The clinical presentation of COVID-19 disease is highly variable, ranging from asymptomatic infection to severe form of pneumonia and respiratory or multi-organ failure. This is intriguing that genetic variability may influence the susceptibility to SARS-CoV-2 infection and its severity. The relevance of germline ACE2 variants as a screening target for the risk assessment of SARS-CoV-2 infection is of interest. Most of such studies have investigated the association between ACE2 variants and SARS-CoV-2 infection or COVID-19 disease severity using bioinformatics approaches while utilizing the frequencies of ACE2 genetic variants from public databases [10, 14, 24–28]. Only two European studies from Germany [17] and Spain [29] investigated the contribution of ACE2 variants to SARS-CoV-2 susceptibility among SARS-CoV-2-positive and SARS-CoV-2-negative individuals. To our knowledge, this is the first Asian study to screen ACE2 germline variants in the peptidase domain in the Pakistani population.
No germline variant was detected in any of the 20 ACE2 residues, previously inferred for binding of SARS-CoV-2 spike protein. This is consistent with previous reports [11], 16, 18, 20, 29, 30]. Variants in most of these residues are absent or present with a low frequency (MAF < 0.0006) in the general population (gnomAD database). These findings highlight that coding variants in the ACE2 peptidase domain are very rare and may not confer an increased susceptibility or resistance to SARS-CoV-2 infection.
In the present study, only one ACE2 missense variant c.77A > G (p.Lys26Arg) was identified with a frequency of 0.44% (1/225) in SARS-CoV-2-positive and 1.38% (3/217) in SARS-CoV-2-negative Pakistani individuals. The allele frequency of c.77A > G among SARS-CoV-2-positive (MAF = 0.3%) and SARS-CoV-2-negative (MAF = 0.9%) individuals was not significantly different (P = 0.624). In consistent with our results, this variant has been reported in a German population with a frequency of 0.34% (1/297) in SARS-CoV-2-positive and 1.18% (3/253) in SARS-CoV-2-negative individuals [17]. Recently, a high prevalence of this variant (10/164; 6.1%) was reported of in SARS-CoV-2-positive patients from Serbia [15]. However, it was not detected in SARS-CoV-2-positive patients from Italy [11, 18], Spain [29], and Russia [20]. This variant has been reported in gnomAD with MAF of 0.388% and 0.131% in the general population and South Asians, respectively. In our study the solitary SARS-CoV-2-positive patient carrying p.Lys26Arg variant showed mild COVID-19 disease symptoms in agreement with previous reports from Serbia [31] and Germany [17]. Collectively, these findings suggest that the p.Lys26Arg variant may neither be associated with increased SARS-CoV-2 infection nor depict a protective effect.
In the current study, p.Lys26Arg was predicted as a benign variant using VarCards in silico analysis tool. Consistently, this variant has been previously classified as a benign variant using various in silico analysis tools including SIFT, MUTTASTER, PROVEAN, PolyPhen-2, CADD, and REVEL [24, 32, 33]. Other studies, based on structural modeling and molecular docking simulations suggested that the p.Lys26Arg increased binding affinity of SARS-CoV-2 spike protein to the ACE2 receptor and showed increased SARS-CoV-2 infection as compared to the wildtype ACE2 [9, 14, 24, 26, 34, 35]. However, this is contradictory to the p.Lys26Arg frequencies in public databases or previous studies on SARS-CoV-2-negative and SARS-CoV-2-positive individuals [17, 29]. Other computational chemistry reports showed that p.Lys26Arg decreased the SARS-CoV-2/ACE2 electrostatic attraction and binding affinity to the host receptor [25, 27, 28]. Further, functional assays and screening of the p.Lys26Arg variant in larger populations of diverse ethnicities and geographical origin may address this discrepancy.
We restricted our analysis to the ACE2 peptidase domain variants screening, which have been established as RBD contact residues for SARS-CoV-2. Previous studies investigated the few ACE2 variants [17, 19, 36–38] or screened the complete gene [11, 20, 29, 30, 39, 40] and reported conflicting results. No association was found in Spanish [29, 39], Turkish [41], Russian [20], and British [40] populations. Contrary, different ACE2 variants were identified in Saudi [37], German [17], Italian [36], Spanish [19], and Polish [38] populations, that were linked with genetic susceptibility to SARS-CoV-2 or severity of the COVID-19 disease. However, most of these variants were in the noncoding region of the ACE2. Previously, it has been shown that noncoding variants may perturb the ACE2 gene activity [18]. These reports suggest that ACE2 variants, particularly in noncoding regions, may have an impact on infection and disease severity of COVID-19 among diverse populations. Complete ACE2 gene screening of in larger populations would be helpful for the clear understanding of its genetic association with SARS-CoV-2 infection and the course of COVID-19 disease.
Strength of the current study includes the size of study population that was well calculated though Online Sample Size Estimator, complying by scientific standards for sample size. Another strength is that all study participants were tested by RT-PCR and stratified as SARS-CoV-2-positive or SARS-CoV-2-negative individuals. Whereas, previous studies reported the prevalence of ACE2 variants in SARS-CoV-2-positive individuals in comparison with healthy controls who were not tested for SARS-CoV-2 [11, 18, 20, 30, 36, 37, 39]. Limitation of this study includes the screening of only partial coding sequence (exons 2, 3, 9, and 10) of ACE2 peptidase domain. Variant screening was performed using HRM and DHPLC assay with variant detection sensitivity < 100%, that may have missed some of the ACE2 variants. Functional variants in other coding region of ACE2 may influence COVID-19 disease occurrence and severity in the Pakistani population.