760 565
Total Bilirubin (mg/dL) 0.6 0.425
Follow-up 5 ALT (IU/L) 50.8 116.9
AST (IU/L) 37 42.1
WBC/mm3 3,360 1700
Neutrophils/mm3 1,850 1,707.5
Lymphocytes/mm3 840 465
Total Bilirubin (mg/dL) 0.6 0.465
Follow-up 6 ALT (IU/L) 65.1 101.9
AST (IU/L) 39.8 36.2
WBC/mm3 3370 1,920
Neutrophils/mm3 1,830 1,674.5
Lymphocytes/mm3 910 636
Total Bilirubin (mg/dL) 0.6 0.5

This table resumes the general clinical characteristics and follow-up data of the patients included in the study. For each laboratory data, the measurement system used has been noted. The data presented in the "Measure" columns corresponds to the n or medians (subcolumn n or median) and in the other subcolumn its percentage or interquartile range (IQR) is evidenced.

Allele and genotype frequencies

Of the 70 patients included in the study, genetic material with adequate quality and quantity was obtained in 68 cases (concentrations from 10 to 129 ng/µL and an average 260/280nm ratio of 1.87; optimal values between 1.8 and 2.0- 33,34). Although DNA extraction was carried out twice for the other two cases, no genetic material was obtained with the quality conditions required to carrying out the polymerase chain reaction assays. A total of 14 patients carried a susceptibility allele: 4 for NUDT15, 4 for rs1800462 (TPMT), 6 for rs1800460 (TPMT), and 4 for rs1142345 of TPMT. Figure 2 shown representative allelic discrimination.

Figure 2 Representative allelic discrimination plots for TaqMan genotyping assays. There were no homozygous patients for the mutated alleles of any of the polymorphisms studied. The graphs represent the groups of samples with the wild type allele aligned towards the X or Y axes according to the probe used for their recognition. The non-aligned dots correspond to the mutated allele. 

Table 2 shows the summary of allelic and genotype frequencies for each of the SNP's tested. All variants were found to be in Hardy-Weinberg equilibrium (HWE). Table 3 summarizes the typing of TPMT following the standards of the Clinical Pharmacogenetic Implementation Consortium" in the 42 patients in which the typing of the 3 SNPs was controlled 13. Briefly, TPMT haplotypes were determined based on the combinations of SNPs rs1800462, rs1800460 and rs1142345 as follows: CCT = * 1, GCT = * 2, CTC = * 3A, CTT = * 3B and CCC = * 3C.

Table 2 Allelic and genotypic frequencies of the polymorphisms of interest. 

Gene Annotation n Frequency HWE (p Value) LatinAmerica1 LatinAmerica2
NUDT15 Alleles rs116855232 68 0.802 n= 804 n= 974
C 132 0.97 0.994 0.953
T 4 0.03 0.006 0.047
Genotypes C/C 64 0.94
C/T 4 0.06
T/T 0 0
TPMT Alleles rs1800462 68 0.802 n= 500 n= 628
C 132 0.970 0.992 0.997
G 4 0.03 0.008 0.003
Genotypes C/C 64 0.94
C/G 4 0.06
G/G 0 0
Alleles rs1800460 68 0.703 n= 1,430 n= 7,010
T 6 0.044 0.026 0.045
C 130 0.956 0.974 0.955
Genotypes T/T 0 0
T/C 6 0.09
C/C 62 0.91
Alleles rs1142345 42 0.746 n= 1,232 n= 5,254
C 4 0.0476 0.041 0.052
T 80 0.9524 0.959 0.948
Genotypes C/C 0 0
C/T 4 0.10
T/T 38 0.90

This table shows the allelic and genotypic frequencies of the polymorphisms studied. All variants were found to be in Hardy-Weinberg equilibrium (column HWE). Additionally, the allelic frequencies identified in our study were compared with those reported in Latin American population noted in the ALFA project. There were no statistically significant differences between our frequencies and those reported in the ALFA project.

Table 3 TPMT typing 

Haplotypes *1 78
*2 2
*3A 4
Types *1/*1 36
*1/*2 2
*1/*3A 4

Frequencies of TPMT haplotypes based on the standards of the "Clinical Pharmacogenetic Implementation Consortium" are listed. Given that genotyping of rs1142345 was only possible in 42 subjects and that to carry out these classifications, it is necessary to have genotyping of the 3 TPMT loci. These data correspond only to the 42 patients in whom genotyping was completed.

