Role of ACE2 and TMPRSS2 polymorphisms in clinical severity and outcomes of COVID-19 in Egypt

Background The clinical presentations of coronavirus disease 2019 (COVID-19) exhibit significant variation, ranging from asymptomatic cases to mortality resulting from severe pneumonia. Host genetics can partially explain this variation. Objective This study evaluated possible associations between severity and outcome of COVID-19 and single nucleotide polymorphism (SNP) rs2285666 in the ACE2 gene and SNP rs2070788 in the TMPRSS2 gene. Methods The study included a sample of 100 consecutive adult patients admitted to the COVID-19 Isolation and Intensive Care Units of the Zagazig University Hospitals, Zagazig, Egypt from July 2021 to November 2021. For rs2285666, polymerase chain reaction-restriction fragment length polymorphism was carried out. For rs2070788, real-time polymerase chain reaction was performed. Results For rs2285666, the GA genotype was the most frequent among female patients (39% [16/41]) and the A genotype was more prevalent among male patients (54.2% [32/59]). For rs2070788, the AA genotype was the most frequent among all patients (46% [46/100]). No rs2285666 or rs2070788 genotypes or allele frequencies had significant associations with either severity or outcomes of patients. Conclusion This study found no significant associations of COVID-19 severity or outcomes of patients with genotypes or allele frequencies of the rs2285666 SNP in the ACE2 gene or the rs2070788 SNP of the TMPRSS2 gene. The search for other genetic associations with COVID-19 infection is still required. What this study adds The study reveals that host genetics explain the variation observed in the disease. Specific genetic variants can confer either increased susceptibility or resistance to the disease.


Introduction
Coronavirus disease 2019 (COVID- 19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in more than 6 million fatalities and 603 million confirmed cases since its identification in 2019 until 07 September 2022. 1 The clinical presentations of the COVID-19 infection showed significant variation, ranging from asymptomatic cases to mortality resulting from severe pneumonia.Host genetics can account for part of this variation, as some genetic variants can confer either increased susceptibility or resistance to the disease. 2

Role of ACE2 and TMPRSS2 polymorphisms in clinical severity and outcomes of COVID-19 in Egypt
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The SARS-CoV-2 virus binds to host cell surface receptors by utilising spike (S) protein to mediate the fusion of the viral envelope with the cell membrane.The S protein is composed of S1 and S2 subunits.The subunit S1 contains the receptor-binding domain, that interacts with the peptidase domain of the angiotensin-converting enzyme 2 (ACE2) protein.This step is critical for the viral entry into the cell.The ACE2 protein is a well-characterised negative regulator of the renin-angiotensin system, which catalyses the conversion of angiotensin II to the heptapeptide angiotensin (1-7), which produces the opposite effect to that of angiotensin II. 4 The ACE2 protein plays two opposing roles in COVID-19 infection.Firstly, it acts as a potent receptor for the virus to enter the cell.Secondly, it has a protective function in maintaining the balance of angiotensin II, a hormone involved in regulating blood pressure.However, when there is an excessive buildup of angiotensin II, it can lead to the release of large amounts of cytokines, causing a cytokine storm and subsequent tissue damage. 5The interaction between SARS-CoV-2 and ACE2 protein receptors results in a decrease in ACE2 protein expression and a reduction in its protective function on the angiotensin II pathway.Therefore, the activation of ACE2 has been proposed as a potential therapy for COVID-19.This emphasises the significance of ongoing research on ACE2 gene polymorphisms and their functional impacts.This is important not only because the ACE2 protein receptor is involved in the development of infections, but also because it is suggested to be a potential target for therapy. 6The ACE2 gene is located on chromosome Xp22; a functional single nucleotide polymorphism (SNP) in the ACE2 gene 'G8790A (rs2285666)' in intron 3 could alter mRNA splicing and so affect ACE2 gene expression. 7In addition, rs2285666 is suggested to alter the binding affinity of the ACE2 protein receptor for SARS-CoV-2. 8ceptor binding is facilitated by the human enzyme transmembrane protease serine 2 (TMPRSS2), 9 a family of membrane-anchored proteases expressed on the airway epithelial cell surface.It acts as a viral entry cofactor by cleavage of the viral spike S protein to S1 and S2 subunits to facilitate the interaction with the ACE2 protein receptor's extracellular domain. 10Therefore, genetic variations in ACE2 and TMPRSS2 nucleotide sequences may change the interaction of receptor-binding domain and peptidase domains, altering the host's susceptibility to the virus and influencing the disease's severity and outcome.The TMPRSS2 gene is located on chromosome 21q22.Rs2070788 is a G to A transition SNP located in intron 11-12 of the TMPRSS2 gene and was found to be associated with reduced TMPRSS2 gene expression. 11sed on that, it was proposed that the aforementioned SNPs can influence the course of COVID-19 infection.This work was performed to evaluate the possible association between the ACE2 (rs2285666) and TMPRSS2 (rs2070788) SNPs and clinical severity and outcomes among COVID-19 patients.

