TNF-α-308A allele Carrier Induced to Development of Chronic Lymphocytic Leukemia in Sudanese Population at Earlier Age

se han realizado varios estudios para investigar la asociación de TNF-α-308G > ASNP y la susceptibilidad a la LLC. Sin embargo, los resultados son inconsistentes. Este estudio tuvo como objetivo investigar la asociación entre TNF-α-308G > ASNP del gen TNF-α y el riesgo de LLC en la población sudanesa y correlacionó los genotipos con las características clinicopatológicas. Se realizó un estudio de casos y controles en el estado de Jartum, durante el período de abril de 2017 a abril de 2018, en el que participaron 110 pacientes con LLC y 50 voluntarios sanos. Se realizó un examen físico, hemograma completo e inmunofenotipo en todos los pacientes para confirmar el diagnóstico. Se estudió la estadificación clínica como Rai y Binet. CD38 y ZAP70 se realizaron por citometría de flujo. Se recolectaron muestras de sangre de todos los participantes; el ADN se extrajo mediante el uso del kit de extracción de ADN de sangre ANALYTIKJENA y se analizó TNF-α-308G>ASNP mediante el uso de AS-PCR. El análisis estadístico se realizó utilizando las frecuencias de genotipo SPSS.TNF-α-308G >A fueron GG (10.0%), GA (87.3%) y AA (2.7%) entre los pacientes con LLC, y GG (14.0%), GA (80.0%) y AA (6.0%) en el grupo de control. La comparación de los pacientes con LLC con el grupo de control no mostró ninguna relación estadísticamente significativa para las frecuencias genotípicas y alélicas. Además, no se observó asociación entre el TNF-α-308G > ASNP y el género, los parámetros hematológicos, los sistemas de estadios clínicos, la expresión de CD38 y la expresión de ZAP-70. La presencia del alelo TNF-α-308 se asoció con una edad media más baja. Estos resultados indican que los genotipos TNF-α-308G >A no están involucrados en la predisposición al desarrollo de la LLC. El portador del alelo TNF-α-308A indujo el desarrollo de CLL a una edad más temprana.

section_content: Chronic lymphocytic leukemia is a chronic lymphoproliferative malignancy characterized by progressive lymphocytosis caused by the clonal accumulation of B-cells in peripheral blood, bone marrow, and lymphoid organs (Swerdlow et al., 2016). Many pleiotropic proinflammatory cytokines play a role in the activation, growth, and apoptosis of leukemic B-cells (Cordingley et al., 1988). Tumor Necrosis Factor-α is one of the most important pro-inflammatory and tumor-related cytokines for regulating immune response, inflammation, Th1/Th2 balance, and lymphomagenesis (Sethi et al., 2008). B-CLL cells can produce TNF, which is known to stimulate their proliferation in an RESEARCH ARTICLE TNF-α-308A allele Carrier Induced to Development of Chronic Lymphocytic Leukemia in Sudanese Population at Earlier Age autocrine and paracrine manner (Cordingley et al., 1988). TNF-α increases the proliferation and growth of CLL cells, but it may also induce cell apoptosis and necrosis (Younes and Aggarwall, 2003). 

The TNF-α gene is located in the short arm of chromosome 6p21. 3, in the class III region of the major histocompatibility complex, and consists of four exons and three introns (Makhatadze, 1998). TNF-α production is generally regulated at the transcriptional level (Raabe et al., 1998). 

Several single nucleotide polymorphisms of the TNF-α gene have been described inside the TNF-α promoter positioned (Elahi et al., 2009). Among the various SNPs of TNF-α, the -308G>A polymorphism; exchange of guanine by adenine at position -308 of the TNF promoter region is associated with higher serum levels of soluble TNF (Wilson et al., 1997). TNF-α-308A allele is associated with higher constitutive and inducible TNF-α expression by affecting a consensus binding site of a transcription factor named activator protein-2 (AP-2) (Kroeger et al., 1997). Approximately 60% to 70% of the white population are homozygous for the wild-type TNF1 allele, 30% to 40% are heterozygous, and 1. 5% to 3% are homozygous for the variant TNF2 allele (Wihlborg et al., 1999). 

