key: cord-0724208-zd7idk81
authors: Ardeshirdavani, A.; Zakeri, P.; Mehrtash, A.; Hosseini, S. M.; Li, G.; Mirtavoos-Mahyari, H.; Soltanpour, M. j.; Tavallaie, M.; Moreau, Y.
title: Clinical population genetic analysis of variants in the SARS-CoV-2 receptor ACE2
date: 2020-05-29
journal: nan
DOI: 10.1101/2020.05.27.20115071
sha: ccc1acb136ea3b14ad227606012b4c6fb6210c21
doc_id: 724208
cord_uid: zd7idk81

Purpose: SARS-CoV-2 infects cells via the human Angiotensin-converting enzyme 2 (ACE2) protein. The genetic variation of ACE2 function and expression across populations is still poorly understood. This study aims at better understanding the genetic basis of COVID-19 outcomes by studying association between genetic variation in ACE2 and disease severity in the Iranian population. Methods: We analyzed two large Iranian cohorts and several publicly available human population variant databases to identify novel and previously known ACE2 exonic variants present in the Iranian population and considered those as candidate variants for association between genetic variation and disease severity. We genotyped these variants across three groups of COVID-19 patients with different clinical outcomes (mild disease, severe disease, and death) and evaluated this genetic variation with regard to clinical outcomes. Results: We identified 32 exonic variants present in Iranian cohorts or other public variant databases. Among those, 11 variants are novel and have thus not been described in other populations previously. Following genotyping of these 32 candidate variants, only the synonymous polymorphism (c.2247G>A) was detected across the three groups of COVID-19 patients. Conclusion: Genetic variability of known and novel exonic variants was low among our COVID-19 patients. Our results do not provide support for the hypothesis that exonic variation in ACE2 has a sizeable impact on COVID-19 severity across the Iranian population.

The Coronavirus Disease 2019 (COVID- 19) pandemic, which is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), continues to spread around the world. At the time of writing of this article, the pandemic had spread to all but a handful of countries worldwide, and the number of COVID-19 cases has sharply risen to 5 million cases, claiming the lives of more than 330,000 people. Iran has one of the highest rates of SARS-CoV-2 infection in the world. As of May 21, 2020, Iran had the tenth-highest number of confirmed cases globally and the ninth-highest number of deaths in the world. With close to 130,000 confirmed COVID-19 cases and more than 7,200 deaths, Iran is currently the country most affected by the pandemic in West Asia.

Four human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-HKU1, and HCoV-OC43) cause respiratory infections with mild symptoms a.k.a. the common cold 1 . However, three other coronaviruses (the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and SARS-CoV-2), which have been transmitted from animals to humans, cause respiratory tract infections that are potentially severe with significant morbidity and mortality 2 . They predominantly affect people with already weak immune systems. While both SARS-CoV and MERS-CoV had higher mortality rates than SARS-CoV2, SARS-CoV2 has already claimed more significantly more lives, possibly because presymptomatic and asymptomic transmission makes epidemic control significantly more difficult 3 .

The Angiotensin-Converting-Enzyme protein 2 (ACE2) was previously identified as the main host cell receptor of SARS-CoV and the human respiratory coronavirus HCoV-All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 29, 2020. . https://doi.org/10.1101/2020.05.27.20115071 doi: medRxiv preprint 5 NL63 1,4,5 . Recent studies have shown that ACE2 also plays a similar role in the entry of the SARS-CoV2 into the cell 6, 7 . The novel SARS-CoV2 binds to membrane-bound ACE2 proteins in their target cells through ACE2 for cellular entry. It has also been reported that ACE2 is mainly expressed by epithelial cells of the lung, intestine, kidney, heart, and blood vessels (it is also lowly expressed in non-vascular cells within the brain) 8 . Previous studies have shown that ACE2 expression is positively correlated with the infection of SARS-CoV and HCoV-NL63 in vitro [9] [10] [11] . In particular, it has been confirmed that several human ACE2 variants could decrease the affinity between ACE2 and the spike (S) protein in coronaviruses (HCoV-NL63 and SARS-CoV) 12, 13 . Moreover, a recent study provides biophysical and structural evidence that the SARS-CoV-2 spike manifests a 10 to 20 times higher binding affinity to human ACE2 than does the spike of SARS-CoV 14 . This characteristic may also allow the virus to pass more easily from human to human than other β-CoV strains, such as SARS-CoV. Thereby, analyzing allele frequencies (AFs) in different populations, the functional impact of ACE2 gene variants, and gene expression levels in different tissues could provide insight into the variability of disease severity among COVID-19 patients.

As a result, one of the major questions in COVID-19 research today revolves around understanding the role of genetic variation in the pathogenesis of COVID-19. More specifically, which genes and pathways are affected by the underlying genetic variation?

Is SARS-CoV2 infection of respiratory epithelia only ACE2-dependent? Do some ACE2 variants provide immunity from infection, or to the contrary increase susceptibility? Do some ACE2 variants contribute to milder or more severe disease progression? More specifically, could some ACE2 variants affect the interaction between the ACE2 and S All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 29, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

To identify exonic variants in ACE2 among the Iranian population, we comprehensively investigate the genomic characteristics of ACE2 in two large cohorts from Iranian genome databases. This gives us insight into the genetic heterogeneity between or within Iranian patient cohorts. To provide a more holistic picture of ACE2 rare and common variant frequencies in the Iranian population, we cross-reference the relevant data in major variant data repositories. All ACE2 gene variants are collected from in- We also considered the protein structure of ACE2 and SARS-CoV-2, and investigate the consequences of any of the protein coding variants. To model the structural complex of ACE2 homodimer and the trimeric spike glycoprotein of SARS-CoV-2, two All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

After examining all variants (see Supplementary Table 2 ) within the exonic coding region of ACE2, we found 32 variants in the Iranian genome variation databases (GenAP and Iranome). Figure 1 shows all of the 32 variants within the coding region of ACE2 (regardless of their effects on the protein structure). 11 of these 32 variants are novel ACE2 variants and have not been reported in the three other databases previously. In particular, we observed that only 3 exonic variants (ACE2: c.2247G>A, All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 29, 2020. We analyzed the frequency of 32 candidate variants -within the exonic coding region of ACE2 obtained using Sanger sequencing technology-between three groups of patients with different clinical manifestations. In particular, 11 of these 32 genetic ACE2 variants are only reported in Iranian genome data banks: Iranome and GenAP. Table 2 shows that ACE2 exonic variation does not appear to be a key determinant of disease severity or mortaliy among Iranian COVID-19 patients. In fact, in all three groups of patients with different clinical features, no evidence for an association between ACE2 and COVID-19 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 29, 2020. . https://doi.org/10.1101/2020.05.27.20115071 doi: medRxiv preprint is detected (see Table 2 ). In particular, the ACE2 single-nucleotide polymorphism (SNP) K26R -located on exon 2, codon 77 (ACE2:c.77A>G) and common in Caucasians-is not associated with COVID19 clinical outcomes in Iranian patients. A recent study has suggested that the SNP may affect the interaction with SARS-CoV-2 spike glycoprotein and could potentially contribute to the severity of disease 24 .

Moreover, we observed that four individuals -out of forty-five patients considered in this study-carried a variant in exon 18, codon 2247 (ACE2: c.2247G>A -ACE2:c.2247G>A). This included two individuals carrying a heterozygous G-to-A variant and two patients with a homozygous variant (AA). In particular, we found two AG heterozygous individuals and one AA homozygous with mild symptoms. The AA homozygous variant was also observed in a 50-year-old woman with severe symptoms, and was hospitalized but has since recovered. This variant is not seen in patients who did not survive. The ACE2 coding variant, namely ACE2: c.2247G>A, identified in Iranian genome variation databases also appeared in other publicly available variant databases. The variant was associated neither with COVID-19 severity (p=0.299, Fisher's exact test) nor with mortality (p=0.112, Fisher's exact test). Also, no significant difference is found in the ACE2: c.2247G>A frequencies between individuals with mild symptoms (3 out of 15) and the combined patients of the other two groups (1 out of 29 (p=0.101, Fisher's exact test)).

