Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 19 March 2020
© 2020 by the author(s). Distributed under a Creative Commons CC BY license.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 19 March 2020

doi:10.20944/preprints202003.0295.v1

Short Report
DNA Methylation Analysis of the COVID-19 host cell receptor, Angiotensin I
Converting Enzyme 2 gene (ACE2) in the Respiratory System Reveal Age and
Gender Differences

AUTHORS:
1Michael J. Corley and 1Lishomwa C. Ndhlovu*
AFFILIATION:
1Division of Infectious Diseases, Department of Medicine, Weill Cornell Medical
College, New York, NY, USA. *Corresponding Author Correspondence:
Lishomwa C. Ndhlovu MD, PhD,
Division of Infectious Diseases, Department of Medicine, Weill Cornell Medical College,
413E 69th St, New York, NY, USA. Email: lndhlovu@med.cornell.edu
Keywords:
Wuhan 2019-nCoV, ACE2, DNA methylation, epigenetics, profiling, lung tissue, age,
gender, COVID-19, coronavirus
CONFLICT OF INTEREST: Authors declare no conflict of interest.
Preprints

ABSTRACT:

Background: Coronavirus disease 2019 (COVID-19) has emerged as a global threat to
human health and disease risk increases with advancing age. The regulation of the ACE2
gene that codes for COVID-19 host receptor ACE2 has been shown to be under
epigenetic regulation. Here, we examined whether intensive DNA methylation profiling
of the ACE2 gene differed by human host tissue and cell type, gender, and age.

Results: Accessing four public datasets, we observed unique human cell-type-specific
ACE2 DNA methylation patterns. In human lung tissues, gender differences in DNA
methylation at 2 sites related to the ACE2 gene were identified. Further, in freshly
isolated airway epithelial cells, DNA methylation near the transcription start site of the
ACE2 gene associated with biological age.

Conclusion: Epigenetic profiling of host tissue may permit discovery of age and gender
related potential risk factors for COVID-19. How perturbations in ACE2 methylation
relate to clinical severity across the ages and gender needs to be determined to guide
screening tools and potential epigenetic modification targeting to alleviate COVID-19
morbidity in the elderly.

INTRODUCTION:

An outbreak of COVID-2019 emerged in Wuhan, China in December 2019 as a
highly contagious coronavirus[1] capable of human infection and transmission [2,3], and
possible mortality following infection similar to the SARS coronavirus [4]. As of March
11, 2020, COVID-19 case fatality rate globally is estimated by the WHO at 3.48% based
on 109,578 confirmed cases and 3,809 deaths and this infection has evolved into a
pandemic[5]. In the United States (US) as of March 16, 2020 a total of 3,487 cases have
been reported with total 68 deaths across 49 states including the District of Columbia as a
multipronged surveillance and containment strategy has been initiated across the country
with a reported mean incubation period of 6.4 days[6]. Recent analysis of
epidemiological data suggests that as many as 86% of all COVID-19 infections are
undocumented and are contributing to the rapid worldwide increase in cases[7].
Clinical research has described COVID-19 as an acute respiratory tract infection
with varied severity of symptoms including fever onset by dry cough. Clinical treatment
of patients infect with COVID-19 has evolving management guidelines of the
disease[2,3,8,9]. Epidemiology research has documented the spread and fatality rates for
the virus[10], virology research has characterized the genomic features and evolution of
COVID-2019[11–14], and vaccine research has started development and testing of
candidate vaccines[15,16]. The global spread of the virus and diversity of human host
capable of infection highlight the importance of understanding host biological differences
related to COVID-2019. There remain numerous key questions on biological factors that
contribute to varying host responses and clinical outcomes. Data from other human patho-

