Skip to main content

Angiotensin-converting enzyme 2 (ACE2) polymorphisms and susceptibility of severe SARS-CoV-2 in a subset of Pakistani population


Science is digging for the varied presentation of COVID-19 patients exposed to the same risk factors, and medical conditions may be influenced by the presence of polymorphic genetic variants. This study investigated the link between ACE2 gene polymorphisms and the severity of SARS-CoV-2. This cross-sectional study recruited COVID-19 PCR-positive patients by consecutive sampling from Ziauddin Hospital from April to September 2020. DNA was extracted from whole blood, followed by gene amplification and Sanger’s sequencing. Most of the patients, 77: 53.8%, were serious. Males were higher (80; 55.9%) with age more than 50 years (106: 74.1%). We found 22 ACE2 SNPs. rs2285666 SNP was most prevalent with 49.2% CC, 45.2% TT, 4.8% CT heterozygosity, and 0.8% AA genotypes. Variants with multiple genotypes were also insignificantly associated with the severity of COVID-19 in the analysis of the dominant model. Only rs2285666 had a significant statistical link with gender (p-value 0.034, OR; 1.438, CI; 1.028–2.011) while rs768883316 with age groups (p-value 0.026, OR; 1.953, CI; 1.085–3.514). Haplotypes ATC of three polymorphisms (rs560997634, rs201159862, and rs751170930) commonly found in 120 (69.77%) and TTTGTAGTTAGTA haplotype consisting of 13 polymorphisms (rs756737634, rs146991645, rs1601703288, rs1927830489, rs1927831624, rs764947941, rs752242172, rs73195521, rs781378335, rs756597390, rs780478736, rs148006212, rs768583671) in 112 (90.32%) had statistically significant association with the severity having p = value 0.029 and 0.001 respectively. Males of old age and diabetics are found to have more severe COVID-19 infection in the current study. We also found that common ACE2 polymorphism rs2285666 influences the susceptibility of acquiring the severe SARS-CoV-2 infection.


The SARS-CoV-2 virus infects some patients severely and fatally, primarily, but not solely, older people with serious underlying medical conditions [1]. One of the most reliable indicators of mortality is hypertension. Angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers may help the first stage of viral infection because SARS-CoV-2 needs the ACE2 protein to permeate the cell membrane and because hypertension people have a higher incidence of sequelae [2]. However, possibly the inflammatory stage of the disease would benefit from these same medications [3].

Blocking ACE2 has been proposed by several authors as a viable method to lower the viral SARS-CoV-2 burden in the pneumocytes and stop the virus from spreading to other organs [4]. In contrast, blocking ACE2 in COVID-19 individuals who are already infected could be harmful because it would lead to less angiotensin 1–7 synthesis, which has been shown to have anti-inflammatory and antifibrotic properties through its receptor [5]. Humans infected with the SARS-Cov-2 virus develop a very varied illness with unpredictably high mortality rates. Some people experience no symptoms at all, while others experience distress, microvascular thrombosis, multi-organ failure, and death as a result of a chain of infections and inflammatory processes. Although prognostic indicators have been established, there is still a lot of unexplained variation [6].

It is likely that changes in the activity of receptor-specific proteins, influenced by the presence of many polymorphic genetic variants in the population, contribute to increased susceptibility to infection or enhanced viral replication efficiency. The existence of multiple polymorphisms in the ACE (I/D), ACE2 (rs2074192, rs1978124, rs2074809, rs2074666) genes could explain both the tendency to infection, the expansion to different organs, and the severity of COVID-19 clinical manifestations [7]. Therefore, we investigated the scientific links between ACE2 gene polymorphisms and the severity of COVID-19.


Patient recruitment and specimen collection

This cross-sectional study was conducted following the approval of the research and ethics review committees of Ziauddin University (Reference code: 2650920SKPAT) as per institutional guidelines. A total of 143 PCR-positive patients presented to COVID-19 outpatient and inpatient departments and the intensive care unit (ICU) at Ziauddin Hospital Clifton, KDLB, and North Nazimabad sites from April 2020 to September 2020, using the non-probability consecutive sampling technique were included. Written informed consent was obtained from the patients or from the guardian of each participant under 18 years of age. Patients under 14 years of age, mentally challenged, patients, post chemotherapy and radiotherapy, and patients with any malignant condition were excluded.

