Exploring the Potential Association Between Frozen Shoulder and Gastroesophageal Reflux Disease Using Mendelian Randomisation

Exploring the Potential Association Between Frozen Shoulder and Gastroesophageal Reflux Disease Using Mendelian Randomisation | 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 Research Article Exploring the Potential Association Between Frozen Shoulder and Gastroesophageal Reflux Disease Using Mendelian Randomisation Bin Luo, Zongyuan Huang, Shuai Zhang, Yanting Chen, Mingde Cao, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5333407/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: Frozen shoulder (FS) has been suggested as a potential risk factor for gastroesophageal reflux disease (GERD). However, observational studies are limited in their ability to establish a causal relationship between FS and GERD. Methods: This study aimed to investigate the genetic causal association between FS and GERD using a two-sample Mendelian randomization (MR) approach. Genome-wide association study (GWAS) summary data for FS were obtained from the UK Biobank, while GWAS summary data for GERD were sourced from the FinnGen Consortium. Multiple MR methods, including inverse-variance weighted (IVW), MR Egger, weighted median, simple mode, and weighted mode, were applied to estimate the causal effect. To assess heterogeneity, Cochrane's Q test was performed. Horizontal pleiotropy was evaluated using the MR-Egger intercept test and MR pleiotropy residual sum and outliers (MR-PRESSO) test. Sensitivity analyses, including leave-one-out analysis, were conducted to ensure the robustness of the findings. Results: The IVW method indicated a potential causal relationship between FS and GERD (P = 0.018). There was no evidence of horizontal pleiotropy (P > 0.05), and heterogeneity among genetic variants was not significant (P > 0.05). Furthermore, leave-one-out analysis demonstrated that the association was consistent and reliable. Limitations: All participants included in the GWAS data were of European ancestry, which may limit the generalizability of the results. Additionally, due to data constraints, stratified analyses based on different populations, ethnicities, or age groups were not performed. Conclusion: This two-sample MR analysis provides evidence of a genetic causal association between FS and GERD, highlighting the need for further research to explore this relationship in diverse populations. Frozen Shoulder Gastroesophageal Reflux Disease Mendelian randomization Bidirectional Figures Figure 1 Figure 2 Figure 3 1. Introduction Adhesive Capsulitis is a prevalent shoulder joint disorder characterised by restricted movement and pain [ 1 , 2 ]. Gastroesophageal reflux disease (GERD) is a common digestive condition marked by symptoms such as acid reflux, heartburn, and chest pain [ 3 ]. Both conditions impose substantial emotional, physical, and economic burdens on patients, often leading to a significant decline in their quality of life. Despite their distinct clinical presentations, emerging research suggests a potential connection between adhesive capsulitis and GERD. Cucchi D et al. demonstrated that patients with GERD have an increased likelihood of developing adhesive capsulitis, indicating a possible causal relationship [ 4 ]. However, traditional data analysis methods are limited in their ability to elucidate causal links due to confounding [ 5 ]. Mendelian randomisation (MR), a method based on the principle of independent assortment, uses genetic variations as instrumental variables (IVs) to evaluate the causal relationship between exposure and outcome [ 6 ]. Since an individual's genotype is fixed at conception and unaffected by environmental factors, MR minimises the risk of reverse causality. With the increasing availability of detailed phenotypic data from large-scale genome-wide association studies (GWAS), MR has become a valuable tool for investigating the etiological mechanisms of various diseases [ 7 ]. Previous studies, such as those by Millard et al., employed MR to demonstrate that GERD's causal effect on shoulder periarthritis is independent of body mass index (BMI) [ 8 , 9 ]. Liu Lin et al. further identified an association between GERD and an increased incidence of shoulder periarthritis. However, the site-specific causal relationship and underlying signalling pathways remain unclear[ 10 , 11 ]. Therefore, the objective of this study is to evaluate the causal relationship between shoulder periarthritis and GERD through a two-sample bidirectional MR framework. This analysis will consider multiple GERD measures (frequency of acid reflux, heartburn frequency, and chest pain frequency) alongside various shoulder periarthritis measures (presence of shoulder periarthritis at any site, early shoulder periarthritis and advanced shoulder periarthritis) [ 12 ]. Employing a two-sample MR approach allows for the exploration of causal relationships using data from independent GWAS, thus enhancing the method's effectiveness and reliability compared to single-sample MR [ 13 ]. In this study, the exposure variable is frozen shoulder, while the outcome variable is GERD. Single-nucleotide polymorphisms (SNPs) will be selected as IVs for further analysis. Utilising the Inverse Variance Weighted (IVW) method, we found a p-value of less than 0.05, indicating a significant causal association between frozen shoulder and GERD. This finding enhances our understanding of the potential role of a frozen shoulder in the pathogenesis of GERD and provides new insights for clinical interventions. Our investigation aims to clarify the causal relationship between shoulder periarthritis and GERD, revealing potential links between these two diseases and guiding future clinical prevention and treatment strategies. Additionally, pleiotropic effects may exist among variables influencing shoulder periarthritis, such as BMI, and genetic factors related to GERD. 2. Materials and Methods 2.1 Study Design and Data Resources This study employed a two-sample MR approach to investigate the causal relationship between shoulder periarthritis and GERD. Shoulder periarthritis was considered the exposure, and GERD was the outcome. SNPs were selected as instrumental variables (IVs) for analysis. The study adheres to the three key assumptions of MR: (1) the IVs are strongly associated with the exposure (P 10); (2) the IVs are independent of confounders; and (3) the IVs influence the outcome solely through the exposure. GWAS summary data for shoulder periarthritis were obtained from the EBI database, comprising 627,998 participants of European ancestry. All participants provided informed consent. GWAS summary data for GERD were accessed from ( https://gwas.mrcieu.ac.uk/datasets/ebi-a-GCST90000514/ ), with 129,080 cases and 473,524 controls. Cases due to alcohol or psychoactive substances were excluded. 