Association of IFNAR2 rs2236757 and OAS3 rs10735079 Polymorphisms with Susceptibility to COVID-19 Infection and Severity in Palestine

The clinical course and severity of COVID-19 vary among patients. This study aimed to investigate the potential correlation between the gene polymorphisms of the interferon receptor (IFNAR2) rs2236757 and oligoadenylate synthetase 3 (OAS3) rs10735079 with the risk of COVID-19 infection and its severity among Palestinian patients. The study was conducted between April and May 2021 on 154 participants who were divided into three groups: the control group (RT-PCR-negative, n = 52), the community cases group (RT-PCR-positive, n = 70), and the critically ill cases (ICU group; n = 32). The genotyping of the investigated polymorphisms was performed using amplicon-based next-generation sequencing. The genotypes distribution for the IFNAR2 rs2236757 was significantly different among the study groups (P = 0.001), while no statistically significant differences were found in the distribution of genotypes for the OAS3 rs10735079 (P = 0.091). Logistic regression analysis adjusted for possible confounding factors revealed a significant association between the risk allele rs2236757A and critical COVID-19 illness (P  <  0.025). Among all patients, those who carried the rs2236757GA were more likely to have a sore throat (OR, 2.52 (95% CI 1.02–6.24); P = 0.011); the presence of the risk allele rs2236757A was associated with an increased risk to dyspnea (OR, 4.70 (95% CI 1.80-12.27); P  <  0.001), while the rs10735079A carriers were less likely to develop muscle aches (OR, 0.34 (95% CI 0.13–0.88); P = 0.0248) and sore throat (OR, 0.17 (95% CI 0.05–0.55); P  <  0.001). In conclusion, our results revealed that the rs2236757A variant was associated with critical COVID-19 illness and dyspnea, whereas the rs10735079A variant was protective for muscle aches and sore throat.


Introduction
Te coronavirus disease 2019 (COVID- 19) is a respiratory and systemic disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), frst reported in Wuhan, China, in December 2019, and then rapidly spread across the globe.Worldwide, more than 613 million coronavirus patients were reported with more than 6.5 million total deaths (Worldometers.info:Dover, Delaware, USA) (accessed date 10-09-2022).It is transmitted predominately from person to person mainly through inhalation of small, exhaled respiratory droplets containing infectious virions [1].
Te clinical manifestations of COVID-19 vary widely, ranging from asymptomatic to severe and life-threatening acute respiratory distress syndrome (ARDS), multiorgan failure, and ultimately death [2].Te major symptoms of the disease include fever, cough, fatigue, sore throat, headache, and shortness of breath, with progression to pneumonia [3].Households are favorable venues for viral transmission, where family members may crowd and be in close contact without adhering to social distancing rules or using masks.Although all household contacts of a positive COVID-19 patient are exposed to the virus, not all necessarily get infected.Previous reports showed that male gender, elder age group, and the existence of comorbidities (e.g., cardiovascular, pulmonary, and renal diseases) are risk factors for severe COVID-19 infection [4].However, the wide range of the reported symptoms suggested that genetic risk factors may also play a crucial role in disease progression.Although the virus's new mutations have emerged (e.g., UK, South Africa, and India), few studies have described interindividual genetic diferences in the immune response to these new versions of coronavirus.
It was reported that variants of the angiotensin-converting enzyme 2 (ACE2) gene encode the cellular receptor for SARS-CoV-2, and polymorphisms of serine protease TMPRSS2 afect viral entry and invasion, thereby increasing COVID-19 severity [5,6].Moreover, a genome-wide association study (GWAS) conducted in the UK compared the genetic variants in critically ill patients (n � 2244) with severe COVID-19 to variants found in a healthy control group.Te study revealed signifcant associations between the severity of COVID-19 and the genetic variants in fve loci including chromosome 3p21.31,spanning the SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6, and XCR1 genes; chromosome 12q24.13, in the OAS gene cluster; chromosome 19p13.2,near tyrosine kinase 2 (TYK2); chromosome 19p13.3,within dipeptidyl peptidase 9 (DPP9); chromosome 21q22.1,within the interferon receptor gene INFAR2.Of these genes, IFNAR2 and OAS are important in the early stages of the disease, whereas the DPP9, TYK2, and CCR2 genes drive infammatory processes in the late stages of critical COVID-19 [7].It is well-established that SARS-CoV-2 infection activates innate and adaptive immune responses.Te failure of this system and dysregulated massive pro-infammatory host response would cause harmful tissue damage [8].
Te aim of this study was to explore the correlation between four specifc SNPs (IFNAR2 rs2236757, DPP9 rs2109069, OAS3 rs10735079, and LZTFL1 rs73064425 variants) and the susceptibility to COVID-19 infection among Palestinian household contacts.Additionally, the research sought to examine how these SNPs are linked to the clinical manifestations and severity of COVID-19, employing amplicon-based next-generation sequencing (NGS) techniques.

