We investigated how the ApaI rs7975232 and BsmI rs1544410 affected the susceptibility to COVID-19 and showed that they might be used as genetic indicators for infection by different SARS-CoV-2 variants.
Alleles A (0.37) for the ApaI rs7975232 and G (0.34) for the BsmI rs1544410 polymorphisms as MAF were directly related to mortality in patients with COVID-19.
The MAF results in our study for ApaI rs7975232 were almost similar to different Asian populations, including Asian (0.313), East Asian (0.314), and other Asian (0.310), while it was different from South Asian (0.615), European (0.537), Latin American (0.589), and African (0.630) (www.ncbi.nlm.nih.gov/snp/rs7975232). The MAF for ApaI rs7975232 in deceased patients (0.42) was slightly higher than recovered patients (0.32) in our study.
The MAF result in our study for BsmI rs1544410 were similar to a study in Iran and South Asian (0.443), Latin American (0.366), European (0.398), and African (0.262), but was different from other regions including Asian (0.060), East Asian (0.056), and other Asian (0.076) (www.ncbi.nlm.nih.gov/snp/rs1544410). The MAF for BsmI rs1544410 in deceased patients (0.39) was slightly higher than recovered patients (0.30) in our study.
In this study, the levels of vitamin D in COVID-19 patients, especially those infected with the Delta variant with a higher mortality rate, were lower than the other two variants. It has been found that vitamin D can play an antiviral inhibitory role in nasal epithelial cells in SARS-CoV-2 infection16. This virus enters the host cells after binding to its receptors on the cell’s surface called ACE2 by Spike protein. Type II alveolar cells, in which ACE2 receptors are strongly expressed, are the virus’s primary target17. Calcitriol, a vitamin D agonist, increases the ACE2 expression and soluble ACE2, which may lead to virus trapping and inactivation. The renin–angiotensin–aldosterone system, altered by SARS-CoV-2 infection, is negatively regulated by calcitriol, inhibiting renin expression. This increased availability of angiotensin II leads to tissue damage, inflammation, and multi-organ failure18.
Active forms of vitamin D and lumisterol have been shown to block SARS-CoV-2 replication machinery enzymes (main protease and RNA-dependent RNA polymerase), implying that novel vitamin D and lumisterol metabolites are potential antiviral therapeutic candidates. Moreover, these metabolites may prevent SARS-CoV-2 receptor binding domain from attaching to ACE2 by interacting with transmembrane serine protease 2 (TMPRSS2) and ACE2. The structural and dynamical motion alterations brought on by these interactions could impact TMPRSS2's ability to prime the SARS-CoV-2 spike proteins19. As a result, novel CYP11A1-derived vitamin D3 hydroxyderivative, including 20(OH) vitamin D3 and 20,23(OH)2 vitamin D3, and lumisterol hydroxymetabolites can inhibit COVID-19 via both independent and nuclear receptor-dependent mechanisms, making them excellent candidates for antiviral drug research as well as the informed use of their precursors as nutrients or supplements in the prevention and attenuation of COVID-19 disease20,21.
Vitamin D's active hydroxyl forms have anti-inflammatory and antioxidant effects, and they also boost innate defense to infectious agents. These characteristics are shared by non-calcemic hydroxyderivatives produced by CYP11A1 and calcitriol. They exhibit inverse agonism on the retinoic acid‐related orphan receptors-γ (ROR-γ), suppress the synthesis of pro-inflammatory cytokines, downregulate NF‐κΒ, and combat oxidative stress by activating transcription factor NF‐E2‐related factor 2 (NRF2). As a result, a direct delivery of vitamin D hydroxyderivatives deserves consideration in the therapy of COVID19 of various etiologies22.
Human VDR has more than 14 distinct identified polymorphisms. These polymorphisms may affect how VDR binds to calcitriol to modulate its response. FokI rs2228570, BsmI rs1544410, ApaI rs7975232, and TaqI rs731236 are the four SNPs that are most commonly examined. They were demonstrated independently modifying vitamin D status and in haplotypes23.
The COVID-19 death rate was considerably more significant in patients with the ApaI rs7975232 AA genotype than in other genotypes. The COVID-19 mortality rate was related to ApaI rs7975232 CA in the Alpha variant and with AA and CA in the Delta variant and with AA in the Omicron BA.5 variant. In agreement with our results, Apaydin et al. showed that the AA genotype was common among patients with severe COVID-1924.
Cohorts from Nigeria, Egypt, Ethiopia, Pakistan, Saudi Arabia, Lebanon, Turkey, and Italy were found to frequently have the AA genotype, according to the frequencies of the ApaI rs7975232 polymorphism. In contrast, the cohorts from Iran, the US, Poland, Greece, Mexico, India, the Netherlands, Czechia, Croatia, Russia, Spain, Finland, Brazil, and Tunisia frequently had the AC genotype. The CC genotype of the ApaI rs7975232 gene was most common in deceased patients from Korea, Japan, and China25. Studies with hepatitis B virus demonstrated that CA/AA genotypes of ApaI rs7975232 polymorphism trigger T helper 2 (Th2) cells proliferation, but there are no studies on ApaI rs7975232 and respiratory system viral infection. On the other hand, AA genotypes result in Th1 proliferation and anti-inflammatory cytokine production, which accelerates the progression of liver disease progression to cirrhosis14.
The fact that participants with the AA genotype of the ApaI rs7975232 polymorphism in this study had a greater death rate suggests that Th2 can also release Interleukin-6 (IL-6), which is related to COVID-19 prognosis. IL-6 is one of the key factors in the cytokine storm caused by COVID-19. IL-6 induces endothelial dysfunction with expression of tissue factor and adhesion molecules via upregulation of angiotensin converting enzyme-2 receptor. These negative effects of IL-6 were mitigated by vitamin D and VDR polymorphisms. As a result, it is possible that this is one of the putative mechanism(s) by which vitamin D exerts its positive effects in COVID-19 infection26. Subjects with the severe and moderate disease who had the "CA" genotype compared to "CC and AA" genotypes demonstrated a more severe risk, according to the study by Abdollahzadeh et al. Contrary to "CA and AA" and "CA" genotypes, symptomatic-asymptomatic and moderately-asymptomatic patients with the CC genotype were more likely to have signs and symptoms. In contrast to our findings, none of the deceased participants had the AA genotype15.
This study's patients with the GG genotype in this study exhibited a higher COVID-19 mortality rate in the BsmI rs1544410 polymorphism. The COVID-19 mortality rate was related to BsmI rs1544410 GA in the Alpha variant, BsmI rs1544410 AA and GA in the Delta variant, and GG in the Omicron BA.5 variant. It has been demonstrated that the BsmI rs1544410 G allele can be a risk factor for COVID-19 severity15, while no such relationship was seen in the study of Apaydin et al.24. The BsmI rs1544410 polymorphism's diversity revealed that the cohorts of the US, China, Poland, Turkey, Egypt, Italy, Saudi Arabia, Russia, Czechia, India, Greece, the Netherlands, Croatia, Brazil, Spain, Tunisia, Nigeria, and Lebanon frequently had the BsmI rs1544410 AG genotype. In contrast, the cohorts from Iran, Korea, Japan, Finland, Pakistan, and Mexico frequently had the BsmI rs1544410 GG genotype in deceased patients, but this was not significant25.
The association of BsmI rs1544410 and viral infections such as HIV has been investigated. It has been demonstrated that BsmI rs1544410 A-allele was strongly correlated with the rapid progression of HIV disease. It is unclear exactly how the BsmI rs1544410 G-allele confers protection, while the BsmI rs1544410 A-allele raises the likelihood of disease27. The BsmI rs1544410 G to A polymorphism alteration occurs in the 3' untranslated regions (3' UTRs) of the VDR gene and is hypothesized to affect the VDR messenger RNA stability. This polymorphism has been linked to an increased HIV infection susceptibility and faster rate of HIV disease development28,29.
Strong LD exists between BsmI rs1544410 and another 3′ UTR polymorphism (ApaI rs7975232), which has also been linked to the course of HIV illness. Given that the BsmI rs1544410 polymorphism is a synonymous mutation, the relationships seen may be explained by LD with one or more functional polymorphisms at other locations in the VDR gene30. However, synonymous rather than silent mutations could cause alternations in the protein's expression, conformation, and function. Therefore, BsmI rs1544410 polymorphisms might also directly change the VDR31. The findings that the BsmI rs1544410 A-allele is more influential in disease progression in the Delta variant than the other two may be explained by the difference in the serum vitamin D level, as these levels in patients with the Delta variant were much higher. Also, in this study, there was a strong LD between BsmI rs1544410 and ApaI rs7975232.
According to our findings, the C-A haplotype was more common among all SARS-CoV-2 variations. The A-G haplotype was linked with COVID-19 mortality in both the Alpha and Delta variants. The A-A haplotype for the Omicron variants was statistically significant. These two SNPs may likely function differently in distinct SARS-CoV-2 variants. However, the mechanism underlying this divergence remains unknown.
There were several limitations in our study that should be considered. We did not have any healthy controls who had not previously suffered from COVID-19. Besides, previous vaccination information of all patients was not available. Moreover, this study was conducted in only one population with the same ethnicity. To generalize the relationship between these two polymorphisms to the whole society, more studies should be done on different races in Iran.
In conclusion, our study showed that the serum vitamin D level and BsmI rs1544410 and ApaI rs7975232 polymorphisms were related to the mortality rate of SARS-CoV-2 with different variants. The COVID-19 mortality rate was related to ApaI rs7975232 CA genotype in the Alpha variant and with AA and CA genotypes in the Delta variant and with AA genotype in the Omicron BA.5 variant. Moreover, in BsmI rs1544410 polymorphisms, the mortality rate was correlated with GA genotype in the Alpha variant and with GG and GA genotypes in the Delta variant and with GG genotype in the Omicron BA.5 variant. The A-G haplotype was linked with COVID-19 mortality in both the Alpha and Delta variants. The A-A haplotype for the Omicron BA.5 variants was statistically significant. Further studies in different ethnicities should be done to confirm our results.