This study provides a comprehensive investigation into the genetic basis of ACE2 function and expression variation in the Iranian population. In summary, our data does All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 29, 2020. . https://doi.org/10.1101/2020.05.27.20115071 doi: medRxiv preprint not support the hypothesis that exonic variation in ACE2 has a sizeable impact on COVID-19 severity across the Iranian general population. However, these results should be interpreted cautiously because of the small number of patients included in this study, which is a major limitation of this investigation. This limitation, in turn, may lead to potential biases in our analysis. In particular, our results do not preclude that statistically significant effects could be detected for some variants in larger patient cohorts from Iran, although one can speculate that the effect size is likely to be small and thus of limited clinical relevance. Furthermore, our results do not preclude that some of the rare variants identified could have a sizeable effect for individual carriers, although it appears unlikely that rare variants could have a sizeable contribution to the disease burden of the whole Iranian population. Also, we did not assess non-exonic or regulatory ACE2 variation, so we cannot speculate on its potential clinical relevance in the Iranian population. Thus, there is still a need to perform larger clinical studies assessing the role of ACE2 variants in COVID-19 clinical outcomes, in particular for rare variants. Finally, we cannot exclude that exonic ACE2 variation could be relevant in other populations.

While ACE2 is the main host cell receptor of SARS-CoV-2 and plays a central role in the entry of the virus into the cell, it is only one of the many candidate genes that could be associated with the variability of disease severity among COVID-19 patients. Another important candidate is a cellular protease, called Transmembrane protease, serine 2 (TMPRSS2), which appears to be essential for cell entry and the viral spread of the coronavirus 7 . In particular, it has been shown that SARS-CoV-2 employs TMPRSS2 for spike protein priming 7 . Moreover, it has recently been suggested that SARS-CoV-2 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) 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 29, 2020. . https://doi.org/10.1101/2020.05.27.20115071 doi: medRxiv preprint 12 may use CD147 -also known as Basigin or EMMPRIN-to gain cell entry 25 . Another potential target is a homolog of ACE2, named Angiotensin-converting enzyme (ACE), which is a major component of the renin-angiotensin system (RAS). ACE2 as a counterregulatory enzyme of ACE plays an essential role in balancing the activity of ACE in the regulation of blood pressure by contributing to the formation of the RAS axis. ACE is characterized by the existence of a common insertion/deletion (I/D) polymorphism.

Several studies have suggested a significant positive correlation between ACE D-allele frequency and disease susceptibility and severity in acute respiratory distress syndrome 26, 27 . Accordingly, the assessment of ACE I/D polymorphism alongside clinical outcomes could be helpful to elucidate the variability of disease severity among COVID-19 patients.

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(which was not certified by peer review) 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 29, 2020. . https://doi.org/10.1101/2020.05.27.20115071 doi: medRxiv preprint 

Coronaviruses: an overview of their replication and pathogenesis

Prevalence and genetic diversity analysis of human coronaviruses among cross-border children

Asymptomatic Transmission, the Achilles' Heel of Current Strategies to Control Covid-19

Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus

Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor

A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury

The S proteins of human coronavirus NL63 and severe acute respiratory syndrome coronavirus bind overlapping regions of ACE2

Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2

Highly conserved regions within the spike proteins of human coronaviruses 229E and NL63 determine recognition of their respective cellular receptors

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Comparative genetic analysis of the novel coronavirus (2019-nCoV/SARS-CoV-2) receptor ACE2 in different populations

Iranome: A catalog of genomic variations in the Iranian population

A map of human genome variation from population-scale sequencing

The ExAC browser: displaying reference data information from over 60 000 exomes

The mutational constraint spectrum quantified from variation in 141,456 humans

The expert Variant Interpreter

The Protein Data Bank

The PyMOL Molecular Graphics System

Exploring genomic alteration in pediatric cancer using ProteinPaint

ACE2 polymorphisms and individual susceptibility to SARS-CoV-2 infection: insights from an <em>in silico</em> study. bioRxiv

No reuse allowed without permission. (which was not certified by peer review) 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

SARS-CoV-2 invades host cells via a novel route: CD147-spike protein. bioRxiv

Angiotensin converting enzyme insertion/deletion polymorphism is associated with susceptibility and outcome in acute respiratory distress syndrome

Angiotensin-converting enzymes in acute respiratory distress syndrome

Variant Review with the Integrative Genomics Viewer

Intervene: a tool for intersection and visualization of multiple gene or genomic region sets

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We are grateful to all patients that participated in this study. We 

The authors declare that they have no competing interests. (which was not certified by peer review) 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 29, 2020. All rights reserved. No reuse allowed without permission.(which was not certified by peer review) 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 29, 2020. . https://doi.org/10.1101/2020.05.27.20115071 doi: medRxiv preprint All rights reserved. No reuse allowed without permission.(which was not certified by peer review) 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 29, 2020. . https://doi.org/10.1101/2020.05.27.20115071 doi: medRxiv preprint