Introduction
Anxiety disorders are a significant health problem widespread and are the leading psychiatric causes of the global burden of diseases [
1]. The World Health Organization (WHO) has also ranked anxiety disorders as one of the largest causes of disability worldwide largely due to their high prevalence, chronicity, and comorbidity [
2,
3]. Effective prevention and treatment of anxiety disorders are critical to reduce the morbidity and disability. Notably, the exploration of the biological mechanism is the basis for the prevention and therapy of anxiety disorders [
4]. Multiple factors, such as psychological and genetic factors, are thought to be involved in the biological mechanisms of anxiety disorders [
4,
5]. However, anxiety disorders are complex conditions, and their biological mechanisms are not fully understood. Although progress in genetics (particularly genome-wide association studies (GWASs)) have largely improved the development of etiology research for mental disorders [
6‐
8], there is still a great barrier translating these genetic findings into biological mechanisms.
In recent years, modern omics-based technologies, including metabolomics, have made a positive contribution to the exploration of disease mechanisms. Specifically, metabolomics can provide novel information into the biological mechanisms of diseases by revealing the intermediate metabolites and altered metabolic pathways [
9,
10]. A recent robust study of GWAS of metabolites has identified the disease-relevant loci and suggest mechanisms for diseases and disease-related traits [
11]. Several studies have suggested that metabolites are functional intermediates that can be used to illustrate the potential biological mechanisms related to the genetics of mental disorders [
12‐
14]. It is worth noting that metabolites are the final products or the intermediate of metabolism that can play important in human. The database of genotype dependent metabolic phenotypes (also known as genetically determined metabolites (GDMs)) has recently been established using a GWAS involving non-targeted metabolomics [
15,
16]. The developed GDMs can promote insight of the underlying relationship of human serum metabolites and associative genetic variants in the biological mechanisms of mental disorders by providing functional intermediates [
17‐
19]. Studies have shown the significance of GDMs in the biological mechanism of major depression, bipolar disorders, autism spectrum disorders and hyperactivity disorders [
17]. However, GDMs and pathway analysis geared toward exploring the biological mechanisms of anxiety disorders are still lacking, which calls for a deep analysis to determine the role played by the effects between genetic variation and metabolites in the biological mechanisms of anxiety disorders.
Mendelian randomization (MR) analysis is a useful epidemiological research strategy in which genetic variants are used to connect exposure with outcome as instrumental variables (IV) for assessing causal relationships. Compared to other epidemiological research strategies, MR can provide unbiased estimates on how genotypes are decided at conception, and are commonly not susceptible to confounding factors and reverse causation [
20]. Given this huge advantage, MR has been widely applied in the past decade to infer causality of related risk exposure to disease using publicly available GWAS summary statistics [
21,
22]. Recently, GWAS have extended the metabolic spectrum, from which an atlas of GDMs was developed.
Herein, we speculated that this GDMs atlas could be used to infer the causality of GDMs on anxiety disorders. Consequently, we implemented a two-sample MR approach to: (1) assess the causal effects of human serum metabolites on anxiety disorders; (2) identify the GDMs that have causal effects on four different GWASs of anxiety disorders; and (3) identify potential metabolic pathways which might help to understand the mechanism of anxiety disorders.
Discussion
This Mendelian randomization study provides unbiased evaluation of the causal relationship between GDMs and anxiety disorders using four different GWAS datasets, including anxiety disorders diagnosed by psychiatrists, unspecified anxiety disorders diagnosed by secondary ICD-10, anxiety diagnosed by general practitioners, and self-reported anxiety disorders. We identified 85 metabolites relevant to the risk of anxiety disorders after using genetic variants as probes. Among them, 11 metabolites were overlapped in the four different datasets of anxiety disorders, with Bonferroni correction showing that 1-linoleoylglycerophosphoethanolamine had the most reliable causal relationship. Moreover, pathway enrichment analysis identified two significant metabolic pathways, the “primary bile acid biosynthesis” pathway and the “valine, leucine, and isoleucine biosynthesis” pathway, which are mainly involved in the anxiety disorders.
As far as we know, this is the first MR study that has combined genomics with metabolomics to evaluate the causality of GDMs on anxiety disorders. Herein, results identified a cluster of metabolites in serum showing relationship with anxiety disorders, among which 1-linoleoylglycerophosphoethanolamine had a robust effect on anxiety disorders diagnosed by psychiatrists and anxiety disorders diagnosed by general practitioners. In a previous study, researchers conducted a delivering preterm with or without preeclampsia population-based birth cohort, and found that higher 1-linoleoylglycerophosphoethanolamine density decreased odds of preeclampsia [
36]. The result is corresponding with previous research which revealed that women who developed preeclampsia had lower levels of lysophospholipids compared to healthy term pregnancies [
37‐
39]. In addition, preeclampsia women were more likely to be diagnosed with anxiety disorders. 1-linoleoylglycerophosphoethanolamine is an important member of the phosphatidylethanolamine (PE) family, whose components mainly include fatty acids, ethanolamine, phosphoric acid, and glycerol [
40]. As a lipid chaperone, PE can assist in the folding of certain membrane proteins, and is closely associated with anxiety disorders. A previous study found that stress-prone Wistar-Kyoto rats had lower PE in the anxiety state compared to the non-stress prone rats, suggesting that PE may be associated with the anxiety disorders [
41]. Studies have also found that alcohol-dependent patients are often accompanied by anxiety disorders when they quit drinking, and the concentration of PE in plasma is higher after alcohol-dependent patients quit drinking [
42]. Notably, Yang et al. [
17] explored the relationship between metabolism and some psychiatric disorders, and found that the genetic associations of 1-linoleoylglycerophosphoethanolamine were involved in the risk of major depression. Clinically, the comorbidity of depression and anxiety is common, and the two often influence each other. Although 1-linoleoylglycerophosphoethanolamine seems like a hopeful biomarker for anxiety disorders, further studies should be conducted to clarify the relevant mechanism.