Study Participants.
Te study participants were recruited from 30 Palestinian families-residing in diferent cities in the West Bank, Palestine-between April and May 2021.A family was considered eligible for inclusion in the study if it had at least one clinically and laboratory-confrmed COVID-19 case and one laboratory-negative household contact and exhibited no symptoms of COVID-19.We divided all family members into two groups: infected cases (community cases group) and uninfected household contacts (control group).An infected case was defned by having a positive reverse transcription polymerase chain reaction (RT-PCR) test regardless of having symptoms or not and regardless of being a primary or secondary case.An uninfected contact was defned as a family member who had unprotected contact with a positive case, lives at the same place, and stayed asymptomatic for ten days after symptoms onset or RT-PCR diagnosis of a positive case and was tested negative by RT-PCR.Additionally, we consecutively enrolled patients from an intensive care unit (ICU) at the Palestinian Medical Complex, Ramallah-due to critical COVID-19 illness (ICU cases).
We excluded individuals who received COVID-19 vaccination, regardless of the type of the vaccine.Patients' data, including demographic information, symptoms, RT-PCR test results, and comorbidities, were collected via a well-structured questionnaire supervised by healthcare personnel.

Sampling, DNA Extraction, and
Genotyping.Blood samples (fve ml) were collected in EDTA tubes from all study participants.Te DNA was extracted from each blood sample (200 μl) using a genomic QIAamp DNA purifcation kit as per the manufacturer's instructions (Qiagen, Hilden, Germany) and kept frozen (−20C) for further analysis.All DNA samples were genotyped for the rs2236757 of IFNAR2, rs2109069 of DPP9, rs10735079 of OAS3, and rs73064425 of the LZTFL1 gene using amplicon-based NGS (NGS).Briefy, two primers (forward and reverse) were used to target each single-nucleotide polymorphism (SNP) as described in Table S1.All primers were modifed with over-hanged Illumina adaptor sequences at the 5′ ends (bolded, Table S1), targeting partial sequences of the studied genes.Te fnal product size for each targeted gene is mentioned in Table S1.
Te PCR products were visualized by 1.5% agarose gel, cleaned by Agencourt AMPure XP system (X1, A63881; Beckman Coulter Genomics, Indianapolis, IN, USA), and eluted into a fnal volume of 25 μl.All purifed samples were amplifed by dual indices PCR to barcode each sample using Nextera XT Index Kit (Illumina, San Diego, CA, USA); fve μl from each barcoded sample was pooled together, cleaned again by Agencourt AMPure XP system (X1), and eluted in 50 μl elution bufer.Te concentrations of the prepared Libraries were tested by Qubit ® Fluorometer (Invitrogen, Carlsbad, CA, USA).A concentration of 4 nM was used with a target of 20k reads for each sample.Deep sequencing was performed by NextSeq 500/550 machine using a 150-cycle Mid Output Kit (Illumina, San Diego, CA, USA).

Bioinformatics and Sequence Analysis.
Te sequencing data were uploaded to the Galaxy web platform, and the public server at "usegalaxy.org" was used to analyze the obtained DNA sequences [9].Te fltration workfow included Illumina adaptor trim and quality selection of Q > 20, with a minimal read length of 100 bp.We used eight virtual probe sequences to identify the targeted variants (Table S1).Ultimately, the genotypes were determined based on the ratio between the read counts for wild-type and minor alleles.SNPs were included in the study if they passed our quality measures: Hardy-Weinberg equilibrium (HWE) >0.05 and genotyping rate >95%.