2.2 Instrumental Variable Selection For MR analysis, the relevance, independence and exclusion restriction assumptions must be met. SNPs were selected based on strong associations with the exposure (P < 5×10^-8), and SNPs with an F-value < 10 were excluded to avoid weak instrument bias. The F-value was calculated as F = R² × (N − 2) / (1 - R²), where R² represents the variance explained by each IV. R² was determined using the formula R² = 2 × EAF × (1 - EAF) × Beta², where Beta is the allele effect size, and EAF is the effect allele frequency. [ 14 ] To reduce bias from linkage disequilibrium, we applied clumping (r² < 0.001, physical distance = 10,000 kb) to ensure IV independence. Additionally, we used Phenoscanner V2 to exclude SNPs associated with confounders such as dementia and drug dependence, ensuring the IVs influence the outcome only through exposure. Figure 1 illustrates the relationships allowed (solid lines, A and B) and those excluded (dashed lines, C and D). Finally, to align the effect alleles across datasets, we harmonised the exposure and outcome data and removed palindromic SNPs with intermediate allele frequencies. [ 15 ]As shown in Fig. 1 , the solid lines and arrows labeled A and B indicate valid causal pathways, where the relationship between SNPs associated with Frozen Shoulder (exposure) and Gastroesophageal Reflux Disease (outcome) is explored. In contrast, the dashed lines and arrows labeled C and D represent potential confounding pathways, which are excluded as they violate the assumptions of the instrumental variable approach. To ensure the effect alleles are consistent between the exposure and outcome datasets, a harmonization process was performed, aligning the alleles across both datasets. This step ensures that the alleles correspond to the same genetic variants, minimizing bias and enhancing the reliability of the Mendelian Randomization analysis. 2.3 Statistical Analysis To investigate the genetic association between frozen shoulder and GERD, we employed five MR methods: MR-Egger regression, weighted median, IVW, simple mode and weighted mode. The IVW method, which assumes that all SNPs used are valid, was considered the primary analysis method due to its potential to provide the most accurate estimates. Subsequently, statistical power was calculated using the tool available at https://shiny.cnsgenomics.com/mRnd/ , based on the research of Brion et al. This tool computes statistical power for MR studies using the non-central parameter. The calculation of power requires the total R² value, representing the variance in the exposure explained by each IV, as previously described. To ensure the robustness of our findings, we performed several additional analyses. First, Cochran's Q test was applied to assess heterogeneity in the associations, with the symmetry of the funnel plot providing further validation. To detect pleiotropy, we used the MR-Egger intercept test and the MR Pleiotropy Residual Sum and Outlier (MR-PRESSO) global test, the latter of which also identifies outliers and recalculates estimates after excluding them. Sensitivity analysis was conducted via a leave-one-out test, where each SNP was excluded sequentially to determine if any single SNP had a disproportionate influence on the overall results. All statistical analyses were performed using the TwoSampleMR package in R (version 4.2.0), with statistical significance defined as P < 0.05 [ 16 ]. 3. Results 3.1 IV Selection We initially identified 64 SNPs strongly associated with the exposure data (P 10, r² < 0.001, physical window = 10,000 kb) as potential IVs, with all F-values exceeding 10. Detailed F-value data is provided in Supplementary Table 1 . No SNPs were excluded based on their association with outcomes or confounders (P < 1 × 10⁻⁵) using Phenoscanner V2. After harmonising the exposure and outcome datasets, 64 SNPs were selected for further MR analysis. Using SNPs linked to frozen shoulder as IVs, we thoroughly investigated the causal relationship between frozen shoulder and GERD. Our final set of instrumental variables met the linkage disequilibrium (LD) independence criterion (r² < 0.001) and achieved a significance level of P < 1 × 10⁻⁵ in GWAS data. In analysing the causal relationship, we carefully examined potential confounding factors for the selected IVs. The heterogeneity test revealed no significant heterogeneity among the IVs (Q_P > 0.05), confirming that pleiotropy or genetic heterogeneity did not impact the MR results. Additionally, the negative control analysis demonstrated that frozen shoulder was not associated with the selected IVs, further validating their applicability in this study. In the reverse MR analysis, we explored the effect of frozen shoulder on GERD, again using SNPs associated with frozen shoulder as IVs. We found only one SNP that met the LD independence criterion (r² < 0.001) and a significance level of P 0.05), confirming that pleiotropy or genetic heterogeneity did not affect our MR analysis results. The negative control analysis similarly affirmed that frozen shoulder was unrelated to the selected IVs, reinforcing their suitability for this study. 3.2 MR Analysis The random-effects IVW method served as the primary approach to examine the genetic association between frozen shoulder and GERD [ 17 ]. The analysis demonstrated that frozen shoulder is an independent risk factor for GERD (P = 2.95 × 10⁻⁵, odds ratio [OR] [95% confidence interval (CI)] = 0.725 [0.6281–0.8369]) ( Fig. 2 ) . The weighted median method further supported this genetic causal relationship. Table 1 summarises the results from the five MR methods employed in this study [ 18 ]. The Cochrane’s Q test revealed no heterogeneity in the association between frozen shoulder and GERD (P = 0.924), and the funnel plot exhibited a symmetrical distribution of SNPs ( Fig. 3 A ) . The MR-Egger intercept test and the MR-PRESSO global test confirmed the absence of horizontal pleiotropy (P = 0.532). Additionally, the MR-PRESSO analysis detected no outliers in the Mendelian randomisation analysis. The leave-one-out test showed that the overall results were not influenced by any single SNP ( Fig. 3 B ) , affirming the stability and robustness of the findings. 4. Discussion This study employed a two-sample MR analysis using large-scale GWAS data to investigate the causal relationship between frozen shoulder and GERD. The results indicate that genetic susceptibility to frozen shoulder is associated with an increased risk of GERD. This finding suggests that early intervention targeting frozen shoulder symptoms could help reduce the incidence and severity of GERD, with potential benefits in preventing long-term gastrointestinal complications and improving clinical outcomes for patients [ 19 ]. A study conducted in China similarly reported a positive correlation between frozen shoulder and GERD risk (IVW: P = 2.954804e-05, OR [95% confidence interval (CI)] = 1.482 [1.136–1.933]). This study was also based on large-scale GWAS data and MR analysis [ 20 ]. However, prior observational studies have yielded conflicting evidence regarding the association between frozen shoulder and GERD. Our study demonstrates a potential causal effect of frozen shoulder on GERD, suggesting that musculoskeletal conditions like frozen shoulder may influence gastrointestinal diseases through shared inflammatory pathways or other mechanisms [ 21 ]. Moreover, compared to other musculoskeletal disorders, frozen shoulder is often accompanied by prolonged inflammation and restricted joint mobility, which may trigger a heightened physiological stress response, indirectly increasing the risk of GERD [ 22 ]. The use of genetic variants associated with frozen shoulder in MR analysis allows for more accurate identification of causal relationships. One possible bias in this relationship is medication use. Proton pump inhibitors (PPIs), commonly prescribed for GERD, could obscure the true association between the two conditions. However, according to the GWAS database ( https://www.ebi.ac.uk/gwas ), no significant SNPs have been linked to PPI use, indicating that medication is unlikely to be a confounding factor in the relationship between frozen shoulder and GERD. Nevertheless, further research is required to elucidate the underlying mechanisms connecting these two diseases. Existing observational studies have not established a causal relationship between frozen shoulder and