key: cord-0690885-jfy0iafh
authors: Abdollahzadeh, Rasoul; Shushizadeh, Mohammad Hossein; Barazandehrokh, Mina; Choopani, Sepideh; Azarnezhad, Asaad; Paknahad, Sahereh; Pirhoushiaran, Maryam; Makani, S. Zahra; Yeganeh, Razieh Zarifian
title: Association of Vitamin D receptor gene polymorphisms and clinical/severe outcomes of COVID-19 patients
date: 2021-10-02
journal: Infect Genet Evol
DOI: 10.1016/j.meegid.2021.105098
sha: 074e068c0625c838171afa6224b7814b86cd4f4c
doc_id: 690885
cord_uid: jfy0iafh

INTRODUCTION: Growing evidence documented the critical impacts of vitamin D (VD) in the prognosis of COVID-19 patients. The functions of VD are dependent on the vitamin D receptor (VDR) in the VD/VDR signaling pathway. Therefore, we aimed to assess the association of VDR gene polymorphisms with COVID-19 outcomes. METHODS: In the present study, eight VDR single nucleotide polymorphisms (SNPs) were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 500 COVID-19 patients in Iran, including 160 asymptomatic, 250 mild/moderate, and 90 severe/critical cases. The association of these polymorphisms with severity, clinical outcomes, and comorbidities were evaluated through the calculation of the Odds ratio (OR). RESULTS: Interestingly, significant associations were disclosed for some of the SNP-related alleles and/or genotypes in one or more genetic models with different clinical data in COVID-19 patients. Significant association of VDR-SNPs with signs, symptoms, and comorbidities was as follows: ApaI with shortness of breath (P ˂ 0.001) and asthma (P = 0.034) in severe/critical patients (group III); BsmI with chronic renal disease (P = 0.010) in mild/moderate patients (group II); Tru9I with vomiting (P = 0.031), shortness of breath (P = 0.04), and hypertension (P = 0.030); FokI with fever and hypertension (P = 0.027) in severe/critical patients (group III); CDX2 with shortness of breath (P = 0.022), hypertension (P = 0.036), and diabetes (P = 0.042) in severe/critical patients (group III); EcoRV with diabetes (P ˂ 0.001 and P = 0.045 in mild/moderate patients (group II) and severe/critical patients (group III), respectively). However, the association of VDR TaqI and BglI polymorphisms with clinical symptoms and comorbidities in COVID-19 patients was not significant. CONCLUSION: VDR gene polymorphisms might play critical roles in the vulnerability to infection and severity of COVID-19, probably by altering the risk of comorbidities. However, these results require further validation in larger studies with different ethnicities and geographical regions.

significantly different between other COVID-19 patients' groups, including groups II & III vs. group I, group III vs. group I, group II vs. group I (P > 0.05). We also didn't found remarkable discrepancies in BsmI allelic distribution between all paired groups, except between the severe/critical group and mild/moderate group (P < 0.05).

As it is shown in Table 4 , the genotypic distributions of Tru9I, the third studied SNP located in the 3′ end's VDR gene, were not observed significantly different for any proposed genetic models, between three groups of COVID-19 patients, including symptomatic (II + III) and asymptomatic groups, severe/critical and asymptomatic groups, mild/moderate and asymptomatic groups, and eventually, severe/critical and mild/moderate groups (P > 0.05). Moreover, no significant discrepancies were found in Tru9I allelic distribution between paired groups, excluding in severe/critical group compared to mild/moderate group, in which lower rates of "U" vs. "u" and higher rates of "u" vs. "U" were significantly different between groups. TaqI polymorphism was another selected SNP in the present study that is located in the 3′ end's VDR gene. As is indicated in Table 4 , our data didn't reveal any remarkable discrepancies in genotypic and allelic distributions of TaqI and BglI SNPs, for any recommended genetic models, between various groups of COVID-19 patients (P > 0.05).

We evaluate the potential association of selected VDR SNPs with various demographic and clinical features of patients, including gender, fever, sore throat, dry cough, headache, shortness of breath, diarrhea, myalgia, fatigue, nausea, vomiting, and parageusia (Tables 5 and 6 ). Additionally, the association of VDR gene polymorphisms with multifactorial diseases that are revealed to function as critical prognostic comorbidities including hypertension, diabetes, asthma, cardiovascular disease, chronic renal disease, and malignancy, were measured in three groups of COVID-19 patients (Tables 5 and 6 ).

Our results didn't show any significant associations between studied VDR gene SNPs and the aforementioned demographic/clinical features as well as comorbidities in both asymptomatic and in the mild/moderate COVID-19 patients (P values > 0.05). However, regarding the comorbidities, we found significant associations of EcoRV and BsmI SNPs with diabetes and chronic renal disease, respectively (P identified in both allelic and genotypic distributions of EcoRV between mild/moderate patients with a positive history of diabetes versus cases with no diabetes, in all suggested genetic models. Accordingly, declined ratios of "EE + Ee vs. ee", "EE + ee vs. Ee", and "E vs. e" were seen in group II cases with diabetes versus group II cases without diabetes.

Remarkable associations between VDR gene polymorphisms with more clinical variables and comorbidities were represented in group III of COCID-19 patients (Tables 5 and 6 ). Regarding the signs and symptoms, significant associations were found between ApaI and CDX2 SNPs with shortness of breath, and Tru9I SNP with vomiting (P ˂ 0.001, P = 0.022, and P = 0.031, respectively). Our data showed a significant association of both ApaI genotypes and alleles with shortness of breath in all proposed genetic models except the dominant model ( Table 7) . Our results also revealed remarkable associations of CDX2 genotypes and alleles with shortness of breath in dominant and codominant genetic models ( Table 7) . It was shown that rates of "CC + Cc vs. cc" and "C vs. c" were higher in severe/critical patients with shortness of breath, while the frequency of "cc vs. CC + Cc", "cc vs. CC", and "c vs. C" were lower.

Additionally, significant associations were observed between VDR gene variants and more comorbidities in severe/critical COVID-19 patients, including ApaI and asthma (P = 0.034), BsmI and chronic renal disease (P = 0.014), FokI and hypertension (P = 0.027), CDX2 and both hypertension and diabetes (P = 0.36 and P = 0.42, respectively), EcoRV and diabetes (P = 0.045) ( Tables 5 and 6 ). As presented in Table 7 , a significant association was found between ApaI and asthma in severe/critical patients only in the dominant genetic model, in which diminished proportion of the "AA + Aa vs. aa" and elevated proportion of the "aa vs. AA + Aa" were disclosed. Regarding the BsmI SNP, significant associations were found with chronic renal disease in dominant and codominant genetic models.

Accordingly, a higher amount of "bb vs. BB + Bb" and "bb vs. BB" were found in severe/critical patients with chronic renal disease than those didn't have this comorbidity, while "BB + Bb vs. bb" was lower.

The association of FokI genotypic distribution with hypertension was significant in severe/critical patients in dominant and codominant genetic models. The data revealed a reduced rate of "FF + Ff vs. ff", but increased rates of the "ff vs. FF + Ff" and "ff vs. FF" in group III patients with hypertension compared to negative hypertension history ( Table 7) . The results of the present study showed a significant CDX2 genotypic discrepancies in severe/critical patients with hypertension in dominant and overdominant genetic models, as well as cases with diabetes in dominant and codominant models compared to negative cases for these comorbidities ( Table 7) . Significantly, higher frequency of "cc vs. CC + Cc" and "CC + cc vs. Cc" were observed in group III COVID-19 patients with hypertension than patients with negative history of hypertension, while the frequency of "CC + Cc vs. cc" and "Cc vs. CC + cc" were considered to J o u r n a l P r e -p r o o f be reduced. Additionally, the results showed significantly increased amounts of "cc vs. CC + Cc", "cc vs.

CC", and "c vs. C", and decreased frequency of "CC + Cc vs. cc" and "C vs. c" in severe/critical COVID-19 patients with diabetes compared to patients without diabetes. Finally, we observed significant association of EcoRV with diabetes in severe/critical patients in recessive, overdominant, and codominant genetic models, in which higher proportions of "ee + Ee vs. EE", "Ee vs. EE + ee", and "Ee vs. EE" were found in group III patients with diabetes than negative diabetes cases, while proportions of the "EE vs. ee + Ee" and "EE + ee vs. Ee" were lower ( Table 7) .

To improve the validity of achieved results, we evaluate the potential association of selected VDR SNPs with signs/symptoms and with comorbidities in all symptomatic COVID-19 patients by combining whole data, regardless of the types of COVID-19 (N = 340 cases, N = 500 cases, respectively). As presented in Table 8 , interesting associations of VDR SNPs with symptoms and comorbidities were found that are briefly mentioned: ApaI with fever and asthma (P = 0.001 and P = 0.023, respectively), BsmI with chronic renal disease (P = 0.029), Tru9I with shortness of breath and hypertension (P = 0.040 and P = 0.003, respectively), FokI with fever and hypertension (P = 0.042 and P = 0.045, respectively), CDX2 with headache, hypertension, and diabetes (P = 0.019, P = 0.005 and P = 0.015, respectively), and EcoRV with diabetes (P ˂ 0.001).

As detailed in Table 9 , the observed associations of genotypic and allelic VDR polymorphisms with signs, symptoms, and comorbidities of COVID-19 patients (regardless of the group of disease) strongly depend on the genetic models. For instance, significant associations of both allelic and genotypic distributions with the fever of COVID-19 patients were detected in recessive, overdominant, and codominant genetic models. Additionally, we found a remarkable association of ApaI genotypic distribution with asthma in dominant and overdominant genetic models, but not in recessive and overdominant models, as well as in allelic distribution. Similar to our finding in the earlier section, significant differences in the distribution of genotypes were revealed between COVID-19 patients with the chronic renal disease compared to negative cases only in dominant and overdominant genetic models.

Accordingly, a higher frequency of "bb vs. BB + Bb" and "BB + bb vs. Bb" were found, while the frequency of "BB + Bb vs. bb" and "Bb vs. BB + bb" were decreased. Despite the no significant association of Tru9I polymorphism with clinical characteristics in various groups of COVID-19 patients, significant associations of Tru9I with shortness of breath in the combined population of COVID-19 susceptibility to virus infection (63). This association could be contributed to the changes in TFIIB-VDR interaction, transcription efficiency, the effects of FokI polymorphism on immune cell behavior (64).

Based on a meta-analysis by Laplana et al., FokI polymorphism was associated with viral infections, wherein the TT genotype and T allele were reported to be risk factors for infections with enveloped viruses, including RSV (65). In this line, the risk f-allele may have a lower transcription of VDR decreasing the efficiency of the vitamin D pathway by hampering the binding of vitamin D to VDR and affecting the expression of vitamin D responsive genes. Further, no significant differences were disclosed in FokI allelic and genotypic distributions between mild/moderate and asymptomatic groups, as well as between mild/moderate and severe/critical patients.

The Cdx-2 site in the 1a promoter region of the VDR gene is a functional binding site for the transcription factor Cdx-2. G to A substitution polymorphism at this site has been found to alter the transcription of the VDR gene, whereby the A-allele increases binding to the Cdx-2 protein and transcription activity of the VDR promoter compared with the G allele (66). According to the CDX2 results, "c" minor allele frequency was higher in symptomatic and severe/critical patients against asymptomatic COVID-19 cases, while "C" major allele rates were lower. Thus, the alleles "c" and "C" can be introduced as risk and protective factors, respectively, for signs, symptoms, and maybe the severity of the COVID-19. CDX2 genotypic distributions illustrated more interesting findings based on dominant, recessive, and codominant genetic models in COVID-19 patients, including protective effects of "CC" versus "Cc + cc", susceptible effects of "cc" versus both "CC + Cc" and "CC" to have clinical features and likely severity of the disease. Cdx2 is considered as a functional polymorphism of the VDR gene that has been demonstrated to impact the immune system alter the risk of contracting certain infectious illnesses (e.g., tuberculosis and rubella) (67, 68). Nevertheless, no substantial link has been established between this SNP and autoimmune disorders such as T1D, MS, vitiligo, or psoriasis (69-72). Although it is uncertain why the polymorphism is connected to illnesses like tuberculosis, numerous studies have connected this association to VDR methylation, vitamin D-mediated control of chemokine-positive T cells, and impact adaptive cytokine responses (67, 68, 73).

The EcoRV polymorphism (rs4516035), like CDX2, is found in the promoter region of the VDR gene and is thought to play a role in the anticancer immune response. EcoRV (5' to exon 1a) is a regulatory region SNPs that can affect VDR transcription via TF binding differences (74). In the presented study, EcoRV allelic and genotypic distributions unveiled several intriguing findings. Firstly, EcoRV minor allele "e" frequencies were remarkably inclined to increase in symptomatic, mild/moderate, and severe/critical patients compared to asymptomatic COVID-19 patients, while major allele "E" rates were decreased. Therefore, negative and positive associations of "E" and "e" alleles, respectively, with clinical J o u r n a l P r e -p r o o f outcomes of COVID-19 can be proposed. Nonetheless, no significant discrepancy was found in allelic frequencies between mild/moderate and severe/critical patients. Accordingly, genetic model-based genotypic distributions of EcoRV polymorphism highlighted the protective role of "EE" vs. "Ee + ee", vulnerable effects of "Ee" versus "EE + ee", and "Ee" versus "EE". Amazingly, we didn't found any significant differences in the distribution of "ee" and "EE" genotypes among different clinical groups.

Furthermore, increased frequencies of "Ee" versus "EE + ee" and "Ee" versus "EE" in severe/critical compared to mild/moderate patients, obviously demonstrated the important role of heterozygous "Ee" in the severity of COVID-19 patients. It is previously reported that EcoRv is correlated with optimal bone density, cancer risk, diabetes, and susceptibility to HIV-1 infection (74, 75).

The ApaI (rs7975232) intronic variation is anticipated to impact splice site alterations, which may change VDR translation. This variation is common, as indicated by 734 and 16,751 homozygous mutants in the 1000G and ExAC databases, respectively (76). ApaI allelic frequencies, determined as major "A" and minor "a" alleles, didn't show significant differences between various paired groups of COVID-19. The present study highlighted that the "AA" genotype made COVID-19 affected people more prone to possess signs and symptoms versus both "Aa + aa" and "Aa" genotypes based on paired-groups of the symptomatic-asymptomatic and mild/moderate-asymptomatic comparisons. Additionally, heterozygous "Aa" patients were more protected to show signs and symptoms compared to combined "AA + aa" genotypes. This finding was interestingly opposite between severe/critical and mild/moderate groups, in which a rising risk of severity was demonstrated in patients with "Aa" genotype compared to "AA + aa"

genotypes. This could be explained by the involvement of several factors determining the severity of the disease and might not be directly related to ApaI effects. Association of ApaI with different conditions including cancers, type 1 diabetes, asthma, multiple sclerosis, and several autoimmune diseases has previously been reported (77, 78-80).

BsmI polymorphism was revealed not to have any significant differences in allelic and genotypic frequencies between asymptomatic COVID-19 patients and other groups, including mild/moderate, severe/critical, and also all symptomatic patients. However, remarkable discrepancies were observed in allelic and genotypic distributions between mild/moderate and severe/critical COVID-19 suffered individuals. Our finding disclosed that minor allele "b" acts as a predisposition factor to COVID-19 severity, but major allele "B" has a protective effect. Moreover, genetic model-based genotypic distributions illustrated that patients with the "BB" genotype versus combined "bb + Bb" genotypes have decreased risk to develop more serious forms of COVID-19. However, "Bb" symptomatic heterozygotes showed elevated vulnerability to have more seriously COVID-19 than combined "BB + Bb" genotypes.

VDR has an essential function in regulating the immune system in macrophages, dendritic cells, J o u r n a l P r e -p r o o f neutrophils, B cells, natural killer (NK) cells, and T lymphocyte. Therefore, these findings could be interpreted that VDR BsmI polymorphism has a significant role in susceptibility to and in the progression of viral infections such as COVID-19.

The SNP Tru9I didn't show any significant differences in allelic distribution between paired-group comparisons, except between severe/critical and mild/moderate groups, in which major "U" and minor "u" alleles were described as protective and risk factors, respectively. Tru9I genotypic frequencies didn't exhibit any significant association with clinical manifestations and also severity COVID-19. TaqI and BglI variants-related allelic and genotypic frequencies showed no significant association with clinical manifestations and also severity of COVID-19 affected peoples based on any genetic models in the present study. TaqI is a synonymous mutation at codon 352 in exon 9 at the 3' end of the VDR gene, in which "T" and "t" alleles were identified as absent and presence of the restriction site, respectively. The TT genotype has been reported to be associated with lower circulating levels of active vitamin D3 (81-83). ApaI, BsmI, Tru9I, and BglI are located in intron 8 at the 3' end of the VDR gene, which are considered silent SNPs. These polymorphisms do not change the amino acid sequence of the encoded protein, however, they may affect gene expression through the regulation of mRNA stability or linkage disequilibrium with other SNPs affecting the susceptibility to diseases (84).

Evaluating the potential association of VDR gene SNPs with signs and symptoms of COVID-19 patients, especially respiratory complications, surely highlights the more detailed importance of these variants in the severity of the disease. Despite the significant associations of some VDR gene variants with signs and symptoms of mild/moderate COVID-19 patients, amazing findings were pinpointed in group III. Accordingly, we found a strong association between both allelic and genotypic distributions of ApaI and CDX2 SNPs with shortness of breath. Regarding the ApaI, we found that major "A" and minor allele "a" provide a protective and susceptible effect, respectively, in severe/critical patients. According, our findings disclosed that severe/critical COVID-19 patients with "Aa" genotype and then "aa" genotype are more at risk of shortness of breath than "AA" patients. The minor "c" and major "C" alleles of CDX2 were found to have positive and negative associations with symptomatic and severe/critical COVID-19 groups, respectively. Moreover, negative association of "CC" genotype versus combined "Cc + cc" genotypes, positive associations of "cc" genotype versus both combined "CC + Cc" genotypes, and "CC" genotype to have clinical features and likely severity of disease are suggested. Nevertheless, "cc" versus both combined "CC + Cc" genotypes and "CC" genotype revealed a strong protective effect against shortness of breath. Unfortunately, we can't provide a rational explanation for these contradictory findings, therefore, it needs to be re-evaluated in other studies with larger sample sizes, in other ethnicities, and geographical regions.

Despite the high prevalence of conflicting results in previous investigations, we separately assessed the potential association of these VDR gene SNPs with some comorbidities including hypertension, diabetes, asthma, cardiovascular disease, chronic renal disease, and malignancy in various COVID-19 groups to further clarify how these genetic variants affect the prognosis of COVID-19 patients. No significant association was found between VDR gene variants and comorbidities in the asymptomatic COVID-19 group, while a strong association of VDR gene SNPs was seen with some of these conditions in mild/moderate and severe/critical groups.

Our results revealed that mild/moderate COVID-19 patients with the "BB" genotype are more prone to chronic renal disease, while patients with "Bb" are more protective. Therefore, it can be proposed that homozygotes subjects ("BB" and "bb") are at increased risk of chronic renal disease than heterozygotes in mild/moderate patients. Unlikely, we found an increased risk of the "bb" genotype versus the combined "BB + Bb" and "BB" genotype, and no significant discrepancy was observed between the distribution of the "Bb" and "BB" to have chronic renal disease in severe/critical COVID-19 patients. Consequently, we can suggest that the "Bb" genotype provides a protective role to have chronic renal disease in both mild/moderate and severe/critical COVID-19 patients, but the effects of "BB" and "bb" genotypes entirely depend on the stage of the disease. Regarding the EcoRV variant and diabetes in mild/moderate COVID-19 patients, we observed a negative association of the "E" allele and a positive association of the "e" allele. Also, our data revealed the protective effect of the "EE" genotype, but predisposing impacts of "ee" genotype, as well as increased risk of "Ee" genotype versus combined "EE + ee" and "EE" genotypes against diabetes. Therefore, it can be proposed that mild/moderate COVID-19 patients with 0, 1, and 2 alleles of minor allele "e" have a low, intermediate, and high risk of diabetes, respectively. Similar findings were observed in severe/critical patients, however, the distribution of "EE" and "ee" didn't show any remarkable difference. Overall, it can be argued that how the EcoRV variant is associated with diabetes depends entirely on the stage of COVID-19 disease, wherein the additive and overdominant genetic model better explains the observed findings in mild/ moderate and severe/critical groups, respectively.

In addition to EcoRV, CDX2 polymorphism has also been disclosed to have a significant association with diabetes in severe/critical COVID-19 patients. The major "C" and minor "c" alleles exhibited a negative and positive association with diabetes, respectively. Moreover, it was demonstrated that severe/critical patients with the "cc" genotype are more susceptible to have diabetes. Also, the CDX2 was recognized to have an association with hypertension, in which severe/critical COVID-19 patients with genotype "cc" have an increased risk for hypertension. Collectively, it can be proposed that the "cc" genotype causes an increased risk on severe/critical COVID-19 to exhibit both diabetes and hypertension comorbidities.

Similarly, FokI SNP illustrated a remarkable association with hypertension in severe/critical COVID-19 patients, in which elevated risk of hypertension was detected in "ff" genotype. ApaI genotypes were deciphered to possess a significant association with asthma, in which severe/critical COVID-19 patients with "aa" genotype strongly have increased risk than "AA + Aa" patients. Briefly, our data highlighted that ApaI SNP is associated with respiratory complications, including shortness of breath and asthma in severe/critical COVID-19 patients more likely based on overdominant and dominant genetic models, respectively.

To evaluate the reproducibility of the results and increase the accuracy of the study, the association of VDR gene SNPs with clinical outcomes and comorbidities was examined, regardless of the severity grouping of COVID-19 patients that in turn led to obtaining a larger sample size. Here, we found a significant association of VDR gene polymorphisms with several clinical outcomes of COVID-19 patients, including the association of ApaI and FokI variants with fever, Tru9I with shortness of breath, and CDX2 with the headache. By comparing these findings with the results described earlier, it is clear that these associations are quietly different. ApaI allelic and genotypic frequencies revealed that alleles "A" and "a" contribute to decreased and increased susceptibility of COVID-19 patients to fever, respectively. Our data revealed that patients with genotype "AA", are more protected to exhibit fever than "Aa + aa" patients, but the "Aa" patients are more susceptible to exhibit fever than "AA + aa", "AA" and "aa" genotypes. All of these findings pinpointed that the overdominant genetic model is the most likely model, in which an increased chance to have a fever might be occurred in heterozygotes compared to both dominant and recessive homozygotes. In respect of FokI SNP, we found that the major "F" allele associate with diminished susceptibility to fever, however the minor "f" allele associate with increased risk. Accordingly, we demonstrated that COVID-19 patients with the "ff" genotype have a higher chance to exhibit fever than "FF + Ff", "FF", and "Ff" patients. We didn't find a significant difference in the distribution of "FF" and "Ff" genotypes between patients with positive and negative fever histories.

Consequently, the dominant genetic model is the most likely model, in which "ff" homozygotes are more vulnerable to fever than "Ff" heterozygotes and "FF" homozygotes. Our results disclosed that Tru9I major "U" and minor "u" alleles possess protective and predisposing effects to the shortness of breath, respectively. Further, "UU" COVID-19 patients are more protective to shortness of breath than "Uu + uu", while "Uu" patients are more susceptible to this respiratory complication than COVID-19 subjects with "UU" or "uu" genotypes. Consequently, although no significant difference between "Uu" and combined "UU + uu" was detected, we can propose an overdominant genetic model for this SNP, in which the heterozygotes "Uu" are at elevated risk compared to both "UU" and "uu" homozygotes. The findings of the present study identified the association of CDX2 allelic and genotypic association with J o u r n a l P r e -p r o o f headache. It was highlighted that the "C" major allele was negatively associated with headache, but the "c" minor allele was positively associated in COVID-19 patients. Accordingly, we found an increased risk of headache in COVID-19 subjects with "cc" genotype than combined "CC + Cc", "Cc", and "CC" genotypes. However, any significant differences in the distribution of "CC" and "Cc" genotypes didn't observe between COVID-19 cases with and without headache though.

The results of VDR gene SNPs association with comorbidities in the combined COVID-19 patient samples regardless of severity groups (N = 500 cases) were interestingly almost consistent with associations found in COVID-19 subgroups. ApaI was identified to associate with asthma in the dominant genetic model, in which COVID-19 patients with the "aa" genotype were at higher risk than "AA + Aa" to have asthma. The "bb" homozygotes of BsmI SNP were more susceptible to chronic renal disease in the combined samples (consists of 500 cases) and severe/critical subgroup, while both "BB" and "bb" genotypes increase the risk of chronic renal disease in mild/moderate group. The association of EcoRV polymorphism with diabetes was disclosed in combined COVID-19 samples and the most likely of proposed genetic models is additive genetic model, similar to mild/moderate group, in which the COVID-19 affected individuals with 0, 1, and 2 alleles of minor allele "e" are at low, intermediate, and high risk of diabetes, respectively, nonetheless, the overdominant model works better in the severe/critical group.

Similar to the severe/critical class of COVID-19, we found a significant association of the CDX2 allelic and genotypic distributions with diabetes and hypertension, in which major "C" and minor "c" alleles exhibited a negative and positive association with both diabetes and hypertension, respectively.

According to the results, the strongest genetic model is the dominant model, in which COVID-19 patients with the "cc" genotype have an increased risk of both diabetes and hypertension comorbidities compared to "CC + Cc", "CC", and "Cc" genotypes. Moreover, we found that FokI's major "F" and minor "f" alleles showed protective and susceptible effects on hypertension in combined COVID-19 samples, respectively. Similar to severe/critical patients, COVID-19 patients with "ff" genotype have elevated risk to hypertension versus "FF + Ff", "Ff", and "FF" genotypes. The last detected association between VDR gene variants and comorbidities was an association of Tru9I with hypertension, which was not observed in subtypes of COVID-19 patients. The results disclosed major "U" and minor "u" alleles as susceptible and protective factors for hypertension, respectively. Tru9I genotypic distributions suggested an overdominant genetic model as the most likely model, in which COVID-19 patients with "Uu" genotype had increased risk to hypertension than "UU + uu", "UU", "uu" patients.

To appropriately recognize individuals who may require hospital and/or ICU admission, risk stratification based on clinical, radiographic, and laboratory data appears to be essential. The existence of comorbidities is among the most alarming clinical characteristics. Some underlying illnesses such as J o u r n a l P r e -p r o o f Journal Pre-proof hypertension, diabetes, lung disease, cardiovascular disease, age may be health issues for severe COVID-19 patients who have poorer outcomes than non-severe COVID-19 patients (85). Current evidence from the present study suggests that comorbidities including age, hypertension, diabetes, and chronic renal disease may work as a risk for the worst prognosis of COVID-19 patients. Consistent with previously reported data, our results revealed that severe/critical patients were older than mild/moderate and asymptomatic patients (86). Therefore, a positive association between elder ages and more severity of COVID-19 patients could be proposed. We observed greater frequencies of these diseases in severe/critical patients versus mild/moderate and asymptomatic patients, which is consistent with several reports (87-89). Asthma has been considered as a risk factor that makes people susceptible to more severe COVID-19 illness (90). However, managing COVID-19 in severe asthma is difficult, and it's uncertain if individuals with severe asthma are at a higher risk of having the poorest results, at least partially due to safety concerns about biologics and systemic corticosteroids (SCSs) (91). Our results showed an increased frequency of asthma conditions in severe/critical patients versus mild/moderate patients.