Statistical Analysis.
We performed the statistical analysis using the SPSS package, version 26.0 (SPSS, Inc., Chicago, IL, USA) and the R environment version 4.1.3.All tests were twotailed, and we considered P value <0.05 signifcant unless specifed.We tested for the Hardy-Weinberg equilibrium (HWE) for all SNPs using the "SNPassoc" package [10].Moreover, we examined the genetic susceptibility of SARS-CoV-2 and the genetic association with the critical COVID-19 illness by comparing the community patients with the controls and the ICU patients with the controls, respectively, using fve genetic models (codominant, dominant, over dominant, recessive, and additive).Models were adjusted for: age, gender, smoking, history of hypertension, diabetes mellitus, and coronary artery diseases using the "SNPassoc" package.Adjusted odds ratios (ORs) with the associated 95% confdence intervals (CIs) were calculated for each model.Te same models were used to investigate the association of genetic polymorphism with symptoms/signs among ICU and comunity cases groups.Te best model for each SNP was selected using the Akaike information criterion [11].We used Bonferroni correction for multiple comparisons to correct statistical signifcance (P < 0.05, divided by the number of analyzed SNPs) [12,13].Ultimately, we investigated any potential gene-gene interaction.

Characteristics of Study Participants. A total of 154
Palestinians were included in this study and divided into three groups: COVID-19-infected patients (community cases group, n � 70), uninfected household contacts (control group, n � 52), and critically ill COVID-19 patients (ICU group, n � 32).In each group, the median (IQR) age was 28 (27), 24.5 (21.5), and 61 years (27), respectively.Te characteristics and comorbidities of each study group are shown in Table 1.Te median age, the prevalence of smoking, diabetes mellitus (DM), hypertension, and coronary artery disease (CAD) were signifcantly higher in the ICU group (P < 0.05) compared to the community cases and control groups.Te clinical characteristics of COVID-19 patients in the community and ICU groups with signs and symptoms frequencies are presented in Table 2. Te percentage of symptomatic patients was 93% in the community cases group, whereas 100% in the ICU group.Te frequency of fatigability, headache, and loss of taste and/or smell was signifcantly higher in the community case group (P < 0.05).However, dyspnea and cough were more frequent in the ICU group (P < 0.05).

Genotyping of IFNAR2 rs2236757 and OAS3 rs10735079.
Te minor allele frequency (MAF) of the IFNAR2 rs2236757A and the OAS3 rs10735079A was 27% and 50%, respectively.Te frequency and genotype distribution of the IFNAR2 rs2236757A and the OAS3 rs10735079A among the three study groups are provided in Table 3. Te IFNAR2 rs2236757 genotypes distribution was signifcantly variable among the study groups (P � 0.001), while no signifcant diferences were observed in the distribution of OAS3 rs10735079 genotypes (P � 0.091).Te two SNPs, DPP9 rs2109069 and the LZTFL1 rs73064425, were excluded from the study due to deviation from the HWE (i.e., P < 0.05) and the low genotyping rate (i.e., <95%).