SARS-CoV-2 RNA positivity was determined using qualitative RT-PCR with in vitro diagnostic kits, following the manufacturer’s protocol. The electronic patient records provided demographics, clinical features, and laboratory results. The age, gender, medical history including early symptoms like fever, cough, dyspnea, and outcome of each patient were recorded. According to the CDC’s standard grades of severity were defined as ‘Asymptomatic’ with no signs or symptoms; ‘Mild’ referred to patients with no dyspnea; whether outpatient or inpatients; ‘Moderate’ referred to in-patients admitted to a ward or ICU who showed the signs of dyspnea but did not need oxygen; ‘Severe’ labeled as hospitalized patients who required high flow oxygen; ‘Critical’ considered all patients who required mechanical ventilation or all COVID-19-related deaths that occurred during the hospital stay or both [8]. To assess statistical risk, the study subjects were divided into two categories: serious and non-serious patients. The non-serious group included asymptomatic, mild, and moderate cases, while the serious group consisted of all severe and critical patients who were admitted to the hospital, whether inwards or in the ICU.

DNA extraction, quantification

The DNA was extracted from the entire blood using the Qiagen Kit (QIAamp DNA mini blood Kit, Cat. No. 51306) in accordance with the manufacturer’s guidelines. The concentration of each DNA sample was verified using a Multiskan Sky spectrophotometer, and purity was determined by calculating the A260/280 ratio. Gel electrophoresis was performed to assess DNA integrity.

The NCBI’s Thousand Genome Database offers over 1700 ACE2 gene polymorphism variants across diverse populations worldwide. As of August 2020, 124 of these variants have been observed in the East Asian population, including Pakistan, which shares a similar genetic makeup to our population. Notably, six frequent variants (rs201159862, rs1601703288, rs781378335, rs752242172, rs2285666, and rs768883316) have been identified [9].

Polymerase chain reaction (PCR)

The primers were designed against the more frequent variants correlated with the COVID-19 in East Asian population. Primers for ACE2 were obtained commercially (Macrogen, peniconpk, 25 nmol), forward primer, 5’-TCATGTCCTTGCCCTTATAGTTCC-3’ and reverse primer 5’-CTATACTACCGCATCACTTTTTGGT-3’. The PCR cycling was performed as initial denaturation at 94ºC for 5 min followed by 35 cycles of 1 min at 94ºC, 1 min at 58.5ºC and 1 min at 72ºC with a final extension of 5 min at 72ºC. (SimpliAmp, Thermal Cycler, applied biosystems, Thermo Fisher Scientific, Ref: A24812, SN: 228,007,070) Amplification products were observed using submerged 2% agarose gel electrophoresis. (Supplementary Fig. 1)

Sanger’s sequencing and bioinformatic analysis

Sanger’s sequencing was used to analyze all the ACE2 products (Size 800 bp). However, 19 samples had missed reads and noise and were subsequently filtered out of the analysis. This left 126 samples for genetic association and statistical analysis. The sequencing procedure was outsourced and performed with Lab Genetix in Lahore. MEGA X software was used for sequence alignment and trimming. The datasets generated and analyzed during the study are available at NCBI, GenBank repository, Submission# BankIt2673536 ZU15 OQ443069,

Statistical evaluation

SPSS version 21 was used for all statistical evaluations conducted on COVID-19. Categorical variables were subjected to frequency calculations, while quantitative variables were analyzed using percentage calculations. Fisher’s exact/Chi-square testing was employed to determine the correlation between COVID-19 severity and clinicopathological features. The Hardy-Weinberg equilibrium (HWE) for genotype distributions was computed using the chi-square (χ2) test in Haploview. Regression analysis was utilized to calculate the odds ratio (OR) at a 95% confidence interval (CI) to assess the association between genotype and allele frequencies, as well as other variables, and the likelihood of COVID-19 severity. A P-value less than 0.05 was considered significant for all estimates, which had a two-sided distribution.


Demographic and clinical characteristics of the patients

Out of all patients who tested positive for PCR, 77 of them (53.8%) had serious cases while 66 (46.2%) had non-serious cases. More than half of the patients were males (80, 55.9%) and were over 50 years old (106, 74.1%) when they first presented with symptoms. The majority of patients had diabetes mellitus (71, 49.7%) and hypertension (83, 58%), while some also had other known diseases such as cardiovascular disease (22, 15.4%) or respiratory disease (7, 4.9%). It should be noted that cardiovascular disease in this context refers to ischemic heart disease, coronary artery disease, and valvular disease, but not arterial diseases or hypertension. Endocrine diseases do not include diabetes mellitus. Fever (90, 62.9%) was the most common symptom reported by patients, followed by dyspnea (87, 60.8%). Almost all patients had raised levels of inflammatory markers, with procalcitonin (139, 97.2%) being the most frequently elevated marker.