This MR analysis also identified certain metabolites, some of which had been reported in previous research. Androsterone sulfate whose chemical formula is 3α-hydroxy-5α-androstan-17-one 3α-sulfate, is one of the primary urinary metabolites of androgens. Previous studies reported that metabolite networks enriched in androsterone sulfate, tyrosine, indoxyl sulfate, or caffeine are associated with a negative personality [
43]. Negative personality manifested as inhibition in social situations is often described as distress or anxiety. Moreover, sleep-related impairment was inversely correlated with androgen deprivation therapy-induced reduction in androsterone sulfate [
44]. Given that people with sleep disorders are often accompanied by anxiety disorders, androsterone sulfate might play a role in neurodevelopment of anxiety disorders. Epiandrosterone sulfate is a vital endogenous androstane steroid produced by the adrenal cortex. Notably, the steroid is an important neurosteroid and neurotrophin, which plays an important physiological function in human body. A recent omics investigation into chronic widespread musculoskeletal pain revealed that epiandrosterone sulfate is an important biomarker [
45]. Considering that people with chronic pain are often accompanied by anxiety disorders, epiandrosterone sulfate might play a role in neurodevelopment of anxiety disorders. In addition, p-acetamidophenylglucuronide showed robust association with intelligence, and thus it can be used to predict important health outcomes that may affect anxiety disorders [
15]. It is worth mentioning that our results are consistent with above results and emphasize the significance of genetics in the progression of mental disorders. It is worth noting that the use of drugs also has an effect on metabolite profiles. The baseline characteristics and the drug use of the participants involving in the study are listed in Additional file
2: Table S23–S25. The effect of drugs (such as antihypertensive and lipid lowering drugs) on the human metabolite concentrations is still not fully understood, and further studies are needed.
In this study, the metabolic pathway analysis showed that “primary bile acid biosynthesis”, and “valine, leucine, and isoleucine biosynthesis” pathways are mainly associated with anxiety disorders. Notably, primary bile acids are synthesized in liver cells by cytochrome P450-mediated oxidation of cholesterol, resulting in the synthesis of primary bile acids (such as deoxycholic acids). Primary bile acids have rich functions and are important physiological factors for intestinal nutrient absorption, bile secretion of lipids, toxic metabolites and exotic organisms. At the same time, primary bile acids are signaling molecules and metabolic regulators, which play an important role in activating nuclear receptor and G-protein-coupled receptor (GPCR) signals, regulating liver lipid, glucose and energy homeostasis and maintaining metabolic homeostasis. Studies have proved that primary bile acid metabolites/pathways are involved in several aspects of brain function and behavior [
46]. Moreover, it has been reported that changes in the gut microbiota composition may be associated with alterations in the primary bile acid metabolism that are involved in the biological process of psychological disorders in Crohn’s disease [
47]. A traditional Chinese medicine cohort revealed that amino acid metabolism, such as cysteine and methionine metabolism, might be involved in brain health disorders characterized by alterations in evaluation of bile acid biosynthesis [
48]. Altogether, these findings suggest that biosynthesis of primary bile acids might play an important role in the biological mechanism of anxiety disorders.
Valine, leucine, and isoleucine are structurally associated with branched-chain fatty acids and are important members of the family of 9 essential amino acids. Metabolism of valine, leucine and isoleucine have been proved to be associated with cancer progression in numerous studies, and key proteins in metabolic pathways may act as potentially prognostic and diagnostic biomarkers in human cancers [
49]. Given that people with cancers are often accompanied by anxiety disorders, valine, leucine, and isoleucine might play a role in neurodevelopment of anxiety disorders. Previous studies have proved that plasma concentrations of valine, leucine, and isoleucine are increased significantly in conditions associated with insulin resistance, such as obesity and diabetes mellitus [
50]. Research has demonstrated that anxiety symptoms are prevalent in people with diabetes, and may affect diabetes management and glycemic control [
51,
52]. Chen et al. [
53] established a rat model to explore the potential mechanisms of antidepressant effects, with the mainly enriched pathways being valine, leucine, and isoleucine degradation. It should be noted that this model is built on liver tissue, suggesting that the liver may potentially be connected with mental disease through some metabolites. To sum up, the biosynthesis of valine, leucine, and isoleucine might be relevant to the biological mechanism of anxiety disorders.
However, this study had several limitations. First, given the classification of the original data, we could not further subdivide the pressure type of anxiety disorders in combination with the ICD classification standard, and thus we could only analyze the anxiety disorders as a whole. Second, the power of the IVs depends largely on the sample size of GWASs, therefore, more data are needed to improve the accuracy of the generated GDMs. Third, although Mendelian randomization has been shown to be a powerful method to assess the causality between human blood metabolites and anxiety disorders, the results should be verified by further studies based on experimental data. Fourth, the veracity of the MR analysis relies largely on the explanation of the instrumental variables on exposure. Therefore, it is necessary to expand the sample size to provide a more accurate assessment of the genetic impact on metabolites. Fifth, due to insufficient data, we used metabolites with uncorrected P values for pathway analysis. At last, although this study identified multiple metabolites that contribute to the risk of anxiety disorders, further studies are needed to reveal their roles in the pathogenic mechanism of anxiety disorders.
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