Genetic Variations in the Human Angiotensin-ConvertingEnzyme 2 and Susceptibility to Coronavirus Disease-19

Background Health and economies are both affected by the coronavirus disease-19 (COVID-19) global pandemic. Angiotensin-converting enzyme 2 (ACE2) is a polymorphic enzyme that is a part of the renin-angiotensin system, and it plays a crucial role in viral entry. Previous investigations and studies revealed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and ACE2 have a considerable association. Recently, ACE2 variants have been described in human populations in association with cardiovascular and pulmonary conditions. In this study, genetic susceptibility to COVID-19 in different populations was investigated. Methods and Results We evaluated the identified variants based on the predictive performance of 5 deleteriousness-scoring methods and the 2015 American College of Medical Genetics and Genomics (ACMG) guidelines. The results indicated 299 variants within the ACE2 gene. The variants were analyzed by different in-silico analysis tools to assess their functional effects. Ultimately, 5 more deleterious variants were found in the ACE2 gene. Conclusions Collecting more information about the variations in binding affinity between SARS-CoV-2 and host-cell receptors due to ACE2 variants leads to progress in treatment strategies for COVID-19. The evidence accumulated in this study showed that ACE2 variants in different populations may be associated with the genetic susceptibility, symptoms, and outcome of SARS-CoV-2 infection.


Introduction
Coronavirus disease-19 (COVID- 19) with frst emergence in Wuhan, China, in December 2019 [1,2] is the consequence of infection with a novel coronavirus naming severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recognized as the cause of this new infectious respiratory disease.Te World Health Organization [3] on March 2, 2020, denoted this infection as a pandemic [4].Fever, cough, vomiting, diarrhea, and other symptoms are common among patients with COVID-19.Some cases might develop acute respiratory distress syndrome [5], severe pneumonia, multiple organ failure, and even death [6,7].Te key characteristic laboratory fndings include increased C-reactive protein level, aspartate aminotransferase, lymphopenia, and lactate dehydrogenase [8].Most COVID-19 afected patients manifest mild symptoms or are asymptomatic [9].Moreover, susceptibility to COVID-19 varies among age groups, with older individuals being more vulnerable than children [10,11].Intensive care unit treatment or hospital admission is required in 10-20% of patients afected with severe disease [12].Older age, high body mass index, the male sex, and underlying comorbidities such as cardiovascular disease, hypertension, obesity, diabetes, and chronic respiratory disease are risk factors for unfavorable outcomes [13].
Te main host-cell receptor of the spike glycoprotein (S) of SARS-CoV-2 is angiotensin-converting enzyme 2 [14].Tis receptor plays a vital role in virus entry into the cell and its infection [15,16].Li et al. showed that specifc residues in the human ACE2 (hACE2) receptor are necessary for binding with the pathogen [17].ACE2 is an important component of the renin-angiotensin system (RAS) [18,19], which regulates cardiovascular homeostasis, blood pressure, blood volume, and systemic vascular resistance [20,21].ACE2 is the main enzyme responsible for converting angiotensin II into angiotensin I [1][2][3][4][5][6][7].Te imbalance of the RAS caused by the binding of SARS-CoV-2 to ACE2 is likely to play a role in COVID-19 pathogenesis [22].Furthermore, ACE2 is associated with cardiovascular disease, kidney disease, hypertension, stroke, and dyslipidemia [23][24][25][26].In the severe acute respiratory syndrome (SARS) outbreak in [2002][2003], which was caused by SARS-CoV, ACE2 played the same role as it plays in SARS-CoV-2 infection [27].Te transmembrane protease serine 2 (TMPRSS2) leads to the cleavage of the C-terminal segment of ACE2 and results in the S protein-driven viral entry [28,29].Mutant S proteins can detect host receptors within species [30].Te S protein has 2 subunits: the S1 subunit contains the receptor-binding domain, which targets receptors in the host cells, and the S2 subunit, which regulates