Does TNF- α 308 G/A (rs1800629) gene polymorphism associate with liver and pancreas disorders in Iraqi adults with beta thalassemia major?

1. Introduction

β -thalassemia is an inherited autosomal recessive blood disorder [1] considered most prevalent genetic disease worldwide [2]. Major type of thalassemia causes in severe anemia, which requires frequent and regular blood transfusions [3]. According to estimations, 3% of the global population possesses a phenotype associated with β -thalassemia [4, 5]. The World Health Organization estimates that 68,000 people are born every year with β -thalassemia and the incidence of symptomatic β -thalassemia is thought to be 1 in 100,000 people in the general population [6]. The number of people with thalassemia in 2019 in Iraq is estimated to be over 22,000, according to the ministry of health, it consider a critical health problem in Iraq due to mainly the non-availability of equipment and drugs during different periods of turmoil and war and requires an efficient management strategy including public health education initiatives to promote early detection and treatment [7]. Even in nations where chelation is widely available, iron overload is still a serious issue for those with thalassemia major.

TNF α is a potent inflammatory cytokine that increases cytokines and has a critical role in immune responses [8], in addition being a crucial multifunctional cytokine produced by a variety of immune cells, including macrophages [9]. The gene encoding TNF- α is located in the class III region of the major histocompatibility complex on chromosome 6 between the HLA-B and HLADR genes [10].

Tumor Necrosis Factor- α (TNF- α ) is an important inflammatory cytokine in the development of liver disease. This cytokine can cause hepatic injury, cirrhosis and eventually promote hepatocellular carcinoma [11, 12]. Moreover, TNF is believed to be one of the main pro-inflammatory cytokines involved in the destruction of pancreatic ß-cells [13]. Studies in vitro and in vivo have demonstrated that TNF ligand-inducing apoptosis-related repression of proliferation and differentiation erythrocyte progenitor cell. furthermore, there are significantly increases in the levels of TNF- α which can prevent erythropoiesis in the bone marrow of thalassemia patients. In the promoter of the TNF α gene, many (SNPs) were discovered. A number of disorders have been studied in association to the TNF - 308G/A SNPs. The prevalent allele G, is changed at location - 308 with the rare allele A. The rare allele was related to elevated expression of TNF- α despite its the accuracy of the mechanism are not clear [14]. These SNPs may result in structural changes in the regulatory sites of the gene, which may have an impact on TNF- α production or function [15]. The biological effects of TNF- α are numerous and complex, and they range from conferring disease resistance to causing pathological complications [16].

In fact, TNF- α have contradictory function which may be caused by genetic polymorphism in the genes that regulate its synthesis and effects [17].

The aim of this study was to explore the relationship of (rs1800629) gene polymorphism associated to liver and pancreas disorders in sample of β -thalassemia major adult Iraqi Patients.

2. Material and method

Blood samples were obtained from 40 patients suffered from β TM with pancreas disorder as group A, along with 40 patient suffered from thalassemia with liver disorder as group B, and 40 patient suffered from thalassemia without pancreas or liver disorders as group C, from Ibn Al-Baladi Hospital, Baghdad, and 40 samples from age and gender-matched apparently healthy individuals as a negative control (Group D),the groups of patients include both male and female without hepatitis, all with age more than 18 years and checked the levels of ferritin, ALT and amylase for all groups to confirm liver and pancreas disorders. After obtaining the approval of the Ibn Al-Baladi Hospital and approval patients and under ethics committee on human research for a study conducted by the Institute of Genetic Engineering and Biotechnology for Post Graduate Studies, University of Baghdad (Reference number: 15/5/2022–1304), (1.5 ml) of venous blood was taken from all subjects, put in an anticoagulant tube contain (EDTA), mixed gently and kept in the ( - 20 ∘ C). Genomic DNA from frozen blood samples was extracted by using DNA extraction kit (Geneaid/Taiwan) according to Sambrook and Russell [18]. Purity and concentration of the DNA were measured by using Nanodrop spectrophotometer (Thermo Scientific/UK). Detection of TNF gene polymorphism G/A (rs1800629) by use T-ARMS-PCR. The primers for TNF α were provided by (Bioneer/Korea) depending on NCBI-Gene Bank data base as illustrated in Table 1. The Program ARMS PCR of TNF- α at position 308 G/A given in Table 2. The gel electrophoresis had been performed to determine the quality of the genomic DNA extraction and to visualize the PCR products size after finishing the ARMS PCR experiments.

The results of this study were analyzed by using SPSS [19]. Chi-square test was used to find the differences among numbers of groups and genotypes, also to find the Odds ratio (ORs) with a 95% confidence interval (CI).

The result of distribution of TNF α - 308 (rs1800629) polymorphism and allele frequency in thalassemia with pancreas disorders patients as group (A) and control as group(D) revealed that the wild genotype GG was 10 (25%), heterogeneous genotype GA was 16 (40%) with odds ratio (2.86), and homogeneous genotype AA was 14 (35%) with odds ratio (1.1), while G allele frequency was 36 (45) with Odd Ratio (OR) (0.288) and A allele frequency was 44 (55) with OR (2.76) in patents of (A) group with highly significant differences ( P ⩽ 0.01). In group (D) the wild genotype GG was 30 (75%), heterogeneous genotype GA was 7 (17.5%), homogeneous genotype AA was 3 (7.5%), G allele frequency was 67 (83.75%) and A allele frequency was 13 (16.25%), as shown in Table 3 and Figs 1, 2.

