Research Article | Open Access

Dhafer Abdulmahdi Faisal Al-Koofee1 and Jawad Mohammed Ismael2

1University of Kufa, Faculty of Pharmacy, An-Najaf, Iraq.
2University of Kufa, Faculty of Medicine, An-Najaf, Iraq.
J Pure Appl Microbiol. 2018;12(4):1899-1904 | Article Number: 5292
http://doi.org/10.22207/JPAM.12.4.25 | © The Author(s). 2018
Received: 03/10/2018 | Accepted: 20/11/2018 | Published: 30/12/2018
Abstract

The purpose of this study is to assess the SNP (rs2279115; – 938 C>A) polymorphism in the BCL-2 gene of Iraqi leukemic patients. The B-cell lymphoma 2 (BCL-2) gene is considered to be related with leukemia improvement. The single nucleotide polymorphism “(SNP; -938 C>A)” of BCL-2 was discovered a few years ago. In accordance with the SNP’s (rs2279115) function against leukemia, we evaluated the distribution frequency of the allele in addition to the relationship of the genotype with clinicopathological features. This study involved a case-control design in which the BCL-2 gene promoter polymorphism with the rs2279115 variant was genotyped in a total of 230 individuals, including 120 leukemia patients and 110 healthy subjects, to predict the variation in leukemic Iraqi patients. The patient group was enrolled from the Medical Euphrates Center for Oncology in the Najaf province that is involved in the WHO strategy of leukemia. From whole categories of blood, the DNA was extracted and genotyped using the BccI enzyme and the RFLP- PCR technique. The occurrences of the CC, AC, and AA alleles of the promoter BCL-2 gene polymorphism (C-938A) in leukemia patients were 38 (32%), 55 (46%), and 27 (22.5%), respectively, while control they were 46 (42%), 54 (49.1%), and 10 (9.1%), respectively in the control group (P<0.01). It was shown that the existence of the A allele in the SNP (rs2279115) in the BCL-2 gene promoter was related to an increased risk for the development of leukemia in the Iraqi population. The minor allele (A) in (rs2279115) of BCL-2 gene was significantly elevated (p<0.01) in leukemia patients (22.5%) as compared with healthy individuals (9.1%). Accordingly, the homozygous genotype AA (OR=3.27, CI 95% 1.41-7.60, P= 0.01) significantly augmented the risk of leukemia development by more than three times when compared with those of reference wild-type AA. The results of this study indicated that the AA genotype of BCL-2 (-938C>A) was associated with a predisposition to leukemia in the Iraqi population.

Keywords

Leukemia, BCL-2, SNP, Cancer, Iraq.

Introduction

During our lives, more than one-third of people will develop some type of disease or malignancy. In Western regions, this is the primary cause of death after cardiovascular disease (Davidson, 1953). Heterogeneous causes involving environmental and genetic toxins promote cancer development that involves abnormal cell growth with the opportunity to expand to new locations in the body (American Cancer Society, 2011). Leukemia is one kind of cancer that originates in the bone marrow, in the blood-forming tissues of the body that prevents typical blood function by abnormal cell division (Buffler et al., 2005.). The development of types of leukemia depends on the category of the affected blood cell. Some forms of leukemia are very familiar in children, like acute lymphocytic leukemia (ALL). Other types that arise frequently in adults are chronic myeloid leukemia (CML), as well as acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) (American Cancer Society, 2014). Differentiated features of AML that account for most cancer-related deaths are due to the monoclonal spread of myeloid blasts in blood and other tissues (He et al., 2014).

It is imperative to maintain tissue balance through restricted cell turnover (Moazami-Goudarzi et al., 2016). Apoptosis is a process that involves a chain of biochemical reaction signals for intrinsic or extrinsic processes. Relatives of the BCL2 protein play a central role in regulating apoptosis through these intrinsic or extrinsic pathways on the outer mitochondrial membrane (Zhang T, Saghatelian A, 2013). Regulation of proliferation and apoptosis homeostasis is carried out through pro-apoptotic members such as “BCL2-associated X protein (BAX)” and anti-apoptotic component like BCL2 itself which keeps cells from cell death programming (Bachmann et al., 2011).