Interestingly, a lower frequency of this condition was observed in mild/moderate patients than asymptomatic COVID-19 cases. Similar to our results, many recent studies revealed the strong positive association of cancer with the severity of COVID-19, even though inconsistent findings were also observed (92). Intriguingly, our results didn't show any significant discrepancies of cancer frequency between severe/critical and asymptomatic COVID-19 patients. Despite early studies suggested that cancer might be a separate risk factor for severe COVID-19, recent matched researches comparing outcomes between hospitalized cancer patients and matched controls found no statistically significant differences in death (93, 94). As a result, a history of cancer and cancer-directed treatments might not even be associated with a greater risk of the most serious COVID-19 outcomes in hospitalized individuals. A proinflammatory state and a weakened innate immune response are suggested as the common characteristics between these chronic illnesses and infectious diseases, which may be connected etiologically to its pathogenesis. More importantly, the co-existence of multiple comorbidities in patients seems to increase the risk of severity or death in COVID-19 disease. Regarding the signs and symptoms in symptomatic patients, increased significant frequencies of the shortness of breath, fatigue, and parageusia were illustrated in the severe/critical group compared to the mild/moderate group, which is similar to previous investigations (95). Breathlessness is a distressing and common symptom in patients with severe illness, and it is thought to be caused by physiological and structural abnormalities in the lungs. The increased ventilatory drive may rationalize our findings since individuals with moderate COVID-19 nevertheless respond physiologically to hypoxia.

Vitamin D has been shown to regulate macrophage responses, stopping them from producing excessive amounts of inflammatory cytokines and chemokines, which are common in COVID-19. Therefore, the prevalence and mortality rate of COVID-19 may depend on the modulatory effect of bioavailable Vitamin D levels of individuals, which is determined by the genetic background, such as VDR gene polymorphisms. Therefore, we designed the present study to explore the association of eight VDR gene SNPs with the clinical status and prognosis of COVID-19 patients. We found significant associations of VDR gene variants with several clinical outcomes such as severity and shortness of breath in mild/moderate and severe/critical cases of COVID-19. Nevertheless, the VDR gene SNPs could not be proposed as either independent or dependent risk factors to COVID-19-co-existing conditions, including hypertension, diabetes, asthma, cardiovascular disease, chronic renal disease, and malignancy. Our data showed that some VDR SNPs have a clinical impact on the COVID-19 patients and might be helpful to identify the individuals at high risk of COVID-19 severity in the Iranian population. Moreover, the variations in the prevalence of COVID-19 and its mortality rates among countries may be explained by Table 2 . 2.03 (1.34-3.08), P ˂ 0.001  2.94 (1.69-5.11) , P ˂ 0.001  1.66 (1.01-2.75), P = 0.047  1.76 (1.14-2.74) 0.62 (0.45-0.85), P = 0.004  0.54 (0.36-0.83) 1.61 (1.18-2.22), P = 0.004  1.85 (1.21-2.78) 

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Vitamin D alleviates lipopolysaccharide-induced acute lung injury via regulation of the renin-angiotensin system

Vitamin D/VDR signaling attenuates lipopolysaccharide-induced acute lung injury by maintaining the integrity of the pulmonary epithelial barrier

VDR attenuates acute lung injury by blocking Ang-2-Tie-2 pathway and renin-angiotensin system

Vitamin D attenuates lung injury via stimulating epithelial repair, reducing epithelial cell apoptosis and inhibits TGF-β induced epithelial to mesenchymal transition

Vitamin D deficiency in association with endothelial dysfunction: Implications for patients with COVID-19. Reviews in cardiovascular medicine

Patterns of genome-wide VDR locations

Perspective: Vitamin D deficiency and COVID-19 severity-plausibly linked by latitude, ethnicity, impacts on cytokines, ACE2 and thrombosis

Structural organization of the human vitamin D receptor chromosomal gene and its promoter

Vitamin D receptor polymorphisms and diseases

Vitamin D Receptor polymorphisms and risk of enveloped virus infection: A meta-analysis

The authors would like to thank the participants enrolled in this study. The author(s) received no specific funding for this work. We also thank all of the individuals who provided insight and expertise that greatly assisted the research, although they may not agree with all of the interpretations/conclusions of this paper.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.