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Polymorphisms in the ACE2 Locus Associate with Severity of COVID-19

Infection

Luke Wooster, BS1,2*; Christopher J. Nicholson, PhD2*; Haakon H. Sigurslid, BS2; Christian L.

Lino Cardenas, PhD2; Rajeev Malhotra, MD2‡

1. Case Western Reserve University School of Medicine, Cleveland, Ohio.

2. Cardiovascular Research Center, Division of Cardiology, Department of Medicine,

Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

* Indicates co-first authors who contributed equally to the manuscript

‡
Corresponding author:
Rajeev Malhotra, MD
Massachusetts General Hospital
Yawkey 5700; 55 Fruit Street
Boston, MA 02114
Phone: 617-726-2677, Fax: 617-724-6767
Email: rmalhotra@mgh.harvard.edu

Abstract
Data from clinical studies suggests a strong association between underlying cardiometabolic
disease and worse outcomes in COVID-19. Given that the SARS-CoV-2 virus has a unique
marked affinity to the human angiotensin-converting enzyme 2 (ACE2) receptor, one potential
explanation behind this phenomenon may involve the higher expression of ACE2 receptor in
these patients. Here, we analyzed association between polymorphisms in the ACE2 locus and
COVID-19 severity in 62 patients found to be COVID-19 positive by polymerase chain reaction.
Of these patients, 23 required hospitalization due to COVID-19 infection. Of 61 ACE2 single
nucleotide polymorphisms (SNPs) genotyped in this patient cohort, 10 were significantly
associated with tissue expression of ACE2. Logistic regression adjusted for age and for sex
identified six of these ten SNPs to be significantly associated with hospitalization. These results
provide preliminary evidence of a genetic link between the ACE2 genotype and COVID-19
disease severity and suggest that the ACE2 genotype may inform COVID-19 risk stratification
and need for more intense therapy.
.
medRxiv preprint doi: https://doi.org/10.1101/2020.06.18.20135152.this version posted June 22, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .

Introduction:

The mechanisms linked to severity of coronavirus disease 2019 (COVID-19), which has thus

far taken the lives of more than 400,000 people worldwide, are not yet understood. Data from

clinical studies suggests a strong association between underlying cardiometabolic disease and

worse outcomes in COVID-19.1 One potential explanation behind this phenomenon may involve

the higher expression of angiotensin-converting enzyme 2 (ACE2) receptor in these patients.2

COVID-19 infection is caused by binding of the SARS-CoV-2 virus through its spike (S)

proteins to the ACE2 receptor, principally in Type II alveolar cells of the lung.3 ACE2 is also

highly expressed in cardiac tissue and vascular endothelial cells, which may therefore provide a

mechanism by which SARS-CoV-2 promotes direct injury of these tissues.4,5 Since interactions

between the virus and ACE2 receptor are critical for virus entry into host cells, genetic

polymorphisms that regulate ACE2 expression and/or the strength of these interactions may

affect COVID-19 severity.6 Recent studies aimed to compare the prevalence of ACE2 functional

coding variants in different populations with endemic COVID-19.6-8 However, to date there are

no data showing a relationship between ACE2 genetic variants and clinical outcomes with

COVID-19. The present study aims to analyze novel associations between mRNA expression-

altering genetic variants of human ACE2 and clinical severity in patients with COVID-19.

Methods and Findings:

Consecutive patients diagnosed with COVID-19 throughout the Partners Hospital System

who were previously genotyped as part of the Partners Biobank repository were studied. All

genotyping was performed using the Illumina Infinium MEGA Consortium v1 SNP array. The

Genotype-Tissue Expression (GTEx) project dataset (v8 release) was used to determine SNPs
medRxiv preprint doi: https://doi.org/10.1101/2020.06.18.20135152.this version posted June 22, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .

with a significant association with ACE2 tissue mRNA expression (expression quantitative trait

loci, eQTLs) with a minor allele frequency (MAF) of at least 5%.9 No coding variants were

included in analysis as none met the threshold of 5% MAF in this cohort. ACE2 is located on the

X chromosome but has been shown to escape X inactivation.10 Therefore, logistic regression

analysis adjusted for age and sex (Plink v1.07) was performed using allelic count where males

were 0 or 1 and females 0, 1, or 2. We analyzed the effects of ACE2 minor allele dosage on

requirement for hospitalization due to COVID-19. This study was approved by the Partners

institutional review board (IRB protocol #2020P000982).

