key: cord-0775007-q5xl889n
authors: Novelli, A.; Biancolella, M.; Borgiani, P.; Cocciadiferro, D.; Colona, V. L.; D'Apice, M. R.; Rogliani, P.; Zaffina, S.; Leonardis, F.; Campana, A.; Raponi, M.; Andreoni, M.; Grelli, S.; Novelli, G.
title: No evidence for allelic association between Covid-19 and ACE2 genetic variants by direct exome sequencing in 99 SARS-CoV-2 positive patients
date: 2020-05-26
journal: nan
DOI: 10.1101/2020.05.23.20111310
sha: 3be154c7a0724ea06657aa9c75551ae5f6207265
doc_id: 775007
cord_uid: q5xl889n

Background: Coronaviruses (CoV) are a large family of viruses that are common in people and many animal species. Animal coronaviruses rarely infect humans with the exceptions of the Middle East Respiratory Syndrome (MERS-CoV), the Severe acute respiratory syndrome coronavirus (SARS-CoV), and now SARS-CoV-2, which is the cause of the ongoing pandemic of coronavirus disease 2019 (COVID-19). Many studies suggested that genetic variants in ACE2 gene may influence the host susceptibility/resistance to SARS-CoV-2 virus according to the functional role of ACE2 in human pathophysiology. However, all these studies have been conducted in silico based on epidemiological and population data. We therefore investigated the occurrence of ACE2 variants in a cohort of 99 Italian unrelated individuals clinically diagnosed with coronavirus disease 19 (COVID-19) to experimental demonstrate allelic association with disease severity. Methods: By whole-exome sequencing we analysed 99 DNA samples of severely and extremely severely COVID-19 patients hospitalized at the University Hospital of Rome Tor Vergata and Bambino Gesu Hospital in Rome. Results: We identified three different germline variants, one intronic (c.439+4G>A) and two missense (c.2158A>G, p.Asn720Asp; c.1888G>C, p.Asp630His ), in 26 patients with a similar frequency between male and female and a not statistically different frequency, except for c.1888G>C, (p.Asp630His) with the ethnically matched populations (EUR). Conclusions: Our results suggest that there is not any ACE2 exonic allelic association with disease severity. It is possible that rare susceptibility alleles are located in the non-coding region of the gene able to control ACE2 gene activity. It is therefore of interest, to explore the existence of ACE2 susceptibility alleles to SARS-Co-V2 in these regulatory regions. In addition, we found no significant evidence that ACE2 alleles is associated with disease severity/sex bias in the Italian population.

Coronaviruses (CoV) are a large family of viruses that are common in people and many animal species, including camels, cattle, cats, and bats. Animal coronaviruses rarely infect humans and then spread between people with the exceptions of the Middle East Respiratory Syndrome (MERS-CoV), the Severe acute respiratory syndrome coronavirus (SARS-CoV), and now SARS-CoV-2, which is the cause of the ongoing pandemic of coronavirus disease 2019 (COVID-19) (1; 2). SARS-CoV-2 utilizes an extensively glycosylated spike (S) protein that protrudes from the viral surface to bind to angiotensin-converting enzyme 2 (ACE2) to mediate host-cell entry (3) . ACE2binding affinity of the receptor-binding domain (RBD) in S1 subunit of S protein of SARS-CoV-2 is 10-to 20-fold higher than that of SARS-CoV, which may contribute to the higher infectivity and transmissibility of SARS-CoV-2 (4) . Many studies suggested that genetic variants in ACE2 gene may influence the host susceptibility/resistance to SARS-CoV-2 virus according to the functional role of ACE2 in human pathophysiology (2) . However, all these studies have been conducted in silico based on epidemiological and population data. We therefore investigated the occurrence of ACE2 variants in a cohort of 99 Italian SARS-CoV-2 -positive patients by direct exome sequencing.