genic coronaviruses SARS-CoV and MERS-CoV[17–19] has provided some insight
into potential biological factors underlying varied host responses and clinical outcomes.
Sex-based differences in susceptibility to SARS-CoV infection have been reported in
mice that parallel those observed in patients and this work identified estrogen receptor
signaling as critical for protection in females[20]. Initial reports suggest sex and age
related differences in COVID-2019 that warrant further investigation[17,21]. Research is
needed to understand specific sex-related biological features that underlie clinical
severity and treatment strategies for COVID-2019 in men and women.
Structural analyses have revealed that receptor angiotensin-converting enzyme 2
(ACE2) as a host receptor permitting cell entry and viral infectivity for COVID
2019[22,23]. Notably, ACE2 was also identified as a host receptor for other
coronaviruses including SARS-CoV and NL63[24]. ACE2 has been well studied as a
central regulator of blood pressure in the renin-angiotensin-aldosterone system[25].
Studies have shown that ACE2 protein and mRNA expression occurs in a variety of
human tissues including lung, liver, stomach, ileum, colon, and kidney[26,27]. Recent
single cell analyses of normal human tissues have shown that ACE2 is expressed in cells
of the respiratory and digestive system suggesting the lung and gut body compartments as
routes for COVID-2019 infection, viral replication, and viral shedding[28–30].
Epigenetics research has suggested that the ACE2 gene that codes for ACE2 may be
transcriptionally regulated by DNA methylation[31], which is a covalent chemical
modification of host DNA. Moreover, ACE2 is located on the X chromosome[32] raising
the possibility of gender differences in susceptibility and progression of COVID-2019[17,19,21].

Thus far, no study has examined the epigenetic landscape of ACE2 and whether it differs by

age or gender.

In this study, we accessed four available genome-wide DNA methylation human
datasets to examine whether DNA methylation profiling related to the ACE2 gene
differed by host tissue/cell type, sex, and varied by biological age to begin to understand
and address the hypothesis of whether epigenetic footprints related to ACE2 impact
susceptibility risk for COVID-19, disease progression, mortality and morbidity.


RESULTS AND DISCUSSION:
We examined evidence for varied DNA methylation patterns related to the ACE2
gene in human lung, gut, liver, pancreas, brain, and blood by accessing a subset of
available raw genome-wide DNA methylation array data[33] (GEO accession:
GSE122126). We analyzed the Illumina MethylationEPIC DNA methylation data using
the Chip Analysis Methylation Pipeline[34] and observed that DNA methylation levels at
loci related to the ACE2 gene of various human tissue cell types showed that DNA
methylation was varied across tissue cell types (Fig. 1). Notably, DNA methylation
across three CpGs (cg04013915, cg08559914, cg03536816) assayed for the ACE2 gene
was lowest in lung epithelial cells compared to the other tissue cell types (Fig. 1).,
suggesting transcription and expression to be highest in the lung/respiratory system
compartment which supports emerging single cell RNA-seq analyses of normal human
lung/respiratory system datasets[28–30]. Average total DNA methylation at all probes
related to the ACE2 gene was hypermethylated in cortical neurons and leukocytes
compared to other cell types examined, suggesting excluded ACE2 transcription and
protein expression in these cell types reported previously [26,27].

Figure 1. Distinct tissue cell type DNA methylation profile related to ACE2. DNA
methylation of 11 CpGs surveyed by the Illumina Infinium MethylationEPIC array
related to ACE2 gene from lung epithelial, hepatocytes, colon epithelial, pancreatic
duct, pancreatic beta, pancreatic acinar, cortical neurons, and leukocytes is shown
from GEO accession: GSE122126. DNA methylation level is shown for each CpG as
ranging from 0 to 1.0 (methylation normalized beta-value).