After analyzing demographic and clinical characteristics in relation to COVID-19 severity, we found a significant statistical association between several factors. Fever (p-value, 0.001), cough (p-value, 0.004), dyspnea (p-value, 0.001), loss of taste (p-value, 0.043), de-dimer (p-value, < 0.001), LDH (p-value, < 0.001), CRP (p-value, < 0.001), and outcome (p-value, < 0.001) all showed significant correlations. Fever, cough, and dyspnea were present in all cases ranging from mild to critical severity, while loss of taste and smell were more common in mild to moderate cases. Please refer to Table 1 for more information.

Table 1 Statistical association of demographic and clinical characteristics with severity of COVID-19: Fever, cough, dyspnea, loss of taste, elevated d-dimer, LDH, and CRP levels were all significantly associated with the severity of the disease. Fever, cough, and dyspnea were commonly present in all severity levels, while loss of taste and smell were more frequently associated with mild and moderate cases

Association of ACE2 variants and the severity of the disease

In our recent survey, we discovered 29 ACE2 variants within a specific range of base pairs (position: 15,592,100 to 15,592,699). Out of these, 7 variants had no changes in their sequences while the remaining 22 ACE2 variants had altered genotypes. Among these, 15 variants were intronic, 4 were splice donor region variants, 2 were missense, and one was a synonymous variant. Additionally, two variants had insertion/deletion and one had insertion. Due to the low number of minor homozygotes (MAF < 0.1, Table: 2), we only analyzed these polymorphisms using a dominant model. We used these associations for further analyses and considered the linkage disequilibrium between gene polymorphisms (Figures: 1 and 2). To control the expected proportion of false positives (False Discovery Rate [FDR]) instead of the more stringent Bonferroni correction, we conducted an exploratory selection of associations. Therefore, we used associations with FDR < 0.1 in haplotype analyses. Firstly, we generated haplotype blocks using the algorithm of four-gamete rules observed at a frequency > 0.01. Then, we tested if the observed frequencies of haplotypes deviated from expected under linkage equilibrium for each block. Finally, we assessed the association between haplotypes and phenotypes using a permutation procedure (Supplementary Table: 1).

Table 2 Minor allele frequency (MAF) and results of exact test to assess deviations from Hardy-Weinberg equilibrium (HWE)
Fig. 1
figure 1

Haploview result belonging to block 1 of ACE2 polymorphisms, contains (rs560997634, rs201159862, and rs751170930) and block 2 that contains (rs756737634, rs146991645, rs1601703288, rs1927830489, rs1927821624, rs764947491, rs752242172, rs73195521, rs781378335, rs756597390, rs780478736, rs148006212 and rs768583671) according to genotyping data of this study. The red color means 100% of linkage disequilibrium (D’ = 1). Boxes number referred to linkage disequilibrium (D’) between SNPs, boxes with no number mean 100% linkage (D’ = 1). Color legend: i) Bright red = high D’; White = low D’; iii) Purple = High D’ but low LOD score (see Haploview documentation for further details;

Fig. 2
figure 2

Haplotypes and haplotype frequencies showing association among each other: The bold lines display a favorable correlation between the ATC haplotypes observed in 120 patients, out of which 66 were severely affected, and the presence of TTTGTAGTTAGTA and TTTGTAGTTAATA haplotypes in 112 patients, out of which 64 were also severely affected. It is important to note that the impact of these haplotypes varies across different populations, depending on environmental factors

In COVID-19-positive patients, the most common variant observed was rs2285666. Out of these patients, 49.2% had the CC genotype, 45.2% had the TT genotype, 4.8% had the CT heterozygosity, and 0.8% were genotypic carriers of AA. The second most frequent SNP was rs1391451327, with 93.5% of patients having the AA genotype, 5.7% having the TT genotype, and 0.8% having the GG genotype. Another variant, rs781378335, had a genotypic frequency of 97.6% TT and 2.4% CC. Although these SNPs do not have a significant association with the severity of the disease, changes were observed in more severe and critical patients in the ICU. This suggests that these variants may increase susceptibility to acquiring severe or critical COVID-19 infections. Some genotypic variants of ACE2 were also found to be associated with mild and moderate cases. However, variants with multiple genotyping were not found to be associated with COVID-19 severity in the analysis of the dominant model. Please refer to Tables 3 and 4 for more information.

Table 3 Genetic association study of ACE2 variants with severity of COVID-19. rs2285666 was a commonly altered variant (63 patients) followed by rs139141327 (8 patients). None of the variants had a statistically significant association with the severity of COVID-19 though the variants were found in the serious groups. bBenjamini-Hockberg method. CI, confidence Interval; FDR, false discovery rate; NA, does not apply; OR, odds ratio
Table 4 Dominant model analyses for ACE2 genetic variants with multiple genotypes. Six variants listed have the multiple altered genotyping that had insignificant statistical association with the severity of COVID-19

Additionally, we aimed to determine the correlation between all 22 ACE2 variants and various factors including ethnicity, age groups, comorbidities, and gender in our current study. However, none of the variants displayed any statistical significance when applying the regression model with ethnicity, except for rs2285666, which showed significant statistical linkage with gender (p-value 0.034), and rs768883316, which had a significant statistical association with age groups (p-value 0.026). This information can be found in Tables 5 and 6.

Table 5 Genotype distribution in COVID-19 severity groups disaggregated by age group. rs76888331 was found to have positive statistical association with the age having the 95 patients with age > 50 years, serious (53 patients) with TT genotyping (53 patients)
Table 6 Genotype distribution in COVID-19 severity groups disaggregated by age gender. rs2285666 had the positive association with gender with male dominance (69 patients), having the CC genotyping (21 patients)

Haplotypes ATC of three polymorphisms (rs560997634, rs201159862 and rs751170930) commonly found in 120 (69.77%) patients including the 54; 43.54% non-serious and 66; 53.22% serious and TTTGTAGTTAGTA haplotype consisting of 13 polymorphisms (rs756737634, rs146991645, rs1601703288, rs1927830489, rs1927831624, rs764947941, rs752242172, rs73195521, rs781378335, rs756597390, rs780478736, rs148006212, rs768583671) in 112 (90.32%) with non-serious 48; 38.7%, serious 64; 51.61% had significance statistical association with the severity having p = value 0.029 and 0.001 respectively. Table: 7.

Table 7 Frequency of ACE2 Haplotypes distribution in COVID-19 patients: ATC and TTTGTAGTTAGTA were most common haplotypes found. ATC haplotypes found in 120 patients having 66 serious patients while TTTGTAGTTAGTA found in 112 patients with 64 serious patients


The Coronavirus is a diverse group of viruses that can lead to common colds, as well as severe respiratory illnesses such as pneumonia, and even conditions like SARS and Middle East Respiratory Syndrome (MERS) [10]. There has been limited publication on prognostic variables for COVID-19. Additionally, the investigation has noted a predominance of males, potentially attributable to the localization of the ACE2 gene on the X-chromosome [11]. The prevalent hypothesis suggests that COVID-19 infection rates are higher among males compared to females. Recent research, conducted through a case-control study on a Chinese demographic, reinforces this theory. This study discovered an inverse correlation between ACE2 expression levels and estrogen levels, suggesting that estrogen may contribute to the suppression of ACE2 expression. This indicates that females may possess a protective factor against COVID-19 infection compared to their male counterparts, possibly due to the influence of estrogen on ACE2 expression [12].

The analysis shows that COVID-19 tends to be more severe among individuals over 50 years old. This age group was found to have a higher incidence of severe cases in the study [13]. However, it is currently unknown how many ACE2 receptors are present in the organs of elderly individuals who are at a higher risk of severe illness. Additionally, the immune system tends to weaken with age, and comorbidities like diabetes, hypertension, and others can further impact immune system deterioration, increasing the risk of contracting COVID-19 infection [14]. ACE inhibitors and ARBs are used to treat hypertension, which causes ACE2 to be unregulated. These findings suggest that ACE2 expression is elevated in diabetes, and that therapy with ACE inhibitors and ARBs improves ACE2 expression [15]. COVID-19 infection would be aided by increased ACE2 expression. Diabetes, cerebral stroke, and hypertension have all been linked to ACE2 polymorphisms, particularly in Asian populations. An individual’s sensitivity may be affected by a combination of therapy and the ACE2 polymorphism [16].

The severity of COVID-19 disease may also vary geographically, as rural hospitals and communities sometimes lack services. Ethnicity is influenced by genetic backgrounds, environmental factors, as well as cultural and behavioral norms [17]. Therefore, it is conceivable that these societal factors could influence the severity of COVID-19 disease in populations of people of color [18]. Although it was highlighted that there was conflicting information about the link between ethnicity and mortality, there was consistent evidence that ethnic minorities had higher infection rates. Different people have different cellular amounts of ACE2 expression [19].

