The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, originated in Wuhan, China in late 2019 and rapidly spread globally. Italy was among the first European countries to experience a significant outbreak, characterized by unexpectedly high clinical severity compared to Asian countries. SARS-CoV-2 utilizes the human angiotensin-converting enzyme 2 (ACE2) as its primary receptor for cell entry. The virus's spike protein binds to ACE2, facilitating viral internalization, a process enhanced by proteases like TMPRSS2. Given the crucial role of ACE2 in SARS-CoV-2 infection, genetic variations within the ACE2 gene could potentially contribute to the observed inter-individual variability in COVID-19 susceptibility and disease severity. Age and comorbidities are established risk factors; however, the significant variation in disease severity among middle-aged adults and children suggests a substantial contribution from host genetic factors. High sequence homology exists between SARS-CoV-2 and SARS-CoV, with both utilizing ACE2 as a receptor, although the binding affinity differs. This study leverages the Network of Italian Genomes (NIG) database to investigate the genetic variation of ACE2 in the Italian population and its potential association with COVID-19.

Existing literature established the role of ACE2 as the primary receptor for SARS-CoV-2 entry into host cells. Studies showed the spike protein's receptor-binding domain (RBD) interacts with specific ACE2 residues. Cleavage of the ACE2 C-terminal segment by TMPRSS2 enhances viral entry. However, prior to this study, a comprehensive analysis of ACE2 genetic variation in the Italian population and its correlation with COVID-19 severity was lacking. This research aimed to fill this gap by examining the ACE2 gene's variability within a large cohort of Italian individuals, both patients and controls, to identify variants that might influence susceptibility or disease severity. The high sequence homology between SARS-CoV-2 and SARS-CoV provided a framework for understanding potential ACE2 variant effects, considering the known interaction sites of SARS-CoV with ACE2. The study also considered the location of ACE2 on the X chromosome and potential implications of X-chromosome inactivation (XCI) on expression levels across different tissues.

This study utilized whole-exome sequencing (WES) data from the Network of Italian Genomes (NIG) database. The NIG database contains WES data from 6930 unrelated, healthy Italian individuals from five different centers. Genomic DNA was extracted from peripheral blood samples, and exome capture was performed using various methods (SureSelect, Nextera Rapid Capture, TruSeq Exome, TruSight One). Sequencing was conducted on Illumina platforms (Genome Analyzer, HiSeq2000, NextSeq). A subset of WES data was outsourced to BGI, Mount Sinai, and the Broad Institute. Raw reads were aligned against the Hg19 reference genome, and variant calling and annotation were performed using in-house pipelines incorporating the GATK Best Practices workflow and Annovar/VEP. The gnomAD database was used to assess allele frequencies across different populations. Variants with coverage below 20x were filtered out. A cohort of 131 COVID-19 patients (34 females, 97 males) and 258 controls (129 males, 129 females) were included in the analysis. Patients were categorized into four severity groups based on respiratory impairment and ventilation needs. In silico analyses were performed using DUET to predict the effects of amino acid substitutions on protein structure and function. Molecular dynamics (MD) simulations in GROMACS were used to assess the structural stability of wild-type and variant ACE2 proteins, with RMSD analysis to quantify structural changes. The identified variants were submitted to the LOVD database.

The analysis of 6930 Italian WES identified 28 rare missense variants and three common variants in the ACE2 gene. Three common missense variants, c.2158A>G p.(Asn720Asp), c.77A>G p.(Lys26Arg), and c.631G>A p.(Gly211Arg), were identified. The frequency of c.2158A>G p.(Asn720Asp) was 0.011 in the Italian population, consistent with gnomAD data but lower than the European non-Finnish population frequency (0.025). This variant was notably absent in the East Asian population. The c.77A>G p.(Lys26Arg) and c.631G>A p.(Gly211Arg) variants had frequencies of 0.0011 and 0.0012, respectively, lower than the European non-Finnish population frequencies. These variants were also less frequent in the East Asian population. Ten novel missense variants and nine novel truncating variants not previously reported in gnomAD were identified. Two rare variants, c.1051C>G p.(Leu351Val) and c.1166C>A p.(Pro389His), were predicted to induce conformational changes affecting the interaction with the SARS-CoV-2 spike protein. Molecular dynamics simulations showed that several variants, including c.1517T>C p.(Val506Ala), c.77A>G p.(Lys26Arg), c.631G>A p.(Gly211Arg), c.1051C>G p.(Leu351Val), and c.1166C>A p.(Pro389His), impacted ACE2 protein stability and dynamics. A statistically significant (P<0.029) higher allelic variability was observed in the control group compared to the COVID-19 patient group, suggesting a possible protective effect of some ACE2 variants.

This study provides evidence that ACE2 genetic variation may contribute to inter-individual differences in COVID-19 susceptibility and severity in the Italian population. The identification of several variants, particularly those predicted to affect protein stability or interaction with the SARS-CoV-2 spike protein, warrants further investigation. The lower allelic variability in COVID-19 patients compared to controls suggests that some ACE2 variants might offer a protective effect against severe disease. However, the association between specific variants and disease severity requires larger studies and functional validation. The study's focus on the Italian population highlights the importance of considering population-specific genetic backgrounds when assessing COVID-19 risk. The identified variants, many of which are novel, should be further investigated to elucidate their functional consequences and potential clinical implications. The relatively small sample size of COVID-19 patients may limit the generalizability of the findings. Future research should focus on larger, more diverse populations to confirm and expand upon these findings.

This study revealed a range of ACE2 genetic variants in the Italian population, some with predicted effects on protein structure and function relevant to SARS-CoV-2 interaction. The observed differences in allelic variability between COVID-19 patients and controls suggest a potential association between ACE2 genotype and COVID-19 susceptibility. Further research, including functional studies, is crucial to confirm these findings and determine the clinical significance of these variants for personalized risk assessment and targeted therapies.

The study's limitations include the relatively small sample size of COVID-19 patients, which may limit the statistical power to detect subtle effects. The cross-sectional nature of the study prevents conclusions about causality. The study primarily focused on missense variants and might have missed other types of variants, such as intronic or regulatory variants, that could influence ACE2 expression or function. The in silico predictions of variant effects require experimental validation. Finally, the study focused on the Italian population; further research with diverse populations is necessary to confirm the generalizability of the findings.