Mendelian Randomization Study Suggests Circulating Copper Level as a Risk Factor for Liver Cirrhosis

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Mendelian Randomization Study Suggests Circulating Copper Level as a Risk Factor for Liver Cirrhosis

https://doi.org/10.21203/rs.3.rs-2734433/v1

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Cirrhosis is a serious liver disease with an unclear etiology and pathogenesis. Previous studies suggest a correlation between circulating copper and cirrhosis. However, whether circulating copper is a risk factor for cirrhosis is currently controversial because the liver is a major organ of copper metabolism and cirrhosis affects copper circulation. To address this, we used a mendelian randomization to explore the effect of circulating copper concentration on the risk of cirrhosis. We selected instrumental variables (IVs) of circulating copper from genome-wide association studies and analyzed two datasets from FinnGen, one for cirrhosis in general and the other for cirrhosis caused by non-alcoholic fatty liver disease (NAFLD). The inverse-variance weighted method was primarily used for mendelian randomization analysis. We created two SNP IVs that were associated with circulating copper, and their genetic associations with cirrhosis were extracted from the two datasets. The cirrhosis-related dataset included 811 cirrhosis patients and 273,592 controls, while the dataset for cirrhosis caused by NAFLD included 437 cirrhosis patients and 216,861 controls. Mendelian randomization analysis predicted a significant association between higher levels of circulating copper and increased risk of cirrhosis of liver (OR = 1.82, 95% CI: 1.30 to 2.54, P < 0.001). Additionally, higher levels of circulating copper were also associated with increased risk of cirrhosis of liver caused by NAFLD (OR = 1.68, 95% CI: 1.08 to 2.60, P = 0.021). The study suggests that higher levels of circulating copper may be a pathogenic risk factor for cirrhosis, providing important insights for the prevention and treatment of cirrhosis.

Mendelian randomization analysis

Circulating copper

Cirrhosis

Liver disease

Humans are potentially exposed to and ingest copper through various routes, including drinking water, copper-containing utensils, and food[1]. Copper plays a crucial role in maintaining tissue function and metabolism[2]. Alterations in circulating copper levels in many organs, such as the brain, heart, kidney, and liver, have been associated with trauma-related mortality[3, 4]. Therefore, humans require a highly coordinated mechanism to maintain copper balance and avoid deficiency or excess[5]. Recent studies have shown that circulating copper concentrations are also determined to some extent by genes[6].

In cases of severe copper metabolism disorders, such as Wilson's disease, patients can suffer from an excessive accumulation of copper in the liver, leading to cirrhosis[7]. However, the relationship between circulating copper and cirrhosis in non-pathological situations is still inconclusive. Circulating copper levels are closely related to copper intake. Early research by Tanner, M S et al. studied the family characteristics of Indian children with Indian Childhood Cirrhosis (ICC) and found that the majority of them used copper utensils for storing water, milk, or for boiling milk. It was suggested that early intake of copper-contaminated animal milk feed might be the cause of ICC in Indian children[8]. This viewpoint was widely accepted for a long time. Pandit, A et al. also suggested that environmental copper intake is the most reasonable explanation for ICC, and genetic defects may also be a factor. ICC may be a specific form of copper-related childhood cirrhosis that requires high environmental copper intake[9]. Researchers believe that infantile cirrhosis ICC is caused by excessive copper intake, which leads to the production of free radicals in liver cells, resulting in severe nuclear and cytoplasmic damage. This damage will stimulate immune cells and connective tissue cells to infiltrate and repair liver cell damage, ultimately leading to cirrhosis[8, 9]. A clinical study showed that the serum copper concentration was slightly increased in patients with more severe clinical states of cirrhosis[10]. Recently, a case report documented a 4-month-old infant with cholestasis diagnosed with copper-related liver toxicity on liver biopsy. The use of a copper cup to prepare formula milk was likely an exogenous source of copper, and the child's condition improved after removing the exogenous copper source[11]. These studies suggest that excessive intake of copper from the environment may contribute to the development of liver cirrhosis. However, there are different views on this issue, as Nayak NC et al. pointed out that a large multicenter study sponsored through the Indian Council of Medical Research (ICMR) found that it appears that genetic factors are a more dominant factor in the occurrence of ICC compared to exogenous copper intake, and exposure to exogenous copper in food, milk, and water should not be a prerequisite for this consideration[12]. A survey investigating the impact of copper content in public tap water on infant liver health in Berlin, Germany, found that although about 30% of households had tap water copper levels above 0.8 mg/l, no liver damage caused by drinking tap water was found among the 541 infants studied, and no significant abnormalities were detected in serum indicators[13]. Therefore, the controversy surrounding whether high copper intake leads to liver cirrhosis persists. The most likely explanation for this situation appears to be the combination of genetic copper metabolism defects and high copper intake[14]. Nevertheless, understanding whether circulating copper is a risk factor for liver cirrhosis remains important for the prevention of liver cirrhosis in specific high-risk populations as well as at the therapeutic level for patients with cirrhosis. It therefore deserves further exploration.