To date, several studies have reported that TNF-α-308G>A gene polymorphism is associated with susceptibility to different cancer types such as Cervical Cancer (Hamadani et al., 2017) and CLL (Demeter et al., 1997; Wihlborg et al., 1999; Mainou-Fowler et al., 2000; Au et al., 2006; Bogunia-Kubik et al., 2006; Cerhan et al., 2008; Ennas et al., 2008; Jevtovic-Stoimenov et al., 2008; Bakirov et al., 2009; Abdou et al., 2010; Fernberg et al., 2010; Lech-Maranda et al., 2010; Bakirov et al., 2012; Lech-Maranda et al., 2013; Jevtovic-Stoimenov et al., 2017; Ovsepyan et al., 2017). However, the results are inconsistent and controversial because of the small sample size of individual study, the ethnic origin of the patients, and possibly because of CLL heterogeneity. These findings indicate that there remains a great controversy regarding the association between TNF-α-308G>A polymorphism and CLL risk. 

To the best of the authors' knowledge, no published studies are describing the possible association between TNF-α-308G>A gene polymorphism and CLL susceptibility in the Sudanese population. This study aimed to investigate the association between TNF-α-308G>A SNP of the TNF-α gene and CLL risk in the Sudanese population. We also examined the correlation between this SNP and CLL clinical parameters as well as hematological profile and some poor prognostic markers such as CD38 expression and ZAP-70 expression.
section_name: Introduction

section_name: Materials and Methods

section_content: This study is a case-control study, conducted in Khartoum state, Sudan, in the period from April 2017 to April 2018, 110 patients with CLL and 50 healthy volunteers as a control group were recruited to participate in this study. Patients were obtained at Flow Cytometry Laboratory for Leukemia & Lymphoma Diagnosis, Khartoum; they were referred for immunophenotype diagnosis. 

All patients were diagnosed based on clinical history, physical examination, and complete blood count. The peripheral blood is important to show morphological abnormalities and immunophenotypic criteria. All our patients have ≥5,000×10 9 /L B lymphocyte, considered in our diagnosis according to the International Workshop on Chronic Lymphocytic Leukemia (Hallek et al., 2008). The stage of the CLL was assessed by Rai and Binet classification (Rai et al., 1975; Binet et al., 1981). All patients were newly diagnosed without any previous treatment, whereas patients with other lymphoid neoplasms (both B and T-cell Lineage) were excluded.
section_name: Study Population

section_content: The peripheral blood was collected as samples from both groups included in the study (the patient group and control group). For the patient group, an amount of four milliliters (ml) of blood was collected from each patient in (EDTA) and divided equally into two tubes; one tube for complete blood count and immunophenotype test and the other tube for molecular analysis. For the control group, two (ml) of blood was collected from each healthy individual in (EDTA) for molecular analysis.
section_name: Sample Collection

section_content: Two ml of peripheral blood were withdrawn from each patient; these samples were collected in EDTA tubes and preserved at room temperature (22-24 o C) then processed within 6-24h from the collection. Complete blood count was analyzed by using automated hematology analyzer (SYSMEX KX-21N, Japan). All results such total WBC, Absolute lymphocyte count, Hemoglobin level, RBC, and platelets were recorded.
section_name: Determination of Blood Count

section_content: The diagnosis of CLL was confirmed in each patient by Flowcytometry (EPICS XL Beckman Coulter Flow Cytometry, Miami, FL, USA), standard protocol of Beckman Coulter was used in fluorescent dye-labelled monoclonal antibody for CD45, CD3 CD5, CD10, CD19, CD20, CD22, CD23, FMC7, CD79b, kappa, and lambda light chain. A marker was considered positive at cutoff ≥30%. However, in order to confirm diagnostic CLL, a scoring system was applied depending on Moreau et al., (1997). Absolute B lymphocyte count was obtained by Flow Cytometry and ZAP-70 and CD38 were used as prognostic markers, with a cutoff point of 20% and 30%, respectively, as previously described (Basabaeen et al., 2019).
section_name: Determination of Immunophenotyping and (CD38 & ZAP-70 expression)

section_content: After confirmed immunophenotyping of patients, genomic DNA was extracted from all blood samples of patients and control groups by using ANALYTIKJENA Blood DNA Extraction Kit (Germany) (REF-845-KS-1020050), according to the manufacturer's instructions. The β-globin gene was used to assess the quality of DNA in all extracted samples, the primer sequences of β globin as previously described (Kerr et al., 2000). All specimens for β-globin gene were Successful amplification with product size 268bp. To evaluate the DNA quantification after DNA extraction, we measured DNA by using a NanoDrop spectrophotometer. Then DNA samples were routinely at -20 o C.
section_name: DNA Extraction