GERD. Frozen shoulder often coexists with other conditions, such as cognitive impairment and cardiovascular diseases, which may partially influence the observed positive correlation between the two [ 23 – 25 ]. GWAS serve as a powerful tool for investigating complex diseases by identifying individuals or gene groups that may be difficult to detect in single-gene studies. This approach not only validates existing findings but also opens new avenues for research. Based on large-scale GWAS data, our study provides genetic evidence for a causal relationship between these two interrelated conditions [ 26 , 27 ]. Elevated cortisol levels have been linked to the development of GERD, potentially due to reduced physical activity [ 28 ]. Chronic pain associated with a frozen shoulder could exacerbate GERD symptoms, as it may impact eating habits and contribute to increased stomach discomfort and reflux [ 29 , 30 ]. Moreover, changes in the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased gastric acid secretion, may represent a potential link between frozen shoulder and GERD. This connection could arise through several mechanisms, including GERD-related chronic inflammation, autonomic nervous system dysfunction, and chronic pain and stress responses, which are also implicated in the pathogenesis of frozen shoulder [ 31 ]. While direct evidence for this connection stems from gastrointestinal function studies, indirect evidence comes from etiological studies that suggest the transmission of inflammatory mediators and systemic inflammation may enhance oesophageal sensitivity. These factors might represent a shared neural pathway between frozen shoulder and GERD. Autonomic dysfunction, commonly observed in patients with frozen shoulder, could be attributed to chronic pain, inflammation, or psychological stress [ 32 ]. The autonomic nervous system, particularly in regulating lower oesophageal sphincter (LES) tone, plays a critical role in oesophageal and gastric functions, and its dysfunction could contribute to GERD. Inflammation and cytokines are well-recognised contributors to the onset and progression of frozen shoulder [ 33 , 34 ]. The overlap of these neurobiological mechanisms may explain the close association between frozen shoulder and GERD. Pathophysiological changes, such as neural network damage, neurodegeneration, neuroinflammation, glial cell alterations, and vascular abnormalities during frozen shoulder development, may heighten susceptibility to GERD. When the gastrointestinal system faces additional stress (such as trauma, infection or surgery), GERD symptoms may emerge. Furthermore, interventions targeting frozen shoulder symptoms have been shown to reduce GERD risk in hospitalised patients, supporting the hypothesis that genetic susceptibility to frozen shoulder increases GERD risk [ 35 ]. Treatments such as non-steroidal anti-inflammatory drugs (NSAIDs) and physical therapy, commonly used to alleviate inflammation and pain in frozen shoulder, have been shown to overlap with strategies to manage GERD symptoms [ 36 ]. Additionally, recent studies highlight the benefits of lifestyle and dietary modifications in preventing GERD, particularly in patients with frozen shoulder, further reinforcing the potential causal relationship between these two conditions. This study has several notable strengths. First, it is the first to investigate the causal relationship between frozen shoulder and GERD using large-scale GWAS data, thereby filling a significant gap in the literature. By employing a two-sample MR approach, we effectively addressed common limitations found in observational studies, such as reverse causality, confounding factors, and biases. Second, we rigorously screened all IVs used in the MR analysis, with the lowest F-value being 137.831, ensuring the accuracy of our results. Finally, we employed multiple analytical methods to comprehensively evaluate the sensitivity, horizontal pleiotropy, and heterogeneity of our findings. All tests consistently indicated that the association between frozen shoulder and GERD is stable and reliable. However, this study also has some limitations. Firstly, all participants in the GWAS were of European ancestry, necessitating further validation to determine the applicability of our findings to other populations or regions. Secondly, despite using the MR intercept test and MR-PRESSO global test to detect and adjust for pleiotropy in genetic variants, there may still be uncontrolled confounding factors, such as education level, personality traits, and nutritional status, that could introduce bias. Thirdly, since our MR analysis relies on data from the underlying GWAS meta-analysis, we were unable to perform stratified analyses by country, ethnicity, or age group. Consequently, the observed effects of frozen shoulder in this study may not apply to populations with different characteristics (such as ethnicity and age). 5. Conclusion This study identified a causal relationship between frozen shoulder and GERD, potentially driven by shared biological mechanisms. These findings offer new insights into the genetic and biological pathways linking frozen shoulder and GERD. Declarations Ethics approval and consent to participate Not applicable. Clinical trial number Not applicable. Consent for publication The publication of this manuscript is approved by all authors. Availability of data and materials Not applicable. Competing interests The authors declare that they have no competing interests. Funding Natural Science Foundation of Guangdong Province(2020A1414010368). Guangzhou Municipal Science and Technology Bureau（NO.SL2022A04J00907） Authors' contributions YC S had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: YC S, B L Acquisition of data: B L ZY H RuiZe Chen. Analysis and interpretation of data: B L ZY H . Drafting of the manuscript:B L ZY H. Critical revision of the manuscript for important intellectual content: YC S, B L ZY H. Statistical analysis: B L ZY H, X W, Q G. Supervision: YC S. All authors read and approved the final manuscript. Acknowledgement Not applicable. References NL M, et al. Frozen shoulder. Nat reviews Disease primers. 2022;8(1):59. M, K., et al., Frozen shoulder. BMJ (Clinical research ed.), 2022. 377: p. e068547. R, F., Gastroesophageal Reflux Disease. N Engl J Med, 2022. 387(13): pp. 1207–16. D C, et al. The presence of gastroesophageal reflux disease increases the risk of developing postoperative shoulder stiffness after arthroscopic rotator cuff repair. J Shoulder Elbow Surg. 2020;29(12):2505–13. AS C, et al. Analysis of 90-Day Readmissions After Total Shoulder Arthroplasty. Orthop J sports Med. 2019;7(9):2325967119868964. Levin MG, Burgess S. 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Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files floatimage1.png Table 1: Mendelian Randomisation Analysis MendelianRandomizationORand95CI.csv mrbaseebiaGCST90000514.ebiaGCST90000513forest.pdf mrbaseebiaGCST90000514.ebiaGCST90000513funnel.pdf mrbaseebiaGCST90000514.ebiaGCST90000513leaveoneout.pdf mrbaseebiaGCST90000514.ebiaGCST90000513scatter.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. 