SARS-CoV-2 susceptibility and COVID-19 illness outcome could all be influenced by ACE2 gene polymorphism, human ACE2 mRNA expression, and human ACE2 protein polymorphism [20]. Human ACE2 has been discovered as a host cell receptor responsible for mediating coronavirus infection in studies on host-pathogen interaction (COVID-19). Srivastava et al., in his report found a significant difference in alleles between Europeans and Asians for the ACE2 polymorphism rs2285666. The alternative allele (TT-plus strand or AA-minus strand) of rs2285666 has been found to increase the expression level of this gene by up to 50%, suggesting that it may have a role in SARS-CoV-2 susceptibility [21]. Because rs2285666 has been linked to hypertension, type 2 diabetes, and coronary artery disease, it could be a predisposing factor for the comorbidities seen in COVID-19 individuals. The variant rs2285666 is found near the start of intron 3, and it could potentially affect gene expression and protein levels by alternative splicing mechanisms [22]. Some genetic variants in the ACE2 can bring about variations in binding affinity of ACE-2 for SARS COV-2 RBD. rs2285666 is one of these SNPs whose wild type enhances ACE2 production with a greater affinity for SARS-CoV-2. Wooster et al. identified that six SNPs in the ACE2 gene region that increase the expression level of ACE2 receptors are significantly associated with a higher risk of hospitalization in patients with COVID-19 [23].

Based on the findings of our current study, we discovered that the common SNPs associated with rs2285666 in COVID-19-positive patients were as follows: 55.9% CC, 39.2% TT, 4.2% CT heterozygosity, and 0.7% AA genotypic carrier. These findings align with the percentages projected for the Pakistani population (PJL: Punjabi Lahore) database, which are 59.7% for CC, 40.3% for TT, and 2.7% for CT heterozygosity. We observed CT heterozygosity in six patients, including one asymptomatic, three mild, one severe, and one critical patient. These results are also supported by the NCBI thousand genome project.

This study also demonstrated that the ACE2 rs2285666 CC genotype had increased risk to develop the COVID-19 disease as compared to ACE2 rs2285666 TT genotype [24]. In a study evaluating the association between rs2285666 genotypes and circulating ACE2 in DM patients, it was discovered that the AA genotype has the highest level of expression compared to the other genotypes [25]. The wild genotype and the C allele was substantially related with the prevalence and risk of SARS-CoV-2 infection in our investigation, comparable to the findings in Indian and Caucasian populations [24]. As a result, it is hypothesized that these functional ACE2 variations could influence disease progression. The A-allele frequency was much greater in patients in our study, although it was linked to COVID-19. Given that ACE2 receptor gene expression may influence an individual’s susceptibility to infection, we hypothesize those genetic variations in the noncoding regions of the ACE2 receptor gene or other noncoding DNAs that regulate ACE gene expression levels may play a role in the severity of the disease [26].

A German study showed that G allele and GG genotype of rs2285666 were linked to COVID-19 vulnerability, especially in critically ill individuals. That demonstrated the susceptibility to COVID-19, particularly in seriously ill patients [27]. This observation was also advocated by other studies, the GG genotype of the ACE2 rs2285666 (G8790A) polymorphism was previously linked to a 50% reduction in protein production when compared to the AA genotype [28]. As shown, the correlation of the rs2285666 polymorphisms with COVID-19 susceptibility varies between studies around the world, which could be due to ethnic differences in populations, as these variants show some population-based differences. Given that ACE2 rs2285666 has been linked to hypertension, this polymorphism may influence susceptibility to SARS-CoV-2 infection and the severity of COVID-19. Reduced ACE2 protein levels and the loss of the protective impact of the ACE2/MAS pathway both contributed to the severe effects of SARS-CoV-2 infection, supporting this hypothesis [28]. Following up on the previous point, Möhlendick et al. found that the ACE2 rs2285666 polymorphism caused function loss in 297 COVID-19 positive people. They also discovered that the ACE2 rs2285666 GG genotype or G allele was linked to a two-fold greater risk of infection and a three-fold increased risk of severe disease or fatality [28]. Celik et al., on the other hand, found no link between this polymorphism and the severity of COVID-19. This could be related to epigenetic mechanisms that control ACE2 receptor expression, as well as alterations in other genes such as pro-inflammatory cytokines and coagulation indicators, all of which can affect a patient’s prognosis [29]. ATC and TTTGTAGTTAGTA haplotypes had more association with the risk of COVID-19 than other haplotypes. These haplotypes were profound in serious patients with severe and critical cases. Currently we did not find any study that highlighted the ACE2 haplotypes to compare our results.