membrane fusion between the host cells and the virus [31].After binding to the ACE2 receptor, the S protein of SARS-CoV-2 is cleaved by the TMPRSS2 protease at the S1/S2 and S2 sites, leading to the activation of the S2 domain and the membrane fusion of the viral and host membranes (Figure 1(a)) [32].Te abundance of ACE2 receptors in any organs of the body, including the brain, heart, kidney, nasopharynx, lymph nodes, small intestine, colon, stomach, thymus, skin, spleen, bone marrow, liver, blood vessels, and oral and nasal mucosa, renders them susceptible to infection by SARS-CoV-2 [10,33].Previous in vitro studies have indicated that there exists a positive robust correlation between SARS-CoV infection and ACE2 expression [34,35].Te levels of ACE2 expression in diferent tissues are shown in Figure 1(b).ACE2 is highly expressed in lung alveolar epithelial cells leading to considerable severe lung damage and therefore ARDS acute lung damage and pneumonia as the consequence [36].Te secondary and dimerization structures of the ACE2 protein are shown in Figures 2(a) and 2(b), respectively.Te crystal structure of the ACE2 receptor is illustrated in Figure 2(c).Te binding strength of ACE2 with SARS-CoV-2 is weaker than that with SARS-CoV, and it is regarded as high as the threshold necessary for the infection of the virus.Te S protein is a trimeric glycoprotein expressed in the surface of SARS-CoV-2 virion, which regulates recognition of receptor throughout its membrane fusion and receptorbinding domain [37,38].
Previous investigations have revealed that the SARS-CoV-2 protein binds to hACE2 through Phe486, Leu455, Ala501, Tyr505, and Gln493.Te 31, 41, 82, and 353-357 residues in the ACE2 receptor are important for its interaction with the S protein of SARS-CoV-2 [17].Recent clinical studies have demonstrated that male and female patients with COVID-19 exhibit signifcant diferences in incidence and mortality rates.COVID-19 is associated with underlying conditions such as cardiovascular disease and cancer, as well as in specifc patients with hypertension consuming antihypertensive medicines [39].Genetic variations in the ACE2 gene (Online Mendelian Inheritance in Man (OMIM): 300335) play a critical role in the susceptibility, symptoms, and outcome of SARS-CoV-2 infection in various populations [40].Some ACE2 polymorphisms may decrease the association between ACE2 and the S protein of SARS-CoV [16].Tis suggests that an investigation of the functional ACE2 polymorphisms could promote personalized treatment strategies and precision medicine for COVID- 19.
Te study aimed to search for the most deleterious variants in the ACE2 gene associated with COVID-19 and the pathogenesis of the identifed variants has been evaluated in silico.We highlighted that the ACE2 gene variants could guide personalized treatments.ACE2 polymorphisms could associate with various genetic susceptibility to COVID-19 and treatment outcomes in diferent ethnic groups.Te limitations of this study included that the genomic data in general populations have been examined and the identifed ACE2 variants need to be evaluated in a case-control study.Also, further studies should be done in the future to evaluate the impact of these variants.

Search Strategy and Data Extraction.
In the present study, genetic susceptibility to COVID-19 was investigated by evaluating the variants of the ACE2 gene.Te inclusion criteria for variants selection was the variants of ACE2 which are related to COVID-19.
Te combination of the following keywords ACE2 and COVID-19, ACE2 variants, and ACE2 [title/abstract] was used in searching PubMed and Google Scholar.Totally, 64 articles were collected, and after duplicate removal, 22 articles remained in which the variants were collected from these related articles.Duplicate publications and studies with overlapping or insufcient data were excluded.Te variants were also collected from the Human Gene Mutation Database (HGMD) (https://www.hgmd.cf.ac.uk/ac/index.php) and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/).

Genetic Analysis of hACE2.
Te variations in the ACE2 gene are probably important not only in modulating the host susceptibility to SARS-CoV-2 infection but also in determining the severity of local and systemic tissue damage [49].
