A wide range of clinical symptoms of COVID-19 disease has been reported from the first weeks after the identification of the disease in Wuhan, China[13]. The range of clinical manifestations has been grouped as follows: Asymptomatic or pre-symptomatic infection, mild illness (repetitive), moderate illness, severe illness, and critical illness with multi-organ failure.[12] It is estimated that 80 percent of patients are asymptomatic or have mild to moderate symptoms, 15 percent have severe symptoms and 5 percent are in critical status [14]. In addition to virus genotype, the wide range of the disease manifestations are attributed to host factors like age, sex, smoking, underlying disorders such as hypertension, diabetes mellitus, blood groups and genomic, transcriptomic and epigenomics factors [15]. Among genetic factors, the association of variants of the ACE2 gene and also the expression level of this gene in different tissues have been investigated [16][17]. Furthermore, the expression of TMPRSS2 and associated variants have also been investigated as a contributing factor mainly in sex difference in clinical outcomes in covid-19 patients [19–21].
Given the vast range of clinical morbidity in the patients affected with covid-19, predicting the clinical severity of the disease is of great importance. In this context, we hypothesized the potential relationship between the expression level of ACE2 and TMPRSS2 genes in nasopharyngeal swabs of COVID-19 patients with disease severity.
We found that the process of aging is positively correlated to the severity of the disease. This finding has been reported in previous studies, in which the association of higher age with susceptibility to severe forms of COVID-19, hospitalization and mortality has been determined (Fig. 2) [19] [20].
While a substantial number of studies so far have focused on the higher risk of mortality, mainly in the male sex, despite the influence of sex differences on COVID-19 fatality, no significant association between sex and respiratory distress was observed in this study. This could be attributed to the type of sampling in this study and also relative to the low number of cases examined in this project [24] [25].
However, previous studies have identified the necessity of co-expression of ACE2 and TMPRSS2 in different cell lines for infectivity of SRAS-CoV2, and controversial findings have been obtained regarding the relationship between the expression profile of these two genes and the severity of the disease [22].
According to the obtained results during this survey, comparing the expression levels of the ACE2 gene among SARS-CoV2 positive cases and negative groups showed decreasing in ACE2 expression in positive samples. Indeed, we suggest that the reduction of ACE2 expression is compatible with the molecular mechanism of viral infection, as the virus uses the cell's translational machinery to replicate and assemble virus particles [23]. Moreover, our evidence supports that decreasing ACE2 expression will result in higher morbidities, hospitalization and death in COVID-19 patients. This finding is consistent with previous findings, as many investigations revealed a positive correlation between the downregulation of ACE2 and the respiratory distress in COVID-19 patients [24]. Accordingly, some studies showed that higher expression of ACE2 was significantly associated with protection against respiratory distress by various mechanisms [25].
In contrast, some other studies suggested a correlation between ACE2 or TMPRSS2 mRNA levels and COVID-19 associated clinical manifestation[26]. Preliminary data suggested that the increased severity of COVID-19 in obese young patients may be linked to increased ACE2 expression in lung epithelial cells.[27]
In addition, higher ACE2 expression levels could be expected in individuals with hypertension [32] and also in the respiratory tract of smokers, supporting that increasing available binding site for SARS-CoV-2 could enhance viral entry into the cell and leads to higher viral load and disease morbidities [29].
On the other hand, some studies have compared the expression levels of ACE2 and TMPRSS2 in children and adults [30]. Berni Cannani et al. compared the expression level of ACE2 in nasal epithelium of 30 children with 30 adults. They found no significant difference between ACE2 expression between the two groups.[31] Bunyavanich and Vicencio investigated the expression level of ACE2 in 305 individuals aged 4–60. They found age-dependent ACE2 gene expression in the nasal epithelium, with the lowest expression in younger children (< 10 years) [32]. Felsenstein and Hedrich discussed increased ACE2 expression in young COVID-19 patients facilitate virus entry into the cell, but its anti-inflammatory role causes mild symptoms in children [33].
In addition, ACE2 plays a critical role in counterbalancing Ang2 in RAS, deficiencies of ACE2, which might be due to the aging or induced by a virus infection, can lead to increased Ang2 subsequently [34]. As a result, increasing Ang2, a product of ACE cleaving angiotensin1, can cause vasoconstriction, inflammation and fibrosis by signaling through angiotensin2 type1 receptors (ATR1) [28].
Therefore, the results so far clearly indicate that the equilibrium concentration of ACE2 can abbreviate the harmful effect of vasoconstriction, inflammation, and fibrosis and generate vasodilation by mediating the conversion of angiotensin2 to angiotensin 1–7, which seems to have a protective effect in lung injury. In fact, the complexity towards the role of ACE2 in COVID-19 disease is entirely due to the dual impact of its' actions on the severity of the disease [35].
On the other hand, in the present study, the functional impact of TMPRSS2 in S protein priming facilitates the process of SARS-CoV-2 fusion and entry after its' recognition by the ACE2 receptor, no significant difference in TMRRSS2 transcription levels was observed between mild and severe cases. However, due to the risk effect of TMPRSS2 on SARS-CoV-2 severity, TMPRSS2 was expected as an important player host for SARS-CoV-2 susceptibility in many studies.[36] [37].
Therefore, considering that TMPRSS2 function is depended on the presence of ACE2 during SARS-CoV-2 entry and since ACE2 is subjected to extensive transcriptional modulation by an epigenetic mechanism, we suggest further expression studies on SARS-CoV-2 pathogenesis in larger size of samples to evaluate the co-expression of both genes and clarify the plausible association of TMPRSS2 levels with SARS-CoV-2 severity.
Furthermore, recent studies have shown the expression level of the ACE2 gene is comparable in nasal swab bronchial samples and broncho-alveolar lavage (BAL) [25] [38]. Therefore, it is reasonable to use nasopharyngeal samples instead of bronchial samples.
One important strength of this study is the samples included in the study were obtained at the time of virus detection at the early viremia phase of the disease instead inflammatory phase of the disease. Therefore, the data could be used as a biomarker for the potential prediction of disease severity. Yet, our study has some limitations that should be considered to improve the next studies. One is the lack of samples from children and also the lack of samples from broncho-alveolar lavage (BAL) and bronchial samples to compare the expression profile of studied genes in different tissues.
Another limitation of this study was that our examination was done after COVID-19 infection, so our data does not consist of the expression levels of our target genes before and after the infection. Therefore, we suggest that prospective studies follow a case-control study to evaluate the probable changes in expression levels of these two genes before and during the infection course and after the recovery.