We analysed by exome sequencing 99 DNA samples of COVID-19 patients hospitalized at the University Hospital of Rome "Tor Vergata" in Rome and Bambino Gesù Hospital, Rome. All patients were clinically diagnosed as COVID-19 based on clinical suspicion and confirmed by viral RNA detection at oropharyngeal and nasopharyngeal swabs. All the selected patients were severely or extremely severely affected. Nine patients in the extremely severe group are passed away. The majority of the enrolled patients were males (61males, 38 females). Median age was 63 years (range: 2-92 years), 64 patients were under 65 years old. Ten patients were children (median age was 11.5 years) showing a severe form of the disease but none of them have Kawasaki-like syndrome (6) . The analytical procedure received approval by the local ethics committee at University Hospital of Rome Tor Vergata (protocol no. 50/20).

We identified in ACE2 gene, three different germline variants, one intronic (c.439+4G>A) and two missense ( c.2158A>G, p.Asn720Asp; c.1888G>C, p.Asp630His ), in 26 patients (14 females and 12 males). The frequency of the three variants identified are similar between male and female patients suggesting that there is not gender effects underlying the frequency distribution of ACE2 variants. (Table 1) .

GnomAD database analysis revealed that the identified ACE2 variants existed with a cumulative frequency of 0.2289 in ethnically matched populations (EUR). The frequency of the variants . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.23.20111310 doi: medRxiv preprint detected in our examined cohort (0.2119) was not statistically different (Table 1) . A significant difference was detected only for the c.1888G>C (p.Asp630His) observed in a heterozygous female (p=0.0067) ( Table 1 ). The allelic frequency of this variant in GnomAD for the EUR reference population is 0.000036 confirming that this is a very rare allele. To predict the potential impact of this variant on the protein we used different tools (PolyPhen2, Mutation Taster, SIFT, MetaLR_pred, and MetaSVM_pred.). The in-silico analyses gave conflicting computational verdict because 3 benign predictions vs. 2 pathogenic predictions. The sequence alignment of the ACE2 protein with its orthologous proteins shows that the wild type residue is not highly conserved in species implying an irrelevant functional or structural role of this residue in the ACE protein.

Concerning the others two variants, the recurrent c.439+4A>G (rs2285666) intronic variant has been previously reported by Strafella et al. Numerous in silico data suggested that the ACE2 variants in structural part of the protein could have an impact on the pathogen binding dynamics or increase the quantitative expression of ACE2.

All these studies were carried out on an epidemiological basis of population allele frequencies deposited in the various databases available. We systematically analyzed the ACE2 coding-region variants in a representative cohort of Italian patients severely affected by COVID-19 in order to identify rare and causative predisposing alleles. Although we have identified a variant (p.Asp630His), very rare in European population, in a single patient affected by COVID-19, we do not believe that there is an enrichment of ACE2 coding mutant alleles in the population of Italian patients affected by COVID-19. Our results confirm and extend that ACE2 is a gene with low allelic frequencies of missense variants as expected on the basis of GnomAD population data. In fact, we provide evidence that the rate of amino acid changes in the binding region with SARS-Co-V2 and at the protein cleavage sites is very low. This suggests that these regions have been under evolutionary pressure, probably for the essential catalytic role of ACE2 as transmembrane carboxypeptidase. It is possible that rare susceptibility alleles are located in the non-coding region . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.23.20111310 doi: medRxiv preprint of the gene able to control ACE2 gene activity. Mutant alleles in noncoding DNA can cause alteration in expression levels or disturbing the timing of the expression (1). These variations concern enhancers, promoters, insulators and silencers or regions that provide instructions for producing functional RNA molecules, such as transfer RNA, miRNAs or long non-coding RNA. It is therefore of interest, to explore the existence of ACE2 susceptibility alleles to SARS-Co-V2 in -125b (11-14) . Genes coding for these miRNAs could reveal variations able to modulate the expression and therefore produce significant quantitative differences in the ACE2 protein.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 26, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 26, 2020. . https://doi.org/10.1101/2020.05.23.20111310 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 26, 2020. 

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Thanks to Dr. Paolo Gravina, Dr. Laura Liberatoscioli and Valentina Lanari for their technical