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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-5333407","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":371887576,"identity":"de03e1b4-c7d0-413c-b052-d2eca7a32c6f","order_by":0,"name":"Bin Luo","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangdong Pharmaceutical University, Guangdong Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Luo","suffix":""},{"id":371887577,"identity":"84a9ffe8-f449-4b28-92b9-9b28f5d5e071","order_by":1,"name":"Zongyuan 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Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYBACAwglIcfP3nzgwIcfxGuxMZbsOZZ4cGYP8VrSEg1u+Bgf5mAjQos5/xkziZ87Dicw3OD5cJiBh0GeX+wAfi2WDWfMJHvPHM5jnN274XCBBYPhzNkJBBx2sMdMgrftcDGzzNkNh2fwMCQY3Cak5TCPmeTftsOJbRI5Dw7zsBGj5RiPmTRvW1pij0QOA5FazrAVW8u22RhL8BwzAAayBBF+OX944823bRJy9sebH3/48MNGnl+agBYGBg4DZJ4EIeUgwP6AGFWjYBSMglEwkgEAmEtIfPJ4RKMAAAAASUVORK5CYII=","orcid":"","institution":"The First Affiliated Hospital of Guangdong Pharmaceutical University, Guangdong Pharmaceutical University","correspondingAuthor":true,"prefix":"","firstName":"Yancheng","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2024-10-25 15:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5333407/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5333407/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69246735,"identity":"d0bb7759-002f-428c-a64c-523c9c6247ae","added_by":"auto","created_at":"2024-11-18 11:09:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":87834,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the present bidirectional MR study of the associations of Frozen Shoulder with Gastroesophageal Reflux Disease Black lines show the relationship across instrumental variables, exposure, and outcomes in the MR study examining the effects of Frozen Shoulder on Gastroesophageal Reflux Disease, and purple lines show these relationships in the reverse MR study\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5333407/v1/3d2be2ae81357af5b482d011.png"},{"id":69246737,"identity":"00d2c14f-5adc-4ca9-a3fc-98499b90a6bb","added_by":"auto","created_at":"2024-11-18 11:09:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":152743,"visible":true,"origin":"","legend":"\u003cp\u003eThe association between Frozen Shoulder and Gastroesophageal Reflux Disease.\u003c/p\u003e\n\u003cp\u003e2a Forest plot of causal effects of Frozen Shoulder \u0026nbsp;on Gastroesophageal Reflux Disease\u003c/p\u003e\n\u003cp\u003e2b Funnel plot Scatter plot of causal effects of Frozen Shoulder on Gastroesophageal Reflux Disease. The slope of the line represents the causal effect of each method.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5333407/v1/5f1bac8d7b5e1a3f76dd7814.png"},{"id":69246736,"identity":"fd5093da-1929-422a-8936-27e8c3cd3567","added_by":"auto","created_at":"2024-11-18 11:09:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":148604,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect size for Frozen Shoulder on Gastroesophageal Reflux Disease\u003c/p\u003e\n\u003cp\u003e3A: The funnel plot showed that the SNPs were symmetric, indicating that there was no heterogeneity in the association.\u003c/p\u003e\n\u003cp\u003e3B: The leave-one-out test showed that the result was not affected by single\u003c/p\u003e\n\u003cp\u003einfluential SNP, so this association was stable\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5333407/v1/315fab870f09da9f7f9cd5a8.png"},{"id":69247380,"identity":"e9d9d1c5-fdf3-4ed6-b330-0cdf5b2726cc","added_by":"auto","created_at":"2024-11-18 11:17:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":813006,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5333407/v1/ecd80270-b61b-4a08-be35-29a7d6fa7670.pdf"},{"id":69246730,"identity":"4a45ce9b-bbed-44a8-b619-321a61aa9ea9","added_by":"auto","created_at":"2024-11-18 11:09:33","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1800438,"visible":true,"origin":"","legend":"\u003cp\u003eTable 1: Mendelian Randomisation Analysis\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5333407/v1/a4a2ba5de8d6bf41f2e11f8d.png"},{"id":69246693,"identity":"cf7d075c-288d-495e-9102-27c9ce07fc97","added_by":"auto","created_at":"2024-11-18 11:09:31","extension":"csv","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":499,"visible":true,"origin":"","legend":"","description":"","filename":"MendelianRandomizationORand95CI.csv","url":"https://assets-eu.researchsquare.com/files/rs-5333407/v1/d200df5081fb2499a87386e7.csv"},{"id":69246732,"identity":"b50e2220-3a48-4d71-a4d7-4027029677d2","added_by":"auto","created_at":"2024-11-18 11:09:34","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11645,"visible":true,"origin":"","legend":"","description":"","filename":"mrbaseebiaGCST90000514.ebiaGCST90000513forest.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5333407/v1/d862c7e4a36a6bec9264cdd0.pdf"},{"id":69246741,"identity":"9abba5a8-8612-4207-a95c-86f231c24801","added_by":"auto","created_at":"2024-11-18 11:09:40","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":8904,"visible":true,"origin":"","legend":"","description":"","filename":"mrbaseebiaGCST90000514.ebiaGCST90000513funnel.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5333407/v1/47eabc2bde824e949f241bcc.pdf"},{"id":69246733,"identity":"f9e81c36-15e1-4176-80be-5f83a260ea7b","added_by":"auto","created_at":"2024-11-18 11:09:34","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":11421,"visible":true,"origin":"","legend":"","description":"","filename":"mrbaseebiaGCST90000514.ebiaGCST90000513leaveoneout.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5333407/v1/bca783661341dd0fa646fcbe.pdf"},{"id":69246734,"identity":"bfc0404b-b879-4102-8397-19e0e3066282","added_by":"auto","created_at":"2024-11-18 11:09:34","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":11335,"visible":true,"origin":"","legend":"","description":"","filename":"mrbaseebiaGCST90000514.ebiaGCST90000513scatter.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5333407/v1/e4f38d2400f186f8c207b55c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring the Potential Association Between Frozen Shoulder and Gastroesophageal Reflux Disease Using Mendelian Randomisation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAdhesive Capsulitis is a prevalent shoulder joint disorder characterised by restricted movement and pain [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Gastroesophageal reflux disease (GERD) is a common digestive condition marked by symptoms such as acid reflux, heartburn, and chest pain [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Both conditions impose substantial emotional, physical, and economic burdens on patients, often leading to a significant decline in their quality of life. Despite their distinct clinical presentations, emerging research suggests a potential connection between adhesive capsulitis and GERD. Cucchi D et al. demonstrated that patients with GERD have an increased likelihood of developing adhesive capsulitis, indicating a possible causal relationship [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, traditional data analysis methods are limited in their ability to elucidate causal links due to confounding [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMendelian randomisation (MR), a method based on the principle of independent assortment, uses genetic variations as instrumental variables (IVs) to evaluate the causal relationship between exposure and outcome [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Since an individual's genotype is fixed at conception and unaffected by environmental factors, MR minimises the risk of reverse causality. With the increasing availability of detailed phenotypic data from large-scale genome-wide association studies (GWAS), MR has become a valuable tool for investigating the etiological mechanisms of various diseases [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Previous studies, such as those by Millard et al., employed MR to demonstrate that GERD's causal effect on shoulder periarthritis is independent of body mass index (BMI) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Liu Lin et al. further identified an association between GERD and an increased incidence of shoulder periarthritis. However, the site-specific causal relationship and underlying signalling pathways remain unclear[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, the objective of this study is to evaluate the causal relationship between shoulder periarthritis and GERD through a two-sample bidirectional MR framework. This analysis will consider multiple GERD measures (frequency of acid reflux, heartburn frequency, and chest pain frequency) alongside various shoulder periarthritis measures (presence of shoulder periarthritis at any site, early shoulder periarthritis and advanced shoulder periarthritis) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEmploying a two-sample MR approach allows for the exploration of causal relationships using data from independent GWAS, thus enhancing the method's effectiveness and reliability compared to single-sample MR [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In this study, the exposure variable is frozen shoulder, while the outcome variable is GERD. Single-nucleotide polymorphisms (SNPs) will be selected as IVs for further analysis.\u003c/p\u003e \u003cp\u003eUtilising the Inverse Variance Weighted (IVW) method, we found a p-value of less than 0.05, indicating a significant causal association between frozen shoulder and GERD. This finding enhances our understanding of the potential role of a frozen shoulder in the pathogenesis of GERD and provides new insights for clinical interventions. Our investigation aims to clarify the causal relationship between shoulder periarthritis and GERD, revealing potential links between these two diseases and guiding future clinical prevention and treatment strategies. Additionally, pleiotropic effects may exist among variables influencing shoulder periarthritis, such as BMI, and genetic factors related to GERD.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Study Design and Data Resources\u003c/h2\u003e \u003cp\u003eThis study employed a two-sample MR approach to investigate the causal relationship between shoulder periarthritis and GERD. Shoulder periarthritis was considered the exposure, and GERD was the outcome. SNPs were selected as instrumental variables (IVs) for analysis. The study adheres to the three key assumptions of MR: (1) the IVs are strongly associated with the exposure (P\u0026thinsp;\u0026lt;\u0026thinsp;5\u0026times;10^-8, F-statistic\u0026thinsp;\u0026gt;\u0026thinsp;10); (2) the IVs are independent of confounders; and (3) the IVs influence the outcome solely through the exposure.\u003c/p\u003e \u003cp\u003eGWAS summary data for shoulder periarthritis were obtained from the EBI database, comprising 627,998 participants of European ancestry. All participants provided informed consent. GWAS summary data for GERD were accessed from (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gwas.mrcieu.ac.uk/datasets/ebi-a-GCST90000514/\u003c/span\u003e\u003cspan address=\"https://gwas.mrcieu.ac.uk/datasets/ebi-a-GCST90000514/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with 129,080 cases and 473,524 controls. Cases due to alcohol or psychoactive substances were excluded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Instrumental Variable Selection\u003c/h2\u003e \u003cp\u003eFor MR analysis, the relevance, independence and exclusion restriction assumptions must be met. SNPs were selected based on strong associations with the exposure (P\u0026thinsp;\u0026lt;\u0026thinsp;5\u0026times;10^-8), and SNPs with an F-value\u0026thinsp;\u0026lt;\u0026thinsp;10 were excluded to avoid weak instrument bias. The F-value was calculated as F\u0026thinsp;=\u0026thinsp;R\u0026sup2; \u0026times; (N \u0026minus;\u0026thinsp;2) / (1 - R\u0026sup2;), where R\u0026sup2; represents the variance explained by each IV. R\u0026sup2; was determined using the formula R\u0026sup2; = 2 \u0026times; EAF \u0026times; (1 - EAF) \u0026times; Beta\u0026sup2;, where Beta is the allele effect size, and EAF is the effect allele frequency. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eTo reduce bias from linkage disequilibrium, we applied clumping (r\u0026sup2; \u0026lt; 0.001, physical distance\u0026thinsp;=\u0026thinsp;10,000 kb) to ensure IV independence. Additionally, we used Phenoscanner V2 to exclude SNPs associated with confounders such as dementia and drug dependence, ensuring the IVs influence the outcome only through exposure. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the relationships allowed (solid lines, A and B) and those excluded (dashed lines, C and D). Finally, to align the effect alleles across datasets, we harmonised the exposure and outcome data and removed palindromic SNPs with intermediate allele frequencies. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the solid lines and arrows labeled A and B indicate valid causal pathways, where the relationship between SNPs associated with Frozen Shoulder (exposure) and Gastroesophageal Reflux Disease (outcome) is explored. In contrast, the dashed lines and arrows labeled C and D represent potential confounding pathways, which are excluded as they violate the assumptions of the instrumental variable approach. To ensure the effect alleles are consistent between the exposure and outcome datasets, a harmonization process was performed, aligning the alleles across both datasets. This step ensures that the alleles correspond to the same genetic variants, minimizing bias and enhancing the reliability of the Mendelian Randomization analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Statistical Analysis\u003c/h2\u003e \u003cp\u003eTo investigate the genetic association between frozen shoulder and GERD, we employed five MR methods: MR-Egger regression, weighted median, IVW, simple mode and weighted mode. The IVW method, which assumes that all SNPs used are valid, was considered the primary analysis method due to its potential to provide the most accurate estimates. Subsequently, statistical power was calculated using the tool available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://shiny.cnsgenomics.com/mRnd/\u003c/span\u003e\u003cspan address=\"https://shiny.cnsgenomics.com/mRnd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, based on the research of Brion et al. This tool computes statistical power for MR studies using the non-central parameter. The calculation of power requires the total R\u0026sup2; value, representing the variance in the exposure explained by each IV, as previously described.\u003c/p\u003e \u003cp\u003eTo ensure the robustness of our findings, we performed several additional analyses. First, Cochran's Q test was applied to assess heterogeneity in the associations, with the symmetry of the funnel plot providing further validation. To detect pleiotropy, we used the MR-Egger intercept test and the MR Pleiotropy Residual Sum and Outlier (MR-PRESSO) global test, the latter of which also identifies outliers and recalculates estimates after excluding them. Sensitivity analysis was conducted via a leave-one-out test, where each SNP was excluded sequentially to determine if any single SNP had a disproportionate influence on the overall results. All statistical analyses were performed using the TwoSampleMR package in R (version 4.2.0), with statistical significance defined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 IV Selection\u003c/h2\u003e \u003cp\u003eWe initially identified 64 SNPs strongly associated with the exposure data (P\u0026thinsp;\u0026lt;\u0026thinsp;5 \u0026times; 10⁻⁸, F-value\u0026thinsp;\u0026gt;\u0026thinsp;10, r\u0026sup2; \u0026lt; 0.001, physical window\u0026thinsp;=\u0026thinsp;10,000 kb) as potential IVs, with all F-values exceeding 10. Detailed F-value data is provided in Supplementary \u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e. No SNPs were excluded based on their association with outcomes or confounders (P\u0026thinsp;\u0026lt;\u0026thinsp;1 \u0026times; 10⁻⁵) using Phenoscanner V2. After harmonising the exposure and outcome datasets, 64 SNPs were selected for further MR analysis.