In the present study, we collected variant datasets from 6 databases: ExAC, 1KGP, ESP6500, gnomAD, Iranome, and GME.Given that any frequency databases which were used in our study are due to global standards and their population study and methods were diferent, the minor allele frequency (MAF) of any databases is diferent.Indeed, we used this information to identify variants with MAF below some specifed threshold, which likely relate to disease.ExAC has collected, harmonized, and released exome sequence data from 60706 individuals.1000G is about common genetic variants with frequencies of at least 1% in the populations studied.ESP6500 is a database of genes and mechanisms that contribute to blood, lung, and heart disorders through NGS data in various populations.gnomAD is a coalition of investigators seeking to aggregate and harmonize exome and genome sequencing data from a variety of large-scale sequencing projects and to make summary data available for the wider scientifc community.Iranome is a catalog of genomic variations in the Iranian population.GME generated a coding base reference for the countries found in the Greater Middle East.As we know, the genetic variations of each population are diferent from the other.Our results revealed 299 variants in the ACE2 gene.A list of the identifed variants in the ACE2 gene is summarized in Table 1.Te majority of the ACE2 gene variants have yet to be identifed functionally.To obtain information about the possibility of the deleterious efects of the identifed variants, we evaluated the variants using the in-silico prediction of their functional efects.Ultimately, we identifed the most deleterious variants in the ACE2 gene based on prediction tools (Figure 3, Table 2).

3.2.
Variants of the ACE2 Gene.Cao et al. explored the allele frequency distribution of 1700 ACE2 gene variants using China Metabolic Analytics and 1K1000 Genomes [50].Twenty-fve variants located within the ACE2 gene were collected and cataloged in the Leiden Open Variation Database [14].Single-nucleotide variations (SNVs) with a low allele frequency appear to be more deleterious than SNVs with a high allele frequency according to some scoring methods [51].According to a study by Hou et al., 39% and 54% of deleterious variants in the ACE2 gene are carried by African/African-American and Non-Finnish European populations, respectively.Specifcally, 2-10% of deleterious variants in this gene occur in Latino/Admixed American, East Asian, Finnish, and South Asian populations, while Amish and Ashkenazi Jewish populations do not carry deleterious variants in the ACE2 coding regions [40].Te variants p.Met383Tr, p.Asp427Tyr, and p.Arg514Gly are carried by African/African-American populations, with an allele frequency of 0.003%, 0.01%, and 0.003%, respectively.Additionally, the p.Pro389His variant, with an allele frequency of 0.015%, is carried by Latino/Admixed American populations only [40].According to a previous study, several ACE2 variants and alterations in amino acid residues in ACE2 could afect the association between the ACE2 receptor and the S protein in SARS-CoV, leading to the conversion of ACE2 into an efcient/inefcient receptor [17].Fujikura and Uesaka identifed 8 SNVs-namely p.Ser19Pro, p.Tr27Ala, p.Glu35Lys, p.Glu35Asp, p.Glu37Lys, p.Met82Ile, p.Glu329Gly, and p.Asp355Asn-in the ACE2 gene in the direct contact residues of the S protein of SARS-CoV/SARS-CoV-2 and hACE2 [51].Residues Arg708/710/716, located in the dimeric interface of the ACE2 receptor, are a vital component for cleavage by TMPRSS2.Tis process is required to strengthen the entry of the virus into the host cells [29].Notably, the variants p.Arg708Trp, p.Arg710Cys, p. Arg710His, and p.Arg716Cys with an allele frequency of 0.01∼0.006%are carried by European populations.East Asian and Latino/Admixed American populations only carry the variants p.Arg708Trp and p.Arg710His, which have an allele frequency of 0.04% and 0.01%, respectively [40].Several variants, including p.Met383Tr, p.Pro389His, and p.Asp427Tyr, inhibited the interaction between the ACE2 receptor and the S protein of SARS-CoV-1 in the SARS outbreak in 2002 [17].Tere are natural ACE2 variants that alter the interaction between the virus and the host cells and, as a result, potentially change the susceptibility of the host.In particular, 9 variants-namely, I21V, Q102P, S19P, K26R, E23K, T27A, T92I, N64K, and H378R-were found in the hACE2 gene, which increased viral binding susceptibility, while 17 