1 INTRODUCTION

Parkinson's disease (PD) is the most common type of degenerative motor disease of the central nervous system and the most common symptom of dyskinesia, estimated 6.1 million patients affected worldwide in 2016, characterized by a broad spectrum of both motor and nonmotor symptoms (NMS).^{1, 2} PD has experienced exponential growth as one of the world's fastest-growing neurological disorders for unclear reasons, with a global burden that has more than doubled in the last generation.² Rapid attention is required to a significant medical issue that involves comprehending the etiology of PD and developing efficient preventive and therapeutic strategies.³

Cholelithiasis, known as gallstones, is a widespread and crucial public health issue with a pooled prevalence of 6.1% and an incidence of 0.47 per 100 person-years, and gallstone frequency rises with age.⁴ In industrialized nations, cholelithiasis affects roughly 10%–15% of individuals and has become increasingly prevalent in recent decades.⁵ It is characterized by a solid clot in the gallbladder or biliary tract⁶ and has traditionally been classed as cholesterol, pigmented, or mixed depending on their constitution. Approximately 10%–20% of adults suffer from cholelithiasis and 20% of patients tolerate complications^6-8 include sepsis, pancreatitis, cholecystitis, cholangitis, and gallbladder rupture. The impacts of cholelithiasis raise the expense of healthcare, burden society financially, and potentially endanger lives. Gallstone disease is one of the most costly digestive disorders from a social perspective.⁹

A clinical study conducted by the Patricia Modaine team in 1995 has performed gallbladder ultrasound in 91 patients with PD and 80 controls. The prevalence of cholelithiasis was found to be 38.5% in the PD group and 30% in the control group, with a significant age-adjusted correlation between PD and the frequency of cholelithiasis. Autonomic dysfunction in patients with PD may lead to dysfunction of gastrointestinal peristalsis, which may affect gallbladder emptying and promote gallstone formation.¹⁰ Recently, the gastrointestinal microbiome in cholelithiasis and the nervous system were suggested similar functions because they have similar advantages.¹¹

Furthermore, it was found that intestines of patients in PD contained higher amounts of secondary bile acids which might be a contributing factor to gallstone development.¹² Recent evidence suggest fasting gallbladder volume was increased in PD patients. A series of observational clinical studies provides light on biliary dysfunction in PD and advocates for more investigation into the impact of cholelithiasis.¹³ Interestingly, a nationwide cohort study recently found that individuals who underwent cholecystectomy had a considerably increased risk of PD, a tendency that was more prevalent in males than in women.¹⁴ Therefore, it is currently unclear whether there is causality between cholelithiasis and PD.

Nowadays Mendelian randomization (MR) analyses have emerged as a powerful implement to calculate the causal influence of certain risk variables on an outcome of interest (such as illness) by employing genetic variation as an instrumental variable (IV).¹⁵ MR has several advantages compared with randomized controlled trials, in terms of cost and time savings, the ability to analyze massive amounts of data, and the lack of ethical restrictions.¹⁶

To our knowledge, this is the first study to investigate the causal connection between PD and cholelithiasis using MR techniques. Our investigation attempts to add novel perspectives and factual data to the body of knowledge regarding the risk of PD.

2 MATERIALS AND METHODS

To evaluate the bidirectional relationship between gallstones and PD using MR analysis, we applied three fundamental assumptions to the genetic variants¹⁷: (1) association assumption, indicating that SNPs are strongly correlated with exposure; (2) independence assumption, implying that SNPs are not influenced by other factors in the exposure-outcome pathway; and (3) exclusivity assumption, indicating that SNPs influence outcomes exclusively through exposure and not via other pathways.

3 RESULTS

3.2 MR analysis

Fixed-effects IVW was employed because there was no substantial heterogeneity among SNPs (p > 0.1). The MR findings (Figure 1) indicated a substantial correlation between a genetically determined gallstone risk and an increased chance of PD (OR = 1.146, 95% CI: 1.062–1.236，p < 0.001). These results are consistent with the weighted median approach (OR = 1.131, 95% CI: 1.014–1.261, p = 0.027) and the weighted mode method (OR = 1.143, 95% CI: 1.026–1.274, p = 0.022), suggesting a causal relationship between cholelithiasis and the risk of PD, and a positive association was found. MR-Egger regression (OR = 1.094, 95% CI: 0.970–1.235, p = 0.155) and simple mode means (OR = 1.205, 95% CI: 0.986–1.473, p = 0.078) shows no significant. As shown in the forest plot in Figure 2A, the results of the IVW method indicated that the prevalence of PD gradually increased with the onset of cholelithiasis.

All of the SNPs' F-values were larger than 10, the Cochran Q test, the Funnel plot (Figure 2B), the leave-one-out approach, and other measures demonstrated the validity of our findings. No heterogeneity was detected by Cochran's Q test among the IVs (Q = 28.982, p = 0.466) (Table 2). Moreover, no horizontal pleiotropy was detected using the MR-Egger intercept (p = 0.350) (Table 2). The MR-PRESSO analysis did not perceive any instrumental outlier at the significance level of 0.05 (Table 2). To evaluate the conformance of the included SNPs, a leave-one-out analysis was performed; the findings showed that no single SNP significantly altered the overall MR results (Figure 2C).

Table 2. Heterogeneity and horizontal pleiotropy analysis between cholelithiasis and Parkinson's disease (PD).