\u003c/p\u003e \u003cp\u003eUsing SNPs linked to frozen shoulder as IVs, we thoroughly investigated the causal relationship between frozen shoulder and GERD. Our final set of instrumental variables met the linkage disequilibrium (LD) independence criterion (r\u0026sup2; \u0026lt; 0.001) and achieved a significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;1 \u0026times; 10⁻⁵ in GWAS data. In analysing the causal relationship, we carefully examined potential confounding factors for the selected IVs. The heterogeneity test revealed no significant heterogeneity among the IVs (Q_P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), confirming that pleiotropy or genetic heterogeneity did not impact the MR results. Additionally, the negative control analysis demonstrated that frozen shoulder was not associated with the selected IVs, further validating their applicability in this study.\u003c/p\u003e \u003cp\u003eIn the reverse MR analysis, we explored the effect of frozen shoulder on GERD, again using SNPs associated with frozen shoulder as IVs. We found only one SNP that met the LD independence criterion (r\u0026sup2; \u0026lt; 0.001) and a significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;1 \u0026times; 10⁻⁵ in the GWAS data. The results of the heterogeneity test indicated no significant heterogeneity among the selected IV (Q_P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), confirming that pleiotropy or genetic heterogeneity did not affect our MR analysis results. The negative control analysis similarly affirmed that frozen shoulder was unrelated to the selected IVs, reinforcing their suitability for this study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2 MR Analysis\u003c/h2\u003e \u003cp\u003eThe random-effects IVW method served as the primary approach to examine the genetic association between frozen shoulder and GERD [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The analysis demonstrated that frozen shoulder is an independent risk factor for GERD (P\u0026thinsp;=\u0026thinsp;2.95 \u0026times; 10⁻⁵, odds ratio [OR] [95% confidence interval (CI)]\u0026thinsp;=\u0026thinsp;0.725 [0.6281\u0026ndash;0.8369]) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The weighted median method further supported this genetic causal relationship. Table\u0026nbsp;1 summarises the results from the five MR methods employed in this study [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Cochrane\u0026rsquo;s Q test revealed no heterogeneity in the association between frozen shoulder and GERD (P\u0026thinsp;=\u0026thinsp;0.924), and the funnel plot exhibited a symmetrical distribution of SNPs\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The MR-Egger intercept test and the MR-PRESSO global test confirmed the absence of horizontal pleiotropy (P\u0026thinsp;=\u0026thinsp;0.532). Additionally, the MR-PRESSO analysis detected no outliers in the Mendelian randomisation analysis. The leave-one-out test showed that the overall results were not influenced by any single SNP\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, affirming the stability and robustness of the findings.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study employed a two-sample MR analysis using large-scale GWAS data to investigate the causal relationship between frozen shoulder and GERD. The results indicate that genetic susceptibility to frozen shoulder is associated with an increased risk of GERD. This finding suggests that early intervention targeting frozen shoulder symptoms could help reduce the incidence and severity of GERD, with potential benefits in preventing long-term gastrointestinal complications and improving clinical outcomes for patients [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA study conducted in China similarly reported a positive correlation between frozen shoulder and GERD risk (IVW: P\u0026thinsp;=\u0026thinsp;2.954804e-05, OR [95% confidence interval (CI)]\u0026thinsp;=\u0026thinsp;1.482 [1.136\u0026ndash;1.933]). This study was also based on large-scale GWAS data and MR analysis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, prior observational studies have yielded conflicting evidence regarding the association between frozen shoulder and GERD. Our study demonstrates a potential causal effect of frozen shoulder on GERD, suggesting that musculoskeletal conditions like frozen shoulder may influence gastrointestinal diseases through shared inflammatory pathways or other mechanisms [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, compared to other musculoskeletal disorders, frozen shoulder is often accompanied by prolonged inflammation and restricted joint mobility, which may trigger a heightened physiological stress response, indirectly increasing the risk of GERD [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The use of genetic variants associated with frozen shoulder in MR analysis allows for more accurate identification of causal relationships. One possible bias in this relationship is medication use. Proton pump inhibitors (PPIs), commonly prescribed for GERD, could obscure the true association between the two conditions. However, according to the GWAS database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/gwas\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/gwas\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), no significant SNPs have been linked to PPI use, indicating that medication is unlikely to be a confounding factor in the relationship between frozen shoulder and GERD. Nevertheless, further research is required to elucidate the underlying mechanisms connecting these two diseases.\u003c/p\u003e \u003cp\u003eExisting observational studies have not established a causal relationship between frozen shoulder and GERD. Frozen shoulder often coexists with other conditions, such as cognitive impairment and cardiovascular diseases, which may partially influence the observed positive correlation between the two [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. GWAS serve as a powerful tool for investigating complex diseases by identifying individuals or gene groups that may be difficult to detect in single-gene studies. This approach not only validates existing findings but also opens new avenues for research. Based on large-scale GWAS data, our study provides genetic evidence for a causal relationship between these two interrelated conditions [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eElevated cortisol levels have been linked to the development of GERD, potentially due to reduced physical activity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Chronic pain associated with a frozen shoulder could exacerbate GERD symptoms, as it may impact eating habits and contribute to increased stomach discomfort and reflux [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Moreover, changes in the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased gastric acid secretion, may represent a potential link between frozen shoulder and GERD. This connection could arise through several mechanisms, including GERD-related chronic inflammation, autonomic nervous system dysfunction, and chronic pain and stress responses, which are also implicated in the pathogenesis of frozen shoulder [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. While direct evidence for this connection stems from gastrointestinal function studies, indirect evidence comes from etiological studies that suggest the transmission of inflammatory mediators and systemic inflammation may enhance oesophageal sensitivity. These factors might represent a shared neural pathway between frozen shoulder and GERD.