Ethical considerations
The Institutional Review Board of the Faculty of Medicine, Zagazig University, approved this work (ZU-IRB#6775).
Participants voluntarily signed a written informed consent.Procedures were carried out in agreement with the Declaration of Helsinki.Patients' data files and sample tubes were coded, so laboratory workers were blinded to patients' information.

Diagnosis and routine investigations
Patients were selected based on the COVID-19 case definition provided by the World Health Organization. 12asopharyngeal and oropharyngeal swabs were collected and processed according to the Centres for Disease Control guidelines. 13The diagnosis of COVID-19 patients was made by real-time polymerase chain reaction (PCR) of the collected swabs, at the Scientific and Medical Research Centre according to the Guidelines of the Egyptian Ministry of Health and as described previously. 14Cycle threshold values were used as an indicator of viral load. 15utine Based on the severity, patients were classified as mild, moderate, or severe cases, and based on the outcome patients were classified as survived and died.Demographic criteria, history of vaccination, comorbidities, occurrence of complications, viral load, and laboratory results were compared in between each two groups.
According to the guidelines of the Egyptian Ministry of Health, parenteral prophylactic anticoagulants were prescribed for hospitalised patients to guard against cardiovascular complications.In case of suspected pulmonary embolism, as most of these patients are haemodynamically unstable, and because of the risk of medical staff exposure to infection, diagnosis was based on echocardiography and D-dimer level rather than CT-angiography. 16

Genotyping
According to manufacturers' guidelines, a volume of 2 mL of ethylenediaminetetraacetic acid anti-coagulated blood was obtained for DNA extraction using the QIAamp Blood Kit (Qiagen GmbH, Hilden, Germany).For the ACE2 SNP, rs2285666, PCR and restriction fragment length polymorphism were used with the primers: forward 5′-CAT GTG GTC AAA AGG ATA TCT-3′ and reverse 5′-AAA GTA AGG TTG GCA GAC AT-3′.Polymerase chain reaction mixture included 12.5 μL of DreamTaq Green PCR Master Mix (Thermo Scientific™, Vilnius, Lithuania), 10 pmol of forward and reverse primers, 100 ng of extracted genomic DNA, and nucleases-free water up to 25 μL.The thermal cycling conditions commenced with an initial denaturation at 95 °C for 5 min, then 25 cycles (95 °C for 30 s, 60 °C for 40 s, and 72 °C for 30 s), ended by a final extension for 3 min at 72°C.The PCR products were digested using AluI (Thermo Fisher Scientific Inc., Toronto, Ontario, Canada) at 37 °C for 3 h. 17he amplified and digested products were examined using 2% agarose gel electrophoresis for 30 min and visualized utilising an ultraviolet transilluminator.The amplicon size was 817 bp, after restriction; the G allele appears as one band at 817 bp, and the A allele appears as two bands at 589 bp and 228 bp.In women, the AG genotype appears as three bands at 817 bp, 589 bp, and 228 bp. 18r the TMPRSS2 SNP, rs2070788, a real-time polymerase chain reaction assay was performed, SNP genotyping assay that contains the specific primer-probe mixture and TaqMan Universal PCR Master Mix were purchased from Thermo Fisher Scientific Inc. (Vilnius, Lithuania), amplification and detection were carried out on QuantStdioTM5 real-time polymerase chain reaction system (Applied Biosystems, Siemens Healthcare Diagnostics Inc, Singapore); all procedures were carried out according to manufacturers' guidelines.The reaction mixture was as follows: 10 μL of (2X) TaqMan Universal PCR Master Mix, 20 units (0.5 μL) of the primer-probe assay, 1 μL of the extracted genomic DNA, and 8.5 μL nuclease-free water making the final volume of the mixture 20 μL.The real-time polymerase chain reaction cycling conditions were 60 °C for 2 min, initial denaturation at 95 °C for 10 min then 40 cycles of denaturation at 95 °C for 15 s, and annealing -extension at 60 °C for 1 min. 19ata analysis and results interpretation were carried out automatically by the instrument's software based on the default cycle threshold cutoff.

