The Relation between ACE Gene Polymorphism and the Severity of COVID-19 Infection

Introduction The coronavirus disease 2019 (COVID-19) pandemic, which emerged in China at the end of 2019, rapidly spread worldwide. The angiotensin-converting enzyme (ACE) gene contains an insertion/deletion (I/D) polymorphism that leads to a higher serum ACE level which is associated with several diseases and also with a high mortality rate in SARS. Therefore, this study aimed at assessing the association between ACE gene polymorphism and the risk and severity of COVID-19 disease in patients. Methodology. Forty-five SARS-CoV-2 infected patients and another random control group of 45 healthy individuals were included. The detection of ACE I/D gene polymorphism in both groups was done by PCR. Results 53% of infected patients with SARS-CoV-2 had an ACE deletion/deletion genotype (D/D), 27% had an ACE deletion/insertion genotype (D/I), and 20% had an ACE insertion/insertion genotype (I/I). On the one hand, the D/D variant was significantly detected in the COVID-19 patients compared to the control subjects, whereas the I/I variant was significantly detected in the control subjects compared to the COVID-19 patients (p = 0.004). The D/D variant subgroup showed the lowest lymphocytic count compared to the D/I or I/I subgroups. In addition, the C-reactive protein was significantly higher and the oxygen saturation was significantly lower in patients with the D/D allele compared to the other subgroups. Conclusions ACE gene polymorphism, particularly the DD genotype, was observed to affect the severity of COVID-19 infection.


Introduction
Coronaviruses (CoV) have emerged as the most common pathogens in respiratory illness outbreaks. Tey are a broad group of single-stranded RNA viruses that can be found in a variety of animals [1]. Coronaviruses are classifed into four genera based on the target host; mammals are infected by α and β-CoV, whereas birds are afected by c and δ-CoV [2].
Te world is dealing with the coronavirus disease 2019 (COVID- 19) pandemic, which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that belongs to β-CoV genus of coronaviruses. Tis pandemic began in China at the end of 2019 and quickly spread throughout the world [3]. In Egypt, the SARS-CoV-2 index case was registered by health authorities on February 14, 2020 [4].
Angiotensin-converting enzyme (ACE) is a component of the renin-angiotensin-aldosterone system (RAAS) that is involved in blood pressure homeostasis. ACE converts angiotensin I (Ang I) to angiotensin II (Ang II) which, through its interaction with the angiotensin II type 1 receptor (AT1R), induces a strong vasoconstriction and triggers proinfammatory, proapoptotic, and profbrotic pathways in the lung and other organs [5].
Another homolog of ACE is ACE2, which is encoded by the human X-chromosome. ACE2 is a typical zincmetalloproteinase type I transmembrane protein that is expressed ubiquitously in diferent organs of the human body, such as the heart, lungs, kidneys, intestines, and endothelium [6]. In RAAS, ACE2 contributes to the inactivation of Ang II by hydrolyzing it to Ang-1-7, which stimulates vasodilation and promotes anti-infammatory, antifbrotic, and antithrombotic actions via the Ang-1-7/ Mas receptor axis. Terefore, ACE2 physiologically counters Ang II/AT1R efects [5].
Another major role of ACE2 was identifed by the emergence of severe acute respiratory syndrome (SARS) in 2002-2003, caused by SARS-CoV. Human ACE2 acts as a functional receptor that binds to the SARS-CoV spike (S) protein with high afnity, which is considered a critical step in the virus's entry into human cells [7].
Te ACE insertion (I)/deletion (D) gene polymorphisms are one of the most frequently defned human polymorphisms. D and I polymorphisms in the ACE gene may result in differences in ACE levels. For instance, the ACE D allele causes an increase in ACE1 level and a decrease in ACE2 level, causing an increased level of angiotensin-2 which may predispose the individual to a variety of disorders including obesity, hypertension, increased cardiovascular risk, and thrombophilia [8]. Furthermore, the ACE D/D gene polymorphism causes the progression of pulmonary edema through increased microvascular permeability, which further worsens the clinical course and prognosis of the acute respiratory distress syndrome (ARDS) and has been linked to a high mortality rate in SARS patients [9].
Te lack of any functional ACE gene polymorphisms being linked to SARS infection might be due to a lack of previous studies. Terefore, this study aimed at assessing the association between ACE gene polymorphism and the risk and severity of COVID-19 disease in patients.

