|Year : 2019 | Volume
| Issue : 3 | Page : 157-162
Study of N-acetyl transferase 2 single-gene polymorphism (rs1799931) in patients with acute myeloid leukemia
Muhammad T AbdelGhafar MD 1, Alzahraa A Allam2, Sara Darwish3, Ghada M Al-Ashmawy4, Kareman A Eshra5, Rowida R Ibrahim6
1 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt
3 Department of Oncology, Faculty of Medicine, Tanta University, Tanta, Egypt
4 Department of Biochemistry, Faculty of Pharmacy, Tanta University, Tanta, Egypt
5 Department of Microbiology and Immunology, Faculty of Medicine, Tanta University, Tanta, Egypt
6 Medical Biochemistry and Molecular Biology, Faculty of Medicine, Tanta University, Tanta, Egypt
|Date of Submission||25-May-2019|
|Date of Acceptance||11-Jun-2019|
|Date of Web Publication||05-Dec-2019|
Muhammad T AbdelGhafar
Department of Clinical Pathology, Faculty of Medicine, El-Gash Street, Medical Campus, Tanta University, Tanta, 31511
Source of Support: None, Conflict of Interest: None
Background The genetic background has emerged as a risk factor for acute myeloid leukemia (AML) progression. N-acetyl transferase 2 (NAT2), as an enzyme, plays a pivotal role in detoxifying the carcinogenic compounds. The NAT2 gene is highly polymorphic and is found to be associated with the process of tumorigenesis and the progression of many cancers.
Objective This was a case–control study adopted to explore the possible association of NAT2 gene polymorphism rs1799931 (G857A) with the susceptibility of AML progression in the Egyptian cohort.
Patients and methods This study was performed during the period spanning from February 2017 to December 2018 on 60 AML cases and 80 controls. NAT2 gene rs1799931 (G857A) polymorphism was genotyped by real-time PCR technique. The NAT2 genotype and allele distributions between the cases and controls were compared by χ2-test. Association strength between NAT2 gene single-gene polymorphism (rs1799931) and AML susceptibility was expressed by odds ratios (ORs) and 95% confidence intervals (CIs) and adjusted to the confounding variables.
Results The NAT2 rs1799931 genotypes’ and alleles’ distribution frequencies were significantly different between AML cases and controls (P<0.05). The GG genotype and G allele frequencies were significantly higher than those of the AA genotype (P=0.027) and A allele (P=0.003) in AML cases if compared with the controls. GG genotype presents a higher susceptibility to AML than the AA genotype (OR: 3.765; 95% CI: 1.167–12.15), and the G allele exhibited 2.365-folds increased adjusted risk for AML if compared with the A allele (OR: 2.365; 95% CI: 1.344–4.163).
Conclusion NAT2 gene rs1799931 (G857A) is associated with increased susceptibility to AML in the Egyptian population with the GG genotype and G allele carrying a higher risk for AML. Further studies should be performed to verify the present results.
Keywords: acute myeloid leukemia, genotype, N-acetyl transferase 2 gene, polymorphism, risk
|How to cite this article:|
AbdelGhafar MT, Allam AA, Darwish S, Al-Ashmawy GM, Eshra KA, Ibrahim RR. Study of N-acetyl transferase 2 single-gene polymorphism (rs1799931) in patients with acute myeloid leukemia. Egypt J Haematol 2019;44:157-62
|How to cite this URL:|
AbdelGhafar MT, Allam AA, Darwish S, Al-Ashmawy GM, Eshra KA, Ibrahim RR. Study of N-acetyl transferase 2 single-gene polymorphism (rs1799931) in patients with acute myeloid leukemia. Egypt J Haematol [serial online] 2019 [cited 2020 Jul 8];44:157-62. Available from: http://www.ehj.eg.net/text.asp?2019/44/3/157/272369
| Introduction|| |
Acute leukemia is a clonal hematological neoplasm characterized by the inhibition of differentiation and accumulation of immature leucocytes in the bone marrow . According to the type of affected cells, acute leukemia can be either myeloid or lymphoblastic. Acute myeloid leukemia (AML) is the commonest type affecting adults ,. AML presents the sixth leading cause of death among malignancies. The pathogenesis of AML progression is not yet well understood and has been suggested to be multifactorial, with the involvement of environmental and genetic factors . The environmental factors include smoking, obesity, and exposure to radiation or chemical carcinogens . However, these factors were found to exhibit little influence on AML progression, suggesting the crucial role of genetic factors.
