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Genotyping of the BCL2 Gene Polymorphism rs2279115 Shows Associations with Leukemia Tendencies in the Iraqi Population.

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 posttranscriptional 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[TM] 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[degrees]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 25pl total volume reaction containing 1pl of genomic DNA (100 ng/pl), which was amplified with 10 mM of forward and reverse primers, 0.2mM each dNTP, 2.0 mM Mg[Cl.sub.2], 1.0 and a U Taq DNA polymerase with a 1X reaction buffer (Promega, Cat#M7502). PCR program conditions included initial melting at 95[degrees]C for 5 minutes followed by 30 cycles of amplification with a denaturation step of 25 seconds at 95[degrees]C, an annealing step of 30 seconds at 58[degrees]C, an elongation step of 20 seconds at 72[degrees]C, and one level of elongation of 7 minutes at 72[degrees]C.

A volume of 10 [micro]l 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 HardyWeinberg 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 ([chi square]) 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 [chi square] values were 0.68 and 1.1, respectively.

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 wildtype, the AC allele produced 252 bp and 154bp fragments, and the AA allele produced 154bp and 98bp fragments.

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).

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).

CONCLUSIONS

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).

Abbreviations: SNP, single-nucleotide polymorphism; WHO, World Health Organization; RFLP-PCR, restriction fragment length polymorphism polymerase chain reaction; BMI, body mass index; BCL2, B-cell lymphoma 2; OR, odds ratio.

http://dx.doi.org/ 10.22207/JPAM.12.4.25

ACKNOWLEDGMENTS

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.

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Dhafer Abdulmahdi Faisal Al-Koofee [1] and Jawad Mohammed Ismael [2]

[1] University of Kufa, Faculty of Pharmacy, An-Najaf, Iraq.

[2] University of Kufa, Faculty of Medicine, An-Najaf, Iraq.

* Correspondence: dhafera.faisal@uokufa.edu.iq

(Received: 03 October 2018; accepted: 20 November 2018)

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

                                   Control   Patients   P-Value
                                    n=110     n=120

Age (years)     [less than or        32         47        N.S
                equal to]  35>35     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   Patients   P-Value
                                    n=110     n=120
Age (years)     [less than or        32         47        N.S
                equal to] 35>35      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

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

Genotype              No. of individuals (%)
Allele           Leukemic         Healthy
               Patients (1)   Individuals (1)

CC reference     38 (32)          46 (42)
AC               55 (46)         54 (49.1)
AA              27 (22.5)        10 (9.1)
AA+AC             82(68)         64(58.2)
CC+AC            93(77.5)         100(91)
AA              27 (22.5)        10 (9.1)
2(AA)+AC         109(90)          74(67)
Total              120              110

Genotype           Non-adjusted (2)
Allele         P-Value          OR

CC reference      1
AC              0.01     1.23(0.70-2.18)
AA              0.01     3.27(1.41-7.60)
AA+AC           0.01       1.550.9-2.66
CC+AC             1
AA              0.005      2.91.33-6.32
2(AA)+AC        0.03     1.781.059-3.003
Total

(1) [chi square] 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.
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Title Annotation:RESEARCH ARTICLE
Author:Al-Koofee, Dhafer Abdulmahdi Faisal; Ismael, Jawad Mohammed
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Geographic Code:7IRAQ
Date:Dec 1, 2018
Words:2751
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