Methodology
A case-control prospective study of the relation between ACE gene polymorphism and the severity of COVID-19 infection was planned. A sample of patients who tested positive for SARS-CoV-2 were compared with a sample of controls who tested negative for SARS-CoV-2, assuming an equal number of cases and controls (r = 1). Previous studies have shown that around 73% of positive SARS-CoV-2 patients had the DD allele (p1 = 0.73) [10]. For achieving a 90% power (1 − β = 0.9) at the 5% level of signifcance (α = 0.05), the sample size to detect a proportion of negative tested subjects of SARS COV2 (p2 = 0.304) [11] is 45 cases and 45 controls.
Patients who presented with the COVID-19 characteristic symptoms of fever, cough, and dyspnoea and were admitted to the hospital with an already proven SARS-CoV-2 infection were originally enrolled. Additionally, we looked at 45 healthy subjects who were negative for SARS-CoV-2 using reverse transcription real-time PCR testing. Te following were the exclusion criteria: (1) age over 70; (2) chronic kidney disease (CKD) managed by dialysis; and (3) patients with restrictive or obstructive lung diseases.
All age groups were included. Informed consent was taken from each patient to use their data in the current research work. We obtained the approval of the Ethics Committee of the Faculty of Medicine, Suez Canal University (Ethics approval number: Research 4555#).
Te following data were collected from each patient in both groups: age, gender, and comorbid conditions (diabetes, cardiovascular diseases, history of active malignancy). In addition, initial peripheral oxygen saturation by pulse oximetry and laboratory tests for complete blood count, C-reactive protein (CRP), and D-dimers were recorded in the COVID-19 group.
A computed tomography of the chest was performed and the extent of the lesions was graded according to the COVID-19 Reporting and Data System (CO-RADS) score ( Te DNA yield and purity were assessed by a spectrophotometer at 260 nm (Nanodrop) (Termo Fisher Scientifc Inc. USA). Te ratio of absorbance at 260 and 280 nm (A260/A280) was used to determine the purity of DNA.

Detection of ACE Insertion/Deletion (I/D) Polymorphism by PCR.
According to Tiret et al., the ACE insertion/deletion (I/D) polymorphism was amplifed by PCR using a specifc set of primers (Table 2) [13].
Te DNA was amplifed in the thermal cycler (Eppendorf Co., Germany) using the following protocol: initial denaturation (94°C for 2 minutes), followed by 35 cycles of denaturation at 94°C for 1 min, annealing (58°C for 1 minute), and extension (72°C for 2 minutes), with a single fnal extension of 10 minutes at 72°C. Te amplifed products were visualized by electrophoresis on 2% agarose gels stained with ethidium bromide and then visualized under ultraviolet (UV) illumination. Te amplicons were compared to a molecular-weight DNA ladder with sizes ranging from 100 to 1000 bp (Fermentas, Germany) to visualize three patterns:

Statistical Analysis.
All statistical analyses were performed using SPSS version 22.0 (IBM, Armonk, New York, United States). Signifcance was observed with p value < 0.05.
Variables were tested for pattern of distribution using the Kolmogorov-Smirnov test and visual assessment of histograms. Data with normal distribution were expressed as mean ± standard deviation while non-normally distributed variables were presented as median and interquartile range (IQR). To assess possible diferences between groups for parametric and nonparametric variables, we used unpaired student t, one-way ANOVA (Bonferroni test for in-between group diferences), and Mann-Whitney U, Kruskal-Wallis tests, respectively. To compare categorical variables, chisquared test was used.
For detection of the prediction model of the DD allelic variation, we calculated the odds ratio (OR) and 95% confdence interval (CI) by multivariable logistic regression.

