Association of lipid-lowering drugs with COVID-19 outcomes from a Mendelian randomization study

Background: Lipid metabolism plays an important role in viral infections. We aimed to assess the causal effect of lipid-lowering drugs (HMGCR inhibitiors, PCSK9 inhibitiors, and NPC1L1 inhibitior) on COVID-19 outcomes using two-sample Mendelian randomization (MR) study. Methods: We used two kinds of genetic instruments to proxy the exposure of lipid-lowering drugs, including expression quantitative trait loci of drugs target genes, and genetic variants within or nearby drugs target genes associated with low-density lipoprotein (LDL cholesterol from genome-wide association study). Summary-data-based MR (SMR) and inverse-variance-weighted MR (IVW-MR) were used to calculate the effect estimates. Results: SMR analysis found that a higher expression of HMGCR was associated with a higher risk of COVID-19 hospitalization (odds ratio [OR] = 1.38, 95% confidence interval [CI] = 1.06–1.81). Similarly, IVW-MR analysis observed a positive association between HMGCR-mediated LDL cholesterol and COVID-19 hospitalization (OR = 1.32, 95% CI = 1.00–1.74). No consistent evidence from both analyses was found for other associations. Conclusions: This two-sample MR study suggested a potential causal relationship between HMGCR inhibition and the reduced risk of COVID-19 hospitalization. Funding: Start-up Fund for high-level talents of Fujian Medical University.


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The COVID-19 pandemic has caused millions of infections and deaths, which is caused 55 by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 56 Lacking drugs specifically targeted to SARS-CoV-2 infection has led to a great interest to 57 identify drugs that can be repurposed to reduce the infection and mortality of the disease. 58 Available studies have suggested an important role of lipid metabolism in viral 59 infections, including in the pathogenesis of SARS-CoV-2 infection (Proto et al., 2021). 60 The plausible mechanisms include the involvement of host lipids in the virus life cycle, 61 the influence of cholesterol on the immune cell functions, interfering with the mevalonate 62 pathway, and so on (Proto et al., 2021). Such evidence indicates the potential protective 63 effect of lipid-lowering drugs against COVID-19. HMG-CoA Reductase (HMGCR) 64 inhibitors, known as statins, are the most commonly-used class of lipid-lowering drugs, 65 which have a couple of predominant merits, such as the well-proven safety, low cost, and 66 pleiotropic effects. Proprotein convertase subtilisin/kexin type 9 (PCSK9) and Niemann-67 Pick C1-Like 1 (NPC1L1) are proteins playing a crucial role for the circulating level of 68 low-density lipoprotein cholesterol (LDL-C) (Sabatine, 2019;Williams et al., 2020). 69 Both PCSK9 inhibitors (i.e,. evolocumab and alirocumab) and NPC1L1 inhibitors (i.e., 70 ezetimibe) are FDA-approved lipid-lowering agents (Sabatine, 2019;Williams et al., 71 2020). A number of observational studies have investigated the association between lipid-72 lowering drugs and COVID-19 outcomes, but generated mixed results (Butt et al., 2020;73 Hariyanto and Kurniawan, 2020; Kow and Hasan, 2020;Zhang et al., 2020;Gupta et al., 74 2021). What's more, confounding bias and reverse causation cannot be avoided in most 75 of these studies. 76 Mendelian randomization (MR) study uses genetic variants as an instrument to perform 77 causal inference between an exposure and an outcome, which could indicate whether an 78 observational association is consistent with a causal effect (Davies et al., 2018     For SMR method, the heterogeneity in dependent instruments (HEIDI) test was used to 158 test if the observed association between gene expression and outcome was due to a 159 linkage scenario, which was performed in the SMR software (Zhu et al., 2016). The 160 HEIDI test of P<0.01 indicates that association is probably due to linkage (Chauquet et 161 al., 2021). One SNP could be related to the expression of more than one genes, leading 162 to the presence of horizontal pleiotropy. To assess the risk of horizontal pleiotropy, we 163 identified other nearby genes (within a 1Mb window), the expression of which was 164 significantly associated with the genetic instrumental variant, and performed SMR 165 analysis to examine if the expression of these genes was related to the COVID-19 166 outcomes.

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For IVW-MR method, we tested the heterogeneity by using a Cochran Q test, where 168 P<0.05 indicates the evidence of heterogeneity (Higgins et al., 2003). MR-Egger test indicates the presence of horizontal pleiotropic outliers (Verbanck et al., 2018).

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Besides, a multivariable MR study was further conducted to examine if the observed 177 association was direct association. We first investigated the association of HMGCR-178 mediated LDL cholesterol with the common risk factors of COVID-19 hospitalization, 179 including body mass index, diabetes, hypertension, and coronary heart disease. After that, 180 a multivariable MR study was performed by adjusting for the factors which showed a 181 significant association. All these analyses were implemented in R software, version 4.1.0.

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To account for multiple testing, Bonferroni correction was used to adjust the thresholds 183 of significance level, thus a strong evidence was suggested for P<0.006 (3 exposures and 184 3 outcomes) and a suggestive evidence of 0.006≤P<0.05.

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A total of 168, 24, and 11 cis-eQTLs were identified from eQTLGen or GTEx 188 Consortium for drugs target gene HMGCR, PCSK9, and NPC1L1, respectively, and the 189 most significant cis-eQTL SNP was selected as a genetic instrument for the target gene of 190 each drug (  (Supplementary File 1-Tables 2 and 3). Positive control study showed significant  File 1-Table 7). Only four genes have available eQTLs at a genome-    Compared to developing a new drug, repurposing an old drug is much more economical 256 and time-saving, in particular, the importance is further highlighted during a pandemic.

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COVID-19 pandemic has driven a number of studies of drug repurposing (Fajgenbaum 258 and Rader, 2020; Gaziano et al., 2021). The role of lipid metabolism in viral infections 259 has raised interest regarding the possibility of repurposing lipid-lowering drugs as anti-260 COVID-19 agents (Proto et al., 2021). As one of the most commonly prescribed drugs, 261 statins have received the greatest attention for their pleiotropic effects, including lowering 262 serum cholesterol, anti-inflammatory and immunomodulatory properties and 263 antithrombotic effect, all of which play a role in viral infections (Fajgenbaum and Rader,264 2020; Proto et al., 2021;Rubin, 2021). Emerging observational studies have investigated 265 if statins might benefit patients with COVID-19 (Butt et al., 2020;Hariyanto and 266 Kurniawan, 2020; Kow and Hasan, 2020;Zhang et al., 2020;Gupta et al., 2021). A 267 largest retrospective cohort study including 13,981 patients admitted to hospital due to 268 COVID-19 suggested a significant reduction in 28-day all-cause mortality by 42% in the 269 group with statins than patients without statins (Zhang et al., 2020). A meta-analysis with 270 8,990 COVID-19 patients also found a 30% lower risk of fatal or severe disease (Kow 271 and Hasan, 2020). However, a Danish nationwide cohort study with 4,842 COVID-19 272 patients and a meta-analysis with 3,449 COVID-19 patients did not find the association 273 between statins use and improved COVID-19 outcomes (Butt et al., 2020;Hariyanto and 274 Kurniawan, 2020). In addition, in these studies, considerable differences in clinical 275 characteristics cannot be avoided between patients with and without statins, and causal 276 inference is not allowed due to the retrospective nature of observational studies.

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As a genetic epidemiological method, MR study could overcome the limitations of 278 traditional observational studies. In this MR study, we used genetic variants related to 279 HMGCR expression or HMGCR-mediated LDL cholesterol as instruments to proxy the 280 exposure of statins. Both analyses found a suggestive evidence that HMGCR inhibition 281 could reduce the risk of COVID-19 hospitalization, rather than COVID-19 susceptibility 282 and very severe outcome. Although strong evidence is lacking, these results provided a 283 causal evidence supporting the finding from the largest cohort study (Zhang et al., 2020), 284 which calls for additional observational studies in different populations, mechanistic 285 studies, and randomized controlled studies to examine its potential effect against COVID-286 19. Patients with COVID-19 who already take statins or start to take it for the indication 287 of statins were recommended to continue to take it, which might be beneficial to both its 288 original indication and COVID-19 (Rubin, 2021). And statins might be a prioritized drug The main strength of our study is the use of genetic instruments to proxy drug exposure, 296 which could minimize confounding bias and avoid reverse causation. Besides, we used 297 two different kinds of genetic instruments to proxy the studied drug, which contributes to 298 validating the effect estimates from each other. A number of sensitivity analyses have 299 been performed to test the efficacy of genetic instruments and the assumptions of MR 300 study.

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This study has several limitations. Firstly, there are no available eQTLs in blood for 303 NPC1L1, so we were not able to explore the association between NPC1L1 expression in 304 blood and COVID-19 outcomes. Besides, there are no available eQTLs in liver (the main 305 tissue related to lipid metabolism) for these target genes, which might provide more 306 convincing evidence of the observed association. The sample size of eQTL study for 307 PCSK9 and NPC1L1 in GTEx is relatively smaller, which may affect the statistical 308 power for the results of PCSK9 or NPC1L1 inhibition. Secondly, the effect of statins 309 probably varies between subgroups, for example, it may be more effective in patients 310 with chronic diseases (e.g., coronary heart disease). However, the use of summary-level 311 data did not allow us to perform subgroup analyses, so further MR study with individual-312 level data is needed to provide more detailed information. Thirdly, the Bonferroni 313 correction for multiple tests suggests that we cannot rule out the false-positive possibility 314 for the finding of the protective effect of statins on COVID hospitalization. Fourthly, 315 confounding bias and/or horizontal pleiotropy cannot be completely excluded although 316 we have performed various sensitivity analyses to test the assumptions of MR study.

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Fifthly, both eQTLs and GWAS data used in this study were predominantly obtained 318 from European population ancestry, thus these findings should be interpreted with 319 caution when generalizing to other populations.

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In conclusion, this MR study suggested a causal relationship between HMGCR inhibition