section_content: Detection of the TNF-α-308G>A polymorphism based upon the method of Kamali-Sarvestani et al, three primers were used for AS-PCR was used to detect the polymorphism at position -308 of TNF-α the following primers were used, G allele: forward (5'ATAGGTTTTGAGGGGCATGG-3'), common reverse ratio with a confidence interval for risk estimation. The Hardy-Weinberg equilibrium was tested by the goodness of fit X2 test to compare the observed genotypic frequencies in normal individual to the expected genotypic frequencies, and then calculated from the observed allelic frequencies. A Chi-Square test was used for analyzing associations between categorical variables. One-Way ANOVA was used to compare the means of two groups. All P-values were two-sided, and <0. 05 was considered as the significance level.
section_name: Determination of TNF-α-308G>A rs1800629 Genotype by Allele Specific-Polymerase Chain Reaction

section_content: The mean age was 62. 97 (range: 22-85 years) for patients and 63. 22 (range: 24-83 years) for controls, and male participants accounted for a greater proportion in both groups (71. 8% and 72. 0%, respectively). No significant differences were found in age and gender between cases and controls (age: p 0. 900; sex: p 0. 981); therefore, the patients and controls seemed to be adequately matched in terms of age and gender. 

The TNF-α-308G>A genotype frequencies were GG (10. 0%), GA (87. 3%), and AA (2. 7%) among the CLL patients, and GG (14. 0%), GA (80. 0%), and AA (6. 0%) in the control subjects. The comparison of patients with CLL with the control group did not show any statistically significant relationship for the genotypic and allelic

(5'-TCTCGGTTTCTTCTCCATCG -3') TNF-α Aallele: forward (5'-ATAGGTTTTGAGGGGCATGA-3') (Kamali-Sarvestani et al., 2005). 

Two separated PCR reaction mixtures of 20µl were prepared for each sample. PCR was performed by using 4µl 5× HOT FIREPol Blend Master Mix, (Solis BioDyne,Estonia), Cat. No. 04-25-00125), 2µl of genomic DNA, 0. 5 µl of each primer, and 13 µl distilled water. Thermocycling conditions for both alleles include initial denaturation at 95 o C for 2 minutes followed by 30 cycles of denaturation at 95 o C for 30 seconds, annealing at 57. 2 o C for 30 seconds, and extension at 72 o Cfor 20 seconds, followed by a final extension at 72 o C for 5 minutes. Thermocycling was using TECHNE Tc-412-UK PCR Thermal Cycler 96 well. After amplification, PCR products and 100 bp DNA ladder (iNtRON BIOTECHNOLOGY, KOREA), were run on 2% agarose gel containing ethidium bromide and identified under UV transilluminator using gel documentation system (SYNGENE, JAPAN) (Figure 1 ).
section_name: Results

section_content: Data was analyzed using the statistical package for social sciences version 23. 0 (Chicago, IL, USA). Numerical data were summarized as mean and standard deviation and n (%) of study participants, respectively. Logistic regression was used for the calculation of odds 1, 2 ). Further, the genotype frequencies for TNF-α-308G>A SNP among both the case and control groups were found to be in disagreement with Hardy-Weinberg equilibrium (HWE) (Table 1 ). Frequency heterozygote GA was higher than that for homozygote TNF-α (-308GG &-308AA) in patients and control group. 

The mean ages at diagnosis differ between the three groups: 66. 27, 63. 26, and 41. 67 years in patients with -308GG, -308GA, and -308AA genotypes, respectively. This study found that there was a significant difference for mean age according to genotype frequencies in the case group (Table 2 ). 

We were unable to identify significant differences according to gender, Rai stage, and Binet stage system as well as hematological parameters between patients with variant genotypes of the studied polymorphism (Tables 2, 3 ). 

We divided patients into the following groups (using a 30 % cutoff value for CD38 and 20 % cutoff value for ZAP-70). Next, we compared the genotype distribution of TNF-α-308G>A between CD38 and ZAP-70 in CLL patients, but we did not find any significant differences (Table 2 ).
section_name: Statistical Analysis

section_content: Recently, several gene polymorphisms have been suggested to correlate with clinical outcome in CLL. There are conflicting data about the association of TNF-α-308G>A polymorphism and raised risk of CLL. 

