Whole-exome-sequencing (WES) of patients of Italian ethnicity (n = 6939) found many missence variants, notably, three common ones with highly destabilizing, protein-structure interference predictions (Benetti et. al). These variants are not present in East-Asian populations. Rare variants were also identified.
- p.(Asn720Asp) (n = 103)
- p.(Lys26Arg) (n = 11)
- p.(Gly211Arg) (n = 12)


The image below shows case-control variant distribution. While no variant was significantly enriched in the population, control cases had a significantly higher ACE2 allelic variability compared to that of patients. No causal relationship between ACE2-variation and COVID-19 severity can be made to date.

ACE2 Variance in the Italian Population

This graphic shows the distribution of 61 potentially harmful ACE2 polymorphisms by population using variant assembly of databases, annotation of non-synonymous polymorphisms, and predictive-deleterious scoring. 
- databases: Genome Aggregation Database, Exome Sequencing Project, and 1000 Genomes Project (1KGP, www.internationalgenome.org)
- non-synonymous variant annotation: ANNOVAR
- deleterious assessment: CADD and Polyphen2 

The top bar shows ACE2 functional domains, and the vertical notches represents population counts of the respective variant. The heat map depicts polymorphism frequencies found within the different populations.

- 39% of variations occur in African/African-American (AFR) populations
- 54% of variations occur in non-Finnish European (EUR) populations
- Amish (AMI) and Ashkenazi Jewish (ASJ) populations do not appear to have any of these polymorphisms
- variants such as p.Arg514Gly may influence COVID-19 pathogenesis and its associated cardiovascular conditions by altering the AGT-ACE2 pathway

Population group legend: AFR, African/African-American; AMI, Amish; AMR, Latino/Admixed American; ASJ, Ashkenazi Jewish; EAS, East Asian; FIN, Finnish; EUR, Non-Finnish European; SAS, South Asian; PNA, population not assigned

Nonsynonymous ACE2 Variations by Population

ACE2 is cleaved by TMPRSS2 as a secondary function of the serine protease at at Arginine 697 to 716 to further facilitate viral entry. This suggests that populations such as non-Finnish Europeans that carry ACE2 polymorphisms including p.Arg708Trp, p.Arg710Cys, p.Arg710His, and p.Arg716Cys may have milder COVID-19 symptoms.  These mutations cause ACE2 to lose their cleavage sites, so TMPRSS2 cannot cleave the enzyme in the same way and causes the receptor to change its conformation.  

Hypothesis: Some ACE2 Variants that May Lead to Less Severe SARS-CoV-2 Infection

Allelic variants in the ACE2 gene has been observed in different ethinic populations, as well as in case-control studies within ethnic groups. However, these are present in relatively low frequencies. Functional studies are encouraged to characterize the relationship between ACE2 variants and COVID-19 severity, but no causal relationship can be made to date.

Smith College

Michigan State University

SARS-CoV-2 relies on its spike (S) protein to bind to cellular receptors to enter host cells. SARS-S, the SARS spike protein, uses ACE2 as an entry receptor into cells. Then the virus utilizes the serine protease TMPRSS2 found within the cell to enhance entry and prime future S proteins. SARS‐CoV‐2 is thought to be more transmissible than others CoVs because its S proteins have a greater affinity for ACE2.

ACE2 as a Receptor for SARS-CoV-2

ACE2 is located on the X chromosome (Xp22), and belongs to a subset of genes that escapes X-inactivation, showing a heterogenous sex bias and evolutionary characteristics of tissue specificity and expression (Tukiainen et. al). However, expression differences in distinct tissues suggest presence of X-inactivation, though incomplete.

The figure below shows ACE2 preferential expression in males (blue). 

ACE2 Chromosomal Localization and Activation.

Differences in COVID-19 inter-individual variability and lethality, along with differences among countries, raises the consideration of the role that predisposed genetics may play. Genetic variation may explain why some individuals without COVID-19 risk factors develop severe COVID-19.  Functional polymorphisms in the following genes are of interest due to their coded proteins' role in the pathogenesis of COVID-19:

- ACE2
- TMPRSS2

Understanding these variations in different populations could lead to more individualized treatment in COVID-19 patients.

Genetic Susceptibility to SARS-CoV-2 Infection in the Pathogenesis of COVID-19

Benetti, E., Tita, R., Spiga, O. et al. ACE2 gene variants may underlie interindividual variability and susceptibility to COVID-19 in the Italian population. Eur J Hum Genet (2020). doi: 10.1038/s41431-020-0691-z https://www.nature.com/articles/s41431-020-0691-z#citeas

ACE2 gene variants may underlie interindividual variability and susceptibility to COVID-19 in the Italian population

- Inhibition of the binding between the spike protein present on the surface of the virus and the ACE-2 receptor present in the membrane of the host cell. This interaction mediates the entry of the virus in the cell. 
- Because ACE 2 plays a central role in the pathogenesis of COVID-19, it is thought that it could be an efficient drug target that could either treat or prevent infection. 

ACE2 normal role:
-Critical to regulate processes such as pressure, wound healing, and inflammation. 


-It helps modulate ANGII, a protein that increases blood pressure and inflammation, increasing damage to blood vessel lining and tissue. 

COVID-19:
-In response to COVID-19, ANGII has increased inflammation and the death of alveoli cells which are critical for bringing oxygen into the body...ACE2 prevents this.

-When COVID-19 virus binds to ACE2, it inhibits its action, thus increasing the effects of ANGII
 

ACE 2 Receptor as a Pharmacological Target for Treating or Preventing COVID-19

- HCoV‐NL63

- SARS‐CoV

The receptor binding domain (RBD) of the S protein of these CoVs differ from each other, so it is thought that they utilize different mechanisms to enter host cells.  However, it has been observed that the S protein of SARS-CoV-2 is much more similar to that of SARS-CoV than HCoV‐NL63.  