Results
Te results of this study showed that the females were represented almost similarly in both groups with no signifcant diference (p > 0.05). In addition, diabetes mellitus, cardiovascular diseases, and history of active malignancy were similar in both groups (p > 0.05). Analysis of the markers of severity related to the SARS-CoV-2, the COVID-19 group revealed low means lymphocytic count, high CRP, and D-dimer tests. Additionally, the COVID-19 patients showed reduced mean oxygen saturation (Table 3).
Regarding the results of conventional PCR for testing the presence of ACE I/D gene polymorphism on peripheral blood samples of patients in both groups as shown in Figures 1 and 2, it was found that 53% of infected patients with SARS-CoV-2 had ACE D/D polymorphism, 27% had ACE D/I polymorphism, and 20% had ACE I/I polymorphism. Te control group showed that PCR of blood samples revealed that 29% had ACE D/D polymorphism, 18% had ACE D/I polymorphism, and 53% had ACE I/I polymorphism.
Te comparison between the SARS-CoV-2 group and the control group regarding the ACE gene allelic variants revealed that the D/D variant was signifcantly detected in the COVID-19 patients compared to the control subjects (53% and 29%, respectively), whereas the I/I variant was signifcantly detected in the control subjects compared to the COVID-19 patients (53% and 20%, respectively) (p � 0.004), as shown in Figure 3.
Te COVID-19 patients with diferent ACE gene allelic variants showed that age and D-dimer were not diferent between all variants' subgroups. Regarding the markers of high infammatory reaction, the D/D variant subgroup showed the lowest lymphocytic count compared to the D/I or I/I subgroups. In addition, the C-reactive protein was signifcantly higher in the D/D patients compared to the other subgroups. Moreover, oxygen saturation was Te level of suspicion is high, and most CT fndings, such as unilateral ground-glass, confuent, or multifocal consolidations without a typical site or any other characteristic fndings, are suspicious but not typical CO- RADS 5 Te level of suspicion is high with typical CT fndings CO-RADS 6 RT-PCR positive for SARS-CoV-2 CO-RADS, COVID-19 reporting and data system.   (Table 4). Multivariable logistic regression analysis was performed for the detection of the predictors of the DD allelic variation and revealed that no variable in the model appeared to have a signifcant association with allelic variation (Table 5).