In this study, the haplotypes ATC and TTTGTAGTTAGTA were analyzed. The former is composed of three polymorphisms (rs560997634, rs201159862, and rs751170930), and the latter is composed of 13 polymorphisms (rs756737634-rs768583671). The results showed that these haplotypes had a statistically significant association with the severity of the condition, with p-values of 0.029 and 0.001, respectively. Most of the affected patients required intensive care, which may be attributed to the different haplotypes found in different populations, affecting the body’s immune system, expression levels, and protein structures in varied ways [30]. Each population has unique genetic makeup, influenced by environmental factors and risk exposure, which can result in variations in gene expression and mutations. The impact of mutations within a gene may differ depending on their location on the same chromosome, a haplotype, or opposite homologous chromosomes [31]. In haplotype studies, it is common to analyze multiple closely linked markers as they offer more valuable information compared to a single marker. This may be a significant factor in understanding the severity of COVID-19 in the current study. The immune response’s effectiveness in producing a specific antiviral immunity without damaging the host tissues depends on various environmental and genetic factors, which are also applicable to the present findings [32].

In the current study, it was observed that several patients with severe diseases possessed SNPs at the slicing donor site. These sites hold significant immune regulatory functions in the body. During the splicing process, the transcript eliminates intronic sequences, leaving behind only the coding sequences in mature mRNA [33]. It is crucial to avoid errors during the splicing process since they can result in improper intron removal and alter the open reading frame. Overall, the splicing process is a complex event that plays a vital role in protein synthesis. Any changes in this process can reduce the messenger RNA level, leading to a lack of protein and causing abnormal cellular metabolism or function. The SNPs discovered in this study may impact protein function, structure, expression levels, and binding affinities, leading to different clinical outcomes and presentations [11]. Furthermore, dysregulation and significantly differential expression of transcripts due to the SNPs at the splice donor site are linked to many viral diseases and COVID-19 severity [34].

To the best of our knowledge, based on a comprehensive review of existing literature, this study is the first of its kind to focus on the distinct ACE2 variants related to disease severity, specifically using human samples from a subset of the Pakistani population. While the single nucleotide polymorphisms (SNPs) identified in our study were associated with viral pathogenesis, no evidence was found linking these genetic variations to an increased susceptibility to COVID-19. This could potentially be due to limited evidence and data scarcity, indicating a need for further investigation with larger sample sizes. Apart from genetic predispositions, numerous factors like age, gender, ethnicity, and co-existing health conditions, are recognized to influence a population’s vulnerability to SARS-CoV-2 infection. However, it is crucial to exercise caution in the interpretation of our findings due to the nature of our study. Since it was small and selective, the results may not be generalizable to a larger population or to communities of diverse nationalities. For our hypotheses to gain broader acceptance, they must be substantiated through additional independent research.


A statistically significant predominance of males with severe cases of COVID-19 was observed, with the older age group being primarily affected. As disease severity increased, there was a concurrent sequential escalation in the levels of pertinent inflammatory markers. Certain ACE2 polymorphisms, particularly ACE2 rs781378335, and rs2285666, emerged as independent associations with the severity of COVID-19. Specifically, the ACE2 rs2768883316 polymorphism appears to correlate with a heightened risk of contracting COVID-19 in later life stages. Moreover, male susceptibility to severe manifestations of the disease was particularly associated with the ACE2 rs2285666 polymorphism.


  1. Team GAC-G, Landi F, Barillaro C, Bellieni A, Brandi V, Carfì A, et al. The new challenge of geriatrics: saving frail older people from the SARS-COV-2 pandemic infection. J Nutr Health Aging. 2020;24:466–70.

    Article  Google Scholar 

  2. Huang NX, Yuan Q, Fang F, Yan BP, Sanderson JE. Systematic review and meta-analysis of the clinical outcomes of ACEI/ARB in East-Asian patients with COVID-19. PLoS ONE. 2023;18(1):e0280280.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ragia G, Manolopoulos VG. Inhibition of SARS-CoV-2 entry through the ACE2/TMPRSS2 pathway: a promising approach for uncovering early COVID-19 drug therapies. Eur J Clin Pharmacol. 2020;76:1623–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Beeraka NM, Sadhu SP, Madhunapantula SV, Rao Pragada R, Svistunov AA, Nikolenko VN, et al. Strategies for targeting sars cov-2: small molecule inhibitors—the current status. Front Immunol. 2020;11:552925.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Latil M, Camelo S, Veillet S, Lafont R, Dilda PJ. Developing new drugs that activate the protective arm of the renin–angiotensin system as a potential treatment for respiratory failure in COVID-19 patients. Drug Discovery Today. 2021;26(5):1311–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Orooji Y, Sohrabi H, Hemmat N, Oroojalian F, Baradaran B, Mokhtarzadeh A, et al. An overview on SARS-CoV-2 (COVID-19) and other human coronaviruses and their detection capability via amplification assay, chemical sensing, biosensing, immunosensing, and clinical assays. Nano-micro Lett. 2021;13:1–30.

