|Year : 2020 | Volume
| Issue : 2 | Page : 192-195
Role of CTLA4-49A polymorphism in the pathogenesis of acute leukemia
Mahmoud A Hawass1, Essam A Afifi1, Ibrahim H.M Hazaa1, Gamal T.A Ebid2
1 Department of Biochemistry, Faculty of Medicine, Azhar University, Cairo, Egypt
2 Department of Clinical Pathology, National Cancer Institute, Cairo University, Cairo, Egypt
|Date of Submission||03-Dec-2019|
|Date of Decision||12-Feb-2020|
|Date of Acceptance||20-Feb-2020|
|Date of Web Publication||29-Jun-2020|
Ibrahim H.M Hazaa
Tel: (+2) (013) 3245497, (011)57029989
Source of Support: None, Conflict of Interest: None
Background Acute leukemia is a clonal disorder of both myeloid and lymphoid progenitors with distinctive morphologic, immunophenotypic, and genotypic features and represents the most frequent malignancy of childhood. Acute leukemia is associated with several humoral and cellular immune abnormalities that could lead to an inadequate antitumor response. One of the co-stimulators that regulates immune response is CTLA-4 which has been well established as a negative regulator of T-cell function.
Objective The aim of this study was to investigate the association of an Single-Nucleotide Polymorphism (SNP) in CTLA4 49-A with genetic susceptibility to acute leukemia.
Participants and methods This is a case–control study that included 65 Egyptian patients with acute lymphoblastic leukemia (ALL) and 25 patients with acute myeloid leukemia (AML). The control group comprised 60 healthy unrelated participants with no family history of leukemia or autoimmune disease. Using the PCR-restriction fragment length polymorphism methodology, CTLA4 49 A/G was analyzed in 150 samples representing 90 patients (65 patients with ALL, and 25 patients with AML) and 60 controls.
Results There was no significant differences that were encountered between the different groups with regard to CTLA4 +49 A/G genotype or allele frequencies in ALL and AML cases. Neither was there a relation between the various genotypes and age of onset or the mode of presentation.
Conclusions CTLA4 49 A/G polymorphism was not recognized as a risk susceptibility factor in our case–control study. To our knowledge, this is the first study involving acute leukemia. The authors recommend that additional studies with larger sample sizes and different populations are required to confirm the findings of the present study
Keywords: acute lymphoblastic leukemia, acute myeloid leukemia, CTLA4
|How to cite this article:|
Hawass MA, Afifi EA, Hazaa IH, Ebid GT. Role of CTLA4-49A polymorphism in the pathogenesis of acute leukemia. Sci J Al-Azhar Med Fac Girls 2020;4:192-5
|How to cite this URL:|
Hawass MA, Afifi EA, Hazaa IH, Ebid GT. Role of CTLA4-49A polymorphism in the pathogenesis of acute leukemia. Sci J Al-Azhar Med Fac Girls [serial online] 2020 [cited 2020 Oct 26];4:192-5. Available from: http://www.sjamf.eg.net/text.asp?2020/4/2/192/288260
| Introduction|| |
Acute leukemia is a clonal disorder of the myeloid and lymphoid progenitors with distinctive morphologic, immunophenotypic, and genotypic features and represents the most frequent malignancy of the childhood. Acute leukemia is associated with several humoral and cellular immune abnormalities that could lead to an inadequate antitumor response .
The immune surveillance of tumor cells depends on the recognition of antigens presented in the context of human leukocyte receptor HLA class I molecules by cytotoxic T lymphocytes (CTLs), via their T-cell receptors .
CTLA-4 has been well established as a negative regulator of T-cell function .
CTLA-4 is rapidly expressed on T cells following activation and is highly upregulated by CD28 engagement. CTLA-4 shares B7 ligands with CD28. CTLA-4 binding with its ligands antagonizes early T-cell activation, leading to decreased IL-2 production, inhibition of cell-cycle progression, decreased cyclin expression, and the modulation of T-cell receptor signaling .
TLA-4-deficient mice develop a severe lymphoproliferative disease and die within 3–4 weeks . CTLA-4 is also important in the function of regulatory cells, which suppress effector T-cell activation and function 
The CTLA-4 gene is located between CD28 and ICOS genes . The functional significance of polymorphisms in the CTLA-4 gene have been widely explored and described. The most studied is CTLA-4.49A>G. This nonsynonymous Single-Nucleotide Polymorphism (SNP) causes an amino acid change from threonine to alanine. It influences T-cell activation and could have a role in antitumor immunity .
The presence of the [AA] genotype as opposed to the [GG] genotype has been shown to be associated with significantly lower levels of activation of T lymphocytes and lower proliferation. The protein product is encoded by the CTLA-4.49A>G [AA] genotype, CTLA-417Thr had a higher capacity to bind B7.1 and a stronger inhibitory effect on T-cell activation compared with CTLA-417Ala .
Also, the CTLA-4.49A>G [GG] genotype was found to be prevalent in the mucosa-associated lymphoid tissue lymphoma  and in multiple myeloma . There was no association between CTLA-4.49A>G SNP and colorectal cancer , B-cell chronic lymphocytic leukemia , cervical squamous cell carcinoma , or nonmalignant melanoma . This study aimed to investigate the association of an SNP in CTLA4 49-A with genetic susceptibility to acute leukemia.
| Participants and methods|| |
The study is carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The protocol was revised and approved by the Institutional Review Board of the National Cancer Institute (NCI), Cairo University. A written informed consent was obtained from all participants or their parents.