Since lung epithelial cells were observed to have a hypomethylation at the ACE2
gene, we sought to further examine DNA methylation levels in human lung tissues. We
accessed available genome-wide DNA methylation Illumina HumanMethylation450 data
from human lung tissues containing samples from both males and females and including
data from smokers and patients with chronic obstructive pulmonary disease[35]. Analysis

of DNA methylation at two CpG sites related to the ACE2 gene showed that females
were significantly hypomethylated compared to males (P = 0.0001) (Fig. 2).
Additionally, DNA methylation trended towards being hyper-methylation in lung disease
conditions including smoking and COPD compared to normal lung although not
statistically significant due to limited sample size (P = 0.18). Notably, the ACE2 gene is
located on the X chromosome raising the possibility of methylation differences due to X
chromosome activation[36]. Of note, the genome-wide Illumina HumanMethylation450
array measures DNA methylation in both X chromosomes of the females. These initial
gender-associated differences in DNA methylation related to the ACE2 gene in lung
tissues warrant further investigation, especially in the context of ACE2 gene and protein
expression and COVID-19 severity. To examine the variability in DNA methylation
levels related to ACE2 in males and females, we accessed Illumina
HumanMethylation450 DNA methylation data from 244 fresh human lung tissues[37].
We observed that DNA methylation at a CpG in the dataset related to the ACE2 gene
showed a large degree of variability in both men and women suggesting DNA
methylation of ACE2 varies by individual (Fig. 3). Of note, this dataset did not have
metadata for age and the differences in DNA methylation related to ACE2 may be
reflective of cell type differences in the lung tissues.
These gender-related differences in ACE2 DNA methylation observed in the
respiratory system support findings that indicate that Angiotensin II metabolism varies by
gender and may relate to hormonal differences or genetic differences in chromosome
dosage[38]. Our DNA methylation data contrast a recent preprint study that reanalyzed
five bulk transcriptome datasets of normal lung tissue and two single cell transcriptome

dataset reported no significant differences in ACE2 between racial groups, age groups, or
gender groups[30]. A major limitation of this study was the sample size and multiple
comparisons as well as the focus only on ACE2 transcription. Notably, the ACE2
transcription preprint study did report higher expression in Asian current smokers
compared to non-smokers supporting our observations of a trend in differences in ACE2
in lung of male smokers. Smoking dramatically impacts the epigenome and will be a key
environmental factor to examine in future epigenetic studies of COVID-19[39,40].
Additional research will need to determine whether protein, gene transcription, or
epigenetic landscape of ACE2 is most relevant to COVID-19 infection risk, disease
severity, and transmission.

Figure 3. DNA methylation variability related to ACE2 gene in human lung tissues 

men and women from GEO accession: GSE52401. DNA methylation level is shown
line at median with minimum and maximum (methylation normalized beta-value). 

 Next, based on epigenetics work showing DNA methylation sites associate with age.

These findings suggest and age-related change in the epigenetic regulation of ACE2

in the respiratory system exist and are relevant to studies of ACE2 and COVID-19 in

elderly.  Our findings complement emerging single cell transcriptional profiling data

that is suggesting that nasal goblet/secretory and ciliated cells of the respiratory system

show the highest ACE2 transcription[29] and highlight the need for age-related single

transcriptomic and epigenetic profiling studies of the respiratory system in COVID-19.

Whether this dramatic relationship between ACE2 DNA methylation and aging is

mediated by other aging-related complications or is a major factor contributing to older

individuals being more susceptible or younger individuals being less susceptible to severe

COVID-19 infection needs to be studied.

Figure 4. DNA methylation at a CpG loci in the TSS200 region related to ACE2 

gene in airway epithelial cells harvested via bronchial brushing from non-asthmatic
females from GEO accession: GSE85566) significantly associates with biological age
(r=-0.59, P = 0.001)
0 20 40 60 80
0.40
0.45
0.50
0.55
0.60
0.65
0.70
Age (years)
DNA Methylation
ACE2 (cg08559914)
r=-0.59, P=0.001

We also examined public available data from ENCODE for RNA-Seq from 

human Lung donors including a 3 year old male, 30 year old female, and fetal
samples[42,43]. Interestingly, high levels of transcription were observed in young male
lung compared to fetal lung and female lung (Fig. 5). Moreover, we observed in a
publicly available ENCODE Hi-C dataset for IMR90 cells[42] used to examine genome
wide chromatin organization that chromatin interaction contacts occurred near the site we
observed DNA methylation differences for the ACE2 gene, suggesting a potential
spatiotemporal gene expression program for ACE2 mediated by DNA methylation.
Additional single cell 3D chromatin research in normal respiratory system and during
COVID-19 infection will need to examine whether a promoter-enhancer interaction for
ACE2 exist and modulates differential host and respiratory system cell type expression
patterns.