Discussion
Te RAAS is mainly responsible for regulating blood pressure, fuid volume, and electrolyte balance. Te physiological homeostasis of the RAAS is regulated by the balance of ACE and ACE2. ACE converts Ang I to Ang II which exerts vasoconstrictive, hypoxic, oxidative, hypertrophic, fbrotic, and infammatory actions and is involved in the development of various pathologies. ACE2 is responsible for counteracting the adverse efects of angiotensin II through its proteolytic product, angiotensin (1-7) which activates signalling pathways that lead to vasodilation and inhibition of cell proliferation, but also accounts for strong antiinfammatory, antifbrosing, and antioxidative efects. Terefore, the quantities of the two enzymes have been considered to be the main endogenous regulators of the RAAS [14].
Several observations support the evident association between RAAS imbalance and COVID-19 disease. Tis is because ACE2 is the main cellular receptor for the SARS-CoV-2, and this interaction results in exhaustion and reduced expression of ACE2, resulting in elevated levels of angiotensin II which could explain the deleterious lung injury observed in SARS-CoV-2 infection [15].
Tis study was conducted to demonstrate the association between ACE gene polymorphism and the risk and severity of COVID-19 infection. It was found that 53% of infected   patients with SARS-CoV-2 had ACE D/D polymorphism, 27% had ACE D/I polymorphism, and 20% had ACE I/I polymorphism. On the one hand, the D/D variant was signifcantly detected in the COVID-19 patients compared to the control subjects (53% and 29%, respectively), whereas the I/I variant was signifcantly detected in the control subjects compared to the COVID-19 patients (53% and 20%, respectively) (p � 0.004).
Tere is an evidence that the ACE I/D polymorphism infuences plasma ACE concentration, with D/D genotype individuals having approximately doubled plasma ACE concentrations (as the D allele of the ACE I/D gene polymorphism mediates higher ACE expression) compared to I/I genotype individuals, and the I/D genotype individuals having intermediate concentrations [16].
Te I/D polymorphism in the ACE gene has been previously examined in relation to variable diseases' susceptibility and consequences, with contrasting results. According to a meta-analysis, ACE gene polymorphisms, particularly the D/D genotype, may increase the risk of respiratory disease with pulmonary hypertension [17]. Some researchers have found a link between the prevalence of the ACE gene polymorphism and the progression of SARS [18].
In Italy, Annunziata et al. found that 73% of critically ill COVID-19 patients had the D/D polymorphism, 23% had the I/D polymorphism, and just 8% had the I/I polymorphism [10]. Moreover, Amar and colleagues in Pakistan found a probable correlation between the ACE I/D gene polymorphism and COVID-19 prevalence, fatalities, and recovery rate in a meta-regression analysis. Tey discovered that nations with a higher frequency of the D allele had higher COVID-19 infection prevalence and fatalities, especially when compared to European and Asian countries [19]. In addition, Mir et al. in Saudi Arabia reported that the frequency of the D allele was found to be signifcantly higher among COVID-19 patients than in the healthy controls [20].
Regarding the markers of high infammatory reaction, our study revealed that the D/D variant subgroup showed the lowest lymphocytic count compared to the D/I or I/I subgroups. In addition, the C-reactive protein was signifcantly higher in the D/D patients compared to the other subgroups. Moreover, oxygen saturation was signifcantly lower in the D/D subgroups in comparison with other subgroups.
Similar results were reported by Gómez et al. in Spain, who conducted research on 204 Spanish SARS-CoV-2-infected patients and found a hypertension-dependent link between the ACE-DD genotype and severe COVID-19 infection [21]. Furthermore, Verma et al. and colleagues in India found that the ACE1 DD genotype and the frequency of the D allele were signifcantly higher in severe COVID-19 patients [22]. Similarly, Mir et al. in Saudi Arabia showed that the ACE-DD genotype was strongly associated with increased COVID-19 severity (p < 0.013) and mortality (p < 0.008) [20].
In contrary to our results, Çelik et al. in Turkey investigated the relationship between ACE gene I/D polymorphism, ACE2 receptor gene rs2106809, and rs2285666 polymorphism, and COVID-19 severity in 155 COVID-19 patients who were divided into three groups (mild, moderate, and severe) according to clinical symptoms. Tey concluded that ACE gene I/D, ACE2 receptor gene rs2106809, and rs2285666 polymorphisms were not associated with the severity of COVID-19 infection [23]. Also, Möhlendick et al. in Germany found that the ACE gene polymorphism was not related to infection risk or severity of COVID-19 [11].
Functionally, COVID-19 patients with the D/D genotype may have an unopposed abundance of angiotension II protein in their blood as a result of the following: increased D allele-mediated excessive ACE expression on one hand and downregulation of ACE2 receptor because of SARS-CoV-2 engagement on the other, thus triggering downstream deleterious efects, the most notable of which is acute lung injury [15].
Tis RAAS imbalance caused by ACE abundance could possibly explain why COVID-19 infection complications are more severe with the D/D genotype.  n/a n/a n/a 0.996 Lymphocyte count n/a n/a n/a 0.989 D-dimer n/a n/a n/a 0.987 CRP n/a n/a n/a 0.983 Constant n/a n/a n/a 0.985 International Journal of Microbiology In our study, multivariable logistic regression analysis was performed for the detection of the predictors of the DD allelic variation and revealed that no variable in the model appeared to have a signifcant association with allelic variation (Table 5). Gómez et al. in Spain conducted a multiple logistic regression analysis and showed that hypertension (p � 0.02; OR � 2.26, 95% CI � 1.12-4.63) and male gender (p � 0.002; OR � 3.15, 95% CI � 1.56-6.66) remained as independent signifcant predictors of COVID-19 severity [21]. Also, Delanghe et al. concluded that the ACE1 D/I polymorphism independently contributes (p � 0.0076) to COVID-19 mortality in a multivariate regression mode [24].
Te study has certain limitations including the relatively smaller sample size. Tis is attributed to limited access to data obtained from COVID-19 hospitalized patients and difculty importing kits and chemicals during the COVID-19 era. Nevertheless, more future research with a larger sample size is required to better understand the association of this polymorphism with the severity of COVID-19 infection.

Conclusions
ACE gene polymorphism, particularly the DD genotype, was observed to afect the severity of COVID-19 infection.

Data Availability
Te datasets generated during and/or analyzed during the current study are available from the corresponding author upon request.

Ethical Approval
Approval was obtained from the Research Ethics Committee of Faculty of Medicine Suez Canal University (Ethics approval number: Research 4555#). Te procedures used in this study adhere to the ethical standards of the Declaration of Helsinki.

Consent
Informed consent was obtained from all individual participants included in the study. Patients signed informed consent regarding publishing their data.

Conflicts of Interest
Te authors declare that they have no conficts of interest.