The BCL2 proto-oncogene is firstly recognized in B-cell lymphoma rather than in a regulator of apoptosis process (Hockenbery et al., 1990). It also contributes to the healing of corrupted DNA and cell cycles (Deng and Gao, 2003; Zhang et al., 2007; Adams et al., 1999; Hara et al., 2008). The BCL2 gene has six exons and two different functional promoters localized on chromosome 18q21.33, and the first promoter is affected by negative regulation of the second promoter (Young, 1993).

The expression of BCL2 gene is dependent on endogenous and environmental stimuli that regulate the results of transcription and post-transcriptional levels. (Young, 1993; Donnini et al, 2004). At the transcriptional level, the regulation of BCL2 gene is carried out by positive or negative elements existing within the promoter, encoding the section and 32 -UTR (Ma et al., 2007).

Materials and Methods

Study subjects
In the current work, 120 leukemic patients in addition to 110 individuals as a control group were genotyped. All details of patients with leukemia were obtained from the Middle Euphrates Center for Oncology in Al-Sader Medical City in Najaf, Iraq. Leukemic patients were newly diagnosed, with histological confirmation and were not previously treated with radio- or chemotherapy. The classification of histology for all patients was performed by the center’s senior pathologists. Both subject groups under study were from Najaf and its surrounding regions. There were no criteria restrictions of their age or gender for participation.

Exclusion criteria involved all cases of previous cancer diagnoses and metastasis from other organs or previous treatments by radiotherapy or chemotherapy. Healthy individuals were cancer-free with no family history of cancer.

Genotyping BCL2
The genomic DNA was extracted from peripheral blood drawn both from patients and control subjects. Approximately 3ml of blood was collected in a Vacutainer Potassium-salt EDTA tube based on the protocol from the ReliaPrep™ Blood gDNA Miniprep System (Promega, USA, Cat# A5081). The concentration and purity of the extracted DNA were measured by using a Biodrop spectrophotometer (England) based on absorbance at 260 and 280 nm. Extracted DNA specimens were stored at -20°C for the genotyping analysis. BCL2 genotypes at the promoter region  (-938) SNP were evaluated by restriction fragment length polymorphism polymerase chain reaction technique (RFLP- PCR). The primers used for amplification of the target fragment were designed depending on the sequence of rs2279115. All primer sequence orders are in direction of 5′ to 3′ forward BCL2 (TTA-TCC-AGC-AGC-TTT-TCG-G’) and reverse BCL2 (GGC-GGC-AGA-TGA-ATT-ACA-A) with a 252bp amplicon size. The PCR reaction was carried out with a 25µl total volume reaction containing 1µl of genomic DNA (100 ng/µl), which was amplified with 10 mM of forward and reverse primers, 0.2mM each dNTP, 2.0 mM MgCl2, 1.0 and a U Taq DNA polymerase with a 1X reaction buffer (Promega, Cat#M7502). PCR program conditions included initial melting at 95°C for 5 minutes followed by 30 cycles of amplification with a denaturation step of 25 seconds at 95°C, an annealing step of 30 seconds at 58°C, an elongation step of 20 seconds at 72°C, and one level of elongation of 7 minutes at 72°C.

A volume of 10 µl of PCR product reaction from each sample was incubated with BccI Enzyme (NEB, England) for 1 hour for digestion. To visualize the digested and undigested products, 2% agarose was used in gel electrophoresis, and images were taken under an ultraviolet light transilluminator.

Statistical Analysis
BCL2 promoter (SNP -938C>A) genotyping based on an allele frequency was assessed by a statistical online web program (http://www.orge.org/software/hwe-mr-calc.shtml) for the Hardy-Weinberg Equilibrium (HWE). Significant levels were considered as p<0.05.