In the present study, we did not find any significant association for the genotype nor the allelic frequency in patients with CLL regarding the TNF-α-308G>A polymorphism. Also, dichotomizing by dividing the TNF-α-308G>A genotypes as A+ genotypes (combining the GA and AA genotypes as one group) and G genotype (GG) also failed to indicate any significant associated with an increased risk of CLL development. In a similar result, most previous studies did not find any relationship between risk of CLL and the TNF-α-308G>A genotype frequency (Wihlborg et al., 1999; Mainou-Fowler et al., 2000; Au et al., 2006; Bogunia-Kubik et al., 2006; Cerhan et al., 2008; Ennas et al., 2008; Jevtovic-Stoimenov et al., 2008; Abdou et al., 2010; Fernberg et al., 2010; Lech-Maranda et al., 2010; Lech-Maranda et al., 2013). 

However, some studies found a significant increase in the frequency of the GG Genotypes and G allele in a polymorphic locus-308G>A gene TNF-α has appeared as markers of the raised risk in CLL development (Demeter et al., 1997; Bakirov et al., 2009; Bakirov et al., 2012). 

In contrast, Ovsepyan et al. and Jevtovic-Stoimenov et al. suggested that the presence of TNF-α-308A allele (-308AG & -308AA) may be associated with an increased risk of CLL development (Jevtovic-Stoimenov et al., 2017; Ovsepyan et al., 2017). Zhai et al., (2014) were performed a meta-analysis study to assess the effect of TNF-α-308G>A polymorphism on the risk of NHL and various subtypes in Caucasian and Asian populations, this meta-analysis study indicated that TNF-α-308G>A polymorphism is not associated with CLL risk in overall and each ethnic subgroup. No available studies in the literature describing the possible impact of TNF-α-308G>A polymorphism on susceptibility to CLL development in the African population, especially in the Sudanese population. 

Surprising the distribution of TNF-α-308G>A genotypes among the healthy controls in our study is dissimilar to those obtained in the previous study conducted in healthy unrelated individuals in Sudan (Bereir et al., 2003). In the present study, the genotype distribution of TNF-α-308G>A polymorphism had shown significant (HWE p<0. 001) deviation in both controls and CLL cases from the Hardy-Weinberg equilibrium. Similar to a previous study conducted in Sudan that included four Sudanese populations of different geographic origins (Bereir et al., 2003). This study suggested TNF-α-308G>A SNP departure from HWE in One population namely Nilotics (Bereir et al., 2003). This indicated inter-population variation for genotype frequencies at this locus (Bereir et al., 2003). 

Reports have demonstrated that the TNF-α -308GG genotype frequency ranged in between 43. 6% to 89. 0%, while TNF-α -308AA (TNF-α -308GA or AA) genotype is present in 0. 0% to 55. 1% among patients with CLL (Demeter et al., 1997; Wihlborg et al., 1999; Mainou-Fowler et al., 2000; Au et al., 2006; Bogunia-Kubik et al., 2006; Cerhan et al., 2008; Ennas et al., 2008; Jevtovic-Stoimenov et al., 2008; Bakirov et al., 2009; Abdou et al., 2010; Fernberg et al., 2010; Lech-Maranda et al., 2010; Bakirov et al., 2012; Lech-Maranda et al., 2013; Jevtovic-Stoimenov et al., 2017; Ovsepyan et al., 2017). No available studies in the literature describing the frequency of TNF-α-308G>A polymorphism among patients with CLL in the African population, especially in the Sudanese population. The present study showed a higher frequency of TNF-α -308GA heterozygote in patients and control group. Also some studies performed in Egypt and Northern Ivory Coast demonstrated a higher frequency of the TNF-α-308GA genotype 86. 7% and 60. 0%, respectively (Settin et al., 2007; Santovito et al., 2012). The variations found in the literature compared to those obtained from our study could be justified by the high heterogeneity degree seen in the African population. This observation suggests that the distribution of this polymorphism varies because of ethnic factors. 

In our study, the presence of theTNF-α-308A allele (TNF-α -308GA or AA) was associated with a lower mean age. In fact, most studies have failed to show a statistically significant correlation between age and TNF-α-308G>A polymorphism in CLL patients (Wihlborg et al., 1999; Au et al., 2006; Jevtovic-Stoimenov et al., 2008; Lech-Maranda et al., 2010; Lech-Maranda et al., 2013). Interestingly, Wihlborg et al., (1999) showed the mean age at diagnosis of Hodgkin's disease patients differed between the groups and were 36. 5 and 28. 2 years for TNFα-308GG and TNF-α-308GA, respectively. This indicated TNF-α-308A allele carrier induced to development of lymphoproliferative disorders at earlier age. Since no reports of the TNF-a gene polymorphism in CLL exist in the literature, this is a finding requiring further study. 