IFNAR2 rs2236757 and OAS3 rs10735079
Polymorphisms and Susceptibility to COVID-19 Infection.Logistic regression analysis under fve genetic models adjusted for age, sex, smoking history, DM, hypertension, and CAD was used to investigate the role of IFNAR2 rs2236757 and OAS3 rs10735079 polymorphisms in the susceptibility of COVID-19 infection and severity.Te community cases group and the ICU group were compared to the control group separately.As shown in Table 4, none of the studied polymorphisms had a statistically signifcant association with SARS-CoV-2 infection among community cases (P > 0.025) after Bonferroni correction.However, the risk allele rs2236757A of the IFNAR2 gene was signifcantly associated with critical COVID-19 illness in all genetic models (P < 0.025) except for the recessive (P � 0.4).According to the Akaike information criterion, the dominant model was the best to explain the association (OR, 8.65 (95% CI 1.60-46.68);P � 0.005).No signifcant relationship between the OAS3 rs10735079 polymorphism and critical COVID-19 illness was observed (P > 0.025; Table S2).For all patients (the community cases group and ICU group), the IFNAR2 rs2236757 GA carriers were more likely to have a sore throat (OR, 2.52 (95% CI 1.02−6.24);P = 0.011) (Table 5).In addition, patients who developed dyspnea were more likely to have the risk allele rs2236757A; the association was best explained by the additive model (OR, 4.70 (95% CI 1.80-12.27);P < 0.001).On the other hand, patients with the risk allele rs10735079A were less prone to develop muscle aches (OR, 0.34 (95% CI 0.13-0.88);P = 0.0248) and sore throat (OR, 0.17 (95% CI 0.05-0.55);P < 0.001); both associations were best explained by the recessive model (Table 5).Further analyses were performed to investigate the association between genetic polymorphisms and signs and symptoms among community cases and ICU cases seperatly as shown in Table 6.None of the community cases group was homozygous for the risk allele IFNAR2 rs2236757A (Table 6).However, the risk allele rs2236757A was associated with loss of taste or smell (OR, 3.57 (95% CI 1.19-10.72);P = 0.019), muscle aches (OR, 3.65 (95% CI 1.12-11.86);P = 0.025), and dyspnea (OR, 4.84 (95% CI 1.45-16.13);P = 0.006).We also found that patients with sore throat in the community cases group were unlikely to be homozygous (AA) for the risk allele rs10735079A of OAS3; the association was only explained by the recessive model (OR, 0.19 (95% CI 0.05-0.79);P = 0.012).Among the ICU group, muscle aches were the only symptoms that had a genetic association; patients with the risk allele rs10735079A were less prone to muscle aches in two models (recessive and additive), and best explained by the additive (OR, 0.22 (95% CI 0.05-0.99);P = 0.014) (Table 6).We tested gene-gene interaction in all models that had a signifcant association with the clinical manifestations; we did not fnd a statistically signifcant interaction between the SNPs.However, heterozygous rs2236757 (GA) carriers with sore throat were less likely to be homozygous (AA) for the risk allele rs10735079A (OR, 0.06 (95% CI 0.01-0.57))(data not shown).
In the current study, we found that the risk allele IFNAR2 rs2236757A is signifcantly associated with critical COVID-19 illness.Such an association was not present for the rs10735079 variant.Te rs2236757A variant was found to be related to critical COVID-19 illness in genome-wide signifcant associations.Additionally, IFNAR2 has a causal role based on Mendelian randomization result; increased expression of the interferon receptor subunit IFNAR2 reduced the odds of severe COVID-19 (P = 0.0043) [7].Type 1 interferons bind IFNAR2, which leads to activation and signal transduction involving the JAK-STAT pathway [21].Consequently, this pathway initiates antiviral activity in the target cells and induces apoptosis in infected cells [22].Te role of IFNAR2 expression was further replicated in other Mendelian randomization studies [23][24][25].OAS is a family of antiviral proteins consisting of four members, OAS1, OAS2, OAS3, and OAS-like protein [26].Both interferon and virus infection stimulate the transcription of OAS genes in the cell [27,28].Ribonuclease L is activated through the OAS1 to OAS3 proteins; products with 2′-5′ oligoadenylate synthetase activity.Ribonuclease L activation leads to the degradation of the cellular and viral RNA, resulting in the inhibition of protein synthesis and terminating viral  Interdisciplinary Perspectives on Infectious Diseases replication [29][30][31].Te variant rs10735079 lies in the interferon-inducible OAS gene cluster (OAS1, OAS2, and OAS3) and was associated with critical COVID-19 illness (P = 1.65 × 10 −8 ) in genome-wide signifcant associations [7].However, a similar association was not present in our study.Diferent symptom clusters have diferences in-hospital outcomes [32].In a cross-sectional study conducted by Reis et al., involving nearly 60,000 COVID-19 patients, it was found that fever and breathing difculty signifcantly increased the likelihood of hospitalization and death.Conversely, runny nose, sore throat, diarrhea, and headache were associated with reduced odds of hospitalization and death.Based on these fndings, the researchers concluded that the latter symptoms may indicate a protective efect against severe outcomes from COVID-19 [33].Sadeghifar et al. used a binary logistic regression model to analyze disease outcomes based on disease symptoms.Tey showed that shortness of breath and abnormal chest radiographic fndings were strong predictors of higher mortality.However, patients with sore throats showed lower mortality rates [34].Moreover, Chang et al. indicated that body temperature, chills, initial chest Xray fndings, and the presence of diabetes were signifcant predictors of progression to severe COVID-19 [35].
Studies that evaluate the role of human genetics in the development of diferent signs and symptoms of COVID-19 are scarce.Williams et al. conducted a study involving 3261 same-sex twins to investigate the presence of heritable components in developing the diferent symptoms of COVID-19.Tey found that heritability elements infuence certain symptoms, including delirium, diarrhea, fatigue, anosmia, and meal skipping [36].
Herein, we investigated the role of the rs2236757 and the rs10735079 variants in developing the diferent signs and symptoms of COVID-19 in all COVID-19 patients, and in the community patients and ICU patients, separately.Our results indicated that patients with the risk allele rs2236757A were more likely to have dyspnea and sore throat.Among the community patients, the risk allele rs2236757A was associated with dyspnea, loss of taste or smell, and muscle aches.Surprisingly, patients with the risk allele rs10735079A were unlikely to have a sore throat or muscle aches.In particular, the community cases group was less likely to have a sore throat, and the ICU subgroup was less prone to muscle aches.Given that the risk allele rs10735079A was found to be associated with critical COVID-19 illness in multiple previous studies [7,20], the inverse association of this risk allele and the presence of a sore throat may indicate that having a sore throat is associated with a lower risk of COVID-19 hospitalization and death, which consistent with the fndings of Reis et al. and Sadeghifar et al. [33,34].
It was reported that interferon-beta inhibits SARS-CoV-2 virus replication in vitro [37].However, clinical trials did not reveal a clear beneft from interferon therapy for hospitalized patients with severe COVID-19 [38][39][40][41].Yet, a systematic review of fve clinical trials concluded that early administration of interferon-beta, combined with other antiviral drugs, is promising [42].Low expression of IFNAR2 has a causal role in the progression to critical COVID-19 illness, and our study was in line with the fndings of Pairo-Castineira et al., which demonstrated that rs2236757A was associated with the severity of COVID-19 illness [7].Terefore, a randomized controlled trial that examines the role of interferon therapy in COVID-19 patients who have the rs2236757A variant or other reported variants will help to understand the role of interferon therapy and its benefts.
Our study is limited by the small number of included participants, which was in part due to the newly emerged variants of the SARS-CoV-2 virus.We could not continue     [43,44].Moreover, the COVID-19 vaccination was started by the government, which can infuence the severity of COVID-19 disease and SARS-CoV-2 infection [45][46][47].Te limited number of patients in the present study may explain the lack of association between the rs10735079 polymorphism and the critical COVID-19 illness.Nonetheless, targeting families strengthen the certainty of adequate exposure to the SARS-CoV-2 virus in the control group.Furthermore, the present study is the frst in Palestine and one of the limited numbers of studies globally to investigate the role of human genetic factors on signs and symptoms of COVID-19.In conclusion, our study revealed that the IFNAR2 rs2236757A variant was associated with critical COVID-19 illness.Te risk allele rs2236757A was associated with dyspnea and sore throat while patients with the risk allele rs10735079A were less likely to have a sore throat or muscle aches.Our study may provide preliminary results for future genetic association studies aimed at elucidating the role of human genetics in various signs and symptoms of COVID-19 and enhancing our understanding of the pathophysiology of SARS-CoV-2 infection and its complications.