RESULTS

Leukemia patients and healthy subjects were all from Iraq. There was no significant statistic variation between the patients and healthy subjects groups in the their biochemical characteristic that involved age, gender, and residency, as presented in Table 1. A Chi-square (c2) test was applied to verify whether all subjects were compatible with the Hardy-Weinberg equilibrium (HWE). Both patient and healthy groups were harmonious with the HWE. The c2 values were 0.68 and 1.1, respectively.

Table (1):
Distribution of known clinicopathological variables of leukemia patients and control groups.

Control n=110 Patients n=120 P-Value
Age (years) ≤35
>35
32 47 N.S
78 73 N.S
Gender
Male 58 65 N.S
Female 52 55 N.S
Leukemia Type
ALL 0
AML 64
CLL 23
CML 33
Residency
An-Najaf 76 81 N.S
Samawa 16 22 N.S
Dewanea 18 17 N.S
Control
n=110
Patients
n=120
P-Value
Age (years) ≤35

>35

32 47 N.S
78 73 N.S
Gender
Male 58 65 N.S
Female 52 55 N.S
Leukemia Type
ALL 0
AML 64
CLL 23
CML 33
Residency
An-Najaf 76 81 N.S
Samawa 16 22 N.S
Dewanea 18 17 N.S

The amplicon of the promoter region of the variant Bcl2 gene (rs2279115; – 938 C>A) that produced by digestion of BccI restriction enzyme was under asingle band (252bp) for CC wild-type, the AC allele produced 252 bp and 154bp fragments, and the AA allele produced 154bp and 98bp fragments.

Table (2):
Genotyping distribution of BCL2 (rs2279115; – 938 C>A) in leukemic and healthy subjects.

 

Genotype Allele No. of individuals (%) Non-adjusted2
Leukemic Patients1 Healthy Individuals1 P-Value OR
CC reference 38 (32) 46 (42) 1
AC 55 (46) 54 (49.1) 0.01 1.23
(0.70- 2.18)
AA 27 (22.5) 10 (9.1) 0.01 3.27
(1.41-7.60)
AA+AC 82(68) 64(58.2) 0.01 1.55
0.9-2.66
CC+AC 93(77.5) 100(91) 1
AA 27 (22.5) 10 (9.1) 0.005 2.9
1.33-6.32
2(AA)+AC 109(90) 74(67) 0.03 1.78
1.059-3.003
Total 120 110

1 X2 for HWE of leukemia patients and healthy individuals groups is 0.68 and 1.1 respectively (both p > 0.05).
2 Logistic regression model, non-adjusted.

As shown in Table 2, the BCL-2 genotype homozygote CC allele was linked to a decreased risk of leukemia by 3.27 times as in the homozygote AA. Moreover, the genotype homozygote AA was associated with an increased risk of leukemia by 2.9-times as in the shared genotypes AC+CC alleles. Random selection of various samples from leukemia patients was carried out to demonstrate the results of the PCR product and genotyping (Fig. 1).

Fig. 1. Genotyping polymorphism of BCL-2 (-938C > A) with PCR product, A; and RFLP-PCR fragments, B. The wild type CC allele of the BCL-2 promoter region produced a single 252-bp fragment, the AC allele produced 252bp and 154bp fragments, and the AA allele produced 154bp and 98bp fragments.