Data analysis
The
Complications occurred more frequently among severe patients (p < 0.001).Statistically significant increases among severe cases were observed for both kidney function markers (p = 0.03) and CRP (p < 0.001).There were no significant associations between either demographic features or laboratory data and patient outcome (Online Supplementary Table 2).A statistically significant increase in mortality rate was observed in association with severity (p < 0.001) and the occurrence of complications (p = 0.02).
For the ACE2 SNP rs2285666, the GA genotype was the most frequent among female patients (39%), while among male patients the A genotype was more prevalent (54.2%).For the TMPRSS2 SNP rs2070788, the AA genotype was the most frequent among all patients (46%).ACE2 rs2285666 and TMPRSS2 rs2070788 genotypes and allele frequencies had no significant associations with either disease severity or patient outcomes, as shown in Table 2 and Table 3.

Discussion
The mean age of patients who developed severe disease was significantly higher than mild or moderate cases.This aligns with many previous findings that age is a major risk factor for COVID-19, and the most unfavourable outcomes are attributed to age-related comorbidities and immunocompromising status. 20Patients with severe disease showed a slight but significant increase in serum urea and creatinine.Hachim et al. 21 Although it was elevated in 94% of patients, CRP showed a sharp increase in severe cases, and it was the only independent predictor for patients' severity in this work.These results go with those of Wang et al. in 2020 in China, 23 who reported that all patients who developed severe diseases displayed elevated CRP results and that CRP was the only independent predictor of severity.reported that viral load is not associated with either severity or outcome.Abdulrahman et al. 26 explain that the correlation between disease severity and CRP level, rather than viral load, can be attributed to the influence of host factors on disease progression and the contribution of pro-inflammatory cytokines to the severity of the disease.
The mortality rate was higher in severe cases than in mild or moderate cases, particularly in those who encountered complications during the disease course.This result agrees with previous studies in Egypt and worldwide.in India, 29 indicated that severe pneumonia is associated with a higher fatality rate.
Angiotensin-converting enzyme 2 and TMPRSS2 gene polymorphisms have been implicated previously in altering the susceptibility to COVID-19 infection and influencing severity and mortality rates. 30Wang et al., in 2020 in China, 31 demonstrated that ACE2 SNPs-rs4646116, rs267606406, and rs143936283 were associated with higher affinity to the receptor-binding domain.In contrast, ACE2 SNPs-rs1244687367, rs146676783, and rs961360700 were associated with lower binding affinity and hence decreased susceptibility to COVID-19 infection.
Also, TMPRSS2 gene polymorphisms have been associated with susceptibility to COVID-19 infection and outcome. 32In a study on Italian patients in 2020, Asselta et al. revealed that TMPRSS2 SNPs-rs2070788, rs9974589, and rs7364083 were associated with increased severity of COVID-19. 32Moreover, Fuentes et al. reported that rs61735794 and rs61735792 were significantly associated with COVID-19 infection outcome. 33n this work, no significant associations were detected between either the ACE2 rs2285666 SNP or the TMPRSS2 rs2070788 SNP and COVID-19 severity and outcome.
1 showed that rs2070788 and another three SNPs, which affect the expression of the TMPRSS2 gene in lung tissues, were more prevalent in American and European populations than in Asian populations; this finding can explain the higher susceptibility and fatality rates in American and European populations.Schönfelder et al., in 2021 in Germany, 43 observed no significant association between rs2070788 and COVID-19 severity.In addition, they attributed differences among several studies to variations in sample size, ethnicity, and the possibility of the presence of linkage disequilibrium with rs383510.

