Discussion
Previous studies have explored genetic risk factors associated with susceptibility to SARS‐CoV‐2 infection and the severity of COVID-19. It has been indicated that the disease is associated with several variants of different genes such as the OAS1, DPP9, CCR2, and ASHG genes36. The association between susceptibility to COVID‐19 and ACE2 gene variants should be analyzed in various populations. The ACE2 gene is the main functional receptor for SARS-CoV-2 invasion to host cells. The other candidate genes related to COVID‐19 susceptibility are TMPRSS2, TYK2, SLC6A20, and IFNAR2. The TMPRSS2 gene codes its protein that mediates the SARS‐CoV‐2 invasion to cells so that the TMPRSS2 protein functions as a coreceptor for the virus. The TYK2 and IFNAR2 genes are involved in the anti-viral immune system defense. Also, the SLC6A20 gene has a protective role against cytokine storms caused by SARS-CoV-2 infection. Therefore, the variants of mentioned genes may modulate viral infectivity in humans, making some individuals more sensitive to SARS-Cov-2 infection than others. In this study, we have investigated a cohort of 100 Iranian patients with or without a history of COVID-19, to evaluate the association between genetic variants in specific genes and SARS‐CoV‐2 infection and COVID-19. We have divided this section according to the different genes examined. In this study, using the Whole-Exome Sequencing technique, approximately 145,000 variants were identified in each patient. Finally, among this number, 9 variants predisposed to contracting COVID-19 were reported in 40 patients with a history of contracting the disease.
ACE2 gene
2 variants out of the total of 9 variants predisposing to COVID-19 were located in the ACE2 gene, which lead to the facilitation of SARS-CoV-2 virus infection. Angiotensin-converting enzyme 1 (ACE) and angiotensin-converting enzyme 2 (ACE2) are homologous genes that regulate the physiological homeostasis of the renin-angiotensin system (RAS) 37. Since SARS-CoV-2 uses the ACE2 receptor to attack alveolar epithelial cells, it is conceivable that the expression level of ACE2 in different tissues can reveal potential genetic susceptibility to COVID-19 infection. The age-dependent expression of the ACE2 gene in the nasal epithelium was investigated in a group of 305 people and it was found that children (less than 10 years old) have the lowest level of ACE2 gene expression 38. Therefore, it was suggested that the lower risk of developing COVID-19 among children may be due to the lower expression of the ACE2 gene related to age. Two polypyrimidine tract splice variants of the ACE2 gene (rs759499720 delT and rs776459296 insT) were identified, which were significantly more common among SARS-CoV-2 patients than the control population, which indicates that they play a role in the infectivity of the SARS-CoV-2 and the severity of the COVID-19. The genetic variants c.584-8delT and c.584-4dupT are a type of sequence variant located in the polypyrimidine fragment at the 3’ end of intron 4. These variants increase ACE2 gene expression, possibly through alternative mRNA splicing mechanisms39. Furthermore, statistically higher allelic diversity has been reported in the group of patients with a history of COVID-19, suggesting that a predisposing genetic background may determine inter-individual differences associated with COVID-19.
TMPRSS2 gene
1 variant out of the total of 9 variants predisposing to COVID-19 was located in the TMPRSS2 gene, which leads to an increase in the penetration of the SARS-CoV-2 virus into the host cell. The TMPRSS2 gene encodes a serine protease enzyme involved in cleavage and activation of the SARS-CoV-2 spike protein during membrane fusion40. Current study showed that rs386818798 (GGA vs. AGT) of the TMPRSS2 genotype is a polynucleotide substitution associated with a more aggressive pattern of the disorder. The polymorphism associated with COVID-19 in the present study was rs386818798. which could affect the level of mRNA transcripts and influence the splicing and possibly the generation of one of the 20 TMPRSS2 isoforms reported so far 41. SRp40 protein is a pre-mRNA splicing factor. It is expressed in all cells, tissues, and organs, including the lung, where airway epithelial cells are found. The GGA allele of rs386818798 can provide a new binding site for SRp40, a splicing protein involved in the generation of protein isoforms (20 mRNA isoforms have been identified). This allele can increase the expression level of TMPRSS2 protein with the subsequent increase of spike protein priming42. This fact could facilitate the entry of SARS-CoV-2 into the cell and increase the risk of contracting COVID-19.