\u003c/p\u003e \u003cp\u003eAutonomic dysfunction, commonly observed in patients with frozen shoulder, could be attributed to chronic pain, inflammation, or psychological stress [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The autonomic nervous system, particularly in regulating lower oesophageal sphincter (LES) tone, plays a critical role in oesophageal and gastric functions, and its dysfunction could contribute to GERD. Inflammation and cytokines are well-recognised contributors to the onset and progression of frozen shoulder [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The overlap of these neurobiological mechanisms may explain the close association between frozen shoulder and GERD. Pathophysiological changes, such as neural network damage, neurodegeneration, neuroinflammation, glial cell alterations, and vascular abnormalities during frozen shoulder development, may heighten susceptibility to GERD. When the gastrointestinal system faces additional stress (such as trauma, infection or surgery), GERD symptoms may emerge.\u003c/p\u003e \u003cp\u003eFurthermore, interventions targeting frozen shoulder symptoms have been shown to reduce GERD risk in hospitalised patients, supporting the hypothesis that genetic susceptibility to frozen shoulder increases GERD risk [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Treatments such as non-steroidal anti-inflammatory drugs (NSAIDs) and physical therapy, commonly used to alleviate inflammation and pain in frozen shoulder, have been shown to overlap with strategies to manage GERD symptoms [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Additionally, recent studies highlight the benefits of lifestyle and dietary modifications in preventing GERD, particularly in patients with frozen shoulder, further reinforcing the potential causal relationship between these two conditions.\u003c/p\u003e \u003cp\u003eThis study has several notable strengths. First, it is the first to investigate the causal relationship between frozen shoulder and GERD using large-scale GWAS data, thereby filling a significant gap in the literature. By employing a two-sample MR approach, we effectively addressed common limitations found in observational studies, such as reverse causality, confounding factors, and biases. Second, we rigorously screened all IVs used in the MR analysis, with the lowest F-value being 137.831, ensuring the accuracy of our results. Finally, we employed multiple analytical methods to comprehensively evaluate the sensitivity, horizontal pleiotropy, and heterogeneity of our findings. All tests consistently indicated that the association between frozen shoulder and GERD is stable and reliable.\u003c/p\u003e \u003cp\u003eHowever, this study also has some limitations. Firstly, all participants in the GWAS were of European ancestry, necessitating further validation to determine the applicability of our findings to other populations or regions. Secondly, despite using the MR intercept test and MR-PRESSO global test to detect and adjust for pleiotropy in genetic variants, there may still be uncontrolled confounding factors, such as education level, personality traits, and nutritional status, that could introduce bias. Thirdly, since our MR analysis relies on data from the underlying GWAS meta-analysis, we were unable to perform stratified analyses by country, ethnicity, or age group. Consequently, the observed effects of frozen shoulder in this study may not apply to populations with different characteristics (such as ethnicity and age).\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study identified a causal relationship between frozen shoulder and GERD, potentially driven by shared biological mechanisms. These findings offer new insights into the genetic and biological pathways linking frozen shoulder and GERD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe publication of this manuscript is approved by all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNatural Science Foundation of Guangdong Province(2020A1414010368).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Guangzhou Municipal Science and Technology Bureau（NO.SL2022A04J00907）\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYC S had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: YC S, B L Acquisition of data: B L ZY H RuiZe Chen. Analysis and interpretation of data: B L ZY H . Drafting of the manuscript:B L ZY H. Critical revision of the manuscript for important intellectual content: YC S, B L ZY H. Statistical analysis: B L ZY H, X W, Q G. Supervision: YC S. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNL M, et al. Frozen shoulder. Nat reviews Disease primers. 2022;8(1):59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM, K., et al., \u003cem\u003eFrozen shoulder.\u003c/em\u003e BMJ (Clinical research ed.), 2022. 377: p. e068547.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR, F., Gastroesophageal Reflux Disease. N Engl J Med, 2022. 387(13): pp. 1207\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD C, et al. The presence of gastroesophageal reflux disease increases the risk of developing postoperative shoulder stiffness after arthroscopic rotator cuff repair. J Shoulder Elbow Surg. 2020;29(12):2505\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAS C, et al. Analysis of 90-Day Readmissions After Total Shoulder Arthroplasty. Orthop J sports Med. 2019;7(9):2325967119868964.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLevin MG, Burgess S. Mendelian Randomization as a Tool for Cardiovascular Research: A Review. JAMA Cardiol. 2024;9(1):79\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBL P, A. H, and, TJ V. Power and instrument strength requirements for Mendelian randomization studies using multiple genetic variants. Int J Epidemiol. 2011;40(3):740\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLAC M, et al. Searching for the causal effects of body mass index in over 300 000 participants in UK Biobank, using Mendelian randomization. PLoS Genet. 2019;15(2):e1007951.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHD G, et al. A genome-wide association study identifies 5 loci associated with frozen shoulder and implicates diabetes as a causal risk factor. PLoS Genet. 2021;17(6):e1009577.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT Y, et al. RAGE-dependent NF-kB inflammation processes in the capsule of frozen shoulders. J Shoulder Elbow Surg. 2020;29(9):1884\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC C, et al. Genetic variants involved in extracellular matrix homeostasis play a role in the susceptibility to frozen shoulder: A case-control study. J Orthop research: official publication Orthop Res Soc. 2019;37(4):948\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eY Z. The causal role of gastroesophageal reflux disease in anxiety disorders and depression: A bidirectional Mendelian randomization study. Front Psychiatry. 2023;14:1135923.