This study included 65 newly diagnosed acute lymphoblastic leukemia (ALL) patients and 25 newly diagnosed acute myeloid leukemia (AML) patients who had attended the Adult and Pediatric Oncology Departments, NCI, Cairo University during the period from March 2013 to March 2015. The recruited patients comprised 37 men and 28 women in cases of ALL between the ages of 1 and 22 years with a median of 5 years. In cases of AML the patients comprised 11 men and 14 women in the age range of 9–72 years with a median of 34 years.
A general population of the control group composed of 60 individuals comprising 25 men and 35 women in the age range of 18–52 years. They were randomly selected from blood donors in the NCI laboratory. They were healthy unrelated participants with no family history of leukemia or other autoimmune diseases. The choice of adults was to avoid ethical issues of using healthy children as controls.
DNA analysis and PCR-restriction fragment length polymorphism testing for CTLA-4 +49 A/G
Genomic DNA was extracted from EDTA peripheral blood samples using the salting out technique . Identification of the loci was performed based on the PCR-restriction fragment length polymorphism protocol . Oligonucleotide primers were synthesized (Pharmacia Biotech, Stockholm, Sweden). Amplification of the CTLA-4 gene was performed in 20 11 mix containing 100 ng of genomic DNA, 25 pmol of each primer (forward primer 50-CCACGGCTTCCTTTCTCGTA-30 and a reverse primer 50-AGTCTCACTCACCTTTGCAG-30), 100 M each dNTP, 1.5 mmol/l MgCl2, and 1.2 U Taq polymerase (Promega, Madison, Wisconsin, USA). PCR conditions consisted of initial denaturation at 95 LC for 2 min, followed by 40 cycles at 94 LC for 30 s, 50 LC for 45 s, and 72 LC for 30 s and a final extension step at 72 LC for 10 min. This results in a fragment of 327 bp.
Digestion of the PCR product was performed in a 20 ll mix containing 10 ll of the PCR product, 10 U BbvI (Fermentas), and 1 X buffer. The mix was incubated at 65 LC for 15 min The digested products were separated on 2% agarose. This resulted in no digestion with a 327 bp single band in the wild type (AA) and two bands of 244 and 83 bp with G allele ([Figure 1]) compared with the 100 bp ladder (Thermo-Fermentas).
|Figure 1 CTLA4–49 A/G genotypes after BbvI digestion. Lane 1: 100 bp ladder. Lanes 2, 4, and 5: heterozygous: AG: 327 bp, 244 bp, and 84 bp. Lane 3: wild type: AA: 327 bp. Lane 6: homozygous: GG: 244 bp and 84 bp.|
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SPSS version 17.0 (IBM, New York, USA) was used for data management. Proportions were compared using the χ2-test and Fisher’s exact. Odds ratio of genotype(s) was calculated with 95% confidence interval. Parametric and nonparametric tests compared the means of two or more than two independent groups (t-test and analysis of variance). P value was always two tailed and considered significant at 0.05 level.
| Results|| |
The age of the patient in cases of ALL range from 1 to 22 years with a mean of 6.3±4.1 and a median of 5 years, while in cases of AML the age of patients range from 9 to 72 years with a mean of 35.7±16.8 and a median of 34 years. The mode of presentation was classic symptoms in almost patients. Diagnosis was performed according to clinical, morphological, cytochemical, and immunophenotypic examination. In cases of ALL the CTLA-4 +49 A/G genotype and allele frequency among the patients and the control group are present in [Table 1] and [Table 2].
|Table 1 Comparison between CTLA4-49 genotypes in acute lymphoblastic leukemia patients versus control groups|
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|Table 2 Comparison between CTLA4-49 genotypes in acute myeloid leukemia patients versus control groups|
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Distribution of CTLA-4 genotypes and alleles in patients compared with control did not show any significance in cases of AML.
| Discussion|| |
Gene polymorphisms have been frequently studied by researchers as possible parameters for the evaluation of cancer risk . Some important polymorphic sites of the CTLA4gene have been studied in various diseases, and different alleles and genotypes of this gene are reported to contribute to susceptibility, as well as to some cancer types . However, these studies have reported controversial results about the association of CTLA4 polymorphisms with various cancer types .
In this study, CTLA-4 genotypes and alleles in patients compared with control did not show any significance in cases of ALL and AML) which agreed with the results of Minhas et al. , Dai et al. , and Karabon and Frydecka .
Polymorphisms in genes can influence the level of protein expression . Therefore, genetic variation in genes encoding co-signaling molecules may also alter the antitumor response and influence cancer susceptibility, particularly susceptibility to acute leukemia. The association of CTLA-4 gene polymorphism with different types of malignancies has been studied as the study of association of CTLA4-49A/G in breast cancer. The result of this study shows lack of any association of the CTLA-4 +49 G/A polymorphism with breast cancer risk in a North Indian Population , where  the functional CTLA4-49A>G was evaluated in colorectal cancer patients and healthy controls. The results suggested that the CTLA4-49A>G polymorphism was not associated with the risk of colorectal cancer in the study population. In contrast to others, where CTLA4-49A>G polymorphism was associated with the risk of non-Hodgkin’s lymphoma (NHL) in the Italian population, the analysis of the CTLA4 gene −49A>G polymorphism among an Italian NHL and control groups, they found that the G variant genotype was associated with a significantly decreased risk of NHL .
| Conclusion|| |
While this study did not confirm the association of CTLA4-49A>G SNP with the susceptibility to acute leukemia in Egyptians, it should be investigated in further studies with larger sample sizes and different populations to confirm the findings of the present study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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