The allele frequencies obtained in the present study were compared to the frequencies reported in the dbSNP databases for Latin American populations obtained from the ALFA (Allele Frequency aggregator) (Table 3). The database frequency of genotypes and phenotypes (dbGap) was developed to archive and distribute the data and results from studies that have investigated the interaction of genotype and phenotype in humans 35. The results obtained for each SNP analyzed did not show a statistically significant difference from the frequencies obtained in the ALFA project.

Genetics associations

Table 4 summarizes the results of the bivariate analyzes in which the correlation between the genetic variants of the SNPs of interest in NUDT15 and TPMT and the side effects of interest. No association was found between the outcomes of interest and the genetic variant of NUDT15 or any TPMT polymorphisms evaluated individually. Similar analyses were carried out about the typing of TPMT, using the homozygosity variable for allele *1 versus carrying *2 or *3A as heterozygous, without evidence of any statistically significant association (supplementary table 1S).

Table 4 Association analysis between genotypes and outcomes.  

rs1800462 rs1800460 rs1142345
6-MP Reduction
Yes 21 2 22 1 3 20 2 8
No 43 2 42 3 3 42 2 30
6-MP Interruption
Yes 19 1 18 2 1 19 0 10
No 45 3 46 2 5 43 4 28
Yes 7 1 6 2 0 8 0 5
No 57 3 58 2 6 54 4 33
Yes 8 1 8 1 0 9 0 5
No 56 3 56 3 6 53 4 33
Yes 1 1 2 0 0 2 0 2
No 63 3 62 4 6 60 4 36

Data are presented as absolute frequency values for each outcome evaluated. No statistically significant association was found between any of the polymorphisms studied and the outcomes of interest.

Although no statistically significant associations were identified, two of the four patients heterozygous for NUDT15 required a decrease in 6-MP during the follow-up period. Concerning the genetic variants of TPMT, for rs1800462, two of the four heterozygous patients presented febrile neutropenia, and the same patients also required discontinuation 6-MP during the first six months of maintenance therapy. In the case of rs1800460, of the six heterozygous patients, three required a dosage decrease, and two were also heterozygous for rs1142345. Additionally, differences in the median for the lab values taken in the first 6 months of maintenance treatment were evaluated: aminotransferases, total bilirubin, leukocytes, and differential cell counts; based on genetic variants, no statistically significant differences were found (Supplementary table 2S).


To our knowledge, this is the first study in Colombia that evaluated the the NUDT15 and TPMT genes polymorphisms and their correlation with the side effects of chemotherapy in pediatric patients with acute lymphoblastic leukemia. Although we are considered as a Latino population, different degrees of Caucasian, Amerindian and African ancestry are found in different regions of Colombia. Although, our findings vary significantly from previous reports regarding the association between variants and outcomes 16,19,36,37, those studies were carried out in non-Latino population.

The allelic frequency for the NUDT15 mutation identified in our study (0.03) is lower than previously reported 19,36,38,39, a possible explanation for this finding is that most studies on this polymorphism were done in populations of Asian origin, finding allelic frequencies of up to 0.27. In the sample tested in our study, originated from the central Andean region of Colombia, the caucasian component (European ancestry) could account for near 50-60% of the genetic ancestry 40. Another reason could be related to our sample size 24,41. For the particular case of TPMT polymorphisms, only one study has previously been conducted in the Colombian population, in which Isaza et al. 42, describe frequencies for allele *2 and *3A of 0.004 and 0.035, respectively, which are lower than those found by us (0.02 and 0.05, respectively). When compared to other Latin American results, we found that our frequencies are also slightly different from those described by Garrido et al. 43, in pediatric patients with acute lymphoblastic leukemia in Guatemala. They identified a frequency of 0.005 for *2 and 0.0375 for *3A. However, our *2 frequency is similar to that found in Brazil by Boson et al. 44, who describe a frequency of 0.022 but a much lower frequency for *3A (0.015). More recently, in Uruguayan pediatric patients who have acute lymphoblastic leukemia, a *3A frequency of 0.05, which is similar to ours, has been described, but with a lower frequency of *2 (37.