Heterogeneity horizontal pleiotropy MR-PRESSO
Q	df	p Value	Egger_intercept	SE	p Value		p Value
28.982	29	0.466	0.010	0.010	0.350		0.480

For the purpose of investigating the impact of PD on the risk of cholelithiasis, we conducted a reverse Mendelian randomization analysis to minimize reverse causality (Individual SNP test，Wald ratio: OR = 1.166 (1.036–1.314), p = 0.011).

4 DISCUSSION

In this study, we investigated the causal relationship between cholelithiasis and risk of PD, using two-sample bidirectional MR which helps to reduce residual confounders and various biases, thus enhancing our causal inference ability by employing exposure-related genetic variations as proxies for exposure. Genetic variance is inherited from the parents and remains unchanged from birth, and the relationship with outcome is chronologically rational. Gametes are formed according to the Mendelian law of inheritance, in which parental alleles are randomly assigned to offspring. Therefore, inherited variants are not affected by potential confounders such as environmental exposure, socioeconomic status, and behavioral factors.²⁷

To the best of our understanding, this is the first study to investigate the causal connection between PD and cholelithiasis. The results of our analysis revealed an increased risk effect of cholelithiasis against PD, which provides significant implications for clinical and public health. Meanwhile, we made sure that these correlations had been assessed in two independent populations, and a series of sensitivity tests were executed to evaluate the robustness of the results, which improves the validity of the results. There was no horizontal pleiotropy (p = 0.35) in the selected IVs, according to the MR-Egger regression intercept term results, and there were no outliers (p = 0.48) in the MR-PRESSO global test. The Cochran's Q test results showed that these IVs did not significantly differ from one another (p = 0.466). No single SNP was shown to have a significant impact on the overall calculations by the leave-one-out study. Even after removing the confounding SNPs, the calculated MR values remained significant, and the conclusion's direction aligned with the original study. Moreover, there was no consistent result whether PD causally contributes to the development cholelithiasis in external validation of another cholelithiasis data set. Even though, the casual association found in our study shed new evidence of therapeutic ideas for PD. There is still much to discover about the potential link between cholelithiasis and PD.

In recent decades, an enormous amount of pharmacologic studies have shown the naturally occurring bile acid ursodeoxycholic acid (UDCA), also as a traditional treatment of chronic cholecystitis,²⁸ is demonstrated a promising mitochondrial rescue compound for PD in both clinical and experimental.^29-31 Cholelithiasis can lead to abnormalities in the components of bile as well as poor bile excretion. These modifications may have an indirect impact on the development and course of PD by influencing the intestinal absorption of nutrients, particularly those essential for the synthesis and metabolism of dopamine.^{32, 33} On the other hand, changes in the PD gut microbiome are linked with increase in the bile acid pool.¹² Interestingly, it was demonstrated an increase in bile acid production in patients with cholesterol gallstones by Metabolite Set Enrichment Analysis (MSEA)³⁴ In rats with PD symptoms overexpressing α-synuclein, gut microbial diversity is altered and levels of the associated bile acids are significantly increased, thereby affecting motor symptoms.³⁵ The brain-gut-microbiota axis is crucial to the etiology of PD.³⁶ Changes in the composition and activity of the gut microbiota due to cholelithiasis³⁷ may have an effect on neuroinflammation, oxidative stress, and neurotransmitter homeostasis, all of which have been connected to the pathophysiological mechanisms behind PD.^{11, 38} Cholelithiasis may trigger an inflammatory response with the release of inflammatory mediators, which enter the central nervous system through the circulation, leading to neuroinflammation and neuronal damage, increasing the risk of PD. To date, it was found that cholecystectomy did not raise the risk of colorectal cancer or metabolic disorders.³⁹ In a nationwide retrospective cohort study, it is found that the cholecystectomy group has a higher incidence of PD, which is more significant in males but not in females. Potential processes linked to a higher risk of PD following cholecystectomy could involve dysbiosis of the gut microbiota and metabolic inefficiency stemming from elevated levels of secondary bile acids.¹⁴ It is important to note that these mechanisms are still in the research phase and more studies are needed to confirm their specific role in the relationship between cholelithiasis disease and PD.

Nevertheless, it is important to recognize the limitations of our MR investigation. First of all, the GWAS databases used in our study are only available for European populations begs the question of whether this causal relationship is still substantial in other groups, calling for further study. Second, the MR-Egger and simple mode means method demonstrated no significant association between cholelithiasis and PD in both major study databases and external validation databases. Indeed, the MR-Egger technique may be used to examine horizontal pleiotropy of genetic variation, yielding causal impact estimates consistent with IVW when pleiotropic SNPs linked with the outcome are excluded.^{23, 40} In the presence of multivalence, MR-Egger estimations are favored. In our analysis, the MR-Egger regression intercept term revealed that the selected IVs did not exhibit horizontal multicollinearity. The simple model approach is an unweighted model of causal impact estimate that is less biased than other models but less accurate due to bias reduction.^{41, 42} Third, both cholelithiasis and PD are heterogenic conditions, however the absence of pertinent stratification data prohibits us from analyzing disease subtypes in more detail. Future attentions on the association between PD and cholelithiasis should be implemented in the future.

In conclusion our MR analysis provide tentative initial evidence to support roles of genetic liability to cholelithiasis and risk of PD and offer novel perspectives on preventing PD. In the future, more investigation is required to confirm the existence of cholelithiasis and investigate the possible involvement of bile acid metabolism and its relationship with gut microbiota in the etiology of PD.

AUTHOR CONTRIBUTIONS

Zijiao Yang: Writing—original draft; writing—review and editing; investigation; formal analysis; validation; methodology; software; data curation; conceptualization; visualization. Chengfu Song: Supervision. Chong Lu: Visualization; validation; supervision; software.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.