key: cord-0849352-itknkp9n
authors: Heidari Nia, Milad; Rokni, Mohsen; Mirinejad, Shekoufeh; Kargar, Maryam; Rahdar, Sara; Sargazi, Saman; Sarhadi, Mohammad; Saravani, Ramin
title: Association of polymorphisms in tumor necrosis factors with SARS‐CoV‐2 infection and mortality rate: A case‐control study and in silico analyses
date: 2021-12-02
journal: J Med Virol
DOI: 10.1002/jmv.27477
sha: e37c5fd3cf2433decef681c213375b50c1d1182a
doc_id: 849352
cord_uid: itknkp9n

The present coronavirus disease 2019 (COVID‐19) is spreading rapidly and existing data has suggested a number of susceptibility factors for developing a severe course of the disease. The current case‐control experiment is aimed to study the associations of genetic polymorphisms in tumor necrosis factors (TNFs) with COVID‐19 and its mortality rate. A total of 550 participants (275 subjects and 275 controls) were enrolled. The tetra‐amplification refractory mutation system polymerase chain reaction technique was recruited to detect −308G>A TNFα and +252A>G TNFβ polymorphisms among the Iranian subjects. We demonstrated that carriers of the G allele of TNFβ‐252A/G, rs909253 A>G were more frequent in COVID‐19 subjects compared to the healthy group and this allele statistically increased the disease risk (odds ratio [OR] = 1.55, 95% confidence interval [CI] = 1.23–1.96, p < 0.0001). At the same time, the A allele of TNFα‐311A/G, rs1800629 G>A moderately decreased the risk of COVID‐19 (OR = 0.68, 95% CI = 0.53–0.86, p < 0.002). Also, we analyzed the various genotypes regarding the para‐clinical and disorder severity; we found that in the AA genotype of TNFβ‐252A/G (rs909253 A>G), the computed tomography scan pattern was different in comparison to cases carrying the AG genotype with p (1) < 0.001. In addition, in the severe cases of COVID‐19, leukocyte and neutrophil count and duration of intensive care unit hospitalization in the deceased patients were significantly increased (p < 0.001). Moreover, the TNFα‐311A/G (rs1800629 G>A) variant is likely to change the pattern of splicing factor sites. Our findings provided deep insights into the relationship between TNFα/TNFβ polymorphisms and severe acute respiratory syndrome coronavirus 2. Replicated studies may give scientific evidence for exploring molecular mechanisms of COVID‐19 in other ethnicities.

In December 2019, the coronavirus disease 2019 (COVID-19) emerged in Wuhan, China, and caused acute respiratory distress syndrome (ARDS).

This new virus, later called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rapidly spread around China and other countries, substantially affected human health, global economics, and created a worldwide crisis. 1, 2 In the majority of the cases, SARS-CoV-2 infection is considered an acute self-resolving disease. Yet, until April 27, 2020, COVID-19 induced death in about 6.89% of infected individuals. 2 Initial reports have indicated that COVID-19 has a mortality rate of approximately 2%, and this highly infectious disease might result in death due to extensive alveolar damage and lung failure. 3 Similar to the Middle East Respiratory Syndrome and SARS-CoV, the SARS-CoV-2 belongs to the family of beta-coronaviruses mainly manifested as pneumonia in humans. 4, 5 In addition, COVID-19 variants of concern, including alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and lambda (C. 37) are associated with higher transmissibility while spreading across Asia, Europe, and other continents. 6 A single cohort study by Nikpouraghdam et al. 7 suggested that older age, male gender, and having comorbid conditions were significantly correlated with death rates among Iranian COVID-19 patients.

Soon after the pandemic COVID-19 outbreak, several studies were performed on almost every aspect of SARS-CoV-2 infection, particularly the pathogenesis of this novel beta-coronavirus. 8 It has been shown that the virus uses angiotensin-converting enzyme type 2 (ACE2), transmembrane serine protease 2 (TMPRSS2), and the viral spike protein (S-protein) for entering host cells. 9, 10 These receptors are abundantly expressed in lung cells, making it easier for the virus to replicate throughout the respiratory tract. 10, 11 Besides genetics, psychological distress, smoking, poor sleep quality, and body mass index are among the risk factors associated with COVID-19 susceptibility and incubation time. 12 Increasing evidence has shown that single-nucleotide polymorphisms (SNPs) play an essential role in determining the case-fatality rate of COVID-19 patients and the disease severity. 13, 14 In this respect, Paniri et al. 15 showed that ACE2 SNPs impact the ability of the SARS-CoV-2 virus to enter cells via altering ACE2 function and structure. By performing a case-control study, Chong et al. reported that the interferon γ (IFNγ) +874 A/T, and the tumor necrosis factor α (TNFα)-308G/A polymorphism, is associated with the onset progress of SARS-CoV-2 infection 16 but not the progress of SARS-CoV. 17 When the cytokine release syndrome happened in COVID-19, it caused increasing levels of TNF-α, interleukin 1 (IL-1), IL-6, IL-8, IL-12, and IFN-γ; therefore, increasing some cytokines, for example, IL-6 and TNF-α cause poor prognosis in patients with COVID-19. 18, 19 More recently, Kirtipal and Bharadwaj 20 reported that IL6 SNPs could be considered an indicator of COVID-19 severity in humans. This indicates that SNPs in genes encoding inflammatory cytokines and other innate immune genes might also impact the susceptibility to acute respiratory disorders, including COVID-19.