variants-namely, K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51S, M62V, K68E, F72V, Y83H, G326E, G352V, D355N, Q388L, and D509Y-were predicted to decrease the binding afnity of the S protein of SARS-CoV-2 and were, thus, considered protective variants [52].Te variants rs73635825 and rs143936283 present a relatively low binding afnity for the S protein of SARS-CoV-2, which may be associated with potential resistance to infection [49].Information regarding these variants is not available in Iranome.Tree variants-namely, p.Lys26Arg, p.Gly211Arg, and p.Asn720Asp-were more frequently expressed in the Italian population than in the Eastern Asian population.Tese variants are close to the sequence essential for the binding of the S protein of SARS-CoV-2.Te presence of these variants may explain the high mortality rate in Italy compared with China [49,53].ACE2 gene mutation naming Leu584Ala facilitates the SARS-CoV entry into target cells [54].Cao et al. characterized 32 variants in the ACE2 gene, among which there were 7 hotspot variants-namely, Lys26Arg, Ile486Val, Ala627Val, Asn638Ser, Ser692Pro, Asn720Asp, and Leu731Ile/Phe-in diferent populations [50].Benetti et al. concluded that 3 more common missense variants-namely, p.Gly211Arg, p.Lys26Arg, and p.Asn720Asp-could interfere with both protein structure and its stabilization.Furthermore, the two rare variants of p.Pro389His and p.Leu351Val were predicted to interfere with the binding of the SARS-CoV-2 S protein [4].Based on the fndings of the present study, diferential variants in the ACE2 gene may clarify various susceptibility and outcomes in diferent ethnic groups.

Discussion
Te ACE2 receptor acts as an entry point for the coronavirus [55].In addition to the strategy of using viral replication inhibitors, another strategy in the treatment option is to block the cellular target of the virus, ACE2 [56].Certain genomic variants within the ACE2 gene that modulate its function or expression cause variable susceptibility to SARS-CoV-2 infection [20].Given the possible connection between circulating ACE2 levels and COVID-19 severity, recombinant ACE2 may be a promising treatment option [57].As a result, tissue-specifc ACE2 expression or plasma ACE2 levels are considered 2 important factors in the severity of COVID-19.Te efects of antihypertensive therapy by both angiotensin-converting enzyme inhibitors (ACE-I) and angiotensin receptor blockers (ARBs) may lead to increased expression levels of ACE2.Studies have shown that the increased level of soluble ACE2 may act as a competitor to SARS-CoV-2 and may, thus, reduce viral penetration into cells and lung tissue [58,59].According to a meta-analysis, ACE-I/ARBs reduced the risk of pneumonia and its mortality [60].Te rs2285666 polymorphism may be a predisposing factor for the comorbidities observed in patients with COVID-19 [61,62].Te population-based frequency of this single-nucleotide polymorphism (SNP) is signifcantly higher among the Indian population (∼0.6) than among Europeans (0.2) and East Asians (0.55) [21,50,62].In our study, among the Iranian population, we identifed a frequency of 0.2575 for this SNP.In the present study, we conduct a comprehensive systematic investigation on genetic variations in the human genes associated with the coronavirus.Te reason for choosing the ACE2 gene in this study was that variants of this gene may be able to modulate intermolecular interactions with the S protein of SARS-CoV-2 and are associated with altering virulence, pathogenicity, clinical outcome, and COVID-19 susceptibility.In the present study, we provided the dataset of ACE2 variants (Table 1).Te ACE2 gene variants may be associated with COVID-19 genetic susceptibility which could guide more personalized and individualized treatments for the COVID-19 pandemic [40].Since ACE2 gene variants may cause diferent responses to COVID-19 treatments concerning the components of the RAS system, we recommend case-control studies to investigate the efects of these variants on treatment outcomes.In addition, the testing of the ACE2 gene polymorphisms has been recommended for patients with COVID-19 undergoing clinical trials with ACE-I/ARBs [9].Worldwide study on the genes linked to life-threatening instances is required despite the development of many licensed vaccinations, the mutation of coronaviruses, and the potential for pandemics.It is also necessary to obtain information on variants for populationappropriate vaccines against SARS-CoV-2 infection.
