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Genetic association between serum 25-hydroxyvitamin D levels and functional outcome after ischemic stroke

BMC Neurology volume 24, Article number: 467 (2024) Cite this article

Abstract

Background

Previous observational studies suggest that serum 25-hydroxyvitamin D [25(OH)D] levels may associate with functional outcome after ischemic stroke. However, the causal relationship is not yet defined. Consequently, this study employed two-sample Mendelian Randomization (MR) to elucidate the causal association between serum 25(OH)D levels and functional outcome after ischemic stroke.

Methods

The genome-wide association study (GWAS) for serum 25(OH)D levels included 417,580 patients of European descent. The GWAS for functional outcome after ischemic stroke is from Genetics of Ischemic Stroke Functional Outcome meta-analysis. Post-stroke outcomes were evaluated using two sets of binary categorical variables (0–2 vs. 3–6 and 0–1 vs. 2–6), and also as ordered modified Rankin Scale (mRS) variables at 3 months. Two-sample MR was used to assess whether the exposure causally affects the outcome. Inverse Variance Weighted (IVW) was the primary method for our MR analysis, which was further supported by sensitivity analyses to ensure the robustness of the results.

Results

The primary analysis using IVW method indicated no significant casual association between serum 25(OH)D levels and mRS (0–1) vs. mRS (2–6) at 3 months (OR = 0.914, 95% CI: 0.587–1.422, P = 0.69), mRS (0–2) vs. mRS (3–6) at 3 months (OR = 0.699, 95% CI: 0.477–1.025, P = 0.07), and mRS at 3 months (OR = 1.202, 95% CI: 0.875–1.650, P = 0.26). Additionally, after adjusting for stroke severity, the IVW analysis also showed no significant association between serum 25(OH)D levels and mRS (0–1) vs. mRS (2–6) at 3 months (OR = 0.93, 95% CI: 0.55–1.56, P = 0.78), mRS (0–2) vs. mRS (3–6) at 3 months (OR = 0.73, 95% CI: 0.47–1.13, P = 0.16), and mRS at 3 months (OR = 1.09, 95% CI: 0.77–1.54, P = 0.62). The results of the sensitivity analysis suggest that our findings are robust.

Conclusion

Our MR study offers no genetic evidence supporting the effects of serum 25(OH)D levels on functional outcomes after ischemic stroke. The associations observed in previous observational studies may be attributed to residual confounding factors and reverse causality.

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Introduction

25-hydroxyvitamin D [25(OH)D] is a prehormone that is the primary circulating form of vitamin D in the blood, used as a marker for overall vitamin D status [1]. It is produced in the liver by hydroxylation of vitamin D3 (cholecalciferol) and D2 (ergocalciferol) [2]. The association between serum 25(OH)D levels and stroke risk has been well-documented through large meta-analysis and Mendelian randomization (MR) analyses [3,4,5]. However, previous observational studies present conflicting results regarding the relationship between serum 25(OH)D levels and the functional outcome of stroke. While some research identifies serum 25(OH)D levels as an independent predictor of post-stroke outcome, suggesting that lower levels might confer a worse outcome of stroke, other studies report no significant association, raising concerns about its causal role [6,7,8,9]. These discrepancies can largely be attributed to the inherent limitations of observational research, such as the potential for reverse causation and the presence of confounding factors, which challenge the establishment of a direct causal relationship [10]. Moreover, the observational nature of these studies makes it difficult to discern whether serum 25(OH)D levels are a precursor or a consequence of post-stroke outcome, thereby complicating the interpretation of results.

MR is a methodological approach in epidemiology that leverages genetic variants as instrumental variables (IVs) to infer causal relationships between modifiable risk factors and health outcomes [11]. This method draws on the random assortment of genes from parents to offspring during gamete formation and fertilization, an inherently random process that mimics the random assignment in randomized controlled trials (RCTs) [11, 12]. By using genetic variants associated with potential risk factors as proxies, MR can help estimate the causal effect of a risk factor on an outcome in a way that is less susceptible to confounding. One of the fundamental advantages of MR is its ability to mitigate the issues of reverse causation and confounding factors that frequently plague observational studies [12, 13]. Since genetic variants are fixed at conception and are not influenced by disease states or environmental factors that may affect the outcome, they serve as robust tools for assessing causality. Recently, genome-wide association studies (GWASs) for serum 25(OH)D levels and functional outcome after ischemic stroke have been extensively applied in MR analyses. In the MR analysis of GWAS examining functional outcomes after ischemic stroke, a genetically predicted higher waist-to-hip ratio was associated with unfavorable outcomes [14]. Moreover, additional MR findings on GWAS of serum 25(OH)D levels indicated a nonlinear relationship between serum 25(OH)D levels and risk of sarcopenia [15].

In this study, we employed two-sample MR to investigate the relationship between serum 25(OH)D levels and functional outcome after ischemic stroke.

Methods

Data availability, ethics statement, process software and standard protocol approvals

This MR analysis was conducted using data from GWASs that are both published and publicly accessible. Ethical approval and informed consent were obtained for all participants as per the protocols detailed in the original publications and consortia associated with each study. The MR analysis was executed using the Two-Sample MR (version 0.6.1) in R (version 4.3.3). This study adheres to the guidelines outlined in the Strengthening the Reporting of Observational Studies in Epidemiology–Mendelian Randomization (STROBE-MR) statement [16].

Data sources

For exposure, we obtained summary statistics for serum 25(OH)D concentrations in a cohort of 417,580 European individuals [17]. The study identified 143 independent loci across 112 distinct 1-Mb regions, with adjustments made for factors such as age, sex, assessment month, assessment center, supplement intake, genotyping batch, and principal components. For outcome, the GWAS study analyzed functional outcome after ischemic stroke using data from 12 distinct studies across Europe, the United States, and Australia [18]. The primary endpoint was the assessment of the modified Rankin Scale (mRS) score, conducted as close to 90 days as possible [19]. The majority of the included studies (approximately 80%) evaluated the mRS at three months, with a variation of ± 2 weeks. Post-stroke outcomes were evaluated using two sets of binary categorical variables (0–2 vs. 3–6 and 0–1 vs. 2–6), and also as ordered mRS variables. The sample sizes for these comparisons were: mRS 0–2 vs. 3–6 had 3,741 and 2,280 participants respectively; mRS 0–1 vs. 2–6 had 1,796 and 2,567; and the ordinal mRS included 6,021 participants in total. Lower mRS scores indicate better functional outcomes. The study controlled for age, sex, ancestry, and baseline stroke severity, as measured by the National Institutes of Health Stroke Scale (NIHSS). Additionally, analyses were also conducted without adjusting for baseline NIHSS scores. The detailed clinical features of the patients are thoroughly described in the previous publication [20], which can be accessed at the following URL: https://journals.sagepub.com/doi/https://doiorg.publicaciones.saludcastillayleon.es/10.1177/2396987317704547.

Study design

We employed two-sample MR to examine the causal relationship between serum 25(OH)D levels and the functional outcome after ischemic stroke. The three core hypotheses underpinning two-sample MR are as follows [21]: (1) Relevance Hypothesis—The genetic variants selected as IVs must demonstrate a robust association with the exposure of interest. (2) Independence Hypothesis—These genetic variants should be independent of any confounders that influence both the exposure and the outcome. (3) Exclusion Restriction Hypothesis—The genetic variants must influence the outcome exclusively through their effect on the exposure, without any alternative pathways involved. The study design is shown in Fig. 1.

Fig. 1
figure 1

An overview of the study design

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Selection of genetic instrumental variables

To enhance the selection of robust IVs for our MR analyses, we implemented stringent criteria for the selection of Single nucleotide polymorphisms (SNPs). These criteria were meticulously designed to ensure the reliability and validity of our instruments, detailed as follows [22]: (1) Genome-wide Significance: We required that SNPs meet a threshold of genome-wide significance with a p-value less than 5 × 10− 8. This criterion helps to ensure that the associations detected are not due to random chance and are likely to be true genetic associations. (2) Linkage Disequilibrium (LD) Independence: SNPs were selected to ensure independence in LD. Specifically, we required that SNPs exhibit an r2 less than 0.001 within a 10 Mb window. This condition minimizes the risk of bias from correlated alleles influencing the estimates. (3) Instrument Strength: We imposed an F-statistic threshold, selecting SNPs with an F-statistic greater than 10. This threshold ensures that the genetic instruments are strong and reduce the risk of weak instrument bias, which can lead to biased and inconsistent estimator performance [23, 24]. (4) SNP Harmonization: To further ensure the validity of our analysis, we removed non-concordant and palindromic SNPs [25]. This step prevents issues related to allele mis-specification and ambiguity, which are critical for accurate MR analysis.

Statistical analysis

To rigorously investigate the causal relationships between serum 25(OH)D levels and functional outcome after ischemic stroke, we employed five distinct MR methods, each designed to provide robust insights while addressing specific statistical challenges. These methods are outlined as follows: (1) Inverse Variance Weighted (IVW): This method used the inverse variance of each SNP-associated risk estimate to provide a weighted average of the causal estimates. It is particularly effective under the assumption that all genetic variants are valid instruments, offering a conventional approach to MR analysis. Therefore, IVW method is main method of our MR analysis [26]. (2) MR-Egger: This method extends beyond the standard IVW method by allowing for the presence of pleiotropic effects among the IVs. MR-Egger includes an intercept term in the regression model, which tests for and, if present, corrects for directional pleiotropy, thereby providing an unbiased estimate even when some genetic variants are invalid instruments [27]. (3) Weighted Median: This approach offers a robust estimate of the causal effect that remains consistent even if up to 50% of the information comes from invalid instruments. By using the median of the weighted distribution of causal estimates from each SNP, this method ensures that the result is less sensitive to outliers or skewed data, providing a more reliable estimate under certain violations of the IV assumptions [28]. (4) Simple Mode: This method involves estimating the causal effect based on the most commonly observed causal estimate across the SNPs, serving as a mode-based approach. It is particularly useful when there is heterogeneity in the causal estimates or when the effect sizes are non-normally distributed, as it emphasizes the most frequently occurring estimate across the IVs [29]. (5) Weighted Mode: An extension of the Simple Mode, the Weighted Mode method weights each mode estimate by the inverse of its variance, potentially providing a more accurate estimation when dealing with a heterogeneous set of IVs. This method enhances the robustness of the mode-based estimate by giving greater emphasis to more precisely estimated effects [29].

To comprehensively address heterogeneity, detect potential pleiotropic effects, and ensure the robustness of our causal inferences, we implemented five sensitivity analyses within our MR framework. These sensitivity analyses are outlined as follows: (1) Cochran’s Q Test: This test is used to assess heterogeneity among the IVs. It provides a statistical measure to evaluate whether the variations in the causal estimates across different genetic variants are greater than what would be expected by chance. A significant result suggests the presence of heterogeneity, which may influence the validity of the causal inference [30]. (2) Intercept of MR-Egger: The intercept term in the MR-Egger regression serves as a test for directional pleiotropy. If the intercept significantly deviates from zero, it indicates the presence of pleiotropic effects among the IVs that could bias the MR estimates. This analysis is crucial for assessing the assumption that the genetic variants affect the outcome only through their impact on the exposure [27]. (3) Mendelian Randomization Pleiotropy RESidual Sum and Outlier (MR-PRESSO): This method detects and corrects for outliers in MR analyses that may be due to pleiotropic effects. MR-PRESSO tests for global pleiotropy and, if detected, can adjust the causal estimate by removing outlier SNPs, thereby enhancing the validity of the MR conclusions [31]. (4) Leave-One-Out Analysis: This robustness check involves recalculating the MR estimate multiple times, each time excluding one SNP at a time from the analysis. This method helps to identify if any single SNP disproportionately influences the overall causal estimate, thus ensuring that the results are not unduly driven by a few influential genetic variants [32]. (5) Funnel Plot: Employed to visually assess the presence of asymmetry in the distribution of causal estimates against their precision. A symmetric funnel plot suggests that the MR results are free from bias related to the size or direction of individual SNP effects, while asymmetry might indicate potential biases such as publication bias or pleiotropy [33].

Results

Genetic instruments variables for serum 25(OH)D levels and functional outcome after ischemic stroke

We identified 117 independent (r2 < 0.001 and KB = 10 MB) and genome-wide significant (P < 5 × 10− 8) SNPs as IVs from the serum 25(OH)D levels GWAS. These SNPs exhibited F-statistics ranging from 25.60 to 1267.58. All these SNP data are cataloged in Table 1.

Primary analysis

Figures 2 and 3 display the primary results of our MR analyses. In the MR analyses, the primary analysis using IVW method indicated no significant casual association between serum 25(OH)D levels and mRS (0–1) vs. mRS (2–6) at 3 months (OR = 0.914, 95% CI: 0.587–1.422, P = 0.69), mRS (0–2) vs. mRS (3–6) at 3 months (OR = 0.699, 95% CI: 0.477–1.025, P = 0.07), and mRS at 3 months (OR = 1.202, 95% CI: 0.875–1.650, P = 0.26). The conclusions from MR-Egger, Weighted Media, Simple Mode, and Weight Mode were consistent with the IVW findings (Fig. 2). Similarly, after adjusting for stroke severity, the IVW analysis also showed no significant association between serum 25(OH)D levels and mRS (0–1) vs. mRS (2–6) at 3 months (OR = 0.929, 95% CI: 0.553–1.561, P = 0.78), mRS (0–2) vs. mRS (3–6) at 3 months (OR = 0.733, 95% CI: 0.474–1.133, P = 0.16), and mRS at 3 months (OR = 1.090, 95% CI: 0.773–1.539, P = 0.62), with other MR methods yielding consistent results (Fig. 3).

Fig. 2
figure 2

MR estimates and Forest plots of serum 25(OH)D levels and functional outcome after ischemic stroke without adjusting and adjusting for stroke severity

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Fig. 3
figure 3

The scatter plots visualizing the association between serum 25(OH)D levels and functional outcome after ischemic stroke displays five lines, each representing different MR methods: inverse-variance weighted, MR—Egger, and weighted median, simple mode and weight mode. Serum 25(OH)D levels and mRS (0–1) vs. mRS (2–6) at 3 months without adjust for stroke severity (A) and adjust for stroke severity (D); Serum 25(OH)D levels and mRS (0–2) vs. mRS (3–6) at 3 months without adjust for stroke severity (B) and adjust for stroke severity (E); Serum 25(OH)D levels and mRS at 3 months without adjust for stroke severity (C) and adjust for stroke severity (F)

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Sensitivity analyses

Our sensitivity analysis revealed significant heterogeneity in the association betwenn serum 25(OH)D levels and mRS (0–1) vs. mRS (2–6) at 3 months adjusting for stroke severity (P < 0.05, Table 1). By using a random effects model with the IVW method, we minimized the impact of significant heterogeneity. Furthermore, the MR-PRESSO Global Test indicated significant directional pleiotropy (P = 0.004). Other sensitivity analyses showed no significant heterogeneity or horizontal pleiotropy (Table 1). Both the leave-one-out test and the funnel plots confirmed the reliability of our results (eFigures 1 and 2).

Table 1 Cochran’s Q test, MR-Egger Intercept and MR-PRESSO of MR analysis for serum 25-Hydroxyvitamin D levels and post-stroke outcome at 3 months
Full size table

Discussion

In the present MR study, we initially examined the causal relationship between serum 25(OH)D concentration and functional outcome after ischemic stroke. Our findings indicate that there is no casual association between serum 25(OH)D concentration and post-stroke outcome at 3 months. Sensitivity analyses confirm the robustness of our results.

Research findings from observational studies on serum 25(OH)D levels and functional outcome of ischemic stroke remain inconsistent. An observational study by Zeng et al., involving 668 stroke patients followed for five years, found that patients with serum 25(OH)D levels below 38.4 nmol/L had a higher risk of poor prognosis [6]. Another retrospective study reported that low serum 25(OH) D levels (≤ 50 nmol/L) were associated with poor functional independence at discharge [7]. The potential mechanisms through which 25(OH)D exerts neuroprotective effects include modulating the immune response, reducing inflammation, and protecting against neuronal injury [34]. However, a meta-analysis encompassing 10 studies indicated that supplementation with 25(OH)D did not improve long-term functional recovery post-stroke [8]. Similarly, a prospective clinical study by Merdin et al. found that while admission NIHSS scores correlated with 25(OH)D levels, they were not predictive of 3-month and 6-month mRS outcomes [9]. These studies are consistent with our conclusions. Nevertheless, observational studies face limitations due to confounding factors and reverse causation, where unmeasured variables may affect outcomes, and the inability to establish real causality [10]. In contrast, MR uses genetic variants as IVs to overcome these limitations and establish precise causal relationships [11].

Our MR results suggest that serum 25(OH) D levels, despite their known biological roles, might not directly influence the functional outcome after ischemic stroke. Clinically, this might support prioritizing other established modifiable risk factors for heart management, such as blood pressure control, lipid management, and lifestyle modifications, over vitamin D supplementation which might not have a clear benefit in this context.

Our study has several strengths. The use of a two-sample MR design substantially reduces confounding and reverse causation, which are common limitations of traditional observational studies, and mitigates issues related to sample overlap and population duplication. The inclusion of large sample sizes in the GWAS for serum 25(OH)D concentrations has increased our statistical power, allowing for more precise and reliable estimates. We employed IVs that are not only statistically significant and independent but also demonstrate strong predictive power. This careful selection of instruments further strengthens the validity of our causal inferences. Importantly, our analysis did not reveal evidence of significant horizontal pleiotropy in MR—Egger method, suggesting that the associations observed are likely driven by the exposure of interest rather than by alternative biological pathways.

However, this study also presents several limitations. One limitation is the absence of data on functional outcome of ischemic stroke subgroups. The lack of stratification by ischemic stroke subtypes limits our ability to assess whether the associations observed are consistent across different subtypes of ischemic stroke, which may have distinct etiologies and pathophysiological mechanisms. Second, the sample size of the datasets for functional outcome of ischemic stroke we analyzed may have been insufficient. A larger sample size would likely yield more definitive conclusions. Third, our analysis predominantly focused on populations of European ancestry, which restricts the generalizability of our findings. It is crucial to extend this research to include diverse populations, particularly Asian cohorts, to better understand the relationship between plasma 25(OH)D levels and functional outcome of stroke across different ethnic and genetic backgrounds. Finally, we observed significant heterogeneity in some of our results, which may reflect differences in study design, population characteristics, or other underlying factors. To address this, we applied a random effects model to mitigate potential biases. However, the presence of heterogeneity still warrants cautious interpretation of the findings, as it may indicate underlying complexities in the relationship that are not fully captured by our analysis.

Conclusion

In conclusion, our two-sample MR analysis found no causal effect of serum 25(OH)D levels on functional outcome after ischemic stroke using recent large-scale GWAS. The associations previously reported in observational studies may be attributed to unmeasured confounding factors and reverse causality.

Data availability

In the present study, all GWAS databases were publicly available. These datasets are publicly available and can be found at the following URL: https://cd.hugeamp.org/downloads.html (post-stroke outcome) and https://www.nature.com/articles/s41467-020-15421-7 [serum 25(OH)D levels].

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Acknowledgements

We express our gratitude to all the researchers who contributed to this MR study, and we appreciate the institutions and respective researchers who generously provided the data for this study.

Funding

This work is supported by Medical Science and Technology Project of Zhejiang Province (2022KY506 and 2024KY025), Zhejiang Provincial Natural Science Foundation of China (LTGY24H090014), and Wenzhou Science and Technology Bureau Public Welfare Technology Research Medical Project (Y20190005).

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Author notes

  1. Yudan Wu and Tianyu Jin contributed equally to this work.

Authors and Affiliations

  1. Department of Rehabilitation, Ningbo No.6 Hospital, Ningbo, Zhejiang, China

    Yudan Wu

  2. Department of Rehabilitation Medicine, the Second Affiliated Hospital, Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

    Tianyu Jin & Qiongyi Pang

  3. Center for Rehabilitation Medicine, Department of Neurology, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, Zhejiang, China

    Yifan Cheng

Authors
  1. Yudan Wu
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  2. Tianyu Jin
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Contributions

Yudan Wu and Tianyu Jin were responsible for data extraction, analysis, and drafting the initial manuscript, while the remaining authors were involved in statistical analysis and providing feedback for revising the manuscript. Yifan Cheng designed the study. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Yifan Cheng.

Ethics declarations

Ethics approval and consent to participate

Each participant received ethical approval and informed consent for the respective study. This study used publicly available GWAS data. For serum 25(OH)D levels, the data was obtained from the UK Biobank under the generic approval of the NHS National Research Ethics Service (projects 12505 and 10214). For the functional outcome of ischemic stroke, all participants provided written informed consent, and for those unable to communicate, consent was obtained from their next of kin. Local ethics committees approved the original studies, which ensured that ethical approvals and participant consents were in place. All data were complied with relevant data-sharing policies. Consequently, no additional ethical approval was required for this secondary analysis.

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No conflict of interest exists in the submission of this manuscript, and the manuscript is approved by all authors for publication.

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The authors declare no competing interests.

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Wu, Y., Jin, T., Pang, Q. et al. Genetic association between serum 25-hydroxyvitamin D levels and functional outcome after ischemic stroke. BMC Neurol 24, 467 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12883-024-03972-x

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12883-024-03972-x

Keywords

BMC Neurology

ISSN: 1471-2377

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