TYK2 gene
3 variants out of a total of 9 variants predisposing to COVID-19 were located in the TYK2 gene, which lead to the weakening of anti-viral immunity. The TYK2 gene encodes a member of the intracellular non-receptor tyrosine kinases of the Janus Kinase family, which plays a key role in the immune response against viral infections43. This protein is functionally related to the IFNAR1 receptor subunit. This connection has a positive effect on the binding of the ligand to the receptor complex. Therefore, proper TYK2 activity is an important step to initiate the type I IFN response. In this study, SNVs located within the protein-coding region of the tyrosine kinase 2 gene and clinically important include rs771922681, rs753470142, and a new variant that has not been reported so far. The rs771922681 variant, within exon 10 of the total 25 exons of the TYK2 gene, affects the expression level of this gene, which is associated with the virulence of COVID-19 infection. It is a missense variant and leads to a decrease in the expression of the TYK2 gene by the destruction of the mRNA transcription molecule using the nonsense mutation-mediated decay (NMD) mechanism 44. Another TYK2 variant, rs753470142, is also significantly associated with contracting COVID-19. A known likely pathogenic missense variant of TYK2 reduces its activity by creating another codon at nucleotide 46 of the 436 nucleotides belonging to exon 25. The rs753470142 variant disrupts the function of the leader sequence, thereby causing intracellular accumulation of non-functional TYK2. Another variant, Q225H, which is a novel variant, can disrupt the inclusion of exon 8, which is essential for TYK2 binding to co-receptors involved in signaling pathways 45.
SLC6A20 gene
2 variants out of the total of 9 variants predisposing to COVID-19 were located in the SLC6A20 gene, which lead to the weakening of its protective role against cytokine storms. The protein product of the SLC6A20 gene is sodium-dependent imino transporter 1 (System IMINO transporter (SIT1)), which is involved in the transport of amino acids such as glycine and proline 46. The main link between SLC6A20 and COVID-19 comes from the functional interaction between SLC6A20 and ACE2 in the membrane of small intestinal enterocytes. The expression levels of SLC6A20 and ACE2 in the lungs are positively correlated, and both genes are mainly expressed in alveolar epithelial type 2 (AT2) cells 47. SIT1 heterodimerizes with ACE2, which appears to be required to form quaternary structures that can serve as binding sites for SARS-CoV-2 spike glycoproteins. The expression of ACE2 increases the expression level of SIT1, its location in the plasma membrane, and its function in amino acid transport. SLC6A20 is a novel regulator of glycine levels. ACE2 activation increases plasma glycine levels. Glycine has anti-inflammatory, anti-oxidative, neurological, and metabolic regulation functions. By binding to its receptor, GlyR, glycine induces chloride entry into the cell, which causes hyperpolarization of the cell membrane, which protects cells from pyroptosis and a proinflammatory cytokine storm induced by SARS-CoV-2 infection. Both variants found in this study, rs147760034 and rs139008024, lead to impaired function of SLC6A20 protein and as a result increased plasma concentration of glycine amino acid. Subsequently, the intensity of the cytokine storm does not subside and the severity of COVID-19 is high 48. In particular, this molecule prevents neurological damage due to its effect on the central nervous system (high expression of SIT1 protein in the brain and spinal cord) and due to its beneficial protective effect against the release of pro-inflammatory cytokines caused by SARS-CoV-2 infection.
IFNAR2 gene
1 variant out of the total of 9 variants predisposing to COVID-19 was located in the IFNAR2 gene, which leads to a defect in the interferon signaling pathway to stimulate T helper cells in creating an immune system response. Type I IFN binds to a receptor complex consisting of interferon alpha and beta receptors 1 and 2 (IFNAR1 and IFNAR2, respectively) 49, which are associated with Janus kinases, TYK2 and JAK1, respectively. The activation of these kinases causes the tyrosine phosphorylation of STAT1 and STAT2, which leads to the formation of a heterotrimer with IRF-family member 9 (IRF-9) and thus organizes the interferon-stimulated gene transcription factor (ISGF3) complex. This signaling pathway activates a self-reinforcing positive feedback loop that leads to the induction of the production of high and protective amounts of IFN-α. The P295L mutant may destabilize critical binding to the IFN molecule in response to COVID-1950. Therefore, P295L is likely to have a major effect in disrupting the structure of the IFNAR2 protein. The rs759744926 variant is a missense variant that affects receptor structure and function and limits the antiviral effects of IFN-α/β.