The study findings were published in the peer reviewed journal: Epigenetics.
https://www.tandfonline.com/doi/full/10.1080/15592294.2022.2089471
The epigenetic events triggered during a mild COVID-19 disease course are largely unexplored, despite the fact that these individuals make up a majority of all SARS-CoV-2-infected individuals.
The main finding of our study was an observed DNAm signature that was evident several months after recovery in CC19s compared to non-infected individuals. Although this has, to our knowledge, not previously been described, further investigations are needed to prove whether this particular signature is a remaining epigenetic mark from the time of active infection.
Not surprisingly, most of these studies mainly identify the engagement of several antiviral immune-related pathways as well as inflammatory responses in severely ill COVID-19 patients compared to controls [42,43]. In contrast, our pathway over-representation analyses revealed the involvement of distinct, previously unappreciated pathways such as the Wnt signalling and the muscarinic acetylcholine receptor 1 and 3 signalling pathways.
The Wnt signalling pathway has been implicated in several aspects of COVID-19, including development of inflammation, cytokine storms, as well as pulmonary fibrosis[44]. Furthermore, potential viral hijacking of host Wnt targets has been suggested upon SARS-CoV-2 infection in multi-omics studies[45].
Furthermore, in post-viral fatigue patients, including post-SARS-CoV and myalgic encephalomyelitis/chronic fatigue syndrome patients, this signalling pathway is dysfunctional, which has been tentatively attributed to the development of anti-muscarinic receptor autoantibodies [48,49].
This is interesting in terms of anosmia (loss of smell) in SARS-CoV-2 infected individuals, particularly in those who experience long-term symptoms, as it could indicate that epigenetic mechanisms are at play. Although we cannot draw any conclusions regarding expression from our data, the consequent immersion of these pathways suggests that they are indeed modulated.
Future studies should elaborate on the role of Wnt and muscarinic acetylcholine signalling in the development of post-acute COVID-19 syndrome, as the effects we observe have persisted for months after the initial exposure to the virus.
In the present study, DNA methylome analysis of PBMCs identified a number of genes that were shared between the natural in vivo infection and following in vitro stimulation, which were further confirmed by several module identification methods. One of these genes was tumour protein 53 (TP53), an evolutionarily conserved protein that is one of the most well-studied hub genes in cell signalling due to its central role in cancer [50].
TP53 has in several other studies previously been identified as a hub gene, in whole blood from COVID-19 patients [51], and has been shown to interact with ACE2 in SARS-CoV-2-infected human induced pluripotent stem cell-derived cardiomyocytes[52]. Moreover, transcriptomic analyses of PBMCs from a small group of patients infected with SARS-CoV-2 revealed involvement of apoptosis and TP53 signalling pathways [53], a finding that was further supported by studies of the SARS-CoV-2 interactome, where TP53 was identified as a central player in apoptosis-mediated pathways[54].
Two additional genes, both members of the heat shock protein family, HSP90AA1 and HSPA4 stand out in the network derived from our in vivo and in vitro data. Interestingly, reports on differentially expressed genes overlapping between acute respiratory distress syndrome and venous thromboembolism datasets identified TP53 and HSP90AA1 as central genes, among the top ranked hub genes in their networks[55].
Although our study does not provide any evidence for a protective role of the observed epigenetic alterations, HSP70 family members have been discussed as both anti-viral defence components [57,58], and anti-viral drug targets, against SARS-CoV-2[59].
Altogether, our findings on the network centrality of the hub genes that we derived from the in vivo and in vitro data suggest that they may be of particular importance in the interaction with epigenetically modulated genes upon SARS-CoV-2 infection. Nevertheless, further studies are needed to elucidate the mechanistic role of these genes during infection and recovery from COVID-19.
A limitation of this pilot study is the lack of validation of the DNAm findings on a transcriptional level. Since epigenetic alterations do not necessarily affect basal transcription levels, such studies need to address the transcriptome comparing epigenetically naïve and rewired samples with and without the exposure to a relevant stimulus [60,61].
Only then, when the need for an activation of defense systems seems apparent can differences be detected at the transcriptome level. Hence, whether the observed DNAm patterns are indeed associated or causally linked to host protective or host detrimental immune responses still needs to be addressed in future studies, with more well-designed, larger cohorts, and consecutive sample materials from the onset of SARS-CoV-2 infection.
The investigation of epigenetic modifications in mild-to-moderately ill COVID-19 patients enabled us to discern DNAm differences that otherwise would have been masked by overriding inflammatory responses. Though these subtle changes may not primarily be relevant to immune response severity towards SARS-CoV-2, they may be insightful for the identification of both effective host protective mechanisms at play, or ensuing deliberating conditions such as long-COVID.
In conclusion, we found epigenome-wide differences in DNAm patterns of individuals that had recovered from a mild-to-moderate disease course of COVID-19 compared to non-infected controls. This study suggests that DNAm is one of several epigenetic mechanisms that is altered upon SARS-CoV-2 infection.
Presently, several clinical trials investigating how DNA methylation may impact and predict short- and long-term outcomes of COVID-19 are ongoing (ClinicalTrials.gov-ID: NCT04364828, NCT04411563, NCT04859894) and these studies will, along with our upcoming longitudinal studies of the epigenetic impact of SARS-CoV-2-infection (NCT04368013), further elaborate on whether our observed findings are induced by protective host responses or constitute virally induced hijacking processes. Pinpointing these matters will aid the development of efficacious diagnostic tools and treatments of COVID-19 in the future.
Genetic Alterations and SARS-CoV-2 Infection
The virus uses the angiotensin-converting enzyme 2 (ACE2) type I membrane receptor to enter the host cell. ACE2 receptors were found on different tissues like arterial and venous endothelial cells, alveolar cells, enterocytes of the small intestine, and arterial smooth muscle cells in most organs. 2 While the virus enters the host cell, subgenomic RNAs were transcribed from the genomic RNA that encodes the spike protein (S), an envelope protein (E), membrane protein (M), and nucleocapsid protein (N). 2
This encoded protein was a functional receptor for the human coronaviruses SARS, human coronavirus NL63 , and SARS-CoV-2. 3 Transmembrane serine protease 2 (TMPRSS2), cathepsin L (CTSL), and furin, paired basic amino acid cleaving enzyme (FURIN) were responsible for cleaving of SARS-CoV proteins. 2
The human ACE2 gene contains hot spots for virus binding and mutations occur near these hot spots that were important to the host diversity of the virus. 2 4 5 Studies showed that N501 T mutation at the position significantly enhances the binding capacity of SARSCoV-2 spike protein receptor-binding domain. 6 Up to date, more than 1,700 variants have been identified on the ACE2 gene, which is nonsense, missense, and intron variants in the 3–UTR of the gene. The important point of ACE2 variants may affect intermolecular interaction with the SARS-CoV-2 S protein like interaction-booster between ACE2 and S1 or interaction-inhibitor between ACE2 and S1. 3
The TMPRSS2 gene encoded a serine protease family member and is critical for the entry of viruses. The TMPRSS2 gene encodes a serine protease family member and is critical for the entry of viruses. Because this protease proteolytically cleaved and activated viral envelope glycoproteins during the infection. TMPRSS2 polymorphisms were associated with an increased risk of severe COVID-19. 3 There is no published epigenetic study that shows the interaction TMPRSS gene and COVID-19. Methylation studies of the TMPRSS gene were based on prostate cancer patients. 3
The FURIN gene was located on chromosome 15q26.1 and plays an important role during the process of protein trafficking of the secretory pathway. Papa et al demonstrated that SARS-CoV-2 replication promoted by cleavage of furin. 7 ADAM-17 gene plays a role during viral infection. The virus uses the angiotensin-converting enzyme 2 (ACE2) type I membrane receptor to enter the host cell. 7
An increased number of studies demonstrated that variations in TMPRSS2, FURIN, and ADAM-17 may also have a role in the SARS-CoV-2 infectivity, disease severity, the outcome of the disease, and during the personalization of treatment planning of affected patients. Overall, an increased number of molecular profile studies will help us to better understand the heterogeneity of infection and disease and help us design personalized medicine tools on COVID-19 infection.
Interaction between Epigenetic Modifications and ACE2
The ACE2 gene is located on chromosome Xp22.2. The X-chromosome is one of the important chromosomes in the female that is epigenetically regulated. X-chromosome inactivation is an epigenetics process and regulated by X inactivation center). Because of random X inactivation, females are mosaic but males are hemizygous. 2 8 X inactivation is not complete and nearly 25% of X-chromosomal genes are escaped from X inactivation. ACE2 was one of the genes that escape from X inactivation. 9 Epigenetic regulation of the ACE2 gene was one of the mechanisms related to COVID-19 infection.
Studies showed gender and age-dependent DNA methylation of the ACE2 gene in airway epithelial cells. 10 DNA methylation level was decreased during aging and led to differential methylation patterns of several genes including aging and immune response-related genes. Therefore, DNA methylation is one of the main mechanisms that affects the prognosis of patients affected by SARS-CoV-2. 2 Under the cell energy stress, NAD-dependent histone deacetylase Sirtuin 1 (SIRT1) regulates ACE2. Also, upregulation was identified in the lung of patients with severe COVID-19. 4 In this point of view, we can suggest that increased expression level of ACE2 was correlated with severe COVID-19 infection.
Corley et al analyzed genome-wide methylation profiles of nine terminally ill COVID-19 patients’ peripheral blood. They determined differentiated DNA methylation signature of severe COVID-19 that showed hypermethylation of IFN-related genes and hypomethylation of inflammatory genes that includes a regulatory region of the NLRP3 inflammasome and antiviral MX1 genes.
The methylation pattern of MX1 was associated with plasma SARS-CoV-2 viral load and platelet count. 11 Inactivation of the IFN gene and increased level of chemokine/cytokine gene expression patterns was observed during the SARS-CoV-2 infection. Therefore, studies concluded that SARS-CoV-2 suppressed the innate antiviral response of the host. 1 Alterations of IFN-stimulated genes, antigen presentation genes, and proinflammatory genes were identified on RNA-Seq studies from patients infected with SARS-CoV-2. 12 13
Corley et al demonstrated epigenetic alterations were changed depending on the cell type. According to the study, enhancer regions of primary neutrophils from peripheral blood were hypomethylated, but on the other side transcription start site regions of primary T cells, primary T helper cells, and primary T regulatory were hypermethylated. 11 Based on Corley’s study, aberrant DNA methylation pattern at cell-type-specific regulatory regions of the host genome was observed on severe COVID-19.
RNA type viruses, like SARS-CoV-2, may also be sensitive to RNA modifications, including N6-methyladenosine (m6A) and N6,2′-Odimethyladenosine (m6Am) modifications (m6A/m). Identification of the importance of these RNA modifications will help us to explain the viral life cycle, host response, and severity of COVID-19.
These studies showed the importance of further studies in the field of epigenetics will help us to understand the severity of the infection and host defense of SARS-CoV-2. A full understanding of the epigenetic background of COVID-19 will promote docking studies to discover possible targets on SARS-CoV-2 as an epigenetic-targeted agent of COVID-19. This will bring up the combination therapy of epigenetic and antiviral drugs for viral infection.
reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8837408/