In the present study, the gender ratio was 2. 5:1 men for each woman in patients with CLL. We did not find any significant relationship for the genotype distribution and gender. In a similar result,Jevtovic-Stoimenov et al. and Lech-Maranda et al. did not find any relationship between gender and the TNF-α-308G>A genotypes distributions (Jevtovic-Stoimenov et al., 2008; Lech-Maranda et al., 2013). On contrast, a study conducted in the Chinese population found TNF-α -308A allele is significantly associated with female CLL cases (Au et al., 2006). Moreover, another study conducted in the Serbian population suggested that TNF-α-308A allele associated with male subjects with CLL (Jevtovic-Stoimenov et al., 2017). We are not able to provide an explanation of this observation at this moment, but the variation of male to female ratio and ethnicity heterogeneity cannot be excluded. 

In this report, we note no significant association was observed with the TNF-α -308G>A genotypes and Rai stage system. A previous studies also found no association between the presence of TNF-α-308G>A polymorphism and Rai stage system (Demeter et al., 1997; Mainou-Fowler et al., 2000; Bogunia-Kubik et al., 2006; Jevtovic-Stoimenov et al., 2008; Lech-Maranda et al., 2010; Lech-Maranda et al., 2013). 

In this study no significant difference was found in TNF-α-308G>A genotype distributions in relation to Binet system. Regarding the Binet stage system, findings of this study are in line with previous studies observations in CLL patients (Demeter et al., 1997; Mainou-Fowler et al., 2000; Bogunia-Kubik et al., 2006; Jevtovic-Stoimenov et al., 2008; Ovsepyan et al., 2017). It should be noted there was no indication that theTNF-α-308G>A polymorphism affects disease progression in CLL patients. 

In the present study statistical analysis did not reveal any differences in hematological parameters according to TNF-α-308G>A genotypes frequencies. In a similar study by AU et al. did not appear to be a relationship between TNF-α-308G>A genotype distributions and white cell count at diagnosis (Au et al., 2006). No another study found on literature discussed such this association. 

Our study, did not find any significant association in CD38 expression as well as ZAP-70 expression between CLL samples displaying the various TNF-α-308G>A genotypes. These results are in keeping with previous findings in other reports (Lech-Maranda et al., 2010; Lech-Maranda et al., 2013), confirming he TNF-α-308G>A polymorphism was not associated with any prognostic markers such as CD38 and ZAP-70 expression CLL patients. In contrast Polzonetti et al. suggested TNF-α-308G>A was significantly associated with CD38 activity in healthy individuals by using an RP-HPLC method (Polzonetti et al., 2012). 

In conclusion, the frequency of the TNF-α-308G>A promoter polymorphism was not significantly different between CLL patients and compared to controls. These results indicate that TNF-α-308G>A) alleles are not involved in the predisposition to the development of CLL. Our study showed a higher frequency of TNF-α -308GA heterozygote in patients and control group. The presence of TNF-α-308A allele (TNF-α -308GA or AA) was associated with lower mean age. No association was shown between the TNF-α-308G>A genotypes and gender, hematological parameters, and clinical stages systems as well as CD38 expression or ZAP-70 expression.
section_name: Discussion

section_name: Acknowledgments

section_content: We would like to thank the staff of the Hematology Department at Al Neelain University for facilities and supporting and we are grateful to the staff of Flow Cytometry Laboratory for Leukemia & Lymphoma for their collaboration. Finally special thanks to the patients for being cooperative, despite their pains. This work is licensed under a Creative Commons Attribution-Non Commercial 4. 0 International License.
section_name: Acknowledgments

section_name: Author Contribution Statement

section_content: AAB & EAA & IKI conceived the study design, participated in data collection, performed the statistical analysis, interpreted the results, and revised the manuscript. EAB, NMA, SOA & AYA participated in the statistical analysis and drafted the manuscript. OAA and EAF participated in the data collection, carried out the laboratory work, and prepared the results. AAB performed molecular analysis. All authors read and approved the final manuscript.
section_name: Author Contribution Statement

section_content: The individual data are available in the archives of the Flow Cytometry for Leukemia & Lymphoma Diagnosis, Khartoum, Sudan and can be obtained from the corresponding author on request.
section_name: Availability of data and materials

section_content: Not applicable
section_name: Consent for publication

section_content: Ethical clearance was obtained from the Institutional Review Board at Al Neelain University. The principal investigator obtained written informed consent from all participants prior to their inclusion in the study.
section_name: Ethics approval and consent to participate

section_content: The authors declare that they have no conflict interests.
section_name: Conflict of interest