Assessment and factors affecting quality of life among patients with Wilson's disease.

Wilson's disease is caused by abnormal copper metabolism resulting in deposition in various organs, including the brain, liver, and cornea, thus disrupting organ function. It is characterized by encephalopathy, extrapyramidal symptoms, progressive liver failure, and copper ring deposition in the cornea. Management of this disease should include quality of life maintenance; however, relevant studies on this topic are lacking. This study aimed to assess the factors affecting the quality of life (QoL) of patients with Wilson's disease. A cross-sectional survey using convenience sampling was conducted between July 2020 and March 2021 at the hospital. Data on patient characteristics, 36-item Short-Form General Health Survey, Uniform Wilson Disease Rating Scale, and Hamilton Depression Rating Scale scores were collected. Associations among quality of life depression, anxiety, and Wilson's disease progression were examined using Pearson correlation analysis. Factors affecting the quality of life of patients, including depression, anxiety, liver function, clinical symptoms, diet, liver function, brain magnetic resonance imaging (MRI) findings, disease duration, Barthel Index, and Morse scores were examined using multivariate linear regression analysis. This study included 134 patients with Wilson's disease whose mean age was 29.12 ± 8.59 years. The mean QoL score in the patient group was 71.38 ± 9.55 points and was negatively correlated with anxiety (r = - 0.883, P = 0.000), depression (r = - 0.852 P = 0.000), and clinical symptoms (r = - 0.542, P = 0.000) scores. Anxiety, depression, and clinical symptoms severity are vital factors for the QoL of patients with Wilson's disease. The study provides foundational evidence to design novel interventions, including symptom management, diet, and self-care ability, which can help in improving the quality of life in patients with Wilson's disease and decreasing the burden associated with this disease.

A new Caenorhabditis elegans model to study copper toxicity in Wilson disease.

Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo-lysosomal compartments drives sequestration of excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua-1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua-1 mutant animals exhibited very poor resistance to Cu compared to the wild-type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua-1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA-1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B-H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B-H1069Q reduced Cu toxicity in cua-1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA-1 function. Taken together, our findings suggest that the newly generated cua-1 mutant strain represents an excellent model for Cu toxicity studies in WD.

Hepatic oxylipin profiles in mouse models of Wilson disease: New insights into early hepatic manifestations.

Hepatic inflammation is commonly identified in Wilson disease (WD), a genetic disease of hepatic and brain copper accumulation. Copper accumulation is associated with increased oxidative stress and reactive oxygen species generation which may result in non-enzymatic oxidation of membrane-bound polyunsaturated fatty acids (PUFA). PUFA can be oxidized enzymatically via lipoxygenases (LOX), cyclooxygenases (COX), and cytochrome P450 monooxygenases (CYP). Products of PUFA oxidation are collectively known as oxylipins (OXL) and are bioactive lipids that modulate hepatic inflammation. We examined hepatic OXL profiles at early stages of WD in two mouse models, the toxic milk mouse from The Jackson Laboratory (tx-j) and the Atp7b knockout on a C57Bl/6 background (Atp7b-/-B6). Targeted lipidomic analysis performed by ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry showed that in both tx-j and Atp7b-/-B6 mice, hepatic OXL profiles were altered with higher thromboxane and prostaglandins levels. The levels of oxidative stress marker, 9-HETE were increased more markedly in tx-j mice. However, both genotypes showed upregulated transcript levels of many genes related to oxidative stress and inflammation. Both genotypes showed higher prostaglandins, thromboxin along with higher PUFA-derived alcohols, diols, and ketones with altered epoxides; the expression of Alox5 was upregulated and many CYP-related genes were dysregulated. Pathway analyses show dysregulation in arachidonic acid and linoleic acid metabolism characterizes mice with WD. Our findings indicate alterations in hepatic PUFA metabolism in early-stage WD and suggest the upregulation of both, non-enzymatic ROS-dependent and enzymatic PUFA oxidation, which could have implications for hepatic manifestations in WD and represent potential targets for future therapies.

Metallothionein: a game changer in histopathological diagnosis of Wilson disease.

AIMS: Wilson disease (WD) is a genetic disorder of copper metabolism caused by mutations in the ATP7B gene. Toxic copper accumulation leads to hepatic, neurologic, and psychiatric disorders with variable presentation. Metallothionein (MT) immunohistochemistry was proposed as a diagnostic marker. METHODS: MT immunohistochemistry was performed on liver specimens of WD patients (n = 64) and control cases (n = 160) including acute liver failure, steatotic liver disease, autoimmune hepatitis, normal liver, primary biliary cholangitis, primary and secondary sclerosing cholangitis, and progressive familial intrahepatic cholestasis. The optimal cutoff for detection of WD was determined by receiver operating characteristic (ROC) analysis. RESULTS: At least moderate staining in >50% of hepatocytes was observed in 81% of analysed liver specimens (n = 56/69) of WD patients, while only five control cases showed this staining pattern. The sensitivity, specificity, and accuracy for a new diagnosis of WD were 85.7%, 96.9%, and 94.9%, respectively. Sensitivity in nonfibrotic patients was 70.6% and this MT pattern was robust in small biopsies. The hepatic copper concentration was similar between MT-positive and MT-negative liver samples (P > 0.05). Zinc treatment may induce hepatocellular MT expression. Kayser-Fleischer rings (50% versus 15%) and neurologic disorders (50% versus 13%) were significantly more prevalent in MT-negative compared to MT-positive WD patients, respectively. CONCLUSION: MT immunostaining is an excellent biomarker for histological diagnosis of WD, should be incorporated in the diagnostic work-up of patients with potential WD, and is useful in a modified Leipzig score.