Mendelian randomization is a method for inferring causal relationships between lifestyle factors and disease outcomes[15]. Compared to previous cohort and case-control analyses, mendelian randomization methods are uniquely positioned to avoid the effects of reverse causality and residual confounders[16]. The causal relationship between cirrhosis and circulating copper was also influenced by reverse causality and residual confounders because the liver is a major organ of copper metabolism, and cirrhosis can also lead to disturbances in copper circulation. Mendelian randomization methods provide more reasonable evidence for a causal relationship between exposure and outcome than traditional epidemiological study designs.

In this study, high circulating copper levels were hypothesized to be a risk factor for cirrhosis, and a two-sample mendelian randomization approach was used to investigate whether elevated circulating copper levels increase the risk of developing cirrhosis. This approach allows inference of a causal relationship between high copper levels and cirrhosis by eliminating the effects of reverse causality and confounding factors.

Data Sources

In this study, we confirmed the association of circulating copper-related SNPs through genome-wide association study (GWAS), with 2,603 participants from Australia included in the analysis[17]. The genetic data of liver cirrhosis was obtained from the biomedical European databases of FinnGen. The FinnGen study, which began in 2017, is unique in that it combines genomic information with digital healthcare data[18]. We selected two liver cirrhosis datasets from FinnGen, one for unclassified liver cirrhosis with 811 cases and 213,592 controls, and the other for non-alcoholic fatty liver disease (NAFLD) induced liver cirrhosis with 437 cases and 216,861 controls. Details on the selected datasets are shown in Table 1.

Table 1

Details of the GWASs included in the mendelian randomization.
Consortium	Phenotype	Participants	Ethnics	Web source
Queensland Institute of Medical Research (QIMR)	Circulating Copper	2603	European	https://doi:10.1093/hmg/ddt239
FinnGen	Fibrosis and chirrhosis of liver	case: 811; control: 213592	European	https://risteys.finngen.fi/endpoints/K11_FIBROCHIRLIV
FinnGen	Chirrosis of liver, NAS	case: 437; control: 216861	European	https://risteys.finngen.fi/endpoints/CHIRHEP_NAS

Selection of Copper-Associated SNPs

Since the two SNPs, rs1175550 and rs2769264, met the genome-wide significance threshold (P < 5 ×10 − 8, r² = 0.001, kb = 10000, Table 2) and showed no overlap with the liver cirrhosis risk loci, they were chosen as instrumental variables (IVs). The variance explained by IVs was estimated by calculating the R² using the minor allele frequency (MAF) and β value from the formula R² = 2×MAF×(1 − MAF × β²)[19]. The variance in circulating copper explained by the two SNPs was found to be 5.32%. The F-statistics was also calculated using the formula F = R² × (n − k − 1) ∕ (1 − R²) in order to quantify the strength of the IVs, where n represents the sample size and k is the number of IVs[20].