N-acetyl transferase 2 (NAT2) is a phase II metabolizing enzyme playing a major role in the acetylation and detoxification of many toxic metabolites and carcinogens such as aryl or aromatic amines, and hydrazines, and which is involved in the process of tumorigenesis . It is encoded by a gene located on the chromosome 8q21.3–23.1 . The NAT2 gene is highly polymorphic, and up to 106 different polymorphisms have been identified affecting the NAT2 gene and resulting in the substitution of amino acids with the subsequent variation of biological activity or stability of the NAT2 enzyme, leading to rapid or slow acetylation . Accordingly, the polymorphisms of NAT2 genes are associated with two main phenotypes, which may be either slow or rapid acetylator types .
Growing evidence suggested the association of NAT2 polymorphisms with the increased susceptibility to various disorders, specifically cancers including acute lymphoblastic leukemia, lung squamous carcinoma, gastric cancer, urinary bladder cancer ,,,, and autoimmune diseases ,. Furthermore, several studies suggested the association of different polymorphisms of NAT2 with high risk for acute leukemia progression, specifically AML, with conflicting results ,. This contradiction may be due to ethnic variation.
Taking together these findings, there is much evidence to suggest the possible association of NAT2 gene polymorphisms with AML predisposition. However, to our knowledge, no previous studies have been conducted to assess this potential association in the Egyptian population. Thus, this case–control study was conducted to explore the association of single-gene polymorphism of NAT2 rs1799931 (G857A) with susceptibility to AML in the Egyptian population.
| Patients and methods|| |
This study was performed in accordance with the Helsinki Declaration. The protocol was designed according to the study objectives and approved by the Ethical Committee of Tanta Faculty of Medicine. All participants included were informed about the study plan, the potential benefits and hazards and required to sign an informed consent before participation.
Study design and participants
This was a case–control study that started on first February 2017 and continued until 31 December 2018. It was conducted at the Internal Medicine Department of Tanta University Hospitals. The eligibility criteria for this study had included all patients with AML, recently diagnosed or who were under treatment, according to the WHO criteria for the diagnosis of acute leukemia , based morphologically on the examination of bone marrow aspirate smears and the detection of more than 20% blast cells. According to our eligibility criteria, a series of 60 cases with AML was selected to participate in this study, in addition to 80 healthy participants who served as controls. All patients with a past history of cancer, diabetes, and autoimmune disorders were excluded from this study.
Clinical and laboratory assessment
All participants were asked about their personal and medical history. The patients’ medical records were reviewed for the clinical features, specifically the presence of enlarged lymph nodes or spleen, which were determined clinically and via abdominal ultrasonography. Laboratory investigations of the patients by which the AML was diagnosed, such as bone marrow aspiration cytology, complete blood count, lactate dehydrogenase, and erythrocyte sedimentation rate were also obtained from their medical records.
Form each participant, 3 ml of venous blood was taken via venipuncture and further placed in K3 EDTA tube for NAT2 gene polymorphism rs1799931 (G857A) genotyping. The samples were mixed gently and transferred immediately into appropriate containers in the laboratory, whereby DNA extraction was performed, and the DNA yields were preserved at −20°C for genotyping.
DNA isolation and single-gene polymorphism genotyping
The genomic DNA was extracted from the peripheral leucocytes using human DNA extraction Kit, QIAamp DNA Blood Mini kits (Qiagen, Hilden, Germany), according to the manufacturer protocol. The TaqMan real-time PCR technique was used for amplification and detection of NAT2 rs1799931 (G857A) genotypes. The sequence of primers used were supplied as presented in [Table 1], and the reaction mixture was prepared in 25 μl containing 12.5 ml of 2× TaqMan universal master mix with 75–200 ng of the genomic DNA (2 μl), 1.25 μl of 20× TaqMan SNP Assay Working Stock (containing NAT2 rs1799931-specific primers and FAM and VIC dye-labeled probes; Thermo-Fisher Scientific, Waltham, Massachusetts, USA), and nuclease-free water. The cycling condition was performed on real-time PCR system (Applied Biosystem, step I version, Foster City, California, USA) as follows: 95°C for 10 min, then 45 cycles as 10 s at 95°C, 10 s at 62°C, and 15 s at 72°C. The data were analyzed and presented on the multicomponent plot for the homogenous and heterogeneous genotypes using Applied Biosystem, step I version software analysis modules.
|Table 1 Primer sequences of N-acetyl transferase 2 gene polymorphism (rs1799931)|
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Data analysis was performed using SSPS software (IBM Corp., version 22.0, Armonk, NY, USA). Data were expressed in the form of mean±SD or median and interquartile range. t-Test was used for the numerical data and χ2-test for the nonparametric data. The Hardy–Weinberg equilibrium of our study population was estimated via χ2-test to assess the quality of the study sample. The genotype and allele frequencies were calculated by direct counting and compared between cases and controls using χ2-test. Multiple logistic regressions were used to estimate the strength of NAT2 gene polymorphism association with AML risk via calculation of odds ratio (OR) and 95% confidence intervals (CIs) with adjustment to the confounding variables. All P values were two-sided, and P values less than 0.05 were considered as significant.
| Results|| |
Basic features of the study patients
In this study, the case series comprised 60 AML patients, aged 51.22±8.5 years, and there were 39 male individuals and 21 female individuals. An overall 75% of them presented with splenomegaly and 45% with lymphadenopathy. Peripheral blood findings revealed low hemoglobin level (7.38±0.78 g/dl), low platelet count (57.42±18.09×103/μl), and 18 cases with leukocytosis and 42 cases with leucopenia. All cases presented with mean blast cells of 64.18±24.32% in their bone marrow aspirate. The controls were 80 patients, aged 50.49±9.33 years, and there were 48 male individuals and 32 female individuals. The control participants were age-matched and sex-matched with cases. The confounding, clinical and laboratory variables of the studied series are demonstrated in [Table 2] and [Table 3].
|Table 3 The clinical and laboratory variables of the acute myeloid leukemia cases|
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N-acetyl transferase 2 gene polymorphism (rs1799931) and acute myeloid leukemia cases’ variables
A significant association had been observed between NAT2 (rs1799931) GG genotype and AML in positive smoker patients, with no significant differences being detected among the three genotypes with regard to the other confounding variables, such as age, sex, and clinical variables such as lymphadenopathy, splenomegaly and laboratory variables ([Table 4]).
|Table 4 The distribution of N-acetyl transferase 2 rs1799931 genotypes with regard to the confounding, clinical and laboratory variables in acute myeloid leukemia cases|
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N-acetyl transferase 2 gene polymorphism (rs1799931) distribution
In this study, the distribution of the different genotypes of NAT2 gene polymorphism (rs1799931) in AML cases and controls were in accordance with the Hardy–Weinberg equilibrium. A significant difference had been observed in the frequencies of the NAT2 rs1799931 genotypes and alleles between cases and controls with the GG genotype, and G allele was the dominant in cases and controls. The frequency of GG genotype was found to be significantly higher than that of the AA genotype in AML cases (P=0.027) if compared with the controls. Moreover, the frequency of NAT2 rs1799931 G allele was significantly higher than the A allele in AML cases (P=0.003) if compared with the controls ([Table 5] and [Figure 1]).
|Table 5 The distribution of N-acetyl transferase 2 rs1799931 genotypes and alleles between acute myeloid leukemia cases and healthy controls|
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|Figure 1 The distribution of N-acetyl transferase 2 (NAT2) rs1799931 genotypes and alleles between acute myeloid leukemia (AML) cases and healthy controls.|
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N-acetyl transferase 2 gene polymorphism (rs1799931) and relative acute myeloid leukemia risk
The carriers of GG genotype exhibit an increased risk for AML by 3.111-folds if compared with the AA genotype (OR: 3.111; 95% CI: 1.019–9.50), and the risk is increased to 3.765-folds after adjustment for the confounding variables including age, sex and smoking. Furthermore, the G allele presents an increased risk for AML by 2.161-folds (OR: 2.161; 95% CI: 1.252–3.730) that is increased after smoking adjustment to reach 2.365-folds ([Table 5]).
| Discussion|| |
AML is a heterogeneous disorder with not yet well-understood mechanisms. However, several risk factors have been identified. Environmental factors play a major role involving exposure to drugs, and chemical and ionizing radiation . Moreover, genetic elements cannot be ignored, either polymorphism or mutations of certain genes, for example, NPM1, CEBPA, c-KIT and AML1/RUNX1 .
