key: cord-0946831-44o5l1ir
authors: Ali, Hussein N.; Niranji, Sherko S.; Al‐Jaf, Sirwan M. A.
title: Association of tumor necrosis factor alpha ‐308 single nucleotide polymorphism with SARS CoV‐2 infection in an Iraqi Kurdish population
date: 2022-04-04
journal: J Clin Lab Anal
DOI: 10.1002/jcla.24400
sha: c50185c4aaa37bb4a7234c4f7c0a5c47a06284d0
doc_id: 946831
cord_uid: 44o5l1ir

Uncovering risk factors playing roles in the severity of Coronavirus disease 2019 (Covid‐19) are important for understanding pathoimmunology of the disease caused by severe acute respiratory syndrome Coronavirus 2 (SARS CoV‐2). Genetic variations in innate immune genes have been found to be associated with Covid‐19 infections. A single‐nucleotide polymorphism (SNP) in a promoter region of tumor necrosis factor alpha (TNF‐α) gene, TNF‐α −308G>A, increases expression of TNF‐α protein against infectious diseases leading to immune dysregulations and organ damage. This study aims to discover associations between TNF‐α −308G>A SNP and Covid‐19 infection. Polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) was used for genotyping a general Kurdish population and Covid‐19 patients. The homozygous mutant (AA) genotype was found to be rare in the current studied population. Interestingly, the heterozygous (GA) genotype was significantly (p value = 0.0342) higher in the Covid‐19 patients than the general population. This suggests that TNF‐α −308G>A SNP might be associated with Covid‐19 infections. Further studies with larger sample sizes focusing on different ethnic populations are recommended.

Coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has been considered as a pandemic and public health threat. The immunopathogenesis of Covid-19 requires outstanding research to uncover the reasons behind the severity of the disease in some Covid-19 patients while it is mild or asymptomatic in others. It is known that SARS CoV-2 enters into human cells via interactions between human angiotensinconverting enzyme 2 receptor (ACE2) and viral spike protein.

Additionally, SARS CoV-2 initiates both innate and adaptive immune response through pattern recognition receptors and cytokines, particularly proinflammatory cytokines including interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNFα), which lead to cytokine storm, acute respiratory distress syndrome (ARDS) and death in some Covid-19 patients. 1, 2 Researchers have been continuously looking for discovering risk factors that can be connected to the disease. At the first step, it was suggested that individuals, who are old, male and have previous cardiovascular problems, severely acquire the disease. Furthermore, it was also noticed that genetic variations in human genes (e.g., ACE2, TMPRSS2, cytokines, TLR-7, androgen receptor, and protease genes) may also be related to Covid-19 severity. 3 Consequently, candidate genes, including genetic variants in cytokines, chemokines, and receptors, were selected based on their previous associations with SARS Cov-1, SARS CoV-2, and other infectious diseases. 4, 5 The candidate genes include variations in ABO blood groups, human leukocyte antigen (HLA loci; type I interferon-related genes (e.g., TLR3 ,   TLR4, UNC93B1, TBK1, IRF7, IFNAR2, OAS, IFITM3, and PRKRA; cytokines (e.g., TNFα, TMEM189-UBE2V1, IL-17A, and WSB1;

chemokines (e.g., CCR2, CCR5, CCR9, CXCR6, and XCR1; and other genes (e.g., DPP7, DPP9, MST1R, GOLGA3, GOLGA8B, LAPTM4b, and ApoE. 2 Consequently, Covid-19 Host Genetics Initiative (HGI) (https://www.covid 19hg.org/) announced a global project collaborating among researchers to discover genetic loci in various populations. Analyzing three meta-analysis studies using approximately 50 thousand people from 19 countries, the HGI project has found 13 genetic loci associated with Covid-19. 6 These loci and their clos- Whole-genome sequences of thousand Covid patients have discovered associations between Covid-19 infections and variants in genes including IL10RB and PLSCR (interferon signaling), BCL11A (leukocyte differentiation), FUT2 (blood type antigen secretor status). 7 In a recent systematic review, the TNFα gene was considered as one of the most important candidate genes which are potentially associated with progressions of Tumor necrosis factor alpha (TNFα) gene is located on chromosome 6p21.33 and encodes TNF-a protein secreted by macrophages and expressed in most organ tissue including lungs (https://www. ncbi.nlm.nih.gov/gene/7124). TNFα SNPs are associated with infectious, autoimmune, dermatological, cardiovascular, neurological diseases. 9 TNFα −308 G > A (rs1800629) in the promoter region of the TNF gene impacted on TNF gene expression, has been regarded as an important SNP, in which nucleotide guanine 308 (G) is substituted with Adenine (A). 10 This SNP is found to be associated with septic shock in most ethnic backgrounds, 11 and Caucasian populations. 12 It is also linked with infectious diseases such as malaria in Kenya, parasitic infections in Indonesia, Cytomegalovirus in Finland, 13 a positive single-strand RNA Chikungunya virus in Nicaragua, 14 Deng virus in various ethnicities, 15 and hepatitis C virus in India. 16 Genetic studies of cytokines have not only important for understanding the role of SNPs in the pathophysiology of diseases but are also critical for designing immunomodulatory therapies against infectious diseases as Covid- 19. 17 In the Middle East, particularly in Iraq, studying genetic polymorphisms linking with Covid-19 infection has been unobserved.