Figure 5. Scaled gene view from WashU Epigenome Browser[43] of ACE2 human gene. ACE2 gene track showed in purple
from gencodeV29. RNA-Seq tracks from human lung and fetal lung obtained from ENCODE[42] shown in blue. CpG
methylation data of the seven 450K array probes over the ACE2 gene shown as blue lines at each CpG. Hi-C genome-wide
chromosome organization data from IMR90 cells shown with view of interaction contacts in purple 20kb resolution.
In this study, we observed DNA methylation levels associated with ACE2 gene 

In this study, we observed DNA methylation levels associated with ACE2 gene varied by tissue type,

differed by gender and disease state in lung, and associated with biological age in airway epithelial

cells. Notably, the significant relationship between DNA methylation of ACE2 and biological age suggest

age-dependent differences in host response may be mediated by the dysregulation of ACE2 and increased

expression of ACE2 with aging. While our DNA methylation findings related to the ACE2 gene are compelling,

ACE2 is dynamically regulated in the body transcriptionally, posttranscriptionally, and posttranslationally[44].

Further work will need to determine whether other pre-existing health conditions such as diabetes and

hypertension or medications being taken such as ACE inhibitors and angiotensin II type-I receptor
blockers lead the dysregulation of ACE2 and increased severe outcomes of COVID-19 infection[45].

This notion has recently received attention and controversy and deserves further study[46].

Additionally, the use of ACE inhibitors as therapeutics to prevent COVID-19 viral entry also warrants further

investigation[47]. In summary, our work focused on studying available epigenetic DNA methylation data to

begin to characterize and focus on the tissue/cell type specific epigenetic landscape of the COVID-19 host

receptor gene ACE2. Future work should examine varying host epigenetic landscapes during COVID-19

infection to understand whether COVID-2019 antagonizes antigen presentation through epigenetic modulation

similar to MERS-CoV [48]. This information will be vital to vaccine development and epigenetic therapeutics for

COVID-19. Another compelling question is whether the DNA methylation profile of ACE2 in COVID-19 viral replication

body sites associates with “super spreaders” and enhanced transmissivity.

METHODS
Public dataset acquisition and processing.
Public DNA methylation datasets were downloaded from the Gene Expression Omnibus
website. DNA methylation data for human isolated cell populations from tissues was
obtained from (GEO: GSE122126). DNA methylation data for airway epithelial cells
used to examine the relationship with age was obtained from (GEO: GSE85568). DNA
methylation data for lungs tissues of smokers and patients with COPD by gender was
obtained from (GEO: GSE92511). DNA methylation data for normal lungs tissues by
gender was obtained from (GEO: GSE52401). RAW IDAT files were loaded into
ChAMP pipeline, preprocessed, and normalized[34]. We loaded RNA-Seq and Hi-C
datasets from the ENCODE portal[49] (https://www.encodeproject.org/) into the WashU
Epigenome Browser. Statistical tests were conducted in Graphpad Prism 8 for DNA
methylation data related to the ACE2 gene CONFLICT OF INTEREST
Authors declare no conflict of interest.

ACKNOWLEDGEMENTS
We would like to acknowledge the authors that deposited raw DNA methylation data for
analysis relevant to ACE2. Public access to all formats of data relevant to COVID-19 will
aid in overcoming the pandemic.



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inserted by FC2 system