NAT2 is a phase II metabolizing enzyme that catalyzes the activation of O-acetylation and inactivation of N-acetylation that aid in the biotransformation of aromatic amines coming from industrial waste, air, water pollutants and dietary products , which are considered significant ultimate carcinogens involved in the initiation process of malignancy. The NAT2 gene is present on chromosome 8 (8p23.1–p21.3) and has two exons . Several polymorphic loci have been detected within the coding region (701-bp) of NAT2 gene . Four main NAT2 acetylator alleles, three ‘slow’ NAT2*5 (341T>C, 481C>T), NAT2*6 (590G>A), NAT2*7 (857G>A) and one ‘rapid’ allele NAT2*4 have been currently identified with clinical significance. The coexistence of a rapid allele with a slow one releases an intermediate acetylation pattern . Interestingly, the rapid acetylation phenotype associated with increased frequency of malignancies, mainly lung and colon cancer ,,, may be due to extensive acetylation with subsequent activation of aryl amines as potential carcinogens .
In this study, we aimed to evaluate the possible association of NAT2 (rs1799931) single-gene polymorphism with the risk for AML progression in the Egyptian population. We detected that the GG genotype and the G allele were predominant in AML cases and in the healthy participants with much higher significant frequency in AML cases. This study revealed that the GG genotype was associated with 3.111-folds higher risk for AML. In addition, the G allele carriers had an increased risk for AML by 2.161-folds if compared with A allele carriers. We detected a more increased risk for AML, if the ratio was adjusted to the confounding variables such as age, sex and smoking, suggesting the triggering role of these factors in enhancing the AML progression.
These findings were supported by those of the study by Zou et al. , who detected the increased frequency of ancestral GG genotype in AML cases, with the A allele carrier having a significantly reduced risk for AML by 0.585-fold if compared with the G allele carriers. Contradictory to our findings, a study by Kamel et al. , showed that slow acetylators NAT2*7A GA and GA/AA were overexpressed in AML cases (OR: 2.74 and 2.72, respectively). This inconsistency may be due to differences in the sample size and ethnicity of the studied population. Moreover, Zanrosso et al.  detected the association of NAT2 slow acetylator with enhanced leukemia risk in children below 1 year of age and in children aged from 1 to 10 years old. Furthermore, they detected that NAT2*14E, NAT2*6B, and NAT2*6F haplotypes also significantly associated with the risk of AML progression in the Brazilian population .
The same contradiction also exists between studies with regard to the association of NAT2 gene polymorphisms with solid tumors. Several studies have linked different NAT2 gene polymorphisms with the genetic predisposition for solid malignancies such as colorectal cancer, lung cancer, and bladder cancer ,,, whereas others detected no association such as with breast cancer .
Functionally, rs1799931 presents a G>A substitution of NAT2 gene at position 857, which results in the subsequent substitution of glycine by glutamine at the 286th position of the NAT2 enzyme protein. This alteration may affect the NAT2 enzyme activity, which results in either activation or inhibition of the metabolism of some drugs and carcinogens, leading to increased or decreased cancer incidence .
Limitation of the study
This study was the first to investigate the association of NAT2 gene polymorphism with AML in the Egyptian cohort. However, this study is limited due to several factors. A relatively small sample size was included in our study. Moreover, the mechanisms by which this polymorphism can affect the activity of the enzyme were not investigated. Thus, more studies are required to confirm the findings of our study.