    Article  CAS  Google Scholar 

  7. Sabater Molina M, Nicolás Rocamora E, Bendicho AI, Vázquez EG, Zorio E, Rodriguez FD, et al. Polymorphisms in ACE, ACE2, AGTR1 genes and severity of COVID-19 disease. PLoS ONE. 2022;17(2):e0263140.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Covid C, Team R, Bialek S, Boundy E, Bowen V, Chow N. Severe outcomes among patients with coronavirus disease 2019 (COVID-19)—United States, February 12–March 16, 2020. Morb Mortal Wkly Rep. 2020;69(12):343.

    Article  Google Scholar 

  9. Lippi G, Lavie CJ, Henry BM, Sanchis-Gomar F. Do genetic polymorphisms in angiotensin converting enzyme 2 (ACE2) gene play a role in coronavirus disease 2019 (COVID-19)? Clinical Chemistry and Laboratory Medicine (CCLM). 2020;58(9):1415–22.

  10. McIntosh K, Perlman S. Coronaviruses, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). Mandell, douglas, and bennett’s principles and practice of infectious diseases. 2015:1928.

  11. Asselta R, Paraboschi EM, Mantovani A, Duga S. ACE2 and TMPRSS2 variants and expression as candidates to sex and country differences in COVID-19 severity in Italy. Aging. 2020;12(11):10087.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ciaglia E, Vecchione C, Puca AA. COVID-19 infection and circulating ACE2 levels: protective role in women and children. Front Pead. 2020;8:206.

    Article  Google Scholar 

  13. Liu Y, Mao B, Liang S, Yang J-W, Lu H-W, Chai Y-H et al. Association between age and clinical characteristics and outcomes of COVID-19. Eur Respir J. 2020;55(5).

  14. Cheng H, Wang Y, Wang GQ. Organ-protective effect of angiotensin‐converting enzyme 2 and its effect on the prognosis of COVID‐19. J Med Virol. 2020;92(7):726–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Parit R, Jayavel S. Association of ACE inhibitors and angiotensin type II blockers with ACE2 overexpression in COVID-19 comorbidities: a pathway-based analytical study. Eur J Pharmacol. 2021;896:173899.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Beyerstedt S, Casaro EB, Rangel ÉB. COVID-19: angiotensin-converting enzyme 2 (ACE2) expression and tissue susceptibility to SARS-CoV-2 infection. Eur J Clin Microbiol Infect Dis. 2021;40(5):905–19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Horowitz JE, Kosmicki JA, Damask A, Sharma D, Roberts GH, Justice AE, et al. Genome-wide analysis provides genetic evidence that ACE2 influences COVID-19 risk and yields risk scores associated with severe disease. Nat Genet. 2022;54(4):382–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Patel P, Hiam L, Sowemimo A, Devakumar D, McKee M. Ethnicity and covid-19. British Medical Journal Publishing Group; 2020.

  19. Al-Mulla F, Mohammad A, Al Madhoun A, Haddad D, Ali H, Eaaswarkhanth M, et al. ACE2 and FURIN variants are potential predictors of SARS-CoV-2 outcome: a time to implement precision medicine against COVID-19. Heliyon. 2021;7(2):e06133.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Calcagnile M, Forgez P, Iannelli A, Bucci C, Alifano M, Alifano P. ACE2 polymorphisms and individual susceptibility to SARS-CoV-2 infection: insights from an in silico study. BioRxiv. 2020.

  21. Srivastava A, Bandopadhyay A, Das D, Pandey RK, Singh V, Khanam N et al. Genetic association of ACE2 rs2285666 polymorphism with COVID-19 spatial distribution in India. Front Genet. 2020:1163.

  22. Gemmati D, Bramanti B, Serino ML, Secchiero P, Zauli G, Tisato V. COVID-19 and individual genetic susceptibility/receptivity: role of ACE1/ACE2 genes, immunity, inflammation and coagulation. Might the double X-chromosome in females be protective against SARS-CoV-2 compared to the single X-chromosome in males? Int J Mol Sci. 2020;21(10):3474.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wooster L, Nicholson CJ, Sigurslid HH, Lino Cardenas CL, Malhotra R. Polymorphisms in the ACE2 locus associate with severity of COVID-19 infection. MedRxiv. 2020:2020.06. 18.20135152.