Among 30,752 previously genotyped Caucasian patients in the Partners Biobank, 62 were

found to be COVID-19 positive by polymerase chain reaction between 1st March and 16th May

2020. Hospitalization status was determined on 27th May by review of the electronic medical

records. The median (IQR) age of this cohort was 62 (52, 73) years and 44% were female (Table

1). Of these patients, 23 (37%) required hospitalization due to COVID-19 infection. Across this

cohort, we performed a case/control genetic association analysis on ACE2 polymorphisms.9 Of

61 ACE2 single nucleotide polymorphisms (SNPs) genotyped in this patient cohort, 10 were

significant eQTLs for ACE2 in at least one tissue type (P<1E-4) with some exhibiting linkage

disequilibrium (Table 2). Of the 10 SNPs, five polymorphisms (rs4240157, rs6632680,

rs4830965, rs1476524, and rs2048683), with minor alleles that associated with higher tissue

expression of ACE2,9 were associated with increased need for hospitalization due to COVID-19

even after adjusting for age and sex (odds ratio 2.9-4.3, P<0.05 for all). Similarly, an ACE2

polymorphism (rs1548474) whose minor allele associated with lower tissue expression was
medRxiv preprint doi: https://doi.org/10.1101/2020.06.18.20135152.this version posted June 22, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .

associated with lesser COVID-19 severity not requiring hospitalization (odds ratio 0.27, 95% CI

0.08-0.85, P=0.025, Table 3).

Discussion:

Previous studies have investigated the potential for ACE2 polymorphisms to explain

population-based differences in COVID-19 severity. Cao et al. found no direct evidence of

population differences in coronavirus S-protein binding-resistant mutants. However,

considerably higher allele frequencies for SNPs associated with higher ACE2 expression in

tissues were observed in the East Asian populations.6 Asselta et al. reported no significant

differences in the burden of rare deleterious ACE2 variants when comparing an Italian

population with Europeans and East Asians.7 In contrast, using single cell sequencing, Zhao et al.

demonstrated that ACE2 was more abundantly expressed in type II alveolar cells from an Asian

male donor, when compared to white and African American donors.8 Whilst these studies shed

light on the contribution of ACE2 genetic variation on population-based differences in

coronavirus etiology, our analysis is the first to identify ACE2 polymorphisms that may influence

disease severity. Importantly, genetic variants that have been associated with ACE2 mRNA

expression levels associated with disease severity. Further studies that analyze the expression

altering effects of these polymorphisms in lung and cardiovascular tissue will be informative to

fully understand their role in COVID-19 infectivity and disease severity.

Conclusion:

In summary, in a cohort of patients with COVID-19 and pre-existing genotypic data, we

found that six out of ten eQTL variants in ACE2, that are associated with altered ACE2 tissue

expression, were associated with the need for hospitalization due to COVID-19. These results,
medRxiv preprint doi: https://doi.org/10.1101/2020.06.18.20135152.this version posted June 22, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .

although requiring replication in a validation cohort and in non-Caucasian populations, provide

preliminary evidence of a genetic link between the ACE2 genotype and COVID-19 disease

severity and suggest that the ACE2 genotype may inform COVID-19 risk stratification and need

for more intense therapy.

Acknowledgements:

The authors have no conflicts of interest to disclose. Dr. Malhotra is supported by NHLBI R01

HL142809, American Heart Association grant 18TPA34230025, and the Wild Family

Foundation.
medRxiv preprint doi: https://doi.org/10.1101/2020.06.18.20135152.this version posted June 22, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .

Table 1. Demographic and clinical characteristics among Caucasian patients who did or

did not require hospitalization due to COVID-19

Required Hospitalization
Characteristic Total No Yes P value
No. 62 39 23
Age, median (IQR) 62 (52, 73) 61 (49, 68) 71 (59, 74) 0.061
Female, n (%) 27 (43.5) 18 (46.2) 9 (39.1) 0.609
Diabetes, n (%) 26 (41.9) 15 (38.5) 11 (47.8) 0.596
CVD, n (%) 21 (33.9) 11 (28.2) 10 (43.5) 0.271
Hyperlipidemia, n (%) 43 (69.4) 26 (66.7) 17 (73.9) 0.584
Hypertension*, n (%) 47 (75.8) 26 (66.7) 21 (91.3) 0.034
CKD, n (%) 10 (16.1) 7 (17.9) 3 (13.0) 0.731
CVD = cardiovascular disease; CKD = chronic kidney disease
For age, the Mann Whitney test was used. For the remaining categorical variables, Fisher’s exact test was used.
* The presence of hypertension was not associated with any of the 10 SNPs in Table 2 by Fisher’s exact test.

Table 2. Association of ACE2 eQTLs with ACE2 Tissue Expression.

SNP ID NES Top eQTL P value Tissue

rs4240157┼ 0.37 1.80E-05 Brain - Substantia nigra
Brain - Cortex
Prostate
rs6632680 0.60 7.60E-16
Nerve – (Tibial)
Artery – (Tibial)
rs1548474 -0.34 3.70E-05 Brain - Frontal Cortex
Brain - Caudate (basal ganglia)
rs4830965┼ 0.35 1.80E-06
Nerve – (Tibial)
rs1476524┼ 0.33 6.30E-05 Brain - Anterior cingulate cortex
Brain - Anterior cingulate cortex
rs2048683 0.44 2.80E-07 Nerve – (Tibial)
Artery – (Tibial)
rs5936091Δ 0.18 1.60E-05 Nerve - Tibial
rs112504565 -0.41 7.00E-05 Brain - Hypothalamus
rs705860 Δ 0.18 1.30E-05 Nerve - Tibial
rs1858558 -0.20 6.50E-05 Nerve - Tibial
┼ and Δ: SNPs that demonstrated linkage disequilibrium with R2 value ≥ 0.8 in a European cohort (https://ldlink.nci.nih.gov/).
medRxiv preprint doi: https://doi.org/10.1101/2020.06.18.20135152.this version posted June 22, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .

Table 3. Association of ACE2 eQTLs with need for hospitalization in COVID-19 patients.
Multivariable
MAF in Multivariable
Major Minor MAF in Logistic OR 95% Confidence
SNP ID Non- Logistic Regression
Allele Allele Hospitalized Regression Interval
Hospitalized P value*
OR*
rs4240157┼ C T 0.56 0.33 4.28 (1.34, 13.69) 0.014
rs6632680 A C 0.62 0.31 4.05 (1.32, 12.37) 0.014
rs1548474 G T 0.19 0.42 0.27 (0.08, 0.85) 0.025
rs4830965┼ A G 0.50 0.28 3.45 (1.15, 10.29) 0.027
rs1476524┼ T C 0.50 0.30 3.41 (1.12, 10.28) 0.030
rs2048683 T G 0.50 0.30 2.94 (1.02, 8.55) 0.047
rs5936091Δ G A 0.28 0.17 1.63 (0.56, 4.73) 0.371
rs112504565 C T 0.14 0.17 0.85 (0.24, 2.95) 0.797
rs705860 Δ A G 0.19 0.16 1.14 (0.37, 3.53) 0.819
rs1858558 T A 0.13 0.11 1.09 (0.26, 4.59) 0.910

MAF = minor allele frequency; OR = odds ratio for requiring hospitalization; NES = Normalized effect size
*adjusted for Age and Sex
┼ and Δ: SNPs that demonstrated linkage disequilibrium with R2 value ≥ 0.8 in a European cohort (https://ldlink.nci.nih.gov/).
medRxiv preprint doi: https://doi.org/10.1101/2020.06.18.20135152.this version posted June 22, 2020. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .

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