| Conclusion|| |
Our findings suggested that NAT2 gene polymorphism rs1799931 (G857A) is associated with the genetic susceptibility for AML progression in the Egyptian population, with the GG genotype and G allele carrying a higher risk for AML.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Zhou L, Zhu YY, Zhang XD, Li Y, Liu ZG. Risk effects of GST gene polymorphisms in patients with acute myeloid leukemia: a prospective study. Asian Pac J Cancer Prev
Meenaghan T, Dowling M, Kelly M. Acute leukaemia: making sense of a complex blood cancer. Br J Nurs
Tomizawa D. Acute leukemia in adolescents and young adults. Rinsho Ketsueki
Filippini T, Heck JE, Malagoli C, Del Giovane C, Vinceti M. A review and meta-analysis of outdoor air pollution and risk of childhood leukemia. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev
Ilhan G, Karakus S, Andic N. Risk factors and primary prevention of acute leukemia. Asian Pac J Cancer Prev
Yarosh SL, Kokhtenko EV, Churnosov MI, Ataman AV, Solodilova MA, Polonikov AV. Synergism between the N
-acetyltransferase 2 gene and oxidant exposure increases the risk of idiopathic male infertility. Reprod Biomed Online
Blum M, Grant DM, McBride W, Heim M, Meyer UA. Human arylamine N
-acetyltransferase genes: isolation, chromosomal localization, and functional expression. DNA Cell Biol
Walker K, Ginsberg G, Hattis D, Johns DO, Guyton KZ, Sonawane B. Genetic polymorphism in N
-acetyltransferase (NAT): population distribution of NAT1 and NAT2 activity. J Toxicol Environ Health B Crit Rev
Stanley LA, Sim E. Update on the pharmacogenetics of NATs: structural considerations. Pharmacogenomics
Kamel AM, Ebid GT, Moussa HS. N-acetyltransferase 2 (NAT2) polymorphism as a risk modifier of susceptibility to pediatric acute lymphoblastic leukemia
. Tumour Biol
Tian F, Zhang Y, Ren Y, Shen L, Wu W, Zhou B. N-acetyltransferase 2 (NAT2) gene polymorphism and exposure to smoking in lung cancer of Chinese males
. Med Oncol
Hein DW. N-acetyltransferase 2 genetic polymorphism: effects of carcinogen and haplotype on urinary bladder cancer risk
Yu J, Deng Y, Chen JP. N-acetyltransferase 2 status and gastric cancer risk: a meta-analysis
. Tumour Biol
Dudarewicz M, Rychlik-Sych M, Baranska M, Wojtczak A, Trzcinski R, Dziki A et al.
Significance of the genetic polymorphism of CYP2D6 and NAT2 in patients with inflammatory bowel diseases. Pharmacol Rep
Santos EC, Pinto AC, Klumb EM, Macedo JM. Polymorphisms in NAT2 (N
-acetyltransferase 2) gene in patients with systemic lupus erythematosus. Rev Bras Reumatol Engl Ed
Majumdar S, Mondal BC, Ghosh M, Dey S, Mukhopadhyay A, Chandra S et al.
Association of cytochrome P450, glutathione S-transferase and N
-acetyl transferase 2 gene polymorphisms with incidence of acute myeloid leukemia. Eur J Cancer Prev
Zanrosso CW, Emerenciano M, Faro A, Goncalves BA, Mansur MB, Pombo-de-Oliveira MS. Genetic variability in N
-acetyltransferase 2 gene determines susceptibility to childhood lymphoid or myeloid leukemia in Brazil. Leuk Lymphoma
Arber DA, Orazi A, Hasserjian R, Thiele J, Borowitz MJ, Le Beau MM et al.
The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood
Poynter JN, Richardson M, Roesler M, Blair CK, Hirsch B, Nguyen P et al.
Chemical exposures and risk of acute myeloid leukemia and myelodysplastic syndromes in a population-based study. Int J Cancer
Goryainova NV. The clinical significance of genetic mutations in acute myeloid leukemia. Lik Sprava
Kozhekbaeva ZhM, Glotov AS, Gra OA, Goldenkova-Pavlova IV, Bruskin SA, Agafonova EE et al.
Identification of functionally significant mutations in NAT2 gene using biological microchips. Mol Biol (Mosk)
Gra OA, Glotov AS, Kozhekbaeva Z, Makarova OV, Nasedkina TV. Genetic polymorphism in GST, NAT2, and MTRR and susceptibility to childhood acute leukemia. Mol Biol (Mosk)
Hein DW. Molecular genetics and function of NAT1 and NAT2: role in aromatic amine metabolism and carcinogenesis. Mutat Res
Osian G, Procopciuc L, Vlad L. NAT2 gene polymorphism and sporadic colorectal cancer. Prevalence, tumor stage and prognosis. A preliminary study in 70 patients. J Gastrointestin Liver Dis
Zou Y, Dong S, Xu S, Gong Q, Chen J. Genetic polymorphisms of NAT2 and risk of acute myeloid leukemia: a case-control study. Medicine (Baltimore)
Zhu Z, Zhang J, Jiang W, Zhang X, Li Y, Xu X. Risks on N
-acetyltransferase 2 and bladder cancer: a meta-analysis. Onco Targets Ther
Ochs-Balcom HM, Wiesner G, Elston RC. A meta-analysis of the association of N-acetyltransferase 2 gene (NAT2) variants with breast cancer. Am J Epidemiol
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]