Table 2 :
Signs and symptoms of COVID-19 in the community cases and the intensive care unit groups.< 0.05 was considered signifcant.ICU, intensive care unit.P values below the signifcance threshold are highlighted in bold. P

Table 3 :
Genotypes distribution of the IFNAR2 rs2236757 and OAS3 rs10735079 among the studied groups.

Table 4 :
Association of IFNAR2 rs2236757 polymorphism with COVID-19 infection among the studied groups.Best model to explain the association according to the Akaike information criterion.Te odds ratios and the P values were from logistic regression models adjusted for age, gender, smoking history, history of hypertension, diabetes mellitus, and coronary artery disease.After Bonferroni correction, a P value <0.025 was considered signifcant.ICU, intensive care unit; OR, odds ratio; ref, reference; NA, not applicable.P values below the signifcance threshold are highlighted in bold.

Table 5 :
Association of IFNAR2 rs2236757 and OAS3 rs10735079 polymorphisms with COVID-19 signs and symptoms in all patients.

Table 5 :
Continued.Best model to explain the association according to the Akaike information criterion.Te odds ratios and the P values were from logistic regression models adjusted for age, gender, smoking history, history of hypertension, diabetes mellitus, and coronary artery disease.After Bonferroni correction, a P value <0.025 was considered signifcant.OR, odds ratio; ref, reference; NA, not applicable.P values below the signifcance threshold are highlighted in bold

Table 6 :
Association of IFNAR2 rs2236757 and OAS3 rs10735079 polymorphisms with COVID-19 signs and symptoms in the community patients and ICU patients, separately.Interdisciplinary Perspectives on Infectious Diseases recruiting patients when the new SARS-CoV-2 variants became prevalent in Palestine, as diferent variants may have diferent manifestations and pathogenesis

Table 6 :
Continued.† Best model to explain the association according to the Akaike information criterion.Te odds ratios and the P values were from logistic regression models adjusted for age, gender, smoking history, history of hypertension, diabetes mellitus, and coronary artery disease.After Bonferroni correction, a P value <0.025 was considered signifcant.OR, odds ratio; ref, reference; NA, not applicable.P values below the signifcance threshold are highlighted in bold.