Table 2

Genome-wide association studies and detailed information of SNP of circulating copper.
SNP	chr	pos	Nearest gene	EA	OA	β	se	P
rs1175550	1	3691528	SMIM1	G	A	0.198	0.032	5.03E-10
rs2769264	1	151344741	SELENBP1	G	T	0.313	0.034	2.63E-20

Statistical Analysis

The inverse-variance weighting (IVW) method was applied to evaluate the relationship between genetically predicted circulating copper levels and the risk of liver cirrhosis in two liver cirrhosis-related datasets in FinnGen. Wald estimates for each instrumental SNP were obtained using the IVW method, which calculates the ratio of the beta coefficient of the causal SNP to the beta coefficient of the exposure SNP[21]. If all genetic variations conform to the assumption of effective IVs, the IVW method will demonstrate a consistent estimation of the causal effect of exposure on the outcome. Additionally, we ensured the consistency of the estimated causal effect of circulating copper levels on liver cirrhosis across SNPs using Cochran's Q test[22, 23]. If Cochran's Q test indicates heterogeneity among SNPs (P < 0.05), the IVW method employs a random-effects model; otherwise, a fixed-effects model is selected.

Based on the F-statistic of 14.74 for circulating copper, it is unlikely that the estimated effect was affected by weak instrumental bias. Using the IVW method with a fixed model, we found a significant association between genetically predicted circulating copper and an altered risk of liver cirrhosis (OR = 1.82, 95% CI: 1.30 to 2.54, P < 0.001, Fig. 1A). We further explored the association between circulating copper and liver cirrhosis caused by NAFLD using the IVW method with a fixed model and found a significant association (OR = 1.68, 95% CI: 1.08 to 2.60, P = 0.021, Fig. 1B).

The present study was designed to investigate the causal relationship between circulating copper and the risk of cirrhosis using a two-sample mendelian randomization approach. The results of the mendelian randomization analysis showed support for the hypothesis that there is circulating copper as a possible risk factor for cirrhosis, which further strengthens the possibility of a causal relationship between circulating copper and cirrhosis.

Cirrhosis can be caused by a variety of factors, including obesity, nonalcoholic fatty liver, heavy alcohol consumption, hepatitis B or C infection, autoimmune disease, cholestasis, and iron or copper overload[24]. In patients with Wilson's disease, excessive accumulation of hepatic copper can lead to cirrhosis[24, 25]. Under normal conditions, small amounts of copper intake by the body do not lead to clinical toxicity because the body can maintain copper homeostasis by decreasing absorption and increasing excretion[26]. However, in some cases, diets high in copper are thought to be the main cause of cirrhosis in Indian children (ICC) and similar idiopathic copper toxicity (ICT)[9], suggesting that copper accumulation may be closely associated with the development of cirrhosis under certain circumstances. Cirrhosis is caused by a prolonged inflammatory response in the liver. This prolonged inflammation leads to the replacement of healthy liver tissue by fibrotic tissue and regenerative nodules, which in turn causes portal hypertension and cirrhosis[7]. In the body, copper possesses important redox functions and plays an important role in oxidative stress as it is involved in a variety of redox reactions and also produces harmful substances such as free radicals[27]. Patients with liver cirrhosis are often accompanied by a state of oxidative stress, so copper may play a role in this[28]. In addition, ceruloplasmin are the main transport carriers of copper in the circulatory system. Since ceruloplasmin are rapidly degraded in the absence of metals, the level of ceruloplasmin in the body can reflect the level of copper in the organism[29]. Ceruloplasmin is a multi-copper oxidase and antioxidant that eliminates some of the harmful free radicals and peroxides in the body. The activity and level of ceruloplasmin can directly affect the degree and incidence of oxidative stress[30]. However, copper can also be involved in the production of harmful substances such as superoxide radicals in the body, which can lead to cell and tissue damage[31]. Using a chicken hepatocyte model, Yang, Fan et al. found that excess copper can induce oxidative stress and apoptosis through the mitochondrial pathway and can affect cell morphology and function, increase ROS levels, and show a dose-effect[32]. This is consistent with another study that found in a mouse model that high doses of copper exposure can increase ROS levels in mouse hepatocytes and induce apoptosis through multiple pathways[33]. Thus, the role of copper in oxidative stress is dual, both in protecting cells and tissues by participating in redox reactions, and in exacerbating oxidative stress by producing harmful substances, suggesting copper may have a complex impact on the development and progression of cirrhosis. Due to the complexity of the relationship between copper and cirrhosis, more studies are needed to explore it. We found that elevated circulating copper may be a risk factor for cirrhosis by mendelian randomization analysis, providing a new reference for the relationship between copper and cirrhosis.

