4. Discussion
In this study, genetic evidence is provided that two variants of theIFIH1 gene, rs1990760 and rs2111485, are associated with susceptibility to COVID-19. The mutant alleles and the corresponding homozygous genotypes of SNPs rs1990760 (T and TT, respectively) and rs2111485 (G and GG, respectively) were found to be significantly associated with increased risk of COVID-19. In addition, the results revealed that haplotype containing mutant alleles, rs1990760T -rs2111485 G haplotype, confers a 3.02-fold increased susceptibility to COVID-19. These data suggest that substituting C with T (rs1990760) and A with G (rs2111485) may frame a role for these two SNPs in the risk of COVID-19 infection. In addition, serum IFIH1 levels were significantly elevated in COVID-19 patients, and these levels were positively affected by the mutant-type genotypes of rs1990760 and rs2111485 (TT and GG, respectively).
For rs1990760, a number of studies have investigated its association with COVID-19 risk in populations of different ethnicities with inconsistent findings. Maiti and colleagues showed that African American and Chinese populations with a low frequency of the mutant Tallele were more predisposed to contracting COVID-19. Furthermore, this allele was associated with decreased expression of IFN-β, a known cytokine that plays a role in protection against fatal viral infections (Maiti et al, 2020). Amado-Rodriguez and colleagues reported that COVID-19 patients with the rs1990760 TT genotype showed an attenuated inflammatory response and survived their intensive care unit (ICU) stay (Amado-Rodríguez et al., 2022). Conversely, the rs1990760 TT genotype was associated with worse COVID-19 outcomes and risk of ICU admission, especially in females and non-Caucasian populations (Dieter et al., 2023). Minashkin and colleagues reported a different observation, where the rs1990760 CC wild-type genotype was associated with an increased risk of COVID-19 (Minashkin et al., 2022). Consistent with this observation, the wild-type rs1990760 C allele showed significantly increased frequency in patients with early onset of disease and was associated with COVID-19 severity (Muñiz-Banciella et al., 2023). However, another study reported that rs1990760 was not associated with susceptibility to COVID-19 risk (Feizollahi et al., 2023). In the current study, the rs1990760 T allele and TT genotype were associated with an increased risk of COVID-19 and contributed to higher serum IFIH1 levels. Regardless of these conflicting results, the IFIH1 rs1990760 variant appears to be associated with COVID-19 risk and the observed differences between studies may be related to ethnic diversity. The rs1990760 SNP is a missense variant resulting from a nitrogenous base substitution, C with T, and as a result an amino acid change, alanine to threonine, occurs at codon 946 of the IFIH1 gene. This change may provide a potential molecular link between rs1990760 and viral infections such as COVID-19. In this context, it has been pointed out that the IFIH1 gene encodes the interferon-inducible RNA helicase, which plays an important role in antiviral innate immune responses (Xiao et al., 2023).
This study also revealed that rs2111485 is another SNP of theIFIH1 gene that shows a significant association with susceptibility to COVID-19. This SNP has not been investigated to determine its role in risk of COVID-19, but in other inflammatory diseases, such as type 1 diabetes and vitiligo, rs2111485 has shown an association with risk of developing these diseases (Gootjes et al., 2022; Onan et al., 2019). Regarding viral infections, such as hepatitis B and C virus infections, rs2111485 has either shown an association with their progression or may have a role in viral clearance (Jiang et al., 2019; Yao et al., 2021). SNP rs2111485 is an intergenic variant in LD with rs1990760 and maps to a non-coding region of the IFIH1 gene. Non-coding SNPs in the human genome have been disclosed to be important in conferring susceptibility to complex diseases and determining human traits. In fact, most genome-wide association studies (GWAS) and meta-analysis studies have shown that the majority of disease-associated loci are located in non-coding regions and suggest a potential role for non-coding SNPs in human disease susceptibility, despite their functional interpretation remains to be discovered (Zhang and Lupski, 2015). Due to the accumulation of non-coding SNPs in DNA regulatory elements, it is hypothesized that these SNPs may disrupt the binding sites of transcription factors and thus indirectly participate in regulating the expression levels of genes (Cano-Gamez and Trynka, 2020). In the current study, the expression level of IFIH1 protein was stratified by rs2111485 genotypes in the serum of COVID-19 patients plus HC. It was found that the mutant-type GG genotype was associated with significantly elevated IFIH1 levels in serum compared with the wild-type AA genotype. These findings suggest that rs2111485 is a novel SNP involved in conferring susceptibility to COVID-19 and has a functional role in determining the serum IFIH1 levels. However, further studies are certainly warranted to confirm or refute these findings.
Serum IFIH1 levels were significantly elevated in COVID-19 patients compared to HC, and this elevation was found to be excellent in distinguishing between COVID-19 patients and HC patients as shown by the AUC of 0.999. Functionally, IFIH1 is involved in mediating antiviral responses by acting as a sensor of the early response to cytoplasmic RNA in viral infections by activating host cells to produce IFN (Brisse and Ly, 2019). Thus, IFIH1 is expected to have a role in the control of viral infections. Bioinformatics analysis using STRING database (https://string-db.org/) confirmed this role and demonstrated that IFIH1 interacts with five proteins involved in enhancing the antiviral immune response, including DHX58, MAVS, NLRC5, ISG15, and ATG12. DHX58 is probable ATP-dependent RNA helicase that acts as a regulator of IFIH1-mediated antiviral signaling (Xu et al., 2021). MAVS is a protein required for innate immune defense against viruses through recognition of viral RNA (Ren et al., 2020). NLRC5 is a protein involved in regulating the IFN signaling pathway and plays a role in homeostatic control of innate immunity and in antiviral defense mechanisms (Kienes et al., 2021). ISG15 is ubiquitin-like protein that plays a key role in the innate immune response to control viral infection (Freitas et al., 2020). ATG12 is another ubiquitin-like protein involved in the formation of autophagy vesicles and its role in antiviral innate immune responses has been recognized (Chawla et al., 2022). These data suggest that IFIH1 and its interacting proteins may have a pathophysiological role during the initiation and persistence of COVID-19 infection. Serum IFIH1 levels have not been evaluated in patients with COVID-19 and the current study is probably the first to conduct this evaluation. However, antibodies to MDA5, the alternative name for IFIH1, have been shown in some patients infected with SARS-CoV-2. Anti-MDA5 syndrome is a rare autoimmune disease that shows striking similarities to COVID-19, particularly elevated levels of pro-inflammatory cytokines (including type I IFNs) and the associated acute respiratory distress syndrome. Therefore, a common immunopathological mechanism between COVID-19 and anti-MDA5 syndrome has been proposed where MDA5 can be considered a cornerstone in this context (Giannini et al., 2020; Tonutti et al., 2022). Accordingly, understanding the mechanistic role of IFIH1 protein in the course of SARS-CoV-2 infection may have therapeutic potential and requires further studies.
IFIH1 levels showed a strong positive correlation with ESR and GLR, both non-specific inflammatory markers associated with COVID-19 pathogenesis (Al-Humairi et al., 2022). This may reflect the inflammatory state in COVID-19 patients and IFIH1 could be considered an important inflammatory marker interacting with other inflammatory markers during SARS-CoV-2 infection.
It is necessary to simultaneously evaluate the mRNA expression and genetic polymorphisms of IFIH1 , DHX58 , MAVS ,NLRC5 , ISG15 , and ATG12 genes in patients with COVID-19 and this may represent a notable limitation of the current study.
In conclusion, IFIH1 protein showed up-regulated levels in the serum of patients with mild/moderate COVID-19. In addition, the IFIH1 gene variants rs1990760 C/T and rs2111485 A/G were associated with susceptibility to SARS-CoV-2 infection, and the study suggests that their mutant-type genotypes are not only associated with increased risk of COVID-19 infection but also contributed to higher IFIH1 levels.