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The results of distribution of TNF α - 308 (rs1800629) polymorphism and allele frequency in thalassemia with liver disorders as group (B) and control as group (D) appeared that the wild genotype GG was 6(15%), heterogeneous genotype GA was 14 (35%) with OR (2.57), and homogeneous genotype AA was 20 (50%) with OR (0.012) while G allele frequency was 26 (32.5%) with OR (0.189) and A allele frequency was 54 (67.5%) with OR (3.76) in group(B) with high significant differences ( P ⩽ 0.01) as shown in Fig. 3. while the wild genotype GG was 30 (75%), heterogeneous genotype GA was 7 (17.5%), homogeneous genotype AA was 3 (7.5%), G allele frequency was 67 (83.75%) and A allele frequency was 13 (16.25%) in group (D) as given in Table 4.

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The findings of distribution of TNF α - 308 (rs1800629) polymorphism and allele frequency in thalassemia without pancreas or liver disorders as group (C) and control as group (D) appeared that the wild genotype GG was 6 (15%), heterogeneous genotype GA was 18 (45%) with OR (1.51), and homogeneous genotype AA was 16 (40%)with OR (1.49), while G allele frequency was 30 (37.5%) with OR (0.496) and A allele frequency was 50 (62.5%) with OR (3.015) in group (C) with high significant differences ( P ⩽ 0.01) as shown in Fig. 4. while the wild genotype GG was 30 (75%), heterogeneous genotype GA was 7 (17.5%), homogeneous genotype AA was 3(7.5%), G allele frequency was 67 (83.75%) and A allele frequency was 13 (16.25%) in group (D) as illustrated in Table 5.

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One of the most common complications associated with thalassemia is iron overload resulting from regular blood transfusions and the excess of iron can damage liver and endocrine system [20].

Since red blood cells are degraded in the reticulo-endothelial system iron overload will affect the hepatic macrophages (called Kupffer cells).Thereafter, intramacrophagic iron will be released progressively into the blood stream, in order to reach the bone marrow and lead to the production of new red blood cells. During this releasing process, plasma transferrin – the normal iron transport protein in the blood, whose normal saturation by iron is less than 45% – increases rapidly, reaching often 100%. This will lead to the appearance of plasma non-transferrin bound iron (NTBI) [21], an iron species which has the property to be very rapidly taken up by parenchymal cells (the hepatocytes for the liver), therefore accounting primarily for hepatic iron overload, the liver being, for circulating iron, both the first line target and the main storage organ, thus liver cells are damaged. Pancreatic iron overload is common in thalassemia major patients and pancreatic iron loading in thalassemia major patients begins in early childhood, as well as many studies have documented endocrine pancreas dysfunction in patients with thalassemia. It is frequently observed that the pancreas’ endocrine activity is impaired, which may result in severe diabetes mellitus [22, 23].

Single nucleotide alteration from guanine (G) to adenine (A) at location - 308 is the most of popular in generally populations. This transformation has been shown affect the expression of TNF- α . Unfortunately, there was no previous studies dealing with TNF α - 308 (rs1800629) polymorphism in thalassemia patients, except study of Mohammed [24] which was agreed with present study when it examined the connection between TNF α gene polymorphism with β -thalassemia patients and found that the genotype AA and allele A were related to risk with β -thalassemia susceptibility, and the mutant genotype AA had an O.R. value of 1.83, as well as the A allele had an O.R. value of 1.3. However, there were many studies that delved into the importance of this gene variation, such as study of Zhuang et al. [25] found that under three genetic comparison models, the TNF - 308 G/A polymorphism was substantially correlated with a reduced incidence of psoriasis and the study by Kim et al. [26] referred to there was no association between the risk of Nephrotic Syndrome and the TNF α - 308 polymorphism. Also a study close to this study, which was conducted by Cavalcante et al. [27] who studied the connection between sickle cell anemia and frequent genotype of patients with TNF-alpha-308 G $>$ A polymorphism was GG n = 20; 80%), and GA ( n = 5; 20%). None of the patients with AA ( n = 0; 0%), and this similar to our results for frequency TNF α - 308 (rs1800629) polymorphism. With regard to pancreatic disorders, there are many studies that have focused on this topic, such as study of Zhang et al. [28] in the Chinese population with acute pancreatitis, discovered no correlation between acute pancreatitis and TNF α - 308. But, they suggest that TNF α - 308 A allele is related to a higher risk of severe sepsis complicating of Acute Pancreatitis. And researchers by Sargen et al. [29] and Powell et al. [30] when studied of TNF α - 308 genotyped in acute pancreatitis patients in UK population and found that TNF α - 308 played role in severity and susceptibility to acute pancreatitis.

5. Conclusion

In a sample of adults patient with beta thalassemia major, the prevalence of the TNF- α 308 G/A SNP plus (A) allele may be linked to an increased risk of liver and pancreatic diseases.

Authors’ contributions

H. A. L. played a significant role in the conceptualization of the study, interpretation and analysis of data, software implementation, validation, formal analysis, investigation, resources, data curation, preparation of the manuscript, and writing-review and editing. B. J. M. also made substantial contributions to the research, including interpretation and analysis of data, validation, writing-review and editing, and supervision of the study. The authors confirm that all authors have read and agreed to the published version of the manuscript, and their combined efforts have resulted in the completion of this work.

Availability of data and materials

The authors acknowledge that the data supporting the findings of this study are available and can be obtained from the Institute of Genetic Engineering and Biotechnology for Post Graduate Studies, University of Baghdad in Iraq. Researchers or individuals interested in accessing the data may submit requests to the corresponding author, who will provide further guidance and facilitate the process.

Ethical considerations

Ethical approval or informed consent for a study conducted by the Institute of Genetic Engineering and Biotechnology for Post Graduate Studies, University of Baghdad (Reference number: 15/5/2022–1304).