Tis study aimed to search for the most deleterious variants associated with COVID-19, and the pathogenesis of the identifed variants has been investigated in silico.We selected the variants with the highest CADD score and were considered as deleterious, damaging, and disease causing in at least three prediction tools.Also, the MAF of the selected variants in the frequency databases was very low, and these variants can be very important in the incidence of the disease (Figure 3, Table 2).Finally, we found the fve variants caused the changes in amino acid residues of the extracellular domain of the ACE2 receptor (residues 18-740) that includes a zinc-binding site (residues 374-378, His-Glu-Met-Gly-His). Te mutated residues are located in the extracellular domain which plays an important role in the main activity of the ACE2 protein, and these variants can consequently disturb its normal function.Te S protein of SARS-CoV-2 is identifed by the extracellular peptidase domain of the ACE2 receptor and leads to the binding of the virus to the host cell.Probably, each of these fve deleterious variants mentioned in this study caused a disturbance in the structure of the ACE2 receptor, which may be efective in the incidence of this disease.Te c.1129G > T variant in the ACE2 gene caused the Gly377Gln substitution within the extracellular domain of the receptor.Tis residue is located in the zinc-binding site (positions 374-378) that is involved in binding.Te E37K variant is in the direct contact residues of hACE2 and the S protein that play a role in the entry of the virus into the host cells.Te initial attachment of the S protein to the receptor has caused the exposure of the most important amino acids for binding (residues 22-57).Te main functional domains of the ACE2 receptor that interact with SARS-CoV-2 are illustrated in Figure 2(c).Te c.109G > A variant in the ACE2 gene caused the Glu37Lys substitution within the main functional domains of ACE2 (residues 30-41).Also, amino acid glycine at position 37 is the main residue at the interface.Genetics Research According to this study, the fve deleterious variants in the ACE2 gene may clarify various susceptibility and outcomes in diferent ethnic groups.Tese ACE2 variants and alterations in amino acid residues in the receptor alter the interaction between the virus and host cells, resulting in altering the host susceptibility.Terefore, we recommend further research to identify the efect of the most pathogenic variants on the binding affnity.Also, the identifed pathogenic variants in the ACE2 gene may afect the clinical efcacy of drugs for COVID-19, which is better investigated.We suggest that the frequency of these deleterious variants in diferent populations is investigated in the future so that the necessary preparations for the disease are considered in populations carrying these variants.
Te tissue-specifc ACE2 expression and plasma ACE2 levels, and density of ACE2 receptors are key factors of the diference in the severity and incidence of the disease in various countries.Also, the levels of ACE2 expression vary in diferent populations and various human tissues (Figure 1(b)).SNPs afect gene expression and lead to a change in the outcome of the disease.We recommend that these factors be investigated in individuals with these variants in diferent populations that could promote personalized treatment strategies and precision medicine for COVID-19.Such studies may afect accurate medical interventions and the design of specifc diagnostic and therapeutic methods for coronavirus.Te present study can be useful for better understanding interindividual clinical variability, and the severity and susceptibility of this disease in diferent ethnic groups.
Te mechanisms resulting from the functional foodsbased treatments included the reduced expression of ACE2 receptors in cells, inhibiting necessary enzymes in SARS-CoV-2, and decreased proinfammatory cytokines that can help the body fght during illness [63].Te mentioned variants that modulate the ACE2 function and expression cause variable susceptibility to SARS-CoV-2 infections.It seems to be benefcial for patients carrying these variants to use the functional foods-based treatments that lead to the reduced expression of ACE2 receptors in the cells.Terefore, we recommend further research to identify the efect of the most pathogenic variants in diferent populations on the ACE2 tissue expression, plasma ACE2 levels, and binding afnity, leading to improved therapeutic strategies and precision medicine for COVID-19.We suggested that the testing of the polymorphisms and the most pathogenic variants in the ACE2 gene should be considered when determining the type of drugs in patients with more severe symptoms.According to the studies, numerous polymorphisms are associated with high ACE2 tissue expression and higher severity, whereas some polymorphisms are associated with low ACE2 tissue expression and lesser severity.As a result, the treatment outcomes in COVID-19 patients are infuenced by the ACE2 variants.Te spike protein mutations increased the viral attachment and subsequent entry into host cells.Te structural target for available drugs and treatments is the high binding afnity of the spike protein and the receptor.It appears that some of the identifed variants and their cumulative efects of them cause diferent susceptibility to the entry of viral cells and have a signifcant efect on the used therapeutics and vaccination efectiveness.Given the possibility that treatment-resistant variants may emerge that could lead to destructive and irrecoverable impacts on global health, continuous viral surveillance of new variants should be performed using viral genomic sequencing.Both the virus and receptor variants are two important factors in the susceptibility and severity of this disease.Terefore, we suggest that both factors should be considered to select the proper therapeutic strategy.Despite the production of several approved vaccines, mass vaccination, recommending vaccine boosters, the latest novel therapeutics available, and food-based treatments, the signifcant progress made so far in stopping the spread of SARS-CoV-2 is threatened by the continued emergence of new variant strains of SARS-CoV-2.It also highlights further investigation on genes associated with life-threatening cases is necessary due to adaptive mutations in the viral genome that can change the pathogenic potential of this virus.Te evaluation of pathogenic variants in the ACE2 gene in male and female genders and diferent populations with the appropriate therapeutic strategies can be efective to prevent infections among populations at risk of SARS-CoV-2 infections resulting from possible viral variants.

Conclusions
Te detection of SNP genotypes is urgently needed to discover likely genetic risk factors for severe outcomes.Te identifcation of variants may have a signifcant impact on the variability of the COVID-19 course and may confer precision medicine interventions, treatment individualization and design, and inexpensive and accurate DNA-based tests for the coronavirus.Our genetic analysis of variants in the hACE2 gene suggests that the ACE2 variants may be associated with COVID-19 susceptibility and clinical outcomes.

Figure 2 :
Figure 2: (a) Te image depicts the secondary structure of the angiotensin-converting enzyme 2 protein.(b) Te image illustrates the dimerization structure of the ACE2 protein with SWISS-MODEL (https://swissmodel.expasy.org/)ID Q9BYF1.ACE2 dimerizes via 2 domains: peptidase-M2 and collectrin, which are shown in color.(c) Te image demonstrates the crystal structure of ACE2 with PDB (https://www.rcsb.org/)ID 1R42.Te main functional domains of ACE2 that interact with SARS-CoV-2 are illustrated in the box.
reports the genomic position, the nucleotide, and amino acid change of identifed variants in the ACE2 gene.Tese data are based on the Genome Reference Consortium Human Build 37 (GRCh37).1 CADD, Phred ≤20: neutral; Phred >20: damaging; 2 SIFT, score ≤0.05: deleterious; score >0.05: tolerable; 3 polyphen-2, score � 0-0.15: benign; score � 0.15-0.85:possibly damaging; score � 0.85-1: probably damaging; 4 PROVEAN, score ≤ −2.5: deleterious; score > −2.5: neutral; TO: tolerable; DE: deleterious; NE: natural, DC: disease causing; NA: not available.PRD: probably damaging; POD: possibly damaging; P: polymorphism.12GeneticsResearch among Americans (0.15), followed by Europeans (0.055), Caucasians (0.051), and Central Asians (0.021).In the current study, we also detected a frequency of 0.0325 for this SNP among the Iranian population.It appears that some of the identifed variants or the cumulative efect of a few of them cause diferent susceptibility to the entry of viral cells and have a signifcant efect on the onset and progression of the disease.Terefore, systematic identifcation of the genetic determinants of COVID-19 susceptibility and the clinical outcome could further explain the current epidemiologic observations, disease pathophysiology, diferent susceptibilities, and disease severities in diferent ethnic groups.

Figure 3 :Table 2 :
Figure 3: Te most pathogenic variants of the ACE2 gene are displayed by arrows.
Te results of another study conducted by Srivastava et al. indicated that the frequency of a synonymous coding region variant, rs35803318, was high Genetics Research