DISCUSSION

The purpose of this study was to identify a polymorphism in the promoter region (-938C>A) of the BCL-2 gene. Many types of cancers are affecting the health of the global population and leukemia is considered to be one of the most damaging blood cancers. Depending on what kind of blood cell is affected, different types of leukemia (ALL, AML, CLL, CML) can  develop (Arber et al., 2018). Due to the past three decades of war against Iraq, many types of cancers involving leukemia have been reported; especially following the last war in 2003 and the next seven years of occupations when many kinds of weapons were used (Al-hashimi & Wang, 2013). Most leukemic Iraqi patients were subjected to many crises in the last three decades. Therefore, it is essential to understand the correlation between the promoters of BCL-2 gene polymorphism and leukemia development, as unfortunately there is no information on this in Arab, particularly Iraqi populations, specifically as regards to the role of gene polymorphisms as predictors for leukemia. The BCL-2 gene is identified as an antiapoptotic regulatory protein that may serve as an inhibitor of proliferation (Bachmann et al., 2011). Hence, many studies examine the role of BCL-2 gene variants in altering gene expression and/or protein function that could affect the sensitive equilibrium of pathways regulating cell death. This could represent a potential biomarker that would offer the best treatment option with drugs targeting BCL-2 (Zhang and Ming, 2008). The purpose of this work was to detect the risk association between a BCI-2 gene polymorphism and leukemia in Iraqi subjects. The Hardy-Weinberg equilibrium (HWE) was measured. The single nucleotide polymorphism (rs2279115) was consistent with HWE (p>0.05). In other words, this SNP is associated with the development of leukemia in the cases under study. This finding was also seen in Asian inhabitants, but not in Caucasian populations (Zhang et al., 2014). BCl-2 gene variant polymorphism (rs2279115), genetic power, and HWE primarily rely on the sample magnitude, project design, and allele frequency in all groups (Evans & Purcell, 2012). An intended SNP of BCL-2 gene genotyping exhibits strong links with leukemia, as the presence of the minor A allele in (rs2279115) was three times more prevalent in leukemia homozygote and recessive patterns when compared with reference wild-types, as this reduces apoptosis and the improves proliferation rate. The failure of apoptosis due to enhanced expression of the anti-apoptotic protein BCL-2 culd thus promote malignant cell growth (Meka et al., 2015).

CONCLUSION

A BCL-2 gene polymorphism (rs2279115) is correlated with the occurrence of leukemia in Iraqi population. The presence of the (AA) homozygous genotype of BCL-2 (-938C > A) is associated with a three-fold increase in the likelihood of leukemia development. On the other hand, carriers of the heterozygous (AC) allele have about a 50 % risk level of (AA) genotype, as compared with reference genotypes (CC).

Declarations

Acknowledgements
I would like to express my gratitude to medical team at the Middle Euphrates Center for Oncology in Al Sader Medical City (Najaf, Iraq) for their support in obtaining blood specimens from all leukemic patients. I am also thankful to all the patients who made this study possible.