Before the Covid-19 pandemic emerged, our group has been working on genetic polymorphisms in both Toll-like receptor-4 (TLR4) and

Apolipoprotein Epsilon (ApoE) in a general Kurdish population 18 

One hundred and twenty-five (125) blood samples were also collected in Covid-19 patients, whose real-time reverse transcriptase polymerase chain reaction (rRT PCR) was positive and seeking treatment in Kalar Polyclinics, Kurdistan Regional Government, Iraq. The unvaccinated, symptomatic, and Iraqi Kurdish patients 

Total genomic DNA was extracted from the blood samples as de- 

A partial DNA sequence of the TNFα gene, where −308G>A SNP is located, was amplified using a pair of primers as previously described 22 and shown in 

The amplified PCR products were digested using NcoI restriction enzyme (NEB). The reactions were performed as follows: molecular grade water (10 µl), NcoI buffer (4 µl), NcoI enzyme (10 units) and

PCR product (10 µl) incubated at 37°C for 3 h using the thermal cycler. The digested PCR products were inspected on 3% agarose gel.

The mutant genotypes were repeated at least twice.

The differences in the frequency of the TNFα genotypes and alleles between both Covid-19 and general population samples were analyzed using Chi-square (Fisher's exact test) and t test (Graphpad prism 9.1.1 software GraphPad Software). p-values of more than 0.05 were regarded as statistically significant and both odds ratio and confidence interval (% 95 Cl) were also used.

The Covid-19 patients were mostly symptomatic which were indicated by clinical parameters including erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), D-dimer, Ferritin, CT scan, and SpO2 (Table 1 ). More than half of the patients had comorbidities including obesity, asthma, diabetes, hypertension, ischemic heart disease, and stroke. Among the comorbidities, both hypertension and obesity were the most common in the Covid-19 patients. Data also contained both old and young ages, males and females.

The PCR products of both the general population and Covid-19

patients were observed on agarose gel electrophoresis. As shown in Table 3 , the heterozygous genotype (GA) of TNFα −308G>A was significantly higher in Covid-19 patients than the general population, whereas other genotypes and alleles were not statistically significant. There are no significant differences between the ratio of males to females between both the general population and COVID-19 patients. However, there is a highly significant difference between the ages of the studied groups.

Comparisons of both genotypes and allele frequencies between mild and moderate-severe cases were also non-significant (Table 4) .

Similarly, no statistically significant differences were found in both genotypes and allele frequencies between genders (Table 5) . Based on ages, data were broken down to young ages (<45 years old) and old ages (>45 years old) and the results showed that there are no statistical differences between young and old ages ( Table 6 ). As displayed in Table 7 , there were no significant differences in both geno- variants are high in severe patients. 21 In the current study, the frequency of TNFα genotypes and alleles, comparing between mild and moderate/severe groups were not statistically significant (Table 4) . Similarly, no significant differences were found according to genders (Table 5) , ages (Table 6) , comorbidities ( Table 7) . Our cohort data are adequate to support that the heterozygous (GA) genotype, which was statistically significant in comparing between patient and control groups, might be associated with Covid-19 susceptibility ( patients with no comorbidities. 29 It seems more interesting to perform whole genome or exome sequencing in young case reports who were previously healthy and currently suffered from severe Covid-19 for exploring multi genetic variations in the same patient.

In the current study, the heterozygous genotype of the TNFα −308 

All authors have no conflict of interest to declare.

HNA, SSN, and SMAA carried out conceptualization, design analysis, planning, and editing the manuscript. HNA carried out sample collections and clinical data. SSN and SMAA carried out genotyping and drafting the article. SMAA carried out statistical analysis.

All data are available through corresponding author, Sherko S.

Niranji.

Sherko S. Niranji https://orcid.org/0000-0001-9210-0129

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