The host genetics plays a fundamental role in the immune response to the SARS-CoV-2 virus and influences the risk of COVID-19, severity, and outcome in affected patients. 21 Herein, we aimed to study the relationship between TNFβ-252A/G, rs909253 A>G and TNFα-311A/G, rs1800629 G>A polymorphisms, susceptibility, lesions in computed tomography (CT) scan, and duration of hospitalization to COVID-19 in an Iranian population. 

According to protocol, genomic DNA was isolated using a simple salting-out procedure from 500 μl of venous whole blood of each participant. 22 The polymorphisms in TNFβ-252A/G, rs909253 A>G NIA ET AL.

and TNFα-311A/G, rs1800629 G>A were genotyped using the tetra amplification refractory mutation system PCR method. In summary, the DNA of each participant was amplified for SNPs using 1 µl of DNA (∼60 ng/ml), 1 µl of each primer (6 pmol), 12 μl of Taq 2X

Master Mix Red-Mgcl 2 1.5 mM (Ampliqon Inc.), and 5 μl of distilled water. Each reaction mixture was heated to 95°C for 5 min for initial denaturation and underwent 30 cycles at 95°C for 45 s, annealing at different temperatures (according to Supporting Information Table   for each SNP) for 45 s with an extension at 72°C for 45 s, followed by a final extension at 72°C for 5 min. For each reaction, we used a common reverse primer, and one of the two allele-specific forward primers was shown in the Supporting Information Table. The products were analyzed on 1.5% agarose gel stained with safe stain dye and recorded using a gel doc system ( Figure 1 ).

Fasting venous blood was collected from all patients and participants for laboratory measurements, and complete cell blood count, C-reactive protein, and chest CT scan were performed. Symptoms/ signs and duration of hospitalization were also recorded. *p < 0.05 was considered statistically significant, between severe and nonsevere.

SPSS version 22.0 for the windows package was recruited for statistical analysis. Quantitative data were described as mean ± standard deviation for parametric data. In terms of qualitative data, number and percent were the basis of analysis. Qualitative data were analyzed by χ 2 and logistic regression wherever it is appropriate. Student t-test and one-way analysis of variance tests were used to compare parametric quantitative data. The distribution of genotypes in all groups and that of in general population was compared using the Hardy-Weinberg equilibrium (HWE) model. 

We found no deviation from HWE in our population. 3.3 | Genotype distribution, disease severity, and signs/symptoms Damage to T lymphocytes by SARS-CoV-2 might be a contributing item leading to substantial decreases in total lymphocytes count and strengthening of patient's status. 26 As reported in the current study, we found statistically significant abnormalities in paraclinical (including lymphopenia, leukocytosis, neutrophils, and increased GGO, consolidation and mixed pattern in CT scan) and sign/symptoms (including cough, respiratory distress, and increased tracheal intubation chance, duration of hospitalization times) in severe cases of COVID-19 as compared with nonsevere cases (p < 0.001).

Several case-control studies of assorted designs have recently elucidated the association of specific host genetic variants with clinical disease severity or susceptibility to SARS-CoV-2 infection and COVID-19 disease outcome. 25 Devaux et al. 27 suggested that functional polymorphisms in human ACE2, which affects ACE2 expression, might influence COVID-19 risk, severity and outcome.

In another case-control experiment, Karst et al. 28 The rs1800629 polymorphism is the most studied TNFα variation, which is a G/A substitution and is located in the promoter region at position −308. 31 36 proposed that TNFα rs1800629 is a risk factor for asthma. This can be explained by the role of TNFα in the pathophysiology of respiratory diseases. 37 Ding et al. 38 showed that allele A of the TNFα rs1800629 polymorphism is associated with risk of ARDS in a Chinese population, whereas the GG genotype was linked to lower mortality. It has also been shown that the G allele of this variation was overrepresented in patients with influenza A/H1N1 and correlated with disease severity in a Mexican population. 39 In contrast to these findings, Wang et al. 40 TNFα resides approximately 252 base pairs downstream of the transcription start site for the gene coding TNFβ (also known as lymphotoxin alpha). 41 As an inflammatory mediator, TNF-α affects the production of other cytokines. However, the interaction between cytokines might result in antagonistic (TNF-α and TNF-β, for instance) or synergistic (e.g., TNF-α with IL-1 interactions) effects.

TNFs also regulate receptor expression of other cytokines or stabilize cytokine messages by another, and, therefore, play pivotal roles in signal transduction. 42 It has been hypothesized that serum concentration of TNF-α is elevated in subjects with COVID-19; thus, these patients have a greater probability of developing ARDS and death. 43 Karki et al. 44 

The authors declare that there are no conflicts of interest. 

The data presented in this manuscript will be available by the corresponding author upon reasonable request. 

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http://orcid.org/0000-0003-0534-4251 Saman Sargazi http://orcid.org/0000-0002-2255-5977