Discussion
ACE2 has been established as the functional host receptor for SARS‐CoV‐2, responsible for the current devastating worldwide pandemic of coronavirus disease 2019 (COVID‐19) [16]. COVID-19 displays symptoms ranging from a mild cold to cardio-respiratory failure that may lead to death [17,18]. Both ACE and ACE2 receptor genes have numerous genetic variations including functional polymorphism of insertion/deletion (Ins/Del) for ACE and rs2106809 and rs2285666 polymorphisms for ACE2 receptor gene. The del allele shows higher ACE activity and polymorphisms in the ACE2 receptor gene affect circulating ACE2 receptor levels [12,13]. Considering the role of ACE in Covid-19 pathogenesis and the variation in disease severity, ACE I / D and ACE2 receptor gene variants have attracted the attention of researchers. However, the studies were limited to in silico analyzes and epidemiological studies. To the best of our knowledge, our work is the first study including wet lab analysis for investigating ACE I/D, ACE2 rs2106809, rs2285666 polymorphisms. Unfortunately, no association was found between these polymorphisms and the severity of symptoms of Covid-19.
Delanghe et all conducted a multiple regression analysis model to compare the prevalence and mortality of the COVID-19 infection from a number of European countries and frequency data of the geographical variation of I/D polymorphism in the ACE gene. Differently, the authors reported a negative correlation between the frequency of the D allele of the ACE Ins/Del polymorphism and prevalence and mortality rates of COVID-19 in 33 countries. It should be noted that this study does not involve wet lab analysis and the results conflicted with eastern Asian populations data. It was suggested a converse association between ACE D allele frequency and prevalence of Covid-19. Considering that the D allele frequency is lower in Asian populations than in European populations, the mortality rate and prevalence of Covid-19 in the Asia population is expected to be higher. However, the prevalence and mortality of COVID-19 in Europe are higher than in Asia [19,20]. On the contrary to Delangheet all, Pati et al. suggested a significant positive correlation of D allele of ACE polymorphism with SARS-CoV-2 infection and mortality rate in their epidemiological investigation [21].
Hatami et al. conducted a meta-analysis study and showed the recovery rate significantly increased with the I/D allele frequency ratio. However, this study provides an ecological perspective but does not provide a direct clinical relevance between the COVID-19 and ACE I/D polymorphism [22].
In two different populations, genetic variants in the ACE2 receptor gene were analyzed by whole-exome sequencing in hospitalized COVID-19 patients. Differently from our study, they investigated the relation of Covid19 with coding-region variants in the ACE2 receptor gene. These studies also provided no strong evidence that ACE2 receptor gene variants are a consistent association with COVID-19 severity. The ACE2  receptor gene sequencing showed no coding sequence variants that could explain an increased risk of developing COVID-19. Similarly, in our study, no relationship was found between ACE-2 receptor gene intron variants and Covid19 severity [23,24].
Our results showed that ACE I/D, ACE2 rs2106809, rs2285666 polymorphisms have no role in the severity of Covid-19. Novelli et al. (2020) also reported that ACE2 receptor gene coding-region variants have no effects on covid-19 severity [23]. Taking that ACE2 receptor gene expression may affect the susceptibility of individuals to infection into consideration, we speculate that genetic variations in the non-coding regions of the ACE2 receptor gene or in other non-coding DNAs that control the expression levels of ACE genes may have a potential role in the severity of the disease.
A growing number of recent findings point out that epigenetic mechanisms such as DNA methylation and histone modifications, play key roles to control gene expression. A recent analysis of public genomic and transcriptomic data outlined the role of histone methylation to regulate ACE2  receptor gene transcription [25]. Further regulation occurs at the mRNA level. From putative microRNA-binding sites identified in vitro, Lambert et al. (2014) demonstrated that miR-421 downregulates the ACE2 receptor gene [26]. Besides undergoing post-translational modifications by glycosylation and phosphorylation, the ACE2 receptor gene is also post-translationally regulated [27]. Therefore, variations in other genes related to epigenetic mechanisms of ACE2 receptor gene expression may have a potential role in the severity of Covid-19.
Second, mortality or severity of disease in COVID-19 patients might be linked to excessive production of proinflammatory cytokines leads to ARDS aggravation and widespread tissue damage resulting in multi-organ failure and death, not linked to increased ACE2 receptor expression. Wang et al. showed that most of the severely ill patients had viral shedding in a variety of tissues for 20-40 days after onset of disease, while the majority of mildly ill patients had viral shedding restricted to the respiratory tract and had no detectable virus RNA 10 days after onset [28]. Similarly, Liu et al. (2020) reported that mild cases were found to have an early viral clearance [29]. These results may indicate that the nasopharyngeal SARS-CoV-2 RNA load is higher in the severe group, or they may be related to the immune response of the host.  In the current literature impairment of SARS-CoV-2 clearance is related due to genetic and viral features, enhanced levels of interferons, neutrophil extracellular traps and pyroptosis, and possible other unknown mechanisms were reported [30]. Besides, the patients from the severe group showed elevated levels of inflammatory cytokines (such as IL-2, IL-2R, RIL-6, IL-8, and IL-10), significant enhancement in coagulation parameters (such as D-dimer, prothrombin time, and fibrinogen), and increases in myocardial injury indicators. Therefore, inflammatory release, coagulation dysfunction, and myocardial injury correlate with disease severity and rise throughout the course of the disease [31,32]. Thus, the severity of COVID-19 may be related to variations in genes encoding proinflammatory cytokines or other genes that are suggested to be associated with the occurrence of severe COVID-19.
In conclusion, our study does not support the hypothesis that ACE I/D, ACE2 rs2106809, rs2285666 polymorphisms are related to Covid-19 severity. Considering that little is known about the genetic basis of the difference of Covid-19 severity and analysis of genetic polymorphisms reveals important information, the possible relation between ACE2 rs2106809, rs2285666 polymorphisms, and Covid-19 severity required to be investigated. However, in order to explain the personal variations in Covid-19 pathogenesis, further studies are needed to investigate variations in other genes related to epigenetic mechanisms of ACE2 receptor gene expression and variation in genes encoding proinflammatory cytokines, coagulation indicators.