Therapeutic implications of impaired nuclear receptor function and dysregulated metabolism in Wilson's disease.

Copper is an essential trace element that is required for the activity of many enzymes and cellular processes, including energy homeostasis and neurotransmitter biosynthesis; however, excess copper accumulation results in significant cellular toxicity. The liver is the major organ for maintaining copper homeostasis. Inactivating mutations of the copper-transporting P-type ATPase, ATP7B, result in Wilson's disease, an autosomal recessive disorder that requires life-long medicinal therapy or liver transplantation. Current treatment protocols are limited to either sequestration of copper via chelation or reduction of copper absorption in the gut (zinc therapy). The goal of these strategies is to reduce free copper, redox stress, and cellular toxicity. Several lines of evidence in Wilson's disease animal models and patients have revealed altered hepatic metabolism and impaired hepatic nuclear receptor activity. Nuclear receptors are transcription factors that coordinate hepatic metabolism in normal and diseased livers, and several hepatic nuclear receptors have decreased activity in Wilson's disease and Atp7b-/- models. In this review, we summarize the basic physiology that underlies Wilson's disease pathology, Wilson's disease animal models, and the possibility of targeting nuclear receptor activity in Wilson's disease patients.

A fluorometric assay to determine labile copper(II) ions in serum.

Labile copper(II) ions (Cu2+) in serum are considered to be readily available for cellular uptake and to constitute the biologically active Cu2+ species in the blood. It might also be suitable to reflect copper dyshomeostasis during diseases such as Wilson's disease (WD) or neurological disorders. So far, no direct quantification method has been described to determine this small Cu2+ subset. This study introduces a fluorometric high throughput assay using the novel Cu2+ binding fluoresceine-peptide sensor FP4 (Kd of the Cu2+-FP4-complex 0.38 pM) to determine labile Cu2+ in human and rat serum. Using 96 human serum samples, labile Cu2+was measured to be 0.14 ± 0.05 pM, showing no correlation with age or other serum trace elements. No sex-specific differences in labile Cu2+ concentrations were noted, in contrast to the total copper levels in serum. Analysis of the effect of drug therapy on labile Cu2+ in the sera of 19 patients with WD showed a significant decrease in labile Cu2+ following copper chelation therapy, suggesting that labile Cu2+ may be a specific marker of disease status and that the assay could be suitable for monitoring treatment progress.

Stimulation of Liver Fibrosis by N2 Neutrophils in Wilson's Disease.

BACKGROUND & AIMS: Wilson's disease is an inherited hepatoneurologic disorder caused by mutations in the copper transporter ATP7B. Liver disease from Wilson's disease is one leading cause of cirrhosis in adolescents. Current copper chelators and zinc salt treatments improve hepatic presentations but frequently worsen neurologic symptoms. In this study, we showed the function and machinery of neutrophil heterogeneity using a zebrafish/murine/cellular model of Wilson's disease. METHODS: We investigated the neutrophil response in atp7b-/- zebrafish by live imaging, movement tracking, and transcriptional analysis in sorted cells. Experiments were conducted to validate liver neutrophil heterogeneity in Atp7b-/- mice. In vitro experiments were performed in ATP7B-knockout human hepatocellular carcinomas G2 cells and isolated bone marrow neutrophils to reveal the mechanism of neutrophil heterogeneity. RESULTS: Recruitment of neutrophils into the liver is observed in atp7b-/- zebrafish. Pharmacologic stimulation of neutrophils aggravates liver and behavior defects in atp7b-/- zebrafish. Transcriptional analysis in sorted liver neutrophils from atp7b-/- zebrafish reveals a distinct transcriptional profile characteristic of N2 neutrophils. Furthermore, liver N2 neutrophils also were observed in ATP7B-knockout mice, and pharmacologically targeted transforming growth factor ß1, DNA methyltransferase, or signal transducer and activator of transcription 3 reduces liver N2 neutrophils and improves liver function and alleviates liver inflammation and fibrosis in ATP7B-knockout mice. Epigenetic silencing of Socs3 expression by transforming growth factor ß1 contributes to N2-neutrophil polarization in isolated bone marrow neutrophils. CONCLUSIONS: Our findings provide a novel prospect that pharmacologic modulation of N2-neutrophil activity should be explored as an alternative therapeutic to improve liver function in Wilson's disease.

Integrated Metabolomics and Network Pharmacology to Reveal the Mechanisms of Gandouling Tablets Against Copper-Overload-Induced Neuronal Injury in Rats with Wilson's Disease.

Purpose: Gandouling Tablets (GDL), a proprietary Chinese medