Bidirectional analysis of gastroesophageal reflux disease and migraine using two-sample Mendelian randomization

Bidirectional analysis of gastroesophageal reflux disease and migraine using two-sample Mendelian randomization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bidirectional analysis of gastroesophageal reflux disease and migraine using two-sample Mendelian randomization Xin Jin, Jianhua Zhuang, Jin Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4897548/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Epidemiological studies suggest a link between gastroesophageal reflux disease (GERD) and migraine, but the causal relationship remains unclear. This study aimed to clarify this relationship using two-sample Mendelian Randomization (MR). Methods Data on GERD and migraine, including subtypes with aura (MA) and without aura (MO), were collected from genome-wide association studies (GWAS). SNPs were selected as instrumental variables (IVs) by accounting for linkage disequilibrium and removing unbalanced connections. The primary analysis used the inverse variance-weighted (IVW) method with supplementary analyses. Heterogeneity and pleiotropy were assessed using Cochran's Q test, MR-Egger intercept, and MR-PRESSO. Finally, reverse causality was explored. Results The IVW method indicated a causal link between GERD and increased risk of migraine (OR = 1.381, 95% CI: 1.190–1.602, p = 2.04E-05), particularly the MO subtype (OR = 1.600, 95% CI: 1.311–1.953, p = 3.67E-06). No significant association was found for MA (OR = 1.193, 95% CI: 0.983–1.449, p = 0.074). Reverse MR analysis showed no causal relationship between migraine and GERD. Conclusion GERD is causally linked to an increased risk of migraine, especially the MO subtype. No reverse causal relationship was found, highlighting the importance of considering migraine subtypes in understanding their association with GERD. Health sciences/Neurology/Neurological disorders/Migraine Health sciences/Gastroenterology/Gastrointestinal diseases/Oesophageal diseases/Gastro oesophageal reflux disease Migraine mendelian randomization gastroesophageal reflux disease genome-wide association analysis causality bidirectional Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Migraine is a widespread disorder, impacting 10-20% of the global population 1,2 , and is a notable source of disability. It is more common in women, with annual rates reaching up to 17% 3,4 . The condition leads to severe disability 5 , diminished quality of life, and represents a substantial economic strain on society 6 . Migraine without aura, which constitutes about 75% of cases, manifests as intense head pain and is frequently joined by nausea, photophobia, and phonophobia 7 . It is among the most frequently reported issues in neurology, ranking just after lower back pain in terms of years lost to ill-health globally 2,8 . Gastroesophageal reflux disease (GERD) is identified by the symptoms and issues resulting from the backflow of stomach acids into the esophagus. Its typical symptoms are heartburn and acid reflux, but it also presents in various other ways such as chronic cough, a globus sensation, wheezing, posterior pharyngitis, dental erosion, and idiopathic pulmonary fibrosis. The global prevalence of GERD is estimated at 13.3% 9 . The central nervous system and the gastrointestinal (GI) system are intricately connected through a network of neural, hormonal, and immune pathways. Research has indicated a link between headaches, especially migraines, and GI disorders 10-12 . Among GI diseases identified by specialists, GERD is frequently observed in individuals with migraines 13 . Despite the frequent co-occurrence of GERD and migraines, the precise nature of their relationship is not well understood, leaving the causality between GERD and the onset of migraines in question. Lacking randomized controlled trials (RCTs), the existing research on GERD and migraines relies on observational studies, which face issues like reverse causality and small-scale data, hindered by confounding influences. Mendelian randomization (MR) serves to interpret observational biases and infer causal relationships 14, 15 . Utilizing single nucleotide polymorphisms (SNPs) for instrumental variables (IVs), this methodology delineates pathways linking exposure to subsequent outcomes 16 . MR leverages the natural randomness in individual genomic compositions, similar to the design of RCTs, and uses instrumental variable analysis for assessing the effects that modifiable exposures have upon genetic variant outcomes 17 . Furthermore, MR offers distinct advantages over conventional observational research by mitigating susceptibility to environmental confounding, as genetic IVs are believed to affect outcomes solely through exposure, preventing interference from reverse causality and unrelated confounding factors 18, 19 . To establish the causality between Gastroesophageal Reflux Disease (GERD) and migraine, encompassing the subtypes Migraine with Aura (MA) and Migraine without Aura (MO), we conducted a bidirectional Mendelian Randomization (MR) analysis. The data mentioned above were sourced from large-scale Genome-Wide Association Studies (GWAS).This research could pave the way for improved clinical strategies and preventive interventions. Materials And Methods 2.1 Study design MR elucidates causal pathways linking a mutable risk factor to an outcome, employing observational data and capitalizing on the random genetic allocation during embryogenesis as an inherent experimental control. Our investigation into the genetic linkages between GERD and migraines employed a bidirectional MR strategy. To qualify as an instrumental variable in MR research, the genetic variants must satisfy specific conditions: Relevance assumption: The variation must genuinely associate with the exposure (GERD or migraine). Independence assumption: The genetic variation should remain unaffected by confounders that relate to both the exposure and the outcome. Exclusion restriction assumption: Genetic alterations solely determine the outcome via the specified exposure, without any confounding pathways involved. The methodology of our investigation, illustrated in Figure 1, leverages extensive GWAS summary datasets that are publicly accessible and have been previously disseminated, ensuring that all contributing individuals had granted their consent in writing for the initial GWAS studies. 2.2. Data sources 2.2.1 GERD data The GERD data were obtained from GWAS ID ebi-a-GCST90000514, which included 602,604 participants of European ancestry. Among them, there were 129,080 cases with GERD and 473,524 control individuals. This dataset comprised data on over 2 million SNPs. The GERD diagnosis was based on a combination of clinical, endoscopic, and physiological criteria, focusing on pathological oesophageal acid exposure. 2.2.2 Migraine data Migraines are commonly categorized into MA (migraine with aura) and MO (migraine without aura). To investigate the relationship between different migraine subtypes and GERD, we selected three datasets from the same study available in the publicly accessible GWAS database, encompassing more than 16 million SNPs. The compiled datasets encompassed instances of migraine (comprising 8,547 affected individuals and 176,107 unaffected controls), MA (with 3,541 affected individuals and the same number of controls), and MO (involving 3,215 affected individuals and 176,107 controls). Corresponding GWAS identifiers for these conditions were designated as finn-b-G6 MIGRAINE for migraine, finn-b-G6 MIGRAINE WITH AURA for MA, and finn-b-G6 MIGRAINE NO AURA for MO. Notably, the study populations are uniformly of European descent to minimize demographic bias. The diagnostic criteria align with the International Classification of Headache Disorders (ICHD) guidelines. 2.3. Instrumental variable selection The chosen IVs for MR must conform to the established criteria. Initially, SNPs linked to GERD are required to surpass a stringent genome-wide significance level, with p-values below 5 × 10−8. IVs for migraine and its subtypes were selected with a lenient threshold of up to 1 × 10−5 to capture more IVs related to the exposure of interest. This threshold adjustment aligns with similar approaches in previous investigations 20 . Additionally, to mitigate bias introduced by linkage disequilibrium (LD), SNPs associated with the exposure were required to have r 2 10,000. In the MR analysis, we excluded palindromic SNPs with intermediate allele frequencies. The efficacy of genetic instruments was evaluated by the F statistic, given by F = R 2 (N-2)/(1-R 2 ), with N as the sample size and R 2 as the SNP variance. R 2 's computation follows R 2 = 2 × MAF × (1-MAF) × beta 2 , utilizing MAF for minor allele frequency and beta for the SNP's effect size. An F statistic above 10 was considered suitable for subsequent analysis 21 . We excluded IVs for which the corresponding SNPs were absent from the outcome GWAS dataset due to their extremely low frequency. 2.4. Statistical analyses For this research, we conducted data analysis using R, version 4.3.2, equipped with the TwoSampleMR package 22 , version 0.5.8, and applied MRPRESSO 23 , version 1.0, to identify outliers and pleiotropy, using a p-value threshold of < 0.05 for significance. In our causal inquiry, we primarily applied the inverse variance-weighted (IVW) method 24 , complemented by additional MR techniques such as MR-Egger regression, weighted median, weighted mode and simple methods, to assess exposure effects on outcomes 25 . Given the binary nature of outcome indicators, we converted ratio estimates into odds ratios (ORs) with 95% confidence intervals (CIs). The IVW method calculates the weighted average of Wald ratio estimates 25 . MR-Egger regression performs weighted linear regression under the principle of Instrument Strength Independent of Direct Effect (InSIDE) and generates reliable causal estimates despite the potential invalidity of all IVs 26, 27 . Nevertheless, its accuracy is lower and it is vulnerable to the effects of pleiotropic genetic variation. The weighted median regression method computes the weighted median of Wald ratio estimates and remains unaffected by pleiotropy bias 28 . Weighted median has demonstrated superiority to MR-Egger regression, offering reduced type I errors and enhanced causal estimation capability. We also used simple and weighted mode methods for verifying MR results' stability 28 . If these methods yield inconsistent results, priority will be given to IVW as the primary outcome. Additionally, sensitivity analyses will be conducted to assess heterogeneity and pleiotropy, utilizing IVW and MR-Egger regression to evaluate heterogeneity, and Cochran’s Q test for its measurement 29 . In the presence of heterogeneity, IVW with random effects was used for the study. Horizontal pleiotropy was evaluated with the MR-Egger intercept method, and the MR-PRESSO test was applied to identify overall pleiotropy, examining SNPs for potential horizontal pleiotropy 23 . Abnormal SNPs were excluded before MR analysis to enhance the robustness of the analysis. Results We performed a bidirectional two-sample MR analysis to investigate the causal relationship linking GERD and migraine incidence, including subtypes. The results suggest a genetic predisposition to GERD is linked to a heightened likelihood of migraine, specifically the MO subtype, without a corresponding increase for MA. The overall relationship between migraine risk and GERD severity, however, is not definitively established. 3.1. GERD's Impact on Migraine and Subtypes Causality 3.1.1. Selection of instrumental variables The GERD GWAS dataset, accessible to the public, was secured employing the R programming language. A total of 80 SNPs significantly correlated with GERD were identified, adhering to a stringent significance threshold of p < 5E-08 and meeting independence standards (r 2 10,000). These SNPs were then correlated with the final GWAS dataset, leading to the elimination of certain SNPs not detected in the outcome dataset. Specifically, the SNP (rs2106353) was excluded across all three analysis groups: GERD associated with migraine, MA, and MO. Additionally, four palindrome SNPs with moderate allele frequencies (rs2145318, rs2358016, rs9517313, rs957345) were excluded from all three studies. Consequently, in the analysis of GERD with migraine and its subtypes MA and MO, 75 SNPs were identified as IVs (Supplementary Table 1). For the analysis of migraine with GERD, 9 SNPs were identified as IVs, while for MA with GERD analysis, 6 SNPs were identified as IVs, and for MO with GERD analysis, 5 SNPs were identified as IVs (Supplementary Table 1). Importantly, all included IVs exhibited F statistics surpassing the threshold of 10, signifying a low probability of weak instrument bias and fulfilling the relevance assumption. To mitigate the likelihood of SNPs being associated with potential confounders or risk determinants, we meticulously assessed and excluded such correlations using the Phenoscanner tool. Notably, we did not find SNPs strongly associated with identified carcinogenic factors. 3.1.2. Two-sample Mendelian randomization analysis This study primarily used the IVW method as the main analytical approach, revealing a causal link between genetic predisposition to GERD and an increased risk of migraine, particularly for the MO subtype, but not for MA. Specifically, the odds ratios (OR) were 1.381 (95% CI: 1.190–1.602, p = 2.04E-05) for GERD and migraine, 1.600 (95% CI: 1.311–1.953, p = 3.67E-06) for GERD and MO, and 1.193 (95% CI: 0.983–1.449, p = 0.074) for GERD and MA. Secondary analytical methods were also employed, yielding the following results: MR-Egger showed ORs of 1.239 (p = 0.634) for GERD and migraine, 1.369 (p = 0.594) for GERD and MA, and 1.624 (p = 0.421) for GERD and MO; weighted median produced ORs of 1.371 (p = 0.001) for GERD and migraine, 1.301 (p = 0.066) for GERD and MA, and 1.470 (p = 0.009) for GERD and MO; simple mode showed ORs of 1.976 (p = 0.014) for GERD and migraine, 1.383 (p = 0.379) for GERD and MA, and 1.502 (p = 0.320) for GERD and MO; and weighted mode provided ORs of 1.923 (p = 0.012) for GERD and migraine, 1.415 (p = 0.303) for GERD and MA, and 1.577 (p = 0.206) for GERD and MO. After conversion to odds ratios, all OR values were greater than 1 (Figures 2, 3, 4). 3.1.3. Sensitivity analysis and visualization We detected heterogeneity using MR-Egger regression and IVW analysis. MR-Egger regression showed Cochran's Q values of 101.540 (p = 0.015) for GERD-migraine, 77.891 (p = 0.326) for GERD-MA, and 69.987 (p = 0.578) for GERD-MO. Similarly, IVW analysis resulted in Cochran's Q values of 101.623 (p = 0.018) for GERD-migraine, 77.951 (p = 0.354) for GERD-MA, and 69.988 (p = 0.611) for GERD-MO. These results indicated no significant heterogeneity in the study (Supplementary Table 2). The heterogeneity visualization is depicted in Supplementary Figure 1 through funnel plots. For horizontal pleiotropy testing, MR-Egger intercepts suggested no pleiotropy for GERD and migraine studies, with an Egger intercept of 0.003 (p = 0.808). However, MR-PRESSO global testing indicated some degree of pleiotropy for GERD-migraine (global test: p = 0.025), although no outlier SNPs were identified, allowing us to proceed with the adjusted IVW method. Notably, no anomalies were detected in the analysis between GERD and MA and MO risks. The MR-Egger intercepts indicated no horizontal pleiotropy for GERD-MA (intercept = -0.005, p = 0.813) and GERD-MO (intercept = -0.0005, p = 0.980). MR-PRESSO testing identified no outliers, and global tests did not show pleiotropy, with p-values of 0.782 for GERD-MA and 0.623 for GERD-MO (Supplementary Table 2). The leave-one-out approach was applied to sequentially eliminate individual SNPs for assessing the influence of any single instrumental variable on the causal relationship. The outcomes confirmed the stability of the Two-Sample Mendelian Randomization (TSMR) analysis, as illustrated in Figure 5. 3.2. Inverse TSMR Analysis In the context of TSMR, migraine, including both MA and MO subtypes, was considered the exposure, with GERD as the outcome. To include a wider range of instrumental variables, the p-value threshold was set below 1E-05. After confirming compliance with linkage disequilibrium parameters (r² 10,000), we ensured the selected IVs conformed to the fundamental MR assumptions. SNPs absent from the outcome dataset and those palindromic with moderate allele frequencies were excluded. MR analysis was then performed on three distinct exposure datasets for migraine, categorized as MA (9 SNPs), MO (6 SNPs), and a combined group (5 SNPs) (Supplementary Table 1). Notably, all IVs had F statistics significantly exceeding a value of 10. The MR outcomes did not support a causal link between genetic predisposition to migraine (including MA and MO subtypes) and increased risk of GERD, with the following odds ratios (OR) and confidence intervals (CI): migraine-GERD, OR = 1.012 (95% CI: 0.965–1.062, p = 0.502); MA-GERD, OR = 1.014 (95% CI: 0.982–1.048, p = 0.393); MO-GERD, OR = 1.008 (95% CI: 0.954–1.064, p = 0.780). Heterogeneity assessments revealed no significant variability in the migraine-GERD and MA-GERD analyses, with Cochran's Q values and p-values as follows: migraine-GERD, MR-Egger: Q = 6.705, p = 0.460; IVW: Q = 6.792, p = 0.559; MA-GERD, MR-Egger: Q = 6.350, p = 0.174; IVW: Q = 7.236, p = 0.203. However, heterogeneity was present in the MO-GERD analysis (MO-GERD, MR-Egger: Q = 6.532, p = 0.163; IVW: Q = 13.412, p = 0.020). In horizontal pleiotropy evaluation, neither the MR-Egger intercepts nor the global MR-PRESSO tests identified any anomalies in the association between migraine and GERD risks, with results showing: migraine-GERD, Egger intercept = -0.0014, p = 0.776; global test, p = 0.628; MA-GERD, Egger intercept = 0.0049, p = 0.497; global test, p = 0.307. In the analysis of MO level and GERD risk, the MR-Egger intercept prior to the exclusion of the outlier SNP did not suggest pleiotropy (MO-GERD, Egger intercept = 0.0631, p = 0.109). However, the MR-PRESSO global test indicated some degree of pleiotropy (global test, p = 0.037). After removing the outlier SNP (rs1155688), subsequent analyses with the MR-Egger intercept and the global MR-PRESSO test revealed no further signs of pleiotropy in the relationship between MO levels and GERD risk (MO-GERD, Egger intercept = 0.0517, p = 0.175; global test, p = 0.177), leading to the removal of the MO dataset containing the outlier SNP. Discussion We utilized a bidirectional TSMR strategy with available GWAS summary datasets to explore the reciprocal causality between GERD and migraine, encompassing both the general condition and its MA and MO subtypes. The MR study indicates that GERD correlates with a higher probability for migraine and the MO, without a corresponding link to an elevated risk of MA. Furthermore, our research does not corroborate a causal connection between the genetic predisposition to migraine, including all subtypes, and an escalated risk of GERD. While some heterogeneity appeared in the GERD-migraine and MO-GERD studies, random effects IVW analysis remains robust against heterogeneity, suggesting a stable causal effect. Prior research has suggested an association between various GI diseases and primary headache syndromes, including migraine. The brain-gut axis facilitates bidirectional communication linking the brain and the intestinal tract, mediated by neural, hormonal, and immune pathways. Early reports noted improvements in migraine symptoms when gastroesophageal reflux symptoms subsided in patients with comorbidities of both diseases 30 . Retrospective analyses have shown GERD occurs more frequently among migraine sufferers than among those without migraines, with similar rates of gastritis and stomach ulcers in both groups 13, 31 . Notably, a significant association has been observed between primary childhood migraine and infantile gastroesophageal reflux, particularly for migraine without aura 32 . The causal link between migraine and GERD remains ambiguous due to a scarcity of research in this domain. Predominantly, epidemiological investigations have relied on case-control or cross-sectional methodologies, which obscure the temporal progression, thereby complicating the determination of causality. Additionally, prior observational research has faced constraints, including small participant groups, difficulties in addressing reverse causality, and the influence of confounding factors. Utilizing more robust study designs, such as bidirectional TSMR analysis, can facilitate a better understanding of the causal dynamics between exposures and outcomes within the current research framework. We have identified a causal relationship between GERD and migraine, particularly its MO subtype, which may be explained by shared mechanisms involving the NF-κB pathway in the inflammatory processes of both conditions 33, 34 . Research suggests that migraine pathogenesis involves cortical spreading depression (CSD) 35 , a self-propagating wave of depolarization that moves throughout the cerebral cortex. This phenomenon could activate the trigeminal input pathway, leading to changes in blood-brain barrier permeability through matrix metalloproteinase activation and upregulation. These changes may induce inflammatory responses in pain-sensitive meninges, resulting in migraine headaches through central and peripheral reflex mechanisms. Molecular cascades during CSD activation may include pannexin-1 channel opening, caspase-1 activation, pro-inflammatory mediator release, and activation of NF-κB in astrocytes, transmitting inflammatory signals to trigeminal nerve fibers around cranial blood vessels 36 . Studies on GERD mechanisms suggest that inflammatory cell infiltration precedes esophageal epithelial surface erosion 37 , indicating inflammation as a key mediator of erosion. Additionally, research has shown that in esophageal inflammation models, epithelial keratinocytes secrete IL-8, indicating that gastroesophageal reflux could activate inflammatory mechanisms, which may subsequently impair esophageal barrier function 38 . This activation of inflammatory pathways, including the NF-κB pathway, by acidic substance-induced chemical injury to the esophageal mucosa can lead to the upregulation of various inflammatory mediators such as matrix metalloproteinase-3, matrix metalloproteinase-9, IL-1, IL-6, and IL-8. Therefore, we hypothesize that NF-κB may play a role in various stages of GERD pathogenesis and migraine occurrence. The inflammatory pathways involved in GERD development may also contribute to migraine attacks, necessitating further investigation. The second possible explanation is speculated to be due to the phenomenon of delayed gastric emptying or gastric paresis, which is shared by both conditions. A prior study identified that delayed gastric emptying , or gastric paresis, substantially contributes to the onset of GERD 39 . Additionally, research indicates that individuals with migraines experience delayed gastric emptying both during and after their attacks, suggesting a potential association with gastroesophageal reflux disease 40, 41 . Furthermore, dysfunction within the autonomic nervous system (ANS) was previously associated with headaches, especially migraines, and GI disorders 42, 43 . Therefore, ANS dysfunction may also be related to the pathogenesis of these two conditions. Abnormalities in visceral mechanosensation and vagal nerve function have been found to be associated with dyspepsia, and visceral neural dysregulation is also linked to migraines 12 . Additionally, individuals presenting with both headache and gastrointestinal symptoms tend to suffer from more intense and frequent episodes compared to those experiencing headaches in isolation 44, 45 . One possible reason is the increased interaction of shared underlying processes, leading to worse symptoms when headaches and gastrointestinal issues occur together 44 . Currently, here is currently considerable debate about whether MO and MA result from the same pathogenic mechanisms. MO and MA exhibit notable differences in their clinical presentations. For instance, MO is more likely to occur after stressful events, exams, or during the post-menstrual relaxation phase in women. In contrast, MA attacks lack this characteristic and may be triggered by intense natural or artificial visual stimuli in some cases 46 . Regarding associations with key female reproductive events, MO is notably affected by menstruation, unlike MA. Approximately one-fifth of MA cases experience heightened attack risks in the perimenstrual phase, compared to approximately three-quarters of MO cases 47 . During gestation, MO commonly abates in most instances, whereas MA tends to continue or intensify, and occasionally emerges for the first time 47 . Oral contraceptives generally exert no adverse effects on MO, yet in the majority of cases, they can worsen MA. MA is characterized by visual, language, sensory, or brainstem symptoms that typically precede the headache phase. The pathogenesis of MA is widely attributed to CSD, involving neuronal and glial depolarization which then transitions to cortical hyperpolarization, progressing at 3–5 mm/min. This process corresponds to significant alterations in ion homeostasis and neurotransmitter release 48 . Additionally, there is a temporary surge in cerebral blood flow due to the increased energy demands for restoring homeostasis 49 . While the pathophysiology of other aura types (such as speech, sensory, and brainstem) is less studied, evidence suggests that CSD may also underlie their mechanisms 49 . Research has also demonstrated differences in cerebral hemodynamics between MO and MA, with MA characterized by significant vasodilation and trigeminal vascular sensitization (TVS), which can trigger aura 50 . The study findings point to a potential causative link connecting GERD and the occurrence of migraine, particularly the MO subtype, underscoring the importance of closely monitoring migraine symptoms in GERD patients, which could be beneficial in clinical practice. Additionally, it's crucial to be attentive to mixed symptoms of GERD or migraine. On the other hand, MO lacks the premonitory aura symptoms seen in MA, and its pathophysiology is not fully elucidated. Some theories propose that CSD may occur silently in subcortical regions, such as the hypothalamus, in MO patients 48 . However, studies have shown that drugs inhibiting CSD, such as tonabersat, significantly reduce the frequency of aura attacks but not MO attacks 51 , indicating potential differences in the underlying mechanisms between the two types of migraine. Our bidirectional TSMR study offers several advantages. Firstly, we applied MR analysis with SNPs that have a strong association (F>10), akin to the experimental setup of RCTs, to yield robust evidence. Unlike RCTs, which can be constrained by cost and sample size, MR analysis circumvents issues of reverse causation and confounding. Secondly, by relying on GWAS databases with European population samples, our study mitigates bias from demographic variability. Thirdly, the insights from our analysis could inform healthcare policy, especially if a causal link between GERD and migraine, notably the MO subtype, is established, potentially influencing strategies for mitigating and managing the condition. Nevertheless, this research acknowledges certain constraints, primarily the heterogeneity in the analysis of GERD versus migraine, and MO versus GERD. The heterogeneity in the former could be due to the inclusion of both MA and MO subtypes in migraine data, while the subsequent examination of GERD versus MO revealed no heterogeneity. Considering the presence of MA data in migraine may contribute to the heterogeneity. The presence of heterogeneity in the latter may be explained by the small sample size. Additionally, we used GERD data rather than data encompassing all digestive system-related diseases, so the current findings cannot be extrapolated to patients with other digestive system disorders. Further research on this topic is warranted. Secondly, as current investigations primarily focus on the link between GERD and migraine, and the number of such investigations is limited, specific studies addressing the connection between GERD and the MO subtype of migraine are sparse. Therefore, the analysis of potential mechanisms remains somewhat limited in this regard. Thirdly, GERD is categorized into reflux esophagitis (RE) and non-erosive reflux disease (NERD). Our study was unable to ascertain the connection among these subtypes with the severity of migraine, including low-frequency as well as high-frequency episodic forms. Additionally, gastroscopy, an essential diagnostic tool for GERD, may lead to an underestimation of diagnosed cases due to its invasive nature. This bias remains unavoidable. Fourthly, despite no significant difference in GERD prevalence among males and females, migraine prevalence is higher in females. However, our reliance on public databases precluded subgroup analyses based on factors like age and gender. Fifthly, the GWAS data's European focus restricts the generalizability of our findings to non-European demographics. Lastly, expanding GWAS sample sizes could strengthen IVs, highlighting the need for larger-scale studies for more comprehensive research. Therefore, additional research is necessary to confirm the results of our current study. Conclusion The bidirectional Two-Sample Mendelian Randomization analysis suggests an association between GERD susceptibility and an increased likelihood of migraine and its MO subtype, with no observed correlation with the risk of MA. Declarations Data Availability Statement The study's original contributions are detailed in the published article and its Supplementary material. For any additional inquiries, please contact the corresponding author(s). Ethics statement The research involving human participants adhered to the local legislation and institutional guidelines, which did not necessitate ethical review and approval. In line with national regulations and institutional policies, obtaining written informed consent from the patients/participants or their legal guardians/next of kin was not required for this study. Author contributions XJ was instrumental in the study design, data collection, statistical analysis, and preparation of the original draft. JHZ contributed by reviewing and revising the manuscript. JX provided supervision over the review process and gave the final approval of the manuscript. Each author has made significant contributions to the article and has given approval to the version submitted for publication. Funding The authors declare that they did not receive any financial support for the research, authorship, and publication of this article. Additional Information Competing interests statement The authors affirm that the research was carried out without any commercial or financial interests that might be perceived as potential conflicts of interest. Publisher’s note The views and assertions expressed in this article are those of the authors alone and do not necessarily reflect the opinions of their affiliated institutions, the publisher, the editors, or the reviewers. The publisher does not assume responsibility for any product evaluated in the article, nor does it endorse any claims made by the manufacturer. Acknowledgments We are grateful to the genetics consortiums for providing the GWAS summary data through their publicly accessible databases, which facilitated our research. Our data were sourced from these open resources. References Lipton, R. B., Stewart, W. F., Diamond, S., Diamond, M. L. & Reed, M. Prevalence and burden of migraine in the United States: data from the American Migraine Study II. Headache . 41 , 646–657. 10.1046/j.1526-4610.2001.041007646.x (2001). Ashina, M. et al. Migraine: epidemiology and systems of care. Lancet . 397 , 1485–1495. 10.1016/S0140-6736(20)32160-7 (2021). Stewart, W. 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Gastric stasis in migraine: more than just a paroxysmal abnormality during a migraine attack. Headache . 46 , 57–63. 10.1111/j.1526-4610.2006.00311.x (2006). Lorena, S. L., Figueiredo, M. J., Almeida, J. R. & Mesquita, M. A. Autonomic function in patients with functional dyspepsia assessed by 24-hour heart rate variability. Dig. Dis. Sci. 47 , 27–31. 10.1023/a:1013246900041 (2002). Peroutka, S. J. Migraine: a chronic sympathetic nervous system disorder. Headache . 44 , 53–64. 10.1111/j.1526-4610.2004.04011.x (2004). Kim, B. S., Chung, C. S., Lee, C. B. & Rhee, P. L. Migraineurs Initially Visiting the Gastroenterology Department. Headache . 56 , 555–563. 10.1111/head.12775 (2016). Tai, M. L. et al. The impact of dyspepsia on symptom severity and quality of life in adults with headache. PLoS One . 10 , e0115838. 10.1371/journal.pone.0115838 (2015). Russell, M. B., Ulrich, V., Gervil, M. & Olesen, J. Migraine without aura and migraine with aura are distinct disorders. A population-based twin survey. Headache . 42 , 332–336. 10.1046/j.1526-4610.2002.02102.x (2002). Manzoni, G. C. & Torelli, P. Migraine with and without aura: a single entity? Neurol. Sci. 29 (Suppl 1), 40–43. 10.1007/s10072-008-0884-7 (2008). Vgontzas, A. & Burch, R. Episodic Migraine With and Without Aura: Key Differences and Implications for Pathophysiology, Management, and Assessing Risks. Curr. Pain Headache Rep. 22 , 78. 10.1007/s11916-018-0735-z (2018). Kurth, T., Diener, H. C. & Buring, J. E. Migraine and cardiovascular disease in women and the role of aspirin: subgroup analyses in the Women's Health Study. Cephalalgia . 31 , 1106–1115. 10.1177/0333102411412628 (2011). Zupan, M., Zaletel, M., Visocnik, D. & Zvan, B. Calcitonin gene-related peptide-induced hemodynamic changes in migraine with and without aura. Acta Neurol. Scand. 144 , 616–622. 10.1111/ane.13495 (2021). Hadjikhani, N. et al. Mechanisms of migraine aura revealed by functional MRI in human visual cortex. Proc. Natl. Acad. Sci. U S A . 98 , 4687–4692. 10.1073/pnas.071582498 (2001). Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4897548","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":357219060,"identity":"309831db-74b7-4ed7-96b4-8b600be6b77c","order_by":0,"name":"Xin Jin","email":"","orcid":"","institution":"Second Affiliated Hospital of Naval Medical University, Shanghai Changzheng Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Jin","suffix":""},{"id":357219061,"identity":"5eedf6e5-fe46-45df-870d-72000018a6bc","order_by":1,"name":"Jianhua Zhuang","email":"","orcid":"","institution":"Second Affiliated Hospital of Naval Medical University, Shanghai Changzheng Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jianhua","middleName":"","lastName":"Zhuang","suffix":""},{"id":357219062,"identity":"555550f2-3948-4279-95d4-b7e39f5eaf32","order_by":2,"name":"Jin Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYJACCSBmZmNgPnDgww/StLAlHpzZQ4IWIOAxPszBRoRy/vazB2/zth1m55Pu+XCYgYdBnl/sAAEbzuQlW/OcOczMJnN2w+ECCwbDmbMT8GsxYMgxk+apAGqRyN1weAYPQ4LBbUJa+N8AtRiAtOQ8OMzDRowWCbgtOQzEaZG48cbYcs6ZdKCWNANgIEsQ9gt/f47hjbdt1snyM5Iff/jww0aeX5qAFhBg4mFgSIbZSlg5CDACk4kdcUpHwSgYBaNgRAIA2ME+KN69woEAAAAASUVORK5CYII=","orcid":"","institution":"Second Affiliated Hospital of Naval Medical University, Shanghai Changzheng Hospital","correspondingAuthor":true,"prefix":"","firstName":"Jin","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2024-08-12 04:36:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4897548/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4897548/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65109206,"identity":"d59d8ffb-f416-4292-8c3e-4175fb102719","added_by":"auto","created_at":"2024-09-23 17:37:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":244578,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart: This schematic illustrates the bidirectional two-sample MR analysis between GERD and migraine and its subtypes, MA and MO. The foundational MR assumptions are outlined:\u003c/p\u003e\n\u003cp\u003e1. Instrumental variables, based on genetic variation, should have a valid link to the exposure.\u003c/p\u003e\n\u003cp\u003e2. Genetic variations must not be affected by confounding influences.\u003c/p\u003e\n\u003cp\u003eThe effect of the genetic variation on the outcome should occur only via the exposure, excluding any alternative routes.\u003c/p\u003e\n\u003cp\u003eAbbreviations: MR, Mendelian randomization; SNPs, single nucleotide polymorphisms.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4897548/v1/6a6fdf791bdf1b8c46a80b86.jpg"},{"id":65109967,"identity":"daf2af44-0fc5-4e57-a18c-ed4a6e0f7fe4","added_by":"auto","created_at":"2024-09-23 17:53:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1053361,"visible":true,"origin":"","legend":"\u003cp\u003eEstimation of Causality: This figure depicts the causative link connecting GERD and migraine, including its MA and MO subtypes, employing various MR approaches. An OR \u0026gt; 1 implies a risk factor, while an OR \u0026lt; 1 indicates a protective effect.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4897548/v1/4fa1f920fbc42c8733beff8c.jpg"},{"id":65109522,"identity":"f2af2bb5-6845-4a0b-905a-be8de17427d0","added_by":"auto","created_at":"2024-09-23 17:45:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1000796,"visible":true,"origin":"","legend":"\u003cp\u003eCausality Estimation: This figure illustrates the estimated causal relationship between migraine (including MA and MO subtypes) and GERD utilizing various MR techniques.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4897548/v1/05a57946613195cda4e12569.jpg"},{"id":65109523,"identity":"ef34c713-bf2b-46ee-8579-0f4eeb37e8a3","added_by":"auto","created_at":"2024-09-23 17:45:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":896632,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic Correlation Plot: This scatter plot represents the bidirectional genetic correlation between GERD and migraine, encompassing both MA and MO subtypes, as analyzed by various MR methods.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4897548/v1/18908bda402c18e4af38d5d6.jpg"},{"id":65109212,"identity":"aded3c22-3564-49b0-bcb1-175a47aef96f","added_by":"auto","created_at":"2024-09-23 17:37:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4513958,"visible":true,"origin":"","legend":"\u003cp\u003eSensitivity Analysis: This figure presents a bidirectional leave-one-out sensitivity test for the association of GERD with migraine, covering both MA and MO subtypes. The red lines indicate the IVW test estimates. IVW: inverse variance weighted.\u003c/p\u003e","description":"","filename":"Figure5.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4897548/v1/6f93cc184dff165753e70ff5.jpg"},{"id":80609562,"identity":"c16eca20-a83e-4867-86ef-3079c74eda25","added_by":"auto","created_at":"2025-04-15 07:31:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8222633,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4897548/v1/05ae0f55-7685-4cef-9efc-3f6288ea8211.pdf"},{"id":65109211,"identity":"5332791f-e8ea-4abd-8dc5-92a312c2dd78","added_by":"auto","created_at":"2024-09-23 17:37:11","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":535809,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4897548/v1/f63c89976514f2bbfb0c3935.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bidirectional analysis of gastroesophageal reflux disease and migraine using two-sample Mendelian randomization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMigraine is a widespread disorder, impacting 10-20% of the global population\u003csup\u003e1,2\u003c/sup\u003e, and is a notable source of disability. It is more common in women, with annual rates reaching up to 17%\u003csup\u003e3,4\u003c/sup\u003e. The condition leads to severe disability\u003csup\u003e5\u003c/sup\u003e, diminished quality of life, and represents a substantial economic strain on society\u003csup\u003e6\u003c/sup\u003e. Migraine without aura, which constitutes about 75% of cases, manifests as intense head pain and is frequently joined by nausea, photophobia, and phonophobia\u003csup\u003e7\u003c/sup\u003e. It is among the most frequently reported issues in neurology, ranking just after lower back pain in terms of years lost to ill-health globally\u003csup\u003e2,8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eGastroesophageal reflux disease (GERD) is identified by the symptoms and issues resulting from the backflow of stomach acids into the esophagus. Its typical symptoms are heartburn and acid reflux, but it also presents in various other ways such as chronic cough, a globus sensation, wheezing, posterior pharyngitis, dental erosion, and idiopathic pulmonary fibrosis. The global prevalence of GERD is estimated at 13.3%\u003csup\u003e9\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe central nervous system and the gastrointestinal (GI) system are intricately connected through a network of neural, hormonal, and immune pathways. Research has indicated a link between headaches, especially migraines, and GI disorders\u003csup\u003e10-12\u003c/sup\u003e. Among GI diseases identified by specialists, GERD is frequently observed in individuals with migraines\u003csup\u003e13\u003c/sup\u003e. Despite the frequent co-occurrence of GERD and migraines, the precise nature of their relationship is not well understood, leaving the causality between GERD and the onset of migraines in question.\u003c/p\u003e\n\u003cp\u003eLacking randomized controlled trials (RCTs), the existing research on GERD and migraines relies on observational studies, which face issues like reverse causality and small-scale data, hindered by confounding influences. Mendelian randomization (MR) serves to interpret observational biases and infer causal relationships\u003csup\u003e14, 15\u003c/sup\u003e. Utilizing single nucleotide polymorphisms (SNPs) for instrumental variables (IVs), this methodology delineates pathways linking exposure to subsequent outcomes\u003csup\u003e16\u003c/sup\u003e. MR leverages the natural randomness in individual genomic compositions, similar to the design of RCTs, and uses instrumental variable analysis for assessing the effects that modifiable exposures have upon genetic variant outcomes \u003csup\u003e17\u003c/sup\u003e. Furthermore, MR offers distinct advantages over conventional observational research by mitigating susceptibility to environmental confounding, as genetic IVs are believed to affect outcomes solely through exposure, preventing interference from reverse causality and unrelated confounding factors\u003csup\u003e18, 19\u003c/sup\u003e. To establish the causality between Gastroesophageal Reflux Disease (GERD) and migraine, encompassing the subtypes Migraine with Aura (MA) and Migraine without Aura (MO), we conducted a bidirectional Mendelian Randomization (MR) analysis. The data mentioned above were sourced from large-scale Genome-Wide Association Studies (GWAS).This research could pave the way for improved clinical strategies and preventive interventions.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e2.1 Study design\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMR elucidates causal pathways linking a mutable risk factor to an outcome, employing observational data and capitalizing on the random genetic allocation during embryogenesis as an inherent experimental control. Our investigation into the genetic linkages between GERD and migraines employed a bidirectional MR strategy. To qualify as an instrumental variable in MR research, the genetic variants must satisfy specific conditions: Relevance assumption: The variation must genuinely associate with the exposure (GERD or migraine). Independence assumption: The genetic variation should remain unaffected by confounders that relate to both the exposure and the outcome. Exclusion restriction assumption: Genetic alterations solely determine the outcome via the specified exposure, without any confounding pathways involved. The methodology of our investigation, illustrated in Figure 1, leverages extensive GWAS summary datasets that are publicly accessible and have been previously disseminated, ensuring that all contributing individuals had granted their consent in writing for the initial GWAS studies.\u003c/p\u003e\n\u003cp\u003e2.2. Data sources\u003c/p\u003e\n\u003cp\u003e2.2.1 GERD data\u003c/p\u003e\n\u003cp\u003eThe GERD data were obtained from GWAS ID ebi-a-GCST90000514, which included 602,604 participants of European ancestry. Among them, there were 129,080 cases with GERD and 473,524 control individuals. This dataset comprised data on over 2 million SNPs.\u0026nbsp;The GERD diagnosis was based on a combination of clinical, endoscopic, and physiological criteria, focusing on pathological oesophageal acid exposure.\u003c/p\u003e\n\u003cp\u003e2.2.2 Migraine data\u003c/p\u003e\n\u003cp\u003eMigraines are commonly categorized into MA (migraine with aura) and MO (migraine without aura). To investigate the relationship between different migraine subtypes and GERD, we selected three datasets from the same study available in the publicly accessible GWAS database, encompassing more than 16 million SNPs. The compiled datasets encompassed instances of migraine (comprising 8,547 affected individuals and 176,107 unaffected controls), MA (with 3,541 affected individuals and the same number of controls), and MO (involving 3,215 affected individuals and 176,107 controls). Corresponding GWAS identifiers for these conditions were designated as finn-b-G6 MIGRAINE for migraine, finn-b-G6 MIGRAINE WITH AURA for MA, and finn-b-G6 MIGRAINE NO AURA for MO. Notably, the study populations are uniformly of European descent to minimize demographic bias. The diagnostic criteria align with the International Classification of Headache Disorders (ICHD) guidelines.\u003c/p\u003e\n\u003cp\u003e2.3. Instrumental variable selection\u003c/p\u003e\n\u003cp\u003eThe chosen IVs for MR must conform to the established criteria. Initially, SNPs linked to GERD are required to surpass a stringent genome-wide significance level, with p-values below 5\u0026thinsp;\u0026times;\u0026thinsp;10\u0026minus;8. IVs for migraine and its subtypes were selected with a lenient threshold of up to 1\u0026thinsp;\u0026times;\u0026thinsp;10\u0026minus;5 to capture more IVs related to the exposure of interest. This threshold adjustment aligns with similar approaches in previous investigations\u003csup\u003e20\u003c/sup\u003e. Additionally, to mitigate bias introduced by linkage disequilibrium (LD), SNPs associated with the exposure were required to have r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and KB\u0026thinsp;\u0026gt;\u0026thinsp;10,000. In the MR analysis, we excluded palindromic SNPs with intermediate allele frequencies. The efficacy of genetic instruments was evaluated by the F statistic, given by F = R\u003csup\u003e2\u003c/sup\u003e(N-2)/(1-R\u003csup\u003e2\u003c/sup\u003e), with N as the sample size and R\u003csup\u003e2\u003c/sup\u003e as the SNP variance. R\u003csup\u003e2\u003c/sup\u003e\u0026apos;s computation follows R\u003csup\u003e2\u003c/sup\u003e = 2 \u0026times; MAF \u0026times; (1-MAF) \u0026times; beta\u003csup\u003e2\u003c/sup\u003e, utilizing MAF for minor allele frequency and beta for the SNP\u0026apos;s effect size. An F statistic above 10 was considered suitable for subsequent analysis\u003csup\u003e21\u003c/sup\u003e.\u0026nbsp;We excluded IVs for which the corresponding SNPs were absent from the outcome GWAS dataset due to their extremely low frequency.\u003c/p\u003e\n\u003cp\u003e2.4. Statistical analyses\u003c/p\u003e\n\u003cp\u003eFor this research, we conducted data analysis using R, version 4.3.2, equipped with the TwoSampleMR package\u003csup\u003e22\u003c/sup\u003e, version 0.5.8, and applied MRPRESSO\u003csup\u003e23\u003c/sup\u003e, version 1.0, to identify outliers and pleiotropy, using a p-value threshold of \u0026lt; 0.05 for significance.\u003c/p\u003e\n\u003cp\u003eIn our causal inquiry, we primarily applied the inverse variance-weighted (IVW) method\u003csup\u003e24\u003c/sup\u003e, complemented by additional MR techniques such as MR-Egger regression, weighted median, weighted mode and simple methods, to assess exposure effects on outcomes\u003csup\u003e25\u003c/sup\u003e. Given the binary nature of outcome indicators, we converted ratio estimates into odds ratios (ORs) with 95% confidence intervals (CIs).\u003c/p\u003e\n\u003cp\u003eThe IVW method calculates the weighted average of Wald ratio estimates\u003csup\u003e25\u003c/sup\u003e.\u0026nbsp;MR-Egger regression performs weighted linear regression under the principle of Instrument Strength Independent of Direct Effect (InSIDE) and generates reliable causal estimates despite the potential invalidity of all IVs\u003csup\u003e26, 27\u003c/sup\u003e. Nevertheless, its accuracy is lower and it is vulnerable to the effects of pleiotropic genetic variation. The weighted median regression method computes the weighted median of Wald ratio estimates and remains unaffected by pleiotropy bias\u003csup\u003e28\u003c/sup\u003e. Weighted median has demonstrated superiority to MR-Egger regression, offering reduced type I errors and enhanced causal estimation capability. We also used simple and weighted mode methods for verifying MR results\u0026apos; stability\u003csup\u003e28\u003c/sup\u003e.\u0026nbsp;If these methods yield inconsistent results, priority will be given to IVW as the primary outcome.\u003c/p\u003e\n\u003cp\u003eAdditionally, sensitivity analyses will be conducted to assess heterogeneity and pleiotropy, utilizing IVW and MR-Egger regression to evaluate heterogeneity, and Cochran\u0026rsquo;s Q test for its measurement\u003csup\u003e29\u003c/sup\u003e. In the presence of heterogeneity, IVW with random effects was used for the study. Horizontal pleiotropy was evaluated with the MR-Egger intercept method, and the MR-PRESSO test was applied to identify overall pleiotropy, examining SNPs for potential horizontal pleiotropy\u003csup\u003e23\u003c/sup\u003e. Abnormal SNPs were excluded before MR analysis to enhance the robustness of the analysis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eWe performed a bidirectional two-sample MR analysis to investigate the causal relationship linking GERD and migraine incidence, including subtypes. The results suggest a genetic predisposition to GERD is linked to a heightened likelihood of migraine, specifically the MO subtype, without a corresponding increase for MA. The overall relationship between migraine risk and GERD severity, however, is not definitively established.\u003c/p\u003e\n\u003cp\u003e3.1. GERD\u0026apos;s Impact on Migraine and Subtypes Causality\u003c/p\u003e\n\u003cp\u003e3.1.1. Selection of instrumental variables\u003c/p\u003e\n\u003cp\u003eThe GERD GWAS dataset, accessible to the public, was secured employing the R programming language. A total of 80 SNPs significantly correlated with GERD were identified, adhering to a stringent significance threshold of p \u0026lt; 5E-08 and meeting independence standards (r\u003csup\u003e2\u003c/sup\u003e \u0026lt; 0.001 and KB \u0026gt; 10,000). These SNPs were then correlated with the final GWAS dataset, leading to the elimination of certain SNPs not detected in the outcome dataset. Specifically, the SNP (rs2106353) was excluded across all three analysis groups: GERD associated with migraine, MA, and MO. Additionally, four palindrome SNPs with moderate allele frequencies (rs2145318, rs2358016, rs9517313, rs957345) were excluded from all three studies. Consequently, in the analysis of GERD with migraine and its subtypes MA and MO, 75 SNPs were identified as IVs (Supplementary Table 1). For the analysis of migraine with GERD, 9 SNPs were identified as IVs, while for MA with GERD analysis, 6 SNPs were identified as IVs, and for MO with GERD analysis, 5 SNPs were identified as IVs (Supplementary Table 1). Importantly, all included IVs exhibited F statistics surpassing the threshold of 10, signifying a low probability of weak instrument bias and fulfilling the relevance assumption. To mitigate the likelihood of SNPs being associated with potential confounders or risk determinants, we meticulously assessed and excluded such correlations using the Phenoscanner tool. Notably, we did not find SNPs strongly associated with identified carcinogenic factors.\u003c/p\u003e\n\u003cp\u003e3.1.2. Two-sample Mendelian randomization analysis\u003c/p\u003e\n\u003cp\u003eThis study primarily used the IVW method as the main analytical approach, revealing a causal link between genetic predisposition to GERD and an increased risk of migraine, particularly for the MO subtype, but not for MA. Specifically, the odds ratios (OR) were 1.381 (95% CI: 1.190\u0026ndash;1.602, p = 2.04E-05) for GERD and migraine, 1.600 (95% CI: 1.311\u0026ndash;1.953, p = 3.67E-06) for GERD and MO, and 1.193 (95% CI: 0.983\u0026ndash;1.449, p = 0.074) for GERD and MA. Secondary analytical methods were also employed, yielding the following results: MR-Egger showed ORs of 1.239 (p = 0.634) for GERD and migraine, 1.369 (p = 0.594) for GERD and MA, and 1.624 (p = 0.421) for GERD and MO; weighted median produced ORs of 1.371 (p = 0.001) for GERD and migraine, 1.301 (p = 0.066) for GERD and MA, and 1.470 (p = 0.009) for GERD and MO; simple mode showed ORs of 1.976 (p = 0.014) for GERD and migraine, 1.383 (p = 0.379) for GERD and MA, and 1.502 (p = 0.320) for GERD and MO; and weighted mode provided ORs of 1.923 (p = 0.012) for GERD and migraine, 1.415 (p = 0.303) for GERD and MA, and 1.577 (p = 0.206) for GERD and MO. After conversion to odds ratios, all OR values were greater than 1 (Figures 2, 3, 4).\u003c/p\u003e\n\u003cp\u003e3.1.3. Sensitivity analysis and visualization\u003c/p\u003e\n\u003cp\u003eWe detected heterogeneity using MR-Egger regression and IVW analysis. MR-Egger regression showed Cochran\u0026apos;s Q values of 101.540 (p = 0.015) for GERD-migraine, 77.891 (p = 0.326) for GERD-MA, and 69.987 (p = 0.578) for GERD-MO. Similarly, IVW analysis resulted in Cochran\u0026apos;s Q values of 101.623 (p = 0.018) for GERD-migraine, 77.951 (p = 0.354) for GERD-MA, and 69.988 (p = 0.611) for GERD-MO. These results indicated no significant heterogeneity in the study (Supplementary Table 2). The heterogeneity visualization is depicted in Supplementary Figure 1 through funnel plots. For horizontal pleiotropy testing, MR-Egger intercepts suggested no pleiotropy for GERD and migraine studies, with an Egger intercept of 0.003 (p = 0.808). However, MR-PRESSO global testing indicated some degree of pleiotropy for GERD-migraine (global test: p = 0.025), although no outlier SNPs were identified, allowing us to proceed with the adjusted IVW method. Notably, no anomalies were detected in the analysis between GERD and MA and MO risks.\u0026nbsp;The MR-Egger intercepts indicated no horizontal pleiotropy for GERD-MA (intercept = -0.005, p = 0.813) and GERD-MO (intercept = -0.0005, p = 0.980). MR-PRESSO testing identified no outliers, and global tests did not show pleiotropy, with p-values of 0.782 for GERD-MA and 0.623 for GERD-MO (Supplementary Table 2).\u003c/p\u003e\n\u003cp\u003eThe leave-one-out approach was applied to sequentially eliminate individual SNPs for assessing the influence of any single instrumental variable on the causal relationship. The outcomes confirmed the stability of the Two-Sample Mendelian Randomization (TSMR) analysis, as illustrated in Figure 5.\u003c/p\u003e\n\u003cp\u003e3.2. Inverse TSMR Analysis\u003c/p\u003e\n\u003cp\u003eIn the context of TSMR, migraine, including both MA and MO subtypes, was considered the exposure, with GERD as the outcome. To include a wider range of instrumental variables, the p-value threshold was set below 1E-05. After confirming compliance with linkage disequilibrium parameters (r\u0026sup2; \u0026lt; 0.001 and KB \u0026gt; 10,000), we ensured the selected IVs conformed to the fundamental MR assumptions. SNPs absent from the outcome dataset and those palindromic with moderate allele frequencies were excluded. MR analysis was then performed on three distinct exposure datasets for migraine, categorized as MA (9 SNPs), MO (6 SNPs), and a combined group (5 SNPs) (Supplementary Table 1). Notably, all IVs had F statistics significantly exceeding a value of 10. The MR outcomes did not support a causal link between genetic predisposition to migraine (including MA and MO subtypes) and increased risk of GERD, with the following odds ratios (OR) and confidence intervals (CI): migraine-GERD, OR = 1.012 (95% CI: 0.965\u0026ndash;1.062, p = 0.502); MA-GERD, OR = 1.014 (95% CI: 0.982\u0026ndash;1.048, p = 0.393); MO-GERD, OR = 1.008 (95% CI: 0.954\u0026ndash;1.064, p = 0.780). Heterogeneity assessments revealed no significant variability in the migraine-GERD and MA-GERD analyses, with Cochran\u0026apos;s Q values and p-values as follows: migraine-GERD, MR-Egger: Q = 6.705, p = 0.460; IVW: Q = 6.792, p = 0.559; MA-GERD, MR-Egger: Q = 6.350, p = 0.174; IVW: Q = 7.236, p = 0.203. However, heterogeneity was present in the MO-GERD analysis (MO-GERD, MR-Egger: Q = 6.532, p = 0.163; IVW: Q = 13.412, p = 0.020). In horizontal pleiotropy evaluation, neither the MR-Egger intercepts nor the global MR-PRESSO tests identified any anomalies in the association between migraine and GERD risks, with results showing: migraine-GERD, Egger intercept = -0.0014, p = 0.776; global test, p = 0.628; MA-GERD, Egger intercept = 0.0049, p = 0.497; global test, p = 0.307. In the analysis of MO level and GERD risk, the MR-Egger intercept prior to the exclusion of the outlier SNP did not suggest pleiotropy (MO-GERD, Egger intercept = 0.0631, p = 0.109). However, the MR-PRESSO global test indicated some degree of pleiotropy (global test, p = 0.037). After removing the outlier SNP (rs1155688), subsequent analyses with the MR-Egger intercept and the global MR-PRESSO test revealed no further signs of pleiotropy in the relationship between MO levels and GERD risk (MO-GERD, Egger intercept = 0.0517, p = 0.175; global test, p = 0.177), leading to the removal of the MO dataset containing the outlier SNP.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe utilized a bidirectional TSMR strategy with available GWAS summary datasets to explore the reciprocal causality between GERD and migraine, encompassing both the general condition and its MA and MO subtypes. The MR study indicates that GERD correlates with a higher probability for migraine and the MO, without a corresponding link to an elevated risk of MA. Furthermore, our research does not corroborate a causal connection between the genetic predisposition to migraine, including all subtypes, and an escalated risk of GERD. While some heterogeneity appeared in the GERD-migraine and MO-GERD studies, random effects IVW analysis remains robust against heterogeneity, suggesting a stable causal effect.\u003c/p\u003e\n\u003cp\u003ePrior research has suggested an association between various GI diseases and primary headache syndromes, including migraine. The brain-gut axis facilitates bidirectional communication linking the brain and the intestinal tract, mediated by neural, hormonal, and immune pathways. Early reports noted improvements in migraine symptoms when gastroesophageal reflux symptoms subsided in patients with comorbidities of both diseases\u003csup\u003e30\u003c/sup\u003e. Retrospective analyses have shown GERD occurs more frequently among migraine sufferers than among those without migraines, with similar rates of gastritis and stomach ulcers in both groups\u003csup\u003e13, 31\u003c/sup\u003e. Notably, a significant association has been observed between primary childhood migraine and infantile gastroesophageal reflux, particularly for migraine without aura\u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe causal link between migraine and GERD remains ambiguous due to a scarcity of research in this domain. Predominantly, epidemiological investigations have relied on case-control or cross-sectional methodologies, which obscure the temporal progression, thereby complicating the determination of causality. Additionally, prior observational research has faced constraints, including small participant groups, difficulties in addressing reverse causality, and the influence of confounding factors. Utilizing more robust study designs, such as bidirectional TSMR analysis, can facilitate a better understanding of the causal dynamics between exposures and outcomes within the current research framework.\u003c/p\u003e\n\u003cp\u003eWe have identified a causal relationship between GERD and migraine, particularly its MO subtype, which may be explained by shared mechanisms involving the NF-\u0026kappa;B pathway in the inflammatory processes of both conditions\u003csup\u003e33, 34\u003c/sup\u003e. Research suggests that migraine pathogenesis involves cortical spreading depression (CSD)\u003csup\u003e35\u003c/sup\u003e, a self-propagating wave of depolarization that moves throughout the cerebral cortex. This phenomenon could activate the trigeminal input pathway, leading to changes in blood-brain barrier permeability through matrix metalloproteinase activation and upregulation. These changes may induce inflammatory responses in pain-sensitive meninges, resulting in migraine headaches through central and peripheral reflex mechanisms. Molecular cascades during CSD activation may include pannexin-1 channel opening, caspase-1 activation, pro-inflammatory mediator release, and activation of NF-\u0026kappa;B in astrocytes, transmitting inflammatory signals to trigeminal nerve fibers around cranial blood vessels\u003csup\u003e36\u003c/sup\u003e.\u0026nbsp;Studies on GERD mechanisms suggest that inflammatory cell infiltration precedes esophageal epithelial surface erosion\u003csup\u003e37\u003c/sup\u003e, indicating inflammation as a key mediator of erosion. Additionally, research has shown that in esophageal inflammation models, epithelial keratinocytes secrete IL-8, indicating that gastroesophageal reflux could activate inflammatory mechanisms, which may subsequently impair esophageal barrier function\u003csup\u003e38\u003c/sup\u003e. This activation of inflammatory pathways, including the NF-\u0026kappa;B pathway, by acidic substance-induced chemical injury to the esophageal mucosa can lead to the upregulation of various inflammatory mediators such as matrix metalloproteinase-3, matrix metalloproteinase-9, IL-1, IL-6, and IL-8. Therefore, we hypothesize that NF-\u0026kappa;B may play a role in various stages of GERD pathogenesis and migraine occurrence. The inflammatory pathways involved in GERD development may also contribute to migraine attacks, necessitating further investigation.\u003c/p\u003e\n\u003cp\u003eThe second possible explanation is speculated to be due to the phenomenon of delayed gastric emptying or gastric paresis, which is shared by both conditions. A prior study identified that delayed gastric emptying , or gastric paresis, substantially contributes to the onset of GERD\u003csup\u003e39\u003c/sup\u003e.\u0026nbsp;Additionally, research indicates that individuals with migraines experience delayed gastric emptying both during and after their attacks, suggesting a potential association with gastroesophageal reflux disease\u003csup\u003e40, 41\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFurthermore, dysfunction within the autonomic nervous system (ANS) was previously associated with headaches, especially migraines, and GI disorders\u003csup\u003e42, 43\u003c/sup\u003e. Therefore, ANS dysfunction may also be related to the pathogenesis of these two conditions. Abnormalities in visceral mechanosensation and vagal nerve function have been found to be associated with dyspepsia, and visceral neural dysregulation is also linked to migraines\u003csup\u003e12\u003c/sup\u003e. Additionally, individuals presenting with both headache and gastrointestinal symptoms tend to suffer from more intense and frequent episodes compared to those experiencing headaches in isolation\u003csup\u003e44, 45\u003c/sup\u003e. One possible reason is the increased interaction of shared underlying processes, leading to worse symptoms when headaches and gastrointestinal issues occur together\u003csup\u003e44\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCurrently, here is currently considerable debate about whether MO and MA result from the same pathogenic mechanisms. MO and MA exhibit notable differences in their clinical presentations. For instance, MO is more likely to occur after stressful events, exams, or during the post-menstrual relaxation phase in women. In contrast, MA attacks lack this characteristic and may be triggered by intense natural or artificial visual stimuli in some cases\u003csup\u003e46\u003c/sup\u003e.\u0026nbsp;Regarding associations with key female reproductive events, MO is notably affected by menstruation, unlike MA. Approximately one-fifth of MA cases experience heightened attack risks in the perimenstrual phase, compared to approximately three-quarters of MO cases\u003csup\u003e47\u003c/sup\u003e. During gestation, MO commonly abates in most instances, whereas MA tends to continue or intensify, and occasionally emerges for the first time\u003csup\u003e47\u003c/sup\u003e. Oral contraceptives generally exert no adverse effects on MO, yet in the majority of cases, they can worsen MA.\u003c/p\u003e\n\u003cp\u003eMA is characterized by visual, language, sensory, or brainstem symptoms that typically precede the headache phase. The pathogenesis of MA is widely attributed to CSD, involving neuronal and glial depolarization which then transitions to cortical hyperpolarization, progressing at 3\u0026ndash;5 mm/min. This process corresponds to significant alterations in ion homeostasis and neurotransmitter release\u003csup\u003e48\u003c/sup\u003e. Additionally, there is a temporary surge in cerebral blood flow due to the increased energy demands for restoring homeostasis\u003csup\u003e49\u003c/sup\u003e. While the pathophysiology of other aura types (such as speech, sensory, and brainstem) is less studied, evidence suggests that CSD may also underlie their mechanisms\u003csup\u003e49\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eResearch has also demonstrated differences in cerebral hemodynamics between MO and MA, with MA characterized by significant vasodilation and trigeminal vascular sensitization (TVS), which can trigger aura\u003csup\u003e50\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe study findings point to a potential causative link connecting GERD and the occurrence of migraine, particularly the MO subtype, underscoring the importance of closely monitoring migraine symptoms in GERD patients,\u0026nbsp;which could be beneficial in clinical practice.\u0026nbsp;Additionally, it\u0026apos;s crucial to be attentive to mixed symptoms of GERD or migraine.\u003c/p\u003e\n\u003cp\u003eOn the other hand, MO lacks the premonitory aura symptoms seen in MA, and its pathophysiology is not fully elucidated. Some theories propose that CSD may occur silently in subcortical regions, such as the hypothalamus, in MO patients\u003csup\u003e48\u003c/sup\u003e. However, studies have shown that drugs inhibiting CSD, such as tonabersat, significantly reduce the frequency of aura attacks but not MO attacks\u003csup\u003e51\u003c/sup\u003e, indicating potential differences in the underlying mechanisms between the two types of migraine.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur bidirectional TSMR study offers several advantages. Firstly, we applied MR analysis with SNPs that have a strong association (F\u0026gt;10), akin to the experimental setup of RCTs, to yield robust evidence. Unlike RCTs, which can be constrained by cost and sample size, MR analysis circumvents issues of reverse causation and confounding. Secondly, by relying on GWAS databases with European population samples, our study mitigates bias from demographic variability. Thirdly, the insights from our analysis could inform healthcare policy, especially if a causal link between GERD and migraine, notably the MO subtype, is established, potentially influencing strategies for mitigating and managing the condition.\u003c/p\u003e\n\u003cp\u003eNevertheless, this research acknowledges certain constraints, primarily the heterogeneity in the analysis of GERD versus migraine, and MO versus GERD. The heterogeneity in the former could be due to the inclusion of both MA and MO subtypes in migraine data, while the subsequent examination of GERD versus MO revealed no heterogeneity. Considering the presence of MA data in migraine may contribute to the heterogeneity. The presence of heterogeneity in the latter may be explained by the small sample size.\u0026nbsp;Additionally, we used GERD data rather than data encompassing all digestive system-related diseases, so the current findings cannot be extrapolated to patients with other digestive system disorders. Further research on this topic is warranted. Secondly, as current investigations primarily focus on the link between GERD and migraine, and the number of such investigations is limited, specific studies addressing the connection between GERD and the MO subtype of migraine are sparse. Therefore, the analysis of potential mechanisms remains somewhat limited in this regard.\u0026nbsp;Thirdly, GERD is categorized into reflux esophagitis (RE) and non-erosive reflux disease (NERD). Our study was unable to ascertain the connection among these subtypes with the severity of migraine, including low-frequency as well as high-frequency episodic forms. Additionally, gastroscopy, an essential diagnostic tool for GERD, may lead to an underestimation of diagnosed cases due to its invasive nature. This bias remains unavoidable. Fourthly, despite no significant difference in GERD prevalence among males and females, migraine prevalence is higher in females. However, our reliance on public databases precluded subgroup analyses based on factors like age and gender. Fifthly, the GWAS data\u0026apos;s European focus restricts the generalizability of our findings to non-European demographics. Lastly, expanding GWAS sample sizes could strengthen IVs, highlighting the need for larger-scale studies for more comprehensive research.\u0026nbsp;Therefore, additional research is necessary to confirm the results of our current study.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe bidirectional Two-Sample Mendelian Randomization analysis suggests an association between GERD susceptibility and an increased likelihood of migraine and its MO subtype, with no observed correlation with the risk of MA.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eData Availability Statement\u003c/p\u003e\n\u003cp\u003eThe study\u0026apos;s original contributions are detailed in the published article and its Supplementary material. For any additional inquiries, please contact the corresponding author(s).\u003c/p\u003e\n\u003cp\u003eEthics statement\u003c/p\u003e\n\u003cp\u003eThe research involving human participants adhered to the local legislation and institutional guidelines, which did not necessitate ethical review and approval. In line with national regulations and institutional policies, obtaining written informed consent from the patients/participants or their legal guardians/next of kin was not required for this study.\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eXJ was instrumental in the study design, data collection, statistical analysis, and preparation of the original draft. JHZ contributed by reviewing and revising the manuscript. JX provided supervision over the review process and gave the final approval of the manuscript. Each author has made significant contributions to the article and has given approval to the version submitted for publication.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThe authors declare that they did not receive any financial support for the research, authorship, and publication of this article.\u003c/p\u003e\n\u003cp\u003eAdditional Information\u003c/p\u003e\n\u003cp\u003eCompeting interests statement\u003c/p\u003e\n\u003cp\u003eThe authors affirm that the research was carried out without any commercial or financial interests that might be perceived as potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003ePublisher\u0026rsquo;s note\u003c/p\u003e\n\u003cp\u003eThe views and assertions expressed in this article are those of the authors alone and do not necessarily reflect the opinions of their affiliated institutions, the publisher, the editors, or the reviewers. The publisher does not assume responsibility for any product evaluated in the article, nor does it endorse any claims made by the manufacturer.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe are grateful to the genetics consortiums for providing the GWAS summary data through their publicly accessible databases, which facilitated our research. Our data were sourced from these open resources.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLipton, R. B., Stewart, W. F., Diamond, S., Diamond, M. L. \u0026amp; Reed, M. 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U S A\u003c/em\u003e. \u003cb\u003e98\u003c/b\u003e, 4687\u0026ndash;4692. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.071582498\u003c/span\u003e\u003cspan address=\"10.1073/pnas.071582498\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2001).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Migraine, mendelian randomization, gastroesophageal reflux disease, genome-wide association analysis, causality, bidirectional","lastPublishedDoi":"10.21203/rs.3.rs-4897548/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4897548/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEpidemiological studies suggest a link between gastroesophageal reflux disease (GERD) and migraine, but the causal relationship remains unclear. This study aimed to clarify this relationship using two-sample Mendelian Randomization (MR).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eData on GERD and migraine, including subtypes with aura (MA) and without aura (MO), were collected from genome-wide association studies (GWAS). SNPs were selected as instrumental variables (IVs) by accounting for linkage disequilibrium and removing unbalanced connections. The primary analysis used the inverse variance-weighted (IVW) method with supplementary analyses. Heterogeneity and pleiotropy were assessed using Cochran's Q test, MR-Egger intercept, and MR-PRESSO. Finally, reverse causality was explored.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe IVW method indicated a causal link between GERD and increased risk of migraine (OR\u0026thinsp;=\u0026thinsp;1.381, 95% CI: 1.190\u0026ndash;1.602, p\u0026thinsp;=\u0026thinsp;2.04E-05), particularly the MO subtype (OR\u0026thinsp;=\u0026thinsp;1.600, 95% CI: 1.311\u0026ndash;1.953, p\u0026thinsp;=\u0026thinsp;3.67E-06). No significant association was found for MA (OR\u0026thinsp;=\u0026thinsp;1.193, 95% CI: 0.983\u0026ndash;1.449, p\u0026thinsp;=\u0026thinsp;0.074). Reverse MR analysis showed no causal relationship between migraine and GERD.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eGERD is causally linked to an increased risk of migraine, especially the MO subtype. No reverse causal relationship was found, highlighting the importance of considering migraine subtypes in understanding their association with GERD.\u003c/p\u003e","manuscriptTitle":"Bidirectional analysis of gastroesophageal reflux disease and migraine using two-sample Mendelian randomization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-23 17:37:06","doi":"10.21203/rs.3.rs-4897548/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8405b9ab-6c85-4cf8-b225-1e3db392f3e8","owner":[],"postedDate":"September 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":38001469,"name":"Health sciences/Neurology/Neurological disorders/Migraine"},{"id":38001470,"name":"Health sciences/Gastroenterology/Gastrointestinal diseases/Oesophageal diseases/Gastro oesophageal reflux disease"}],"tags":[],"updatedAt":"2025-04-15T07:23:46+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-23 17:37:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4897548","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4897548","identity":"rs-4897548","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}