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL L, et al. Causal associations between gastroesophageal reflux disease and lung cancer risk: A Mendelian randomization study. Cancer Med. 2023;12(6):7552\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanderson E, Spiller W, Bowden J. Testing and correcting for weak and pleiotropic instruments in two-sample multivariable Mendelian randomization. Stat Med. 2021;40(25):5434\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMG L. Mendelian Randomization as a Tool for Cardiovascular Research: A Review. JAMA Cardiol. 2024;9(1):79\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eF Z, et al. No evidence of a causal relationship between miscarriage and 25-hydroxyvitamin D: a Mendelian randomization study. Volume 2024. Human reproduction open; 2024. p. hoae011. 2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eW Z, et al. Association of atopic dermatitis with autoimmune diseases: A bidirectional and multivariable two-sample mendelian randomization study. Front Immunol. 2023;14:1132719.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR H, L. C, and, D L. A Mendelian randomization analysis identifies causal association between sarcopenia and gastroesophageal reflux disease. Aging. 2024;16(5):4723\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIM M, GW M. Genomic characterisation of the overlap of endometriosis with 76 comorbidities identifies pleiotropic and causal mechanisms underlying disease risk. Hum Genet. 2023;142(9):1345\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK A, et al. Mendelian Randomization Analysis Reveals a Complex Genetic Interplay among Atopic Dermatitis, Asthma, and Gastroesophageal Reflux Disease. Am J Respir Crit Care Med. 2023;207(2):130\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRizk TE, Pinals RS. Frozen shoulder. Semin Arthritis Rheum. 1982;11(4):440\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHB P, et al. Association Between High-Sensitivity C-Reactive Protein and Idiopathic Adhesive Capsulitis. J Bone Joint Surg Am Vol. 2020;102(9):761\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eButtgereit F, et al. Polymyalgia Rheumatica and Giant Cell Arteritis: A Systematic Review. JAMA. 2016;315(22):2442\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS, H., et al., Progressive exercise compared with best practice advice, with or without corticosteroid injection, for the treatment of patients with rotator cuff disorders (GRASP): a multicentre, pragmatic, 2 \u0026times; 2 factorial, randomised controlled trial. Lancet (London England), 2021. 398(10298): pp. 416\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBridgman JF. Periarthritis of the shoulder and diabetes mellitus. Ann Rheum Dis. 1972;31(1):69\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC D, et al. Medical relevance of protein-truncating variants across 337,205 individuals in the UK Biobank study. Nat Commun. 2018;9(1):1612.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShaheen NJ, et al. The burden of gastrointestinal and liver diseases, 2006. Am J Gastroenterol. 2006;101(9):2128\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC Y, et al. Association between physical activity and risk of gastroesophageal reflux disease: A systematic review and meta-analysis. J sport health Sci. 2024;13(5):687\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS, P., Scoping review and interpretation of myofascial pain/fibromyalgia syndrome: An attempt to assemble a medical puzzle. PLoS ONE, 2022. 17(2): p. e0263087.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH H, NS A, HB E-S. Meta-analysis: obesity and the risk for gastroesophageal reflux disease and its complications. Ann Intern Med. 2005;143(3):199\u0026ndash;211.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJK P. Rotator Cuff-Related Shoulder Pain: Is It Time to Reframe the Advice, You Need to Strengthen Your Shoulder? J Orthop Sports Phys Ther. 2021;51(4):156\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM, C., et al., \u003cem\u003eGastroparesis.\u003c/em\u003e Nat reviews Disease primers, 2018. 4(1): p. 41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV R, et al. The pathophysiology associated with primary (idiopathic) frozen shoulder: A systematic review. BMC Musculoskelet Disord. 2016;17(1):340.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkbar M et al. \u003cem\u003eTranslational targeting of inflammation and fibrosis in frozen shoulder: Molecular dissection of the T cell/IL-17A axis.\u003c/em\u003e Proceedings of the National Academy of Sciences, 2021. 118(39): p. e2102715118.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJE R. Presentation and Epidemiology of Gastroesophageal Reflux Disease. Gastroenterology. 2018;154(2):267\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaund E et al. Management of frozen shoulder: a systematic review and cost-effectiveness analysis. Health Technol Assess, 2012. 16(11).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\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":"Frozen Shoulder Gastroesophageal Reflux Disease Mendelian randomization Bidirectional","lastPublishedDoi":"10.21203/rs.3.rs-5333407/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5333407/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Background: Frozen shoulder (FS) has been suggested as a potential risk factor for gastroesophageal reflux disease (GERD). However, observational studies are limited in their ability to establish a causal relationship between FS and GERD.\nMethods: This study aimed to investigate the genetic causal association between FS and GERD using a two-sample Mendelian randomization (MR) approach. Genome-wide association study (GWAS) summary data for FS were obtained from the UK Biobank, while GWAS summary data for GERD were sourced from the FinnGen Consortium. Multiple MR methods, including inverse-variance weighted (IVW), MR Egger, weighted median, simple mode, and weighted mode, were applied to estimate the causal effect. To assess heterogeneity, Cochrane's Q test was performed. Horizontal pleiotropy was evaluated using the MR-Egger intercept test and MR pleiotropy residual sum and outliers (MR-PRESSO) test. Sensitivity analyses, including leave-one-out analysis, were conducted to ensure the robustness of the findings.\nResults: The IVW method indicated a potential causal relationship between FS and GERD (P = 0.018). There was no evidence of horizontal pleiotropy (P \u003e 0.05), and heterogeneity among genetic variants was not significant (P \u003e 0.05). Furthermore, leave-one-out analysis demonstrated that the association was consistent and reliable. Limitations: All participants included in the GWAS data were of European ancestry, which may limit the generalizability of the results. Additionally, due to data constraints, stratified analyses based on different populations, ethnicities, or age groups were not performed.\nConclusion: This two-sample MR analysis provides evidence of a genetic causal association between FS and GERD, highlighting the need for further research to explore this relationship in diverse populations.","manuscriptTitle":"Exploring the Potential Association Between Frozen Shoulder and Gastroesophageal Reflux Disease Using Mendelian Randomisation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-18 11:08:55","doi":"10.21203/rs.3.rs-5333407/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":"1d7750ae-3c8b-4902-acbf-dc7f4c8e89a8","owner":[],"postedDate":"November 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-28T04:08:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-18 11:08:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5333407","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5333407","identity":"rs-5333407","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}