Increasing evidence highlights the role of genetic variants of NUDT15 in the development of side effects of 6-MP toxicity 19,20,36,37. However, when evaluating the possible associations in our study population, we failed to identify any statistical significance, possibly because of the sample size. Concerning TPMT, evidence supports its role in both the pharmacokinetics and the toxic effects of mercaptopurine 13,16. Our study did not find any association between the allelic frequencies of each polymorphism or in those 42 cases in which we had the complete typing of TPMT. Among the possible reasons that would explain this results, for the case of NUDT15, could be that the original studies describing such associations were based on GWAS approach 19, in which the representation of the Latino population was low, and this may be due to an effect related to the specific ancestry of the Colombian population 45. On the other hand, in the case of TPMT, an explanation could be that the alleles found in our population (*2 and *3A) correspond to intermediate metabolizers, which usually do not require many adjustments in therapy 13.

In exploratory analyses, we evaluated whether there was an association between the genetic variants studied and the outcomes: relapses or death, reviewed as of October 2018, without showing any correlation, which is consistent with what is described by Lennard et al. (46, who, after genotyping TPMT in 2,387 patients with acute lymphoblastic leukemia, found no association with event-free survival. Within the limitations of the present study, there are the sample size and selection bias since it is a captive sample. However, patients were included prospectively and consecutively, which allowed better follow-up of the development of the adverse events of interest. On the other hand, a great strength of the present study is the translational approach, based on previous clinical observations and the evaluation of basic biomedical aspects that could answer the search for predictive factors for the development of toxicity in the treatment of pediatric patients with oncological conditions.


In summary, the allelic frequencies of the genes of interest were different from those previously described, even when compared with Latin American studies. We did not find any statistical association with toxicity events for any of the polymorphisms studied. These findings highlight the need for future studies with a larger sample size; either through a multicenter study with pediatric ALL patients or even including other pediatric populations receiving management with thiopurines, such as patients with inflammatory bowel disease or juvenile idiopathic arthritis, which may allow us to have an adecuate sample size on which to identify better inferences of associations with toxicity outcomes, as well as, to evaluate the possibility that other genes may be influencing toxicity and side effects and to examine the effect that ancestry could have on these interactions in populations with a genetic admixture like our population.


We thank the patients and families who agreed to participate in this study, and the Genetics Institute of the National University of Colombia in Bogota for providing us their facilities to develop genotyping tests. We want to thank Jun J. Yang and Takaya Moriyama from St. Jude Children's Research Hospital for their valuable help in interpreting TPMT genetic data.


1. PDQ Pediatric Treatment Editorial Board. Childhood Acute Lymphoblastic Leukemia Treatment (PDQ(R)): Health Professional Version. In: PDQ Cancer Information Summaries. Bethesda (MD): National Cancer Institute (US); 2002-2021. [ Links ]

2. Cocce MC, Alonso CN, Rossi JG, Bernasconi AR, Rampazzi MA, Felice MS, et al. Cytogenetic and Molecular Findings in Children with Acute Lymphoblastic Leukemia: Experience of a Single Institution in Argentina. Molecular syndromology. 2015;6(4):193-203. doi: 10.1159/000441046 [ Links ]

3. Gowda C, Dovat S. Genetic targets in pediatric acute lymphoblastic leukemia. Advances in experimental medicine and biology. 2013;779:327-40. doi: 10.1007/978-1-4614-6176-0_15 [ Links ]

4. Lim JY, Bhatia S, Robison LL, Yang JJ. Genomics of racial and ethnic disparities in childhood acute lymphoblastic leukemia. Cancer. 2014;120(7):955-62. doi: 10.1002/cncr.28531 [ Links ]

5. Winestone LE, Aplenc R. Disparities in Survival and Health Outcomes in Childhood Leukemia. Curr Hematol Malig Rep. 2019;14(3):179-86. doi: 10.1007/s11899-019-00515-x [ Links ]

6. Hunger SP, Mullighan CG. Acute Lymphoblastic Leukemia in Children. The New England journal of medicine. 2015;373(16):1541-52. doi: 10.1056/NEJMra1400972 [ Links ]

7. Hunger SP, Lu X, Devidas M, Camitta BM, Gaynon PS, Winick NJ, et al. Improved survival for children and adolescents with acute lymphoblastic leukemia between 1990 and 2005: a report from the children's oncology group. Journal of clinical oncology. 2012;30(14):1663-9. doi: 10.1200/JCO.2011.37.8018 [ Links ]