Limitations
This study has some limitations that should be considered when interpreting the findings.Firstly, this was a singlecentre study with a relatively low sample size.Thus, the results may be insufficiently powered and require further confirmation by future multicentre studies with larger cohorts.Secondly, host genetics is not the only factor that can influence COVID-19 disease: the host immunological status as well as SARS-CoV-2 strain variations have impacts on disease severity and outcome.In addition, an interplay between all of these factors exists; no data were available about patients' antibody response or viral genotypes so these factors could not be included in the analyses.This may have resulted in insufficient data about the impact of these factors on disease behaviour.

Conclusion
The genotypes and allele frequencies of the ACE2 gene SNP rs2285666 and the TMPRSS2 gene SNP rs2070788 showed no significant associations with the severity and outcome of patients.Further investigation into additional genetic correlations with COVID-19 infection is still necessary.
patient selection and data collection.W.S., O.A.G., R.M. and A.F. contributed to sample collection and processing and manuscript revision.R.M.A., A.F. and M.E.A. contributed to data collection and analysis, and manuscript revision.A.A. Ahmed contributed to data analysis and manuscript writing.Finally, the manuscript has been read and approved by all the authors.All authors are responsible for the reported research.

Sources of support
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
cases were hospitalized in intermediate care units, while critically ill patients were hospitalized in intensive care units.Therapy was carried out according to the aforementioned guidelines.Routine laboratory workup was performed in the clinical pathology department laboratories.
14spiratory rate > 30, despite receiving oxygen therapy.14Asymptomatic,mild, and moderate cases without risk were referred to home isolation with close follow-up.Conversely, moderate cases were hospitalized in COVID-19 areas, severe SPSS ® statistical software version 22 (IBM Corp., Armonk, New York, United States) was used for data analysis.Numeric values are presented as mean ± standard deviation if it was normally distributed (age, haemoglobin concentration, and platelet counts), while for not-normally distributed data (total and differential leukocyte counts, prothrombin time, kidney function tests, liver enzymes, lactate dehydrogenase, ferritin, CRP, and cycle threshold value) the median (range) was used.Student's t-test and one-way analysis of variance with least significant differences post hoc test were used to compare means; Mann-Whitney U test and Krusskal-Wallis with Bonferroni post hoc tests were used for medians comparison.Frequencies were expressed as numbers (percentages).Chisquare test and the two-tailed Fisher's exact test for results ≤ 5 were used to compare frequencies.p-values < 0.05 were considered statistically significant differences.After comparing all variables either qualitatively or quantitatively between different groups, all variables that have p < 0.1 were tested by the multivariate logistic regression, and significant results are presented as p-value, odds ratio and 95% confidence interval.Because ACE2 is an X-linked gene, ACE2 G8790A genotypes were analysed separately in female and male patients.
28ber et al.,  in 2022in Egypt,27reported that oxygen saturation and chest computed tomography severity score are predictors of mortality.Furthermore, Assal et al., in 2022 in Egypt,28found that the need for mechanical ventilation and the occurrence of secondary bacterial infections are associated with higher mortality.Additionally,Mahendra et al., in 2021
32mez et al., in 2022in Mexico,38and Sabater et al., in 2022 in Spain,39found that the AA genotype was associated with critical outcome.The current work's results agree with those of Alimoradi et al., in 2022 in Iran,18Karakaş et al., in 2021 in Turkey,40and Elnagdy et al., in 2022 in Egypt,41that there is no significant association between rs2285666 and COVID-19 severity and outcome.Pandey et al., in 2022in India,42and Asselta et al., in 2020 in Italy,32observed that the TMPRSS2 SNP rs2070788 had a statistically significant association with disease severity and fatality rate in American and Italian populations, respectively.Another multicentre study byIrham et al. in 2020