key: cord-0949218-mxu92u4x authors: Cui, Z.; Tian, Y. title: The causal effect of serum vitamin D concentration on COVID-19 susceptibility, severity and hospitalization traits: a Mendelian randomization study date: 2021-03-13 journal: nan DOI: 10.1101/2021.03.08.21252901 sha: 777d48d4e0a07c3f11fd5400190dc25ecc8d4269 doc_id: 949218 cord_uid: mxu92u4x Background Evidence supporting the role of vitamin D in the coronavirus disease 2019 (COVID-19) pandemic remains controversial. Methods We performed a two-sample Mendelian randomization (MR) analysis to analyze the causal effect of the 25-hydroxyvitamin D [25(OH)D] concentration on COVID-19 susceptibility, severity and hospitalization traits by using summary-level GWAS data. The causal associations were estimated with inverse variance weighted (IVW) with fixed effects (IVW-fixed) and random effects (IVW-random), MR-Egger, weighted median and MR Robust Adjusted Profile Score (MR.RAPS) methods. We further applied the MR Steiger filtering method, MR Pleiotropy RESidual Sum and Outlier (MR-PRESSO) global test and PhenoScanner tool to check and remove single nucleotide polymorphisms (SNPs) that were horizontally pleiotropic. Results We found no evidence to support the causal associations between the serum 25(OH)D concentration and the risk of COVID-19 susceptibility (IVW-fixed: odds ratio [OR] = 0.9049, 95% confidence interval [CI] 0.8197~0.9988, p = 0.0473), severity (IVW-fixed: OR = 1.0298, 95% CI 0.7699~1.3775, p = 0.8432) and hospitalized traits (IVW-fixed: OR = 1.0713, 95% CI 0.8819~1.3013, p = 0.4878) using outlier removed sets at a Bonferroni-corrected p threshold of 0.0167. Sensitivity analyses did not reveal any sign of horizontal pleiotropy. Conclusions Our MR analysis provided precise evidence that genetically lowered serum 25(OH)D concentrations were not causally associated with COVID-19 susceptibility, severity or hospitalized traits. Our study therefore did not provide evidence assessing the role of vitamin D supplementation during the COVID-19 pandemic. High-quality randomized controlled trials are necessary to explore and define the role of vitamin D supplementation in the prevention and treatment of COVID-19. The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 4 2 (SARS-CoV-2) has struck globally and is exerting a devastating toll on humans [1] . As of February 12, 2021, 5 this emerging highly infectious disease has spread to six continents at light speed, and there have 6 been 107,252,265 confirmed cases of COVID-19, including 2,355,339 deaths, reported to the World Health 1 susceptibility, severity and hospitalization traits. Our approach relied upon summary-level GWAS data to obtain 2 MR estimates [11, 12] . We used all SNPs that strongly and independently (R 2 < 0.001) predicted exposures at 3 genome-wide significance (p < 5E−08). Proxy SNPs (R 2 > 0.9) from LDlink (https://ldlink.nci.nih.gov/) were 4 used when the SNPs were not available for the outcome [13] . The palindromic SNPs with intermediate allele 5 frequencies (palindromic SNPs referred to the SNPs with A/T or G/C alleles and "intermediate allele 6 frequencies" referred to 0.01 0.01 in 17 both data sets. Genotype data were quality-controlled and imputed to the Haplotype Reference Consortium and 1 performed. Information regarding the quality control and statistical analyses has been reported previously [17] . 2 We obtained estimates of the effect of the 25(OH)D concentration on COVID-19 by obtaining effect 3 coefficients from the above SNPs in GWAS meta-analyses from the COVID-19 Host Genetics Initiative 4 (COVID-19 HGI) [19] . The latest summary statistics were from the fifth round of GWAS meta-analysis shared 5 publicly on January 18,2021. Detailed information on participating studies, quality control, and analyses has 6 been provided on the COVID-19 HGI website (https://www.covid19hg.org/results/). The COVID-19 HGI used 7 different case/control definitions to identify genetic variants associated with COVID-19 susceptibility, disease 8 severity and hospitalized cases of European ancestry and all ancestries. In our study, we only included European 9 ancestry. We used a susceptibility phenotype that compared 38,984 confirmed COVID-19 cases, defined as 10 individuals with laboratory confirmation of SARS-CoV-2 infection based on nucleic acid amplification-or 11 serology-based tests or by electrical health records (using International Classification of Diseases [ICD] or 12 physician notes), chart review or self-reporting, with 1,644,784 controls enrolled in the cohorts and not included 13 as cases. To assess COVID- 19 in Tables S1-3 in the Supplementary Material. We extracted 114 independent SNPs from the 25(OH)D GWAS 20 as IVs for the three COVID-19 phenotypes. One SNP (rs182244780) was not available in the GWAS of the 21 hospitalized phenotype, and we did not find the proxy SNP from LDlink; therefore, it was left out of the analysis. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in [22] . We also calculated the Robust Adjusted Profile Score (MR.RAPS) to estimate the causal effects, which 20 can lead to considerably higher statistical power than the conventional MR analysis [24] . MR.RAPS considers 21 the measurement error in SNP-exposure effects and is unbiased when there are many weak instruments as well 22 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in (Table S7) . Then, to reduce the limitations of SNP numbers, we pruned SNPs to a R 2 of 20 0.01 and extracted 10 SNPs associated with 25(OH)D (Table S8) preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted March 13, 2021. ; https://doi.org/10.1101/2021.03.08.21252901 doi: medRxiv preprint 1 removed sets in the current study were associated with other phenotypes at risk of affecting the three COVID-19 2 phenotypes independent of the 25(OH)D concentration. We assessed SNPs at a threshold of p < 5E-08 for their 3 association with any other phenotypes. Using the PhenoScanner tool, we found that 35 SNPs (rs10454087, 4 rs1047891, rs10822145, rs12056768, rs12317268, rs1260326, rs142004400, rs1800588, rs182050989, 5 rs2037511, rs2131925, rs2229742, rs2418929, rs2642439, rs2756119, rs28407950, rs2952289, rs3745669, 6 rs41301394, rs429358, rs4418728, rs4616820, rs4846917, rs512083, rs532436, rs5771043, rs58387006, 7 rs6834488, rs71297391, rs73413596, rs804281, rs8063565, rs8107974, rs964184 and rs9861009) were 8 significantly associated with hematological traits (e.g., white blood cell count, platelet count and granulocyte perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted March 13, 2021. performed on a population of approximately 173,000 individuals of European ancestry, excluding those with 1 blood cancers or major blood disorders, largely from the UK. The significance threshold was set using 2 Bonferroni correction at p < 4.90E-04 (0.05/[34×3]). We excluded SNPs significantly associated with blood cell 3 traits if causal links were identified between blood cell traits and COVID-19 phenotypes. As we mentioned above, outlier removed sets included 106,109 and 109 SNPs, respectively ( Table 1) Table 1 ). The results did not provide evidence for the causal effects, and 21 were stable and consistent using the Steiger filtered (IVW-fixed: OR = 0.9307, 95% CI 0.8437~1.0268, p = 22 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in We performed "leave-one-out" analysis based on the IVW-random method using the outlier removed sets for 18 three COVID-19 phenotypes and found that there was no potentially influential SNP driving the causal link, and 19 the result was stable (Tables S9-11 (Tables S7-8) . To avoid horizontal pleiotropy, we performed univariable two-sample MR 22 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted March 13, 2021. ; https://doi.org/10.1101/2021.03.08.21252901 doi: medRxiv preprint 1 IVW-fixed and IVW-random methods, we found no evidence to report the significant causal associations 2 between 34 hematological traits and three COVID-19 phenotypes (p＜4.90E-04) (e.g., white blood cell count 3 with COVID-19 susceptibility, beta = 0.0016, IVW-fixed p = 0.0650, IVW-random p = 0.0629) (Tables S12-14). 4 Therefore, we did not remove those 35 SNPs. 5 Table 3 There is no doubt that immune modulation is the fulcrum of most diseases, and COVID-19 is no exception. Vitamin D has many mechanisms to modulate immunity, such as physical barriers and innate and adaptive In this MR study, we used the strong IVs from the summary statistics of the largest GWAS conducted for 9 vitamin D and COVID-19 phenotypes in European populations. We aimed to determine whether the relationship 10 between 25(OH)D and COVID-19 was causal by employing a range of two-sample MR methods. We employed 11 a range of methods known to control for pleiotropy, checked the heterogeneity and obtained highly consistent 12 results. Pleiotropic effects were detected by using the MR-Egger intercept and MR-PRESSO method. Using the 13 MR design, we could mitigate the confounding factors due to the application of Mendel's second law of the 14 random assortment of alleles. Reverse causality was also prevented because genetic variants were fixed at 15 conception and cannot be affected by disease processes. We used the PhenoScanner tool to detect potential 16 pleiotropic SNPs. Some SNPs were found to be associated with hematological parameters. We then performed 17 univariable MR and found no evidence to support the causal association between hematological parameters and 18 COVID-19 traits. The results above showed that the presence of pleiotropic SNPs was minimal. Taken together, 19 our MR results did not support the evidence that the serum 25(OH)D concentration was a causal factor for 20 COVID-19 susceptibility, severity or hospitalization traits. Our study therefore did not provide evidence 21 assessing the role of vitamin D supplementation during the pandemic. All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in Despite the MR design being less susceptible to confounding than other observational studies, limitations still 17 exist. First, potential pleiotropy is the common limitation on all MRs, and our results may still have been 18 affected by unmeasured horizontal pleiotropy. To assess this bias, we assessed potential pleiotropy using the 19 MR-Egger method and MR-PRESSO method. We also used MR Steiger filtering and the PhenoScanner tool and 20 observed no evidence that pleiotropic SNPs existed. Hence, whereas the risk of a residual horizontal pleiotropic 21 effect cannot be ruled out, we still believe it is unlikely to change the conclusions of this study in a clinically 22 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted March 13, 2021. ; https://doi.org/10.1101/2021.03.08.21252901 doi: medRxiv preprint meaningful way. Second, the low power of MR might be the reason for null results rather than a true null. The authors declare that they have no competing interests. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted March 13, 2021. ; https://doi.org/10.1101/2021.03.08.21252901 doi: medRxiv preprint Pathophysiology, transmission, diagnosis, 2 and treatment of Coronavirus disease 2019 (COVID-19): A review London: National Institute for Health and Care Excellence (UK). 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