8. Tasian SK, Loh ML, Hunger SP. Childhood acute lymphoblastic leukemia: Integrating genomics into therapy. Cancer. 2015;121(20):3577-90. doi: 10.1038/leu.2008.251 [ Links ]

9. Nguyen K, Devidas M, Cheng SC, La M, Raetz EA, Carroll WL, et al. Factors influencing survival after relapse from acute lymphoblastic leukemia: a Children´s Oncology Group study. Leukemia. 2008;22(12):2142-50. doi: 10.1038/leu.2008.251 [ Links ]

10. Prucker C, Attarbaschi A, Peters C, Dworzak MN, Potschger U, Urban C, et al. Induction death and treatment-related mortality in first remission of children with acute lymphoblastic leukemia: a population-based analysis of the Austrian Berlin-Frankfurt-Munster study group. Leukemia. 2009;23(7):1264-9. doi: 10.1038/leu.2009.12 [ Links ]

11. Christensen MS, Heyman M, Mottonen M, Zeller B, Jonmundsson G, Hasle H, et al. Treatment-related death in childhood acute lymphoblastic leukaemia in the Nordic countries: 1992-2001. British journal of haematology. 2005;131(1):50-8. doi: 10.1111/j.1365-2141.2005.05736.x [ Links ]

12. O´Connor D, Bate J, Wade R, Clack R, Dhir S, Hough R, et al. Infection-related mortality in children with acute lymphoblastic leukemia: an analysis of infectious deaths on UKALL2003. Blood. 2014;124(7):1056-61. doi: 10.1182/blood-2014-03-560847 [ Links ]

13. Relling MV, Schwab M, Whirl-Carrillo M, Suarez-Kurtz G, Pui CH, Stein CM, et al. Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for thiopurine dosing based on TPMT and NUDT15 genotypes: 2018 update. Clinical pharmacology and therapeutics. 2019;105(5):1095-105. doi: 10.1002/cpt.1304 [ Links ]

14. Mei L, Ontiveros EP, Griffiths EA, Thompson JE, Wang ES, Wetzler M. Pharmacogenetics predictive of response and toxicity in acute lymphoblastic leukemia therapy. Blood reviews. 2015;29(4):243-9. doi: 10.1016/j.blre.2015.01.001 [ Links ]

15. Lopez-Lopez E, Gutierrez-Camino A, Bilbao-Aldaiturriaga N, Pombar-Gomez M, Martin-Guerrero I, Garcia-Orad A. Pharmacogenetics of childhood acute lymphoblastic leukemia. Pharmacogenomics. 2014;15(10):1383-98. doi: 10.2217/pgs.14.106 [ Links ]

16. El-Rashedy FH, Ragab SM, Dawood AA, Temraz SA. Clinical implication of thiopurine methyltransferase polymorphism in children with acute lymphoblastic leukemia: A preliminary Egyptian study. Indian journal of medical and paediatric oncology : official journal of Indian Society of Medical & Paediatric Oncology. 2015;36(4):265-70. doi: 10.4103/0971-5851.171553 [ Links ]

17. Adam de Beaumais T, Fakhoury M, Medard Y, Azougagh S, Zhang D, Yakouben K, et al. Determinants of mercaptopurine toxicity in paediatric acute lymphoblastic leukemia maintenance therapy. British journal of clinical pharmacology. 2011;71(4):575-84. doi: 10.1111/j.1365-2125.2010.03867.x [ Links ]

18. Zgheib NK, Akika R, Mahfouz R, Aridi CA, Ghanem KM, Saab R, et al. NUDT15 and TPMT genetic polymorphisms are related to 6-mercaptopurine intolerance in children treated for acute lymphoblastic leukemia at the Children&apos;s Cancer Center of Lebanon. Pediatric blood & cancer. 2017;64(1):146-50. doi: 10.1002/pbc.26189 [ Links ]

19. Yang JJ, Landier W, Yang W, Liu C, Hageman L, Cheng C, et al. Inherited NUDT15 variant is a genetic determinant of mercaptopurine intolerance in children with acute lymphoblastic leukemia. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2015;33(11):1235-42. doi: 10.1200/JCO.2014.59.4671 [ Links ]

20. Moriyama T, Nishii R, Perez-Andreu V, Yang W, Klussmann FA, Zhao X, et al. NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity. Nature genetics. 2016;48(4):367-73. doi: 10.1038/ng.3508 [ Links ]

