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.