Other CoVs that Utilize the Human ACE2 Receptor for Entry

The receptor binding domain (RBD) is the part the viral S protein that interacts with human ACE2.  It can exist in two conformations: open (B) or closed (A).  Molecular docking assays have shown that SARS-CoV-2 S protein can interact with the  human ACE2 in both of its conformations (D - open; C - closed).  However, the open conformation interacts protease domain (PD) at a more favorable binding energy than the closed domain which interacts with a different region of ACE2.  This confirms the importance of the open conformation of S protein in SARS-CoV-2. infection. 

SARS-CoV-2 S Protein Conformation and ACE2 Receptor Interaction

SARS-CoV-2 S protein has two relevant subunits involved in the pathogenesis of COVID-19: S1 and S2.  S1 has a high binding affinity with the receptor binding domain (RBD) of ACE2.  S2 contains amino acid sequences that are required for the virus to enter host cells.  

ACE2 Interaction with SARS-CoV-2 S Protein by Subunit

A549 are a cell line that do not express ACE2 receptors.  Partridge et al. observed that SARS-CoV-2 S protein interact with these cells even though they do not express ACE2.  Further studies were conducted using two types of 293T cells: cells that express low amounts of ACE2 and cells that over-express ACE2.  S protein interacted with both cell types, but the binding affinity for those expressing low amounts of ACE2 was nearly undetectable.  This suggests that the S protein, specifically the S1 and S2 subunits, may initially interact with host cells via  a different receptor.  However, binding affinity studies suggest that the viral receptor binding domain (RBD) requires higher expression levels of ACE2 to interact with cells.

SARS-CoV-2 S Protein May Interact with Human Cells Independently of ACE2

Five potential binding sites between HCQ/CQ and ACE2 were identified using computerized molecular docking assays.  Reported values are calculated Glide Standard Precision (SP) Glide Extra Precision (XP) measurements which quantify protein/ligand interactions.  These findings support the observation that HCQ is a more effective anti-SARS-CoV-2 drug than CQ in vitro because of HCQ's stronger interaction with the ACE2 allosteric site, which is involved with regulation of the receptor.

ACE2 as a Target for Hydroxychloroquine/Chloroquine in COVID-19 Treatment

ACE2 variants and COVID-19

ADAM17 has been demonstrated to upregulate ACE2 expression in cells via a process known as shedding, where it cleaves the extracellular portion of the receptor off of the cell.  Increased ACE2 receptors increases the number of SARS-CoV-2 viral receptors on the cellular surface, and this may exacerbate the infection or COVID-19 symptoms.  

ADAM17 Induced ACE2 Upregulation May Contribute to SARS-CoV-2 Infections

- subcellular expression
- tissue and organ expression
- mediators that influence expression
- comparing to TMPRSS2 expression
- circulating expression
- expression in embryonic development

ACE2 Expression

Schuler et al. demonstrated using both mouse and human cell lines that TMPRSS2 expression increased with age.  The regulation of the enzyme during development could contribute to why infants and children are less susceptible to severe COVID-19.  Hou et al. writes that is will be important to study age-related polymorphisms of TMPRSS2 in the future using genetic epidemiology methods.

Age-Related Polymorphisms of TMPRSS2

Hou et al. proposed three different models for treating COVID-19 based on three different phenotypes which are found in different populations.  The following drugs were used in the models:

- hydroxychloroquine
- E-64D (a protease inhibitor)
- camostat mesylate (a TMPRSS2 inhibitor approved in Japan for treating chronic pancreatitis) 

These pharmacogenomic models have yet to be tested and validated.  

Hypothesized Combination Therapies to Treat COVID-19 Based on Pharmacogenomics

Because the incidence and mortality rates of COVID-19 are significantly different in different regions of the world, it is thought that human variation may play a greater role than viral variation in determining these outcomes.  Significant variation in the gene TMPRSS2, which codes for the protease TMPRSS2, has been observed in different ethnic groups.  This may contribute to the different rates of infectivity seen in different populations.  

Genetic variation within the TMPRSS2 protease may play a role in COVID-19 infection

This graphic shows the distribution of 63 potentially harmful TMPRSS2 polymorphisms by population using variant assembly of databases, annotation of non-synonymous polymorphisms, and predictive-deleterious scoring.

- databases: Genome Aggregation Database, Exome Sequencing Project, and 1000 Genomes Project (1KGP, www.internationalgenome.org)
- non-synonymous variant annotation: ANNOVAR
- deleterious assessment: CADD and Polyphen2

The top bar shows TMPRSS2 functional domains, and the vertical notches represents population counts of the respective variant. The heat map depicts polymorphism frequencies found within the different populations.

- 35% of variations occur in African/African-American (AFR) populations
- 59% of variations occur in non-Finnish European (EUR) populations
- all populations carry p.Val160Met (~25% allele frequency)
- p.Asp435Tyr, which codes for a key residue for the substrate binding of TMPRSS2, is only carried by non-Finnish European (EUR) populations
- if TMPRSS2 polymorphisms cause gene overexpression, Down Syndrome may be a risk-factor for COVID-19 due to TMPRSS2's localization on chromosome 21.

Population group legend: AFR, African/African-American; AMI, Amish; AMR, Latino/Admixed American; ASJ, Ashkenazi Jewish; EAS, East Asian; FIN, Finnish; EUR, Non-Finnish European; SAS, South Asian; PNA, population not assigned

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