21. Suzuki H, Fukushima H, Suzuki R, Hosaka S, Yamaki Y, Kobayashi C, et al. Genotyping NUDT15 can predict the dose reduction of 6-MP for children with acute lymphoblastic leukemia especially at a preschool age. Journal of human genetics. 2016;61(9):797-801. doi: 10.1038/jhg.2016.55 [ Links ]

22. Chiengthong K, Ittiwut C, Muensri S, Sophonphan J, Sosothikul D, Seksan P, et al. NUDT15 c.415C´T increases risk of 6-mercaptopurine induced myelosuppression during maintenance therapy in children with acute lymphoblastic leukemia. Haematologica. 2016;101(1):e24-6. doi: 10.3324/haematol.2015.134775 [ Links ]

23. Kimura S, Hasegawa D, Yoshimoto Y, Hirabayashi S, Hosoya Y, Yoshihara H, et al. Severe 6-mercaptopurine-induced hematotoxicity in childhood an ALL patient with homozygous NUDT15 missence variants. Rinsho ketsueki. 2016;57(6):748-53. doi: 10.11406/rinketsu.57.748 [ Links ]

24. Liang DC, Yang CP, Liu HC, Jaing TH, Chen SH, Hung IJ, et al. NUDT15 gene polymorphism related to mercaptopurine intolerance in Taiwan Chinese children with acute lymphoblastic leukemia. The pharmacogenomics journal. 2016;16(6):536-9. doi: 10.1038/tpj.2015.75 [ Links ]

25. Choudhry S, Coyle NE, Tang H, Salari K, Lind D, Clark SL, et al. Population stratification confounds genetic association studies among Latinos. Human genetics. 2006;118(5):652-64. doi: 10.1007/s00439-005-0071-3 [ Links ]

26. Kishi S, Cheng C, French D, Pei D, Das S, Cook EH, et al. Ancestry and pharmacogenetics of antileukemic drug toxicity. Blood. 2007;109(10):4151-7. doi: 10.1182/blood-2006-10-054528 [ Links ]

27. Corvol H, De Giacomo A, Eng C, Seibold M, Ziv E, Chapela R, et al. Genetic ancestry modifies pharmacogenetic gene-gene interaction for asthma. Pharmacogenetics and genomics. 2009;19(7):489-96. doi: 10.1097/FPC.0b013e32832c440e [ Links ]

28. Hoang PT, Ambroise J, Dekairelle AF, Durant JF, Butoescu V, Chi VL, et al. Comparative pharmacogenetic analysis of risk polymorphisms in Caucasian and Vietnamese children with acute lymphoblastic leukemia: prediction of therapeutic outcome? British journal of clinical pharmacology. 2015;79(3):429-40. doi: 10.1111/bcp.12481 [ Links ]

29. Suarez-Kurtz G, Pena SD, Struchiner CJ, Hutz MH. Pharmacogenomic Diversity among Brazilians: Influence of Ancestry, Self-Reported Color, and Geographical Origin. Frontiers in pharmacology. 2012;3:191. doi: 10.3389/fphar.2012.00191 [ Links ]

30. Trujillo AM, Linares-Ballesteros A, Sarmiento IC. Intensive chemotherapy in children with acute lymphoblastic leukemia. Interim analysis in a referral center in Colombia. Rev Fac Med 2016;64(3):417-25. doi: 10.15446/revfacmed.v64n3.53961 [ Links ]

31. Yunis LK, Linares A, Cabrera E, Yunis JJ. Systematic molecular analysis of hemophilia A patients from Colombia. Genet Mol Biol. 2018;41(4):750-7. doi: 10.1590/1678-4685-GMB-2017-0072 [ Links ]

32. Ip SC, Lin SW, Lai KM. An evaluation of the performance of five extraction methods: Chelex(R) 100, QIAamp(R) DNA Blood Mini Kit, QIAamp(R) DNA Investigator Kit, QIAsymphony(R) DNA Investigator(R) Kit and DNA IQ. Sci Justice. 2015;55(3):200-8. doi: 10.1016/j.scijus.2015.01.005 [ Links ]

33. Wilfinger WW, Mackey K, Chomczynski P. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques. 1997;22(3):474-6, 8-81. doi: 10.2144/97223st01 [ Links ]

34. Desjardins P, Conklin D. NanoDrop microvolume quantitation of nucleic acids. J Vis Exp. 2010; (45): 2565. doi: 10.3791/2565 [ Links ]