References
  1. Adams JM, Huang DC, Puthalakath H, Bouillet P, Vairo G, Moriishi K, Hausmann G, O’Reilly L, Newton K, Ogilvy S, Bath ML, Print CG, Harris AW, Strasser A, C. S. Control of apoptosis in hematopoietic cells by the Bcl-2 family of proteins. Cold Spring Harb Symp Quant Biol., 1999 ; 64: 351–8.
  2. Al-hashimi, M. M. Y., & Wang, X. Trend of Leukemia in Ninawa / Iraq, 2013; 1(8), 353–362.
  3. American Cancer Society. What Is Liver Cancer? American Cancer Society, (November 2009), 2011; 1–6.
  4. American Cancer Society. Cancer in Young Adults. Retreived from https://www.cancer.org/cancer/cancer-in-young-adults.html 2014.
  5. Arber, D. A., Orazi, A., Hasserjian, R., Borowitz, M. J., Beau, M. M. Le, Bloomfield, C. D., Vardiman, J. W. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia, 2018; 127(20), 2391–2406. doi:10.1182/blood-2016-03-643544.The
  6. Bachmann, H. S., Heukamp, L. C., Schmitz, K. J., Hilburn, C. F., Kahl, P., Buettner, R., … Schulte, J. H. Regulatory BCL2 promoter polymorphism (-938C>A) is associated with adverse outcome in patients with prostate carcinoma. International Journal of Cancer. Journal International Du Cancer, 2011; 129(10), 2390–9. doi:10.1002/ijc.25904
  7. Buffler, P. a, Kwan, M. L., Reynolds, P., & Urayama, K. Y. Environmental and genetic risk factors for childhood leukemia: appraising the evidence., 2005; 23(1), 60–75. doi:10.1081/CNV-200046402
  8. Davidson’s. Principles & Practice of Medicine 20th. (n.d.). 1953.
  9. Deng X1, Gao F, M. W. J. Bcl2 retards G1/S cell cycle transition by regulating intracellular ROS. Blood, 2003; 1;102(9), 3179–85. doi:10.1182/blood-2003-04-1027
  10. Donnini M, Lapucci A, Papucci L, Witort E, Jacquier A, Brewer G, Nicolin A, Capaccioli S, S. N. Identification of TINO: a new evolutionarily conserved BCL-2 AU-rich element RNA-binding protein. J Biol Chem, 2004; 7;279(19), 20154–66. doi:10.1074/jbc.M314071200
  11. Evans, D. M., & Purcell, S. Power Calculations in Genetic Studies, 2012; 664–674. doi:10.1101/pdb.top069559
  12. Hara T1, Omura-Minamisawa M, Kang Y, Cheng C, I. T. Flavopiridol potentiates the cytotoxic effects of radiation in radioresistant tumor cells in which p53 is mutated or Bcl-2 is overexpressed. Int J Radiat Oncol Biol Phys, 2008; 1;71(5), 1485–95. doi:10.1016/j.ijrobp.2008.03.039
  13. He, G., Wang, C., Li, Q., Tan, H., Chen, F., Huang, Z., … Liu, D. Clinical and laboratory features of seven patients with acute myeloid leukemia (AML)-M2/M3 and elevated myeloblasts and abnormal promyelocytes. Cancer Cell International, 2014; 14, 111. doi:10.1186/s12935-014-0111-y
  14. Hockenbery, D., Nuñez, G., Milliman, C., Schreiber, R.D., Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature, 1990; 22;348(6299), 334–6. doi:10.1038/348334a0
  15. Ma, C., Zhang, J., Durrin, L.K., Lv, J., Zhu D., Han X. The BCL2 major breakpoint region (mbr) regulates gene expression. Oncogene, 2007; 19;26(18), 2649–57. doi:10.1038/sj.onc.1210069
  16. Meka, P., Jarjapu, S., & Vishwakarma, S. K. Influence of BCL2-938 C > A promoter polymorphism and BCL2 gene expression on the progression of breast cancer. Tumor Biology 2015; 37(5) doi:10.1007/s13277-015-4554-0
  17. Moazami-Goudarzi, M., Farshdousti-Hagh, M., Hoseinpour-Feizi, A., Talebi, M., Movassaghpour-Akbari, A. A., Shams-Asanjan, K., … Seifi, M. The acute lymphoblastic leukemia prognostic scoring whether it is possible by BCL-2, BAX gene promoter genotyping. Caspian Journal of Internal Medicine, 2016; 7(2), 105–113.
  18. Young RL, K. S. A negative regulatory element in the bcl-2 5´-untranslated region inhibits expression from an upstream promoter. Mol Cell Biol, 1993; 13, 3686–97.
  19. Zhang, L. Ming L. J. Y. NIH Public Access, 2008; 10(6), 207–217.
  20. Zhang,T, Saghatelian A. Emerging roles of lipids in BCL-2 family-regulated apoptosis. Biochim Biophys Acta, 2013; 1831(10), 1542–54. doi:10.1016/j.bbalip.2013.03.001
  21. Zhang, W., Pantschenko, A.G., McCarthy, M.B. Bone-targeted overexpression of Bcl-2 increases osteoblast adhesion and differentiation and inhibits mineralization in vitro. Calcif Tissue Int, 2007; 80(2), 111–22. doi:10.1007/s00223-006-0168-2
  22. Zhang, X., Weng, W., Xu, W., Wang, Y., Yu, W., Tang, X., … Pan, Q. Role of Bcl-2 -938 C . A polymorphism in susceptibility and prognosis of cancer: a meta-analysis, (August), 2–8; 2014. doi:10.1038/srep07241.

Article Metrics

Article View: 1551

Share This Article

© The Author(s) 2018. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.