An observational study found that serum copper levels were significantly elevated in patients with cirrhosis and that serum copper concentrations were slightly elevated in patients with more severe clinical status of cirrhosis[10]. However, since the liver is the main organ of copper metabolism and cirrhosis can also lead to disturbances in copper circulation, this study could not demonstrate a causal relationship between serum copper and cirrhosis. To investigate the causal relationship between the two, we used mendelian randomization. Mendelian randomization uses genetic variation that is already present in nature to simulate randomization experiments and thus assess whether there is a causal relationship between a certain exposure and a certain outcome[16]. Our mendelian randomization analysis suggests that elevated circulating copper may be a risk factor for cirrhosis. Thus, this provides new evidence for a causal relationship between serum copper levels and cirrhosis.

Although the core hypotheses were satisfied in this study, there are still potential limitations. First, the small sample size may lead to insufficient reliability of the results. Mendelian randomization requires the inclusion of a large number of participants with specific genetic variants. Second, we assumed a linear association between circulating copper and cirrhosis, but in fact the biological effects of copper showed a U-shaped dose-effect relationship[34, 35]. In addition, the present study was analyzed only in a population of European ancestry, so extrapolation of the results is limited to this population. Therefore, additional epidemiological studies as well as in vivo and in vitro studies are needed to explore the relationship between circulating copper levels and the risk of developing cirrhosis.

In this study, a mendelian randomization analysis was used to assess the causal relationship between circulating copper and cirrhosis by simulating a randomization experiment using genetic variants already present in nature. The results of the analysis suggest that high circulating copper may be a risk factor for cirrhosis, providing new evidence for an in-depth investigation of this causal relationship.

Acknowledgments

The authors express their sincere gratitude to the esteemed researchers and dedicated participants who contributed to the collection and sharing of large-scale data through their invaluable observational articles in the context of the GWAS study.

Authors' contributions

Xiaolong Tang and Yandong Miao contributed to the conceptualization of the study; Xiaolong Tang, Wuhua Ha, and Zheng Li performed the methodology and formal analysis; Xiaolong Tang and Yandong Miao wrote the writing-original draft; Lixia Yang helped perform the analysis with data curation and validation; Denghai Mi contributed significantly to the writing-review & editing.

Funding

This work was supported by the Special Plan for Condition Construction of [Gansu Provincial Scientific Research Institutes] (Grants No. 20JR10RA432), Nanchong City school science and technology strategic cooperation project of [Nanchong Science and Technology Bureau] (Grants No. 22SXQT0077), and Research Development Fund project of North Sichuan Medical College of [Department of Science and Technology, North Sichuan Medical College] (Grants No. CBY22-QNA12).

Data Availability

All data described in the manuscript and codes would be available upon request pending.

Ethics Approval

Due to the utilization of publicly available data and published literature, no further ethical clearance was deemed necessary for the present study.

Consent to Participate

The written informed consent of all participants was obtained by the respective Institutional Review Boards.

Conflicts of Interest

No potential conflicts of interest were disclosed.

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No competing interests reported.

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Mendelian Randomization Study Suggests Circulating Copper Level as a Risk Factor for Liver Cirrhosis

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Introduction

Materials And Methods

Data Sources

Selection of Copper-Associated SNPs

Statistical Analysis

Results

Discussion

Conclusion

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References

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