35. NIH. ALFA: Allele Frequency Aggregator. National Center for Biotechnology Information, U.S. 2020. Available from: [ Links ]

36. Singh M, Bhatia P, Khera S, Trehan A. Emerging role of NUDT15 polymorphisms in 6-mercaptopurine metabolism and dose related toxicity in acute lymphoblastic leukaemia. Leuk Res. 2017;62:17-22. doi: 10.1016/j.leukres.2017.09.012 [ Links ]

37. Soler AM, Olano N, Mendez Y, Lopes A, Silveira A, Dabezies A, et al. TPMT and NUDT15 genes are both related to mercaptopurine intolerance in acute lymphoblastic leukaemia patients from Uruguay. British journal of haematology. 2018;181(2):252-5. doi: 10.1111/bjh.14532 [ Links ]

38. Yang JJ, Whirl-Carrillo M, Scott SA, Turner AJ, Schwab M, Tanaka Y, et al. Pharmacogene Variation Consortium Gene Introduction: NUDT15. Clinical pharmacology and therapeutics. 2019;105(5):1091-4. doi: 10.1002/cpt.1268 [ Links ]

39. Shah SAV, Paradkar MU, Desai DC, Ashavaid TF. Preemptive NUDT15 genotyping: redefining the management of patients with thiopurine-induced toxicity. Drug Metab Pers Ther. 2018;33(1):57-60. doi: 10.1515/dmpt-2017-0038 [ Links ]

40. Ossa H, Aquino J, Pereira R, Ibarra A, Ossa R, Perez LA, et al. Outlining the Ancestry Landscape of Colombian Admixed Populations. PloS one. 2016;11(10):e0164414. doi: 10.1371/journal.pone.0164414 [ Links ]

41. Tanaka Y, Kato M, Hasegawa D, Urayama KY, Nakadate H, Kondoh K, et al. Susceptibility to 6-MP toxicity conferred by a NUDT15 variant in Japanese children with acute lymphoblastic leukaemia. British journal of haematology. 2015;171(1):109-15. doi: 10.1111/bjh.13518 [ Links ]

42. Isaza C, Henao J, Lopez AM, Cacabelos R. Allelic variants of the thiopurine methyltransferase (TPMT) gene in the Colombian population. Methods Find Exp Clin Pharmacol. 2003;25(6):423-9. doi: 10.1358/mf.2003.25.6.769646 [ Links ]

43. Garrido C, Santizo VG, Mullers P, Soriano DR, Avila GB, Dean M, et al. Frequency of thiopurine S-methyltransferase mutant alleles in indigenous and admixed Guatemalan patients with acute lymphoblastic leukemia. Med Oncol. 2013;30(1):474. doi: 10.1007/s12032-013-0474-2 [ Links ]

44. Boson WL, Romano-Silva MA, Correa H, Falcao RP, Teixeira-Vidigal PV, De Marco L. Thiopurine methyltransferase polymorphisms in a Brazilian population. The pharmacogenomics journal. 2003;3(3):178-82. doi: 10.1038/sj.tpj.6500175 [ Links ]

45. Norris ET, Wang L, Conley AB, Rishishwar L, Marino-Ramirez L, Valderrama-Aguirre A, et al. Genetic ancestry, admixture and health determinants in Latin America. BMC Genomics. 2018;19(Suppl 8):861. doi: 10.1186/s12864-018-5195-7 [ Links ]

46. Lennard L, Cartwright CS, Wade R, Vora A. Thiopurine methyltransferase and treatment outcome in the UK acute lymphoblastic leukaemia trial ALL2003. British journal of haematology. 2015;170(4):550-8. doi: 10.1111/bjh.13469 [ Links ]


Funding Source This research was supported by a grant from the Universidad Nacional de Colombia HERMES Research 2016-2018, awarded to Isabel Sarmiento-Urbina, Project Code 35872.

Supplementary material

Table 1S Association analysis between TPMT diplotypes and outcomes. 

OUTCOMES TPMT diplotypes p Value
*1/*1 *1/another
6-MP Reduction
Yes 8 2 0.616
No 28 4
6-MP Interruption
Yes 10 0 0.308
No 26 6
Yes 5 0 1.000
No 31 6
Yes 4 1 0.557
No 32 5
Yes 2 0 1.000
No 34 6

Data are presented as absolute frequency values for each outcome evaluated. No statistically significant association was found between any of the TPMT diplotypes studied and the outcomes of interest.