  24. Alimoradi N, Sharqi M, Firouzabadi D, Sadeghi MM, Moezzi MI, Firouzabadi N. SNPs of ACE1 (rs4343) and ACE2 (rs2285666) genes are linked to SARS-CoV-2 infection but not with the severity of disease. Virol J. 2022;19(1):1–9.

    Article  Google Scholar 

  25. Bosso M, Thanaraj TA, Abu-Farha M, Alanbaei M, Abubaker J, Al-Mulla F. The two faces of ACE2: the role of ACE2 receptor and its polymorphisms in hypertension and COVID-19. Mol Therapy-Methods Clin Dev. 2020;18:321–7.

    Article  CAS  Google Scholar 

  26. Karakaş Çelik S, Çakmak Genç G, Pişkin N, Açikgöz B, Altinsoy B, Kurucu İşsiz B, et al. Polymorphisms of ACE (I/D) and ACE2 receptor gene (Rs2106809, Rs2285666) are not related to the clinical course of COVID-19: a case study. J Med Virol. 2021;93(10):5947–52.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Huang W, Yang W, Wang Y, Zhao Q, Gu D, Chen R. Association study of angiotensin-converting enzyme 2 gene (ACE2) polymorphisms and essential hypertension in northern Han Chinese. J Hum Hypertens. 2006;20(12):968–71.

    Article  CAS  PubMed  Google Scholar 

  28. Saengsiwaritt W, Jittikoon J, Chaikledkaew U, Udomsinprasert W. Genetic polymorphisms of ACE1, ACE2, and TMPRSS2 associated with COVID-19 severity: a systematic review with meta‐analysis. Rev Med Virol. 2022:e2323.

  29. Çelik SK, Genç G, Piskin N, Acikgoz B, Altınsoy B, İşsiz BK et al. ACE I/D and ACE2 receptor gene (RS2106809, RS2285666) polymorphisms is not related to the clinical course of COVID-19; a case study. Authorea Preprints. 2021.

  30. Choudhary S, Sreenivasulu K, Mitra P, Misra S, Sharma P. Role of genetic variants and gene expression in the susceptibility and severity of COVID-19. Annals of laboratory medicine. 2021;41(2):129–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Khoury MJ, Beaty TH, Cohen BH. Fundamentals of genetic epidemiology: Monographs in Epidemiology and; 1993.

  32. Yamamoto N, Nishida N, Yamamoto R, Gojobori T, Shimotohno K, Mizokami M, et al. Angiotensin–converting enzyme (ACE) 1 gene polymorphism and phenotypic expression of COVID-19 symptoms. Genes. 2021;12(10):1572.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Pouladi N, Abdolahi S. Investigating the ACE2 polymorphisms in COVID-19 susceptibility: an in silico analysis. Mol Genet Genom Med. 2021;9(6):e1672.

    CAS  Google Scholar 

  34. Huffman JE, Butler-Laporte G, Khan A, Pairo-Castineira E, Drivas TG, Peloso GM, et al. Multi-ancestry fine mapping implicates OAS1 splicing in risk of severe COVID-19. Nat Genet. 2022;54(2):125–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


The researchers would like to acknowledge all the patients who participated in the study. The publication of this article was funded by Qatar National Library.



Open Access funding provided by the Qatar National Library.

Author information

Authors and Affiliations



Dr. Santosh Kumar and Dr. Talat Mirza conceived the idea, designed, and supervised the project. Dr. Ambrina Khatoon did the bioinformatics analysis and bench work along with Dr. Rizma Khan. Dr. Fouzia Shaikh co-supervised the project, did the proofreading and editing. Dr. Santosh Kumar conducted bench work and wrote the manuscript with statistical analysis. Dr. Omer Ahmed Shaikh and Abdulqadir Nashwan co-wrote the manuscript with statistical analysis, along with doing the proofreading and editing.

Corresponding authors

Correspondence to Ambrina Khatoon or Abdulqadir J. Nashwan.

Ethics declarations

Conflict of interest

The authors have no known conflicts of interest associated with the study.

Ethical approval

was taken from the institutional approval from Ethical review committee dated November 5th, 2020. (Reference code: 2650920SKPAT).

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Figure: 1

(a): Gel doc for ACE2 demonstrating the bands for PCR products.

Supplementary Table 1

: ACE2 haplotypes, haplotype frequency and permutation test for all haplotypes organized in block 1 and block 2

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sidhwani, S.K., Mirza, T., Khatoon, A. et al. Angiotensin-converting enzyme 2 (ACE2) polymorphisms and susceptibility of severe SARS-CoV-2 in a subset of Pakistani population. Virol J 20, 120 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: