1. Introduction
Coronavirus disease-19 (COVID-19) with first emergence in Wuhan, China, in December 2019 [Reference Zhu, Zhang and Wang1, Reference Huang, Wang and Li2] 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. The World Health Organization [Reference Wallace, Newhouse and Braund3] on March 2, 2020, denoted this infection as a pandemic [Reference Benetti, Tita and Spiga4]. Fever, cough, vomiting, diarrhea, and other symptoms are common among patients with COVID-19. Some cases might develop acute respiratory distress syndrome [Reference Richards, Aziz and Bale5], severe pneumonia, multiple organ failure, and even death [Reference Chen, Zhou and Dong6, Reference Liu, Wang, Zhou, Zhao, Zhang and Li7]. The key characteristic laboratory findings include increased C-reactive protein level, aspartate aminotransferase, lymphopenia, and lactate dehydrogenase [Reference Lake8]. Most COVID-19 affected patients manifest mild symptoms or are asymptomatic [Reference Zheng and Cao9]. Moreover, susceptibility to COVID-19 varies among age groups, with older individuals being more vulnerable than children [Reference Behl, Kaur and Bungau10, Reference Dhochak, Singhal, Kabra and Lodha11]. Intensive care unit treatment or hospital admission is required in 10–20% of patients affected with severe disease [Reference Bourgonje, Abdulle and Timens12]. 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 [Reference Zhou, Yu and Du13].
The main host-cell receptor of the spike glycoprotein (S) of SARS-CoV-2 is angiotensin-converting enzyme 2 [Reference Lovd14]. This receptor plays a vital role in virus entry into the cell and its infection [Reference Hoffmann, Kleine-Weber and Schroeder15, Reference Lu, Zhao and Li16]. Li et al. showed that specific residues in the human ACE2 (hACE2) receptor are necessary for binding with the pathogen [Reference Li, Zhang and Sui17]. ACE2 is an important component of the renin-angiotensin system (RAS) [Reference Donoghue, Hsieh and Baronas18, Reference Tipnis, Hooper, Hyde, Karran, Christie and Turner19], which regulates cardiovascular homeostasis, blood pressure, blood volume, and systemic vascular resistance [Reference Srivastava, Bandopadhyay and Das20, Reference Srivastava, Pandey and Singh21]. ACE2 is the main enzyme responsible for converting angiotensin II into angiotensin I [Reference Zhu, Zhang and Wang1–Reference Liu, Wang, Zhou, Zhao, Zhang and Li7]. The imbalance of the RAS caused by the binding of SARS-CoV-2 to ACE2 is likely to play a role in COVID-19 pathogenesis [Reference Costa, Perez and Palmeira22]. Furthermore, ACE2 is associated with cardiovascular disease, kidney disease, hypertension, stroke, and dyslipidemia [Reference Zhang, Cong and Wang23–Reference Pan, Wang and Li26]. 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 [Reference Chaudhary27]. The 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 [Reference Shulla, Heald-Sargent, Subramanya, Zhao, Perlman and Gallagher28, Reference Heurich, Hofmann-Winkler, Gierer, Liepold, Jahn and Pöhlmann29]. Mutant S proteins can detect host receptors within species [Reference Nemati, Ramezani, Najafi, Sayad, Nazeri and Sadeghi30]. The 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 [Reference Li, Li, Farzan and Harrison31]. 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)) [Reference Hartenian, Nandakumar, Lari, Ly, Tucker and Glaunsinger32]. The 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 [Reference Behl, Kaur and Bungau10, Reference Hamming, Timens, Bulthuis, Lely, Navis and van Goor33]. Previous in vitro studies have indicated that there exists a positive robust correlation between SARS-CoV infection and ACE2 expression [Reference Hofmann, Geier and Marzi34, Reference Imai, Kuba and Penninger35]. The levels of ACE2 expression in different 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 [Reference Zhang and Baker36]. The secondary and dimerization structures of the ACE2 protein are shown in Figures 2(a) and 2(b), respectively. The crystal structure of the ACE2 receptor is illustrated in Figure 2(c). The 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. The 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 receptor-binding domain [Reference Wrapp, Wang, Corbett, Goldsmith, Hsieh, Abiona, Graham and McLellan37, Reference Yan, Zhang, Li, Xia, Guo and Zhou38].
Figure 1 (a) The image illustrates the intracellular interactions between angiotensin-converting enzyme 2 and its ligand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (b) The image shows ACE2 expression in 15 primates and 16 tissues. The level for significantly expressed genes is color-coded in 8 equally sized bins (light-to-dark green). Light gray is for weak not-accurately measured expression (2 to 8 reads above the intergenic background), while dark gray is for no expression or no sequence conservation (0 read in the gene). The plot on the right shows the distribution of the measured expression values in all tissues for all genes (blue) and for this gene in magic index = log2 (1000 sFPKM). HUM: human, CHP: chimpanzee, PTM: pig-tailed Macaque, JMI: Japanese macaque, RMI: rhesus macaque Indian, RMC: rhesus macaque Chinese, CMM: cynomolgus macaque Mauritian, CMC: cynomolgus macaque Chinese, BAB: olive baboon, SMY: sooty mangabey, MST: common marmoset, SQM: squirrel monkey, OWL: owl monkey, MLM: mouse Lemur, RTL: ring-tailed lemur. This information was obtained from the AceView database (https://www.ncbi.nlm.nih.gov/ieb/research/acembly/).
Figure 2 (a) The image depicts the secondary structure of the angiotensin-converting enzyme 2 protein. (b) The 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) The image demonstrates the crystal structure of ACE2 with PDB (https://www.rcsb.org/) ID 1R42. The main functional domains of ACE2 that interact with SARS-CoV-2 are illustrated in the box.
Previous investigations have revealed that the SARS-CoV-2 protein binds to hACE2 through Phe486, Leu455, Ala501, Tyr505, and Gln493. The 31, 41, 82, and 353–357 residues in the ACE2 receptor are important for its interaction with the S protein of SARS-CoV-2 [Reference Li, Zhang and Sui17]. Recent clinical studies have demonstrated that male and female patients with COVID-19 exhibit significant differences in incidence and mortality rates. COVID-19 is associated with underlying conditions such as cardiovascular disease and cancer, as well as in specific patients with hypertension consuming antihypertensive medicines [Reference Guo, Fan and Chen39]. 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 [Reference Hou, Zhao and Martin40]. Some ACE2 polymorphisms may decrease the association between ACE2 and the S protein of SARS-CoV [Reference Lu, Zhao and Li16]. This suggests that an investigation of the functional ACE2 polymorphisms could promote personalized treatment strategies and precision medicine for COVID-19.
The reported variants of concern (VOCs) included B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and B.1.1.529 (Omicron) that have mutations in the receptor-binding domain (RBD) and the N-terminal domain (NTD) of the spike protein [Reference Aleem, Ab and Slenker41]. These variants lead to increased virulence and transmissibility, reduced neutralization by antibodies, and reduced efficacy of the treatment or vaccination [Reference Aleem, Ab and Slenker41]. The development of drugs that target the spike protein is an appropriate therapeutic strategy, which causes an alteration in binding to the ACE2 receptor [Reference Singh, Sharma, Lee and Yadav42]. Antiviral drugs, monoclonal antibodies against SARS-CoV-2, anti-inflammatory drugs, and immunomodulatory agents are available as therapeutic strategies [Reference Coopersmith, Antonelli and Bauer43].
The study aimed to search for the most deleterious variants in the ACE2 gene associated with COVID-19 and the pathogenesis of the identified 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 different ethnic groups. The limitations of this study included that the genomic data in general populations have been examined and the identified 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.
2. Materials and Methods
2.1 Search Strategy and Data Extraction
In the present study, genetic susceptibility to COVID-19 was investigated by evaluating the variants of the ACE2 gene. The inclusion criteria for variants selection was the variants of ACE2 which are related to COVID-19.
The 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 insufficient data were excluded. The 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/).
The Exome Aggregation Consortium (ExAC: https://exac.broadinstitute.org), the 1000 Genomes Project (KGP) (https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/), the Exome Sequencing Project (https://evs.gs.washington.edu/EVS/), the Genome Aggregation Database (gnomAD v3) (https://gnomad.broadinstitute.org/), Iranome (https://www.iranome.ir/), and the Greater Middle East (GME) Variome Project (https://igm.ucsd.edu/gme/) were used to obtain variants’ frequency.
2.2 Variants Evaluation
It seems that most of the ACE2 variants have not been functionally characterized. We evaluated the identified variants based on the 5 prediction tools score according to the threshold value, including Combined Annotation Dependent Depletion (CADD) (https://cadd.gs.washington.edu/home) [Reference Kircher, Witten, Jain, O'roak, Cooper and Shendure44], Sorting Intolerant from Tolerant (SIFT) (https://sift.bii.a-star.edu.sg/) [Reference Kumar, Henikoff and Ng45], Polymorphism Phenotyping v2 (PolyPhen-2) (https://genetics.bwh.harvard.edu/pph2/) [Reference Adzhubei, Jordan and Sunyaev46], Protein Variation Effect Analyzer (PROVEAN) (https://provean.jcvi.org/index.php) [Reference Choi, Sims, Murphy, Miller and Chan47], and Mutation Taster (https://www.mutationtaster.org/) [Reference Schwarz, Cooper, Schuelke and Seelow48]. CADD is the most important prediction tool among all bioinformatics software that was used in our manuscript, and the highest CADD Phred for variants evaluation was considered (Phred ≤ 20). Other prediction tools (SIFT, PolyPhen-2, PROVEAN, and MutationTaster) just were explained as descriptive in the range (SIFT: score ≤ 0.05: deleterious, score > 0.05: tolerable; Polyphen-2: score = 0–0.15: benign, score = 0.15–0.85: possibly damaging, score = 0.85–1: probably damaging; PROVEAN: score ≤ −2.5: deleterious, score > −2.5: neutral). We found the variants in the ACE2 genes that have strong criteria for pathogenesis, i.e., described as a pathogen variant in at least 3 tools. Nomenclature for variants was also confirmed according to the recommendations of the Human Genetic Variation Society (HGVS) (https://varnomen.hgvs.org/). We found the potentially deleterious variants in the ACE2 gene based on the 2015 American College of Medical Genetics and Genomics (ACMG) guidelines for the interpretation of sequence variants [Reference Richards, Aziz and Bale5].
3. Results
3.1 Genetic Analysis of hACE2
The 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 [Reference Lippi, Lavie, Henry and Sanchis-Gomar49]. 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 different, the minor allele frequency (MAF) of any databases is different. Indeed, we used this information to identify variants with MAF below some specified 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 scientific 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 different from the other. Our results revealed 299 variants in the ACE2 gene. A list of the identified variants in the ACE2 gene is summarized in Table 1. The majority of the ACE2 gene variants have yet to be identified functionally. To obtain information about the possibility of the deleterious effects of the identified variants, we evaluated the variants using the in-silico prediction of their functional effects. Ultimately, we identified the most deleterious variants in the ACE2 gene based on prediction tools (Figure 3, Table 2).
Table 1 Genetic variations in ACE2 gene (NM_021804.2).
The table reports the genomic position, the nucleotide, and amino acid change of identified variants in the ACE2 gene. These data are based on the Genome Reference Consortium Human Build 37 (GRCh37). 1CADD, Phred ≤20: neutral; Phred >20: damaging; 2SIFT, score ≤0.05: deleterious; score >0.05: tolerable; 3polyphen-2, score = 0–0.15: benign; score = 0.15–0.85: possibly damaging; score = 0.85–1: probably damaging; 4PROVEAN, 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.
Figure 3 The most pathogenic variants of the ACE2 gene are displayed by arrows.
Table 2 The most pathogenic variants of the ACE2 gene.
The table reports the genomic position, the nucleotide, and amino acid change of the most pathogenic variants in the ACE2 gene. 1CADD, Phred ≤20: neutral; Phred >20: damaging; 2SIFT, score ≤0.05: deleterious; score >0.05: tolerable; 3polyphen-2, score = 0–0.15: benign; score = 0.15–0.85: possibly damaging; score = 0.85–1: probably damaging; 4PROVEAN, 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.
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 [Reference Cao, Li and Feng50]. Twenty-five variants located within the ACE2 gene were collected and cataloged in the Leiden Open Variation Database [Reference Lovd14]. 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 [Reference Fujikura and Uesaka51]. 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. Specifically, 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 [Reference Hou, Zhao and Martin40]. The variants p.Met383Thr, 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 [Reference Hou, Zhao and Martin40]. According to a previous study, several ACE2 variants and alterations in amino acid residues in ACE2 could affect the association between the ACE2 receptor and the S protein in SARS-CoV, leading to the conversion of ACE2 into an efficient/inefficient receptor [Reference Li, Zhang and Sui17]. Fujikura and Uesaka identified 8 SNVs—namely p.Ser19Pro, p.Thr27Ala, 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 [Reference Fujikura and Uesaka51]. Residues Arg708/710/716, located in the dimeric interface of the ACE2 receptor, are a vital component for cleavage by TMPRSS2. This process is required to strengthen the entry of the virus into the host cells [Reference Heurich, Hofmann-Winkler, Gierer, Liepold, Jahn and Pöhlmann29]. 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 [Reference Hou, Zhao and Martin40]. Several variants, including p.Met383Thr, 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 [Reference Li, Zhang and Sui17]. There 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 affinity of the S protein of SARS-CoV-2 and were, thus, considered protective variants [Reference Stawiski, Diwanji and Suryamohan52]. The variants rs73635825 and rs143936283 present a relatively low binding affinity for the S protein of SARS-CoV-2, which may be associated with potential resistance to infection [Reference Lippi, Lavie, Henry and Sanchis-Gomar49]. Information regarding these variants is not available in Iranome. Three variants—namely, p.Lys26Arg, p.Gly211Arg, and p.Asn720Asp—were more frequently expressed in the Italian population than in the Eastern Asian population. These variants are close to the sequence essential for the binding of the S protein of SARS-CoV-2. The presence of these variants may explain the high mortality rate in Italy compared with China [Reference Lippi, Lavie, Henry and Sanchis-Gomar49, Reference Lippi, Mattiuzzi, Sanchis-Gomar and Henry53]. ACE2 gene mutation naming Leu584Ala facilitates the SARS-CoV entry into target cells [Reference Xiao, Zimpelmann, Agaybi, Gurley, Puente and Burns54]. 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 different populations [Reference Cao, Li and Feng50]. 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 [Reference Benetti, Tita and Spiga4]. Based on the findings of the present study, differential variants in the ACE2 gene may clarify various susceptibility and outcomes in different ethnic groups.
4. Discussion
The ACE2 receptor acts as an entry point for the coronavirus [Reference Li, Moore and Vasilieva55]. 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 [Reference Walls, Park, Tortorici, Wall, McGuire and Veesler56]. Certain genomic variants within the ACE2 gene that modulate its function or expression cause variable susceptibility to SARS-CoV-2 infection [Reference Srivastava, Bandopadhyay and Das20]. Given the possible connection between circulating ACE2 levels and COVID-19 severity, recombinant ACE2 may be a promising treatment option [Reference Ciaglia, Vecchione and Puca57]. As a result, tissue-specific ACE2 expression or plasma ACE2 levels are considered 2 important factors in the severity of COVID-19. The effects 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 [Reference Williams, Mancia and Spiering58, Reference Batlle, Wysocki and Satchell59]. According to a meta-analysis, ACE-I/ARBs reduced the risk of pneumonia and its mortality [Reference Caldeira, Alarcão, Vaz-Carneiro and Costa60]. The rs2285666 polymorphism may be a predisposing factor for the comorbidities observed in patients with COVID-19 [Reference Chaoxin, Daili, Yanxin, Ruwei, Chenlong and Yaobin61, Reference Asselta, Paraboschi and Mantovani62]. The population-based frequency of this single-nucleotide polymorphism (SNP) is significantly higher among the Indian population (∼0.6) than among Europeans (0.2) and East Asians (0.55) [Reference Srivastava, Pandey and Singh21, Reference Cao, Li and Feng50, Reference Asselta, Paraboschi and Mantovani62]. In our study, among the Iranian population, we identified a frequency of 0.2575 for this SNP. The results of another study conducted by Srivastava et al. indicated that the frequency of a synonymous coding region variant, rs35803318, was high 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 identified variants or the cumulative effect of a few of them cause different susceptibility to the entry of viral cells and have a significant effect on the onset and progression of the disease. Therefore, systematic identification of the genetic determinants of COVID-19 susceptibility and the clinical outcome could further explain the current epidemiologic observations, disease pathophysiology, different susceptibilities, and disease severities in different ethnic groups.
In the present study, we conduct a comprehensive systematic investigation on genetic variations in the human genes associated with the coronavirus. The 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). The ACE2 gene variants may be associated with COVID-19 genetic susceptibility which could guide more personalized and individualized treatments for the COVID-19 pandemic [Reference Hou, Zhao and Martin40]. Since ACE2 gene variants may cause different responses to COVID-19 treatments concerning the components of the RAS system, we recommend case-control studies to investigate the effects 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 [Reference Zheng and Cao9]. 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 population-appropriate vaccines against SARS-CoV-2 infection.
This study aimed to search for the most deleterious variants associated with COVID-19, and the pathogenesis of the identified 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 five 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). The 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. The S protein of SARS-CoV-2 is identified 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 five deleterious variants mentioned in this study caused a disturbance in the structure of the ACE2 receptor, which may be effective in the incidence of this disease. The c.1129G > T variant in the ACE2 gene caused the Gly377Gln substitution within the extracellular domain of the receptor. This residue is located in the zinc-binding site (positions 374–378) that is involved in binding. The 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. The initial attachment of the S protein to the receptor has caused the exposure of the most important amino acids for binding (residues 22–57). The main functional domains of the ACE2 receptor that interact with SARS-CoV-2 are illustrated in Figure 2(c). The 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.
According to this study, the five deleterious variants in the ACE2 gene may clarify various susceptibility and outcomes in different ethnic groups. These 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. Therefore, we recommend further research to identify the effect of the most pathogenic variants on the binding affinity. Also, the identified pathogenic variants in the ACE2 gene may affect the clinical efficacy of drugs for COVID-19, which is better investigated. We suggest that the frequency of these deleterious variants in different populations is investigated in the future so that the necessary preparations for the disease are considered in populations carrying these variants.
The tissue-specific ACE2 expression and plasma ACE2 levels, and density of ACE2 receptors are key factors of the difference in the severity and incidence of the disease in various countries. Also, the levels of ACE2 expression vary in different populations and various human tissues (Figure 1(b)). SNPs affect 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 different populations that could promote personalized treatment strategies and precision medicine for COVID-19. Such studies may affect accurate medical interventions and the design of specific diagnostic and therapeutic methods for coronavirus. The present study can be useful for better understanding interindividual clinical variability, and the severity and susceptibility of this disease in different ethnic groups.
The mechanisms resulting from the functional foods-based treatments included the reduced expression of ACE2 receptors in cells, inhibiting necessary enzymes in SARS-CoV-2, and decreased proinflammatory cytokines that can help the body fight during illness [Reference Farzana, Shahriar and Jeba63]. The mentioned variants that modulate the ACE2 function and expression cause variable susceptibility to SARS-CoV-2 infections. It seems to be beneficial for patients carrying these variants to use the functional foods-based treatments that lead to the reduced expression of ACE2 receptors in the cells. Therefore, we recommend further research to identify the effect of the most pathogenic variants in different populations on the ACE2 tissue expression, plasma ACE2 levels, and binding affinity, 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 influenced by the ACE2 variants. The spike protein mutations increased the viral attachment and subsequent entry into host cells. The structural target for available drugs and treatments is the high binding affinity of the spike protein and the receptor. It appears that some of the identified variants and their cumulative effects of them cause different susceptibility to the entry of viral cells and have a significant effect on the used therapeutics and vaccination effectiveness. 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. Therefore, 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 significant 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. The evaluation of pathogenic variants in the ACE2 gene in male and female genders and different populations with the appropriate therapeutic strategies can be effective to prevent infections among populations at risk of SARS-CoV-2 infections resulting from possible viral variants.
5. Conclusions
The detection of SNP genotypes is urgently needed to discover likely genetic risk factors for severe outcomes. The identification of variants may have a significant 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.
Data Availability
All the data generated or analyzed during this study are included in this published article. The datasets generated and/or analyzed during the current study are available in the HGMD (https://www.hgmd.cf.ac.uk/ac/index.php), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), and Google Scholar (https://scholar.google.com/).
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
We appreciate the support from the Cardiogenetic Research Center, Rajaie Cardiovascular Medical and Research Center, Iran University of Medical Sciences, Tehran, Iran.
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