Table 2S Comparison of laboratory follow-up according to genotypes.  

Follow-up NUDT15 TPMT
rs1800462 rs1800460 rs1142345
1st ALT (IU/L) 46.20 50.50 49 36 45.15 48.60 49.30 53
AST (IU/L) 34.20 41.80 36.90 30.30 42.90 35.35 42.90 38.55
WBC/mm3 3,315 3,780 3,315 3,610 2,200 3,380 2,030 3,310
Neutrophils/mm3 1,820 1,995 1,820 1,737 980 1,910 1,020 1,980
Lymphocytes/mm3 783 409 780 773 630 783 600 725
Total Bilirubin (mg/dL) 0.50 0.60 0.51 0.40 0.60 0.50 0.45 0.52
2nd ALT (IU/L) 45 74.50 55.40 28.80 26 46 53.15 45
AST (IU/L) 33.55 33.70 33.55 33.15 37 32.70 42.80 38.25
WBC/mm3 3,135 2,170 3,020 3,980 2,160 3,225 2,305 3,245
Neutrophils/mm3 1,835 655 1,691 2,515.50 1,295 1,835 1,440 1,835
Lymphocytes/mm3 630 1,290 640 475 727 630 583.50 746.50
Total Bilirubin (mg/dL) 0.50 0.23 0.50 0.45 0.40 0.50 0.40 0.59
3rd ALT (IU/L) 40 59.10 39.50 89.45 37 54 80.20 54
AST (IU/L) 34.50 33.10 33.30 38.65 42.20 33.65 42.20 35
WBC/mm3 3,070 2,865 3,100 2,085 3,100 3,050 3,130 3,055
Neutrophils/mm3 1,635 1,430 1,645 1,175 1,440 1,635 2,680 1,540
Lymphocytes/mm3 734 1010 753 470 1030 712 875 768
Total Bilirubin (mg/dL) 0.50 0.30 0.50 0.60 0.50 0.50 0.50 0.50
4th ALT (IU/L) 52.40 18.10 52.40 33.70 41.60 52.40 138.30 63
AST (IU/L) 31.10 32.10 31.20 36.30 47.21 31.20 89.90 30.40
WBC/mm3 3,400 3,135 3,400 2,980 2,570 3,510 2,615 3,635
Neutrophils/mm3 1,940 1,720 1,955 860 1,700 1,970 1,735 1,955
Lymphocytes/mm3 760 740 760 1,305 535 800 535 800
Total Bilirubin (mg/dL) 0.70 0.40 0.60 0.70 0.45 0.70 0.40 0.70
5th ALT (IU/L) 54 23.40 55 24 29.10 54 67.10 60.75
AST (IU/L) 40.70 35.40 42.10 25.50 42.10 38.15 55.10 47.30
WBC/mm3 3,470 3,060 3,430 3,435 2,615 3,470 2,615 3,740
Neutrophils/mm3 1,930 1,410 1,905 1,764 1,360 1,930 1,360 1,800
Lymphocytes/mm3 845 560 830 900 793 850 845 870
Total Bilirubin (mg/dL) 0.50 0.45 0.50 0.80 0.30 0.60 0.40 0.65
6th ALT (IU/L) 68.55 38 70.30 35.45 71 66.05 71 70.10
AST (IU/L) 41.50 30.90 43.95 31.35 53.80 40 63 44.90
WBC/mm3 3,385 2,655 3,385 3,145 3,200 3,370 2,755 3,650
Neutrophils/mm3 1,820 1,415 1,790 1,790 1,515 1,800 1,185 1,750
Lymphocytes/mm3 905 1,060 910 810 681 915.50 680 921
Total Bilirubin (mg/dL) 0.60 0.50 0.60 0.71 0.56 0.60 0.38 0.60

Data are presented as median values for each laboratory evaluated. When the medians of each laboratory value were compared according to the genotyping of each SNPs, no statistically significant differences were identified.

Received: December 17, 2020; Revised: April 07, 2021; Accepted: September 27, 2021

Corresponding author: Isabel Sarmiento-Urbina. Department of Pediatrics, School of Medicine, Universidad Nacional de Colombia. Av. Caracas #1-65, Bogotá D.C., Colombia

Conflict of interest:

All authors do not have any possible conflicts of interest

Creative Commons License This is an open-access article distributed under the terms of the Creative Commons Attribution License