Coronavirus disease 2019 (COVID-19) has become a global pandemic 1 . Aimed at delaying disease onset by modulating modifiable risk factors, primary prevention has been proposed as a potentially effective and feasible tool to address the global challenge posed by COVID-19.
Vitamin D is a hormone, produced in the skin during exposure to sunlight, helped regulate the amount of calcium and phosphate in the body, which are needed to keep bones, teeth, and muscles healthy, and played a critical role in the immune system [2] [3] [4] . Vitamin D deficiency is a candidate risk factor for a range of adverse health outcomes, such as cancer, cardiovascular diseases, metabolic disorders, infectious diseases, as well as autoimmune diseases 5, 6 . There were well recognized evidences that vitamin D deficiency contributes to the seasonal increase of virus infections of the respiratory tract, from the common cold to influenza , and now possibly also COVID-19 7, 8 . Some systematic reviews indicated that vitamin D deficiency may increase infection risk of COVID-19 by discussing the biological mechanism of virus infection 4, 9, 10 . In addition, numerous population-based studies have evaluated the Vitamin D deficiency in COVID-19 patients relative to controls; however, the findings have been inconsistent. Some studies showed that COVID-19 patients had a lower 25 hydroxyvitamin D (25OHD) concentration compared with healthy controls 7,11-13 , while others found no association [14] [15] [16] . The inconsistent findings from these epidemiological studies may be due to differences in the study All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted August 13, 2020. . https://doi.org/10.1101/2020.08.09.20171280 doi: medRxiv preprint design, study population, assays used for measuring Vitamin D deficiency. It is important to note that as the nature of the above studies is observational, such traditional epidemiological studies are particularly vulnerable to reverse causality and residual confounding.
A promising approach known as Mendelian randomization (MR), which uses inherited genetic variants as instrumental variables, provides stronger evidence for the causal effect of exposure on the diseases largely overcoming the traditional limitations due to confounding and reverse causality 17,18 . Summary based MR is an excellent strategy to evaluate the causality using summary statistics from Genome Wide Association Study (GWAS) data 19,20 . Therefore, we conducted MR analysis to assess whether 25OHD concentration was causally associated with risk of COVID-19.
The summarized data was obtained from the most recent version of GWAS analyses from the COVID-19 host genetics initiative from UK Biobank individuals, which released on July 1, 2020 (https://www.covid19hg.org/results/) 21 . Summarized data on COVID-19 included 3523 patients and 36634 control participants, and summarized data on severe COVID-19 included 536 patients and 329391 control participants.
Selection of 25OHD concentration -associated single nucleotide polymorphisms (SNPs) All rights reserved. No reuse allowed without permission.
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We selected 143 SNPs as instrumental variables (IVs) for 25OHD concentration based on the recent large-scale GWAS 22 . In addition, we retained independent variants from each other (Linkage disequilibrium [LD], r 2 < 0.001) for sensitivity analysis. When we encountered genetic variants in LD, the SNP with the lowest p-value was selected. The LD proxies were defined using 1000 genomes European samples.
In the analyses, the inverse-variance weighted (IVW) method was used to estimate the overall causal association of 25OHD concentration on COVID-19 susceptibility and severity. We additionally conducted the weighted median, penalized weighted median, and MR-Egger regression to account for potential violations of valid instrumental variable assumptions. The MR-Egger analysis was performed to evaluate pleiotropy based on the intercept. We conducted a heterogeneity test in MR analyses using the IVW Q test. Then, sensitivity analyses were performed to examine the stability of the causal estimate. Firstly, we performed a "leave one out" analysis to further investigate the possibility that the causal association was driven by a single SNP. Then, we retained independent variants from each other (LD, r 2 < 0.001) for further sensitivity analysis. Results were presented as odds ratios (OR) with their 95% confidence interval (CI) of outcomes per genetically predicted increase in each exposure factor.
In terms of various estimates for different measures, we chose the result of main MR method as the following rules: All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted August 13, 2020. . https://doi.org/10.1101/2020.08.09.20171280 doi: medRxiv preprint (1) If no directional pleiotropy in MR estimates (Q statistic: P value > 0.05, MR-Egger intercept: P value > 0.05), the results of the IVW method were reported.
(2) If directional pleiotropy was detected (MR-Egger intercept: P value < 0.05) and P value > 0.05 for the Q test, the results of the MR-Egger method were reported.
(3) If directional pleiotropy was detected (MR-Egger intercept: P value < 0.05) and P value < 0.05 for the Q test, the results of the weighted median method were reported.
All data analyses were performed by the "twosampleMR" package in R (R version 3.6.3, R Core Team, 2017). Table 1 , the MR analysis showed no significant association of genetically instrumented 25OHD concentration with COVID-19 using 89-SNP (OR, 1.168; 95% CI, 0.956-1.427; P=0.128). The association of 25OHD concentration with COVID-19 was robust in the weighted median and penalized weighted median methods, except in the MR-Egger regression (OR, 1.302; 95% CI, 1.011-1.676;
P=0.044). Pleiotropy bias (P for MR-Egger intercept=0.174) and heterogeneity (P=0.656) were also not observed. In terms of various estimates for different measures, we reported the results of the IVW method. In addition, the "leave one out" results showed that by omitting the included 89 SNPs one at a time, no individual genetic variants seem to have any significant effect on the overall results (Figure 1 ).
The association of 25OHD concentration with COVID-19 remained robust using All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted August 13, 2020. . https://doi.org/10.1101/2020.08.09.20171280 doi: medRxiv preprint 57-SNP instrument (Table 1, Figure S1 ).
The MR analysis showed no significant association of genetically instrumented 25OHD concentration with severe COVID-19 using 82-SNP (OR, 0.889; 95% CI, 0.549-1.439; P=0.632) ( Table 2 ). The result in the weighted median, penalized weighted median, and MR-Egger regression methods were robust. There was limited evidence of heterogeneity and horizontal pleiotropy based on the Q test (P=0.793) and
MR-Egger intercept test (P=0.760). Sensitivity analyses using different instruments yielded similar findings, suggesting the robustness of the causal association (Table 2, Figure 2 , Figure S2 ).
In the current study, we performed an MR analysis to investigate the causal association of 25OHD concentration with the risk of COVID-19. Our results indicated that there might be no linear causal relationship of 25OHD concentration with COVID-19 susceptibility and severity.
An observational study based on the UK Biobank data claimed that no link between vitamin D concentrations and risk of COVID-19 infection either overall or separated ethnic groups 16 . However, one study used causal inference analysis, supported the hypothesis that vitamin D plays a causal role in COVID-19 outcomes via modification of host responses to SARS-CoV-2 23 . In addition, there were also systematic reviews and meta-analyses to explore the association between vitamin D All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted August 13, 2020. . https://doi.org/10.1101/2020.08.09.20171280 doi: medRxiv preprint and COVID-19 9,10,24 . These studies provided a biological hypothesis and evolving epidemiological data supporting a role for vitamin D in COVID-19. But these results only based on the observational study design, which may be confounder bias. In our study, the SNPs associated with vitamin D as IVs were used to estimate the overall causal association of 25OHD concentration on COVID-19 susceptibility and severity, based on the MR design. The MR study could potentially avoid many biases and confounding issues existing in conventional observational studies and thus help to identify causally related risk factors. Using MR design, we found no evidence supporting that genetically predicted 25OHD concentration was significantly associated with COVID-19 susceptibility and severity.
There are some possible explanations for these negative findings. First, these null findings suggest that the associations of 25OHD concentration with COVID-19 susceptibility and severity could attribute from the reverse causation bias and confounder bias. Vitamin D from environment with across to adequate sunshine or diet was metabolized in the liver to 25OHD, which was used to determine a patient's vitamin D status 22, 25 . Vitamin D deficiency may be common in COVID-19 patients, as a consequence of quarantined and reduced outdoor behavior. The observed association between 25OHD concentration with increased risk of COVID-19 could be confounded by outdoor behavior which may be corrected with the genetic liability to COVID-19 22 . Our findings suggested that COVID-19 susceptibility and severity are expected to decrease the prevalence of vitamin D deficiency, which are needed to be proved by more bi-directional MR studies. Second, the association between the All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted August 13, 2020. However, we noted that there is a linearity assumption in our Mendelian randomization analyses 22 , then non-linear relationship could not be tested and might equate to the null hypothesis of no effect of the exposure on the outcome. Therefore, our results indicated that there might be no linear causal relationship of 25OHD concentration with COVID-19 susceptibility and severity.
The evidence of findings from MR studies sit at the interface between observational studies and RCTs 30 . Of the three study designs, RCTs provide interventions for disease, while observational studies and MR studies demonstrate the risk factors that increase the risk of disease. It should be focused on the effect of vitamin D supplementation on COVID-19. The previous findings suggest that vitamin D deficiency and treatment has a long-term effect on preventing overall mortality 12,31 .
In addition, future research should pay attention to not only the impact of vitamin D deficiency and treatment on the incidence of COVID-19, but also the impact of All rights reserved. No reuse allowed without permission.
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Using 25OHD concentration-related SNPs as IVs from GWAS data, the MR analysis results indicated that there might be no linear causal relationship of 25OHD All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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concentration with COVID-19 susceptibility and severity. In future, the bi-directional MR and non-linear MR study was needed to further prove these results. In addition, we should pay more attention to the randomization control trials about association between vitamin D treatment and the improvement of the COVID-19 in the long-term benefits.
All data generated or analyzed during this study are included in this published article and its supplementary information files.
All authors have approved the manuscript and its submission. No potential conflicts of interest were disclosed by the authors.
The study was supported by grants from the National Natural Science Foundation of China (81872682 and 81773527), and the China-Australian Collaborative Grant (NSFC 81561128020-NHMRC APP1112767).
The funding organization had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; or decision to submit the manuscript for publication.
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The copyright holder for this preprint this version posted August 13, 2020. Leave-one-out analysis: each row represents a MR analysis of 25OHD concentration on severe COVID-19 using all instruments expect for the SNP listed on the y-axis.
The point represents the odds ratio with that SNP removed and the line represents 95% (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted August 13, 2020. . https://doi.org/10.1101/2020.08.09.20171280 doi: medRxiv preprint
Leave-one-out analysis: each row represents a MR analysis of 25OHD concentration on COVID-19 using all instruments expect for the SNP listed on the y-axis. The point represents the odds ratio with that SNP removed and the line represents 95% Leave-one-out analysis: each row represents a MR analysis of 25OHD concentration on severe COVID-19 using all instruments expect for the SNP listed on the y-axis.
The point represents the odds ratio with that SNP removed and the line represents 95% (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted August 13, 2020. . https://doi.org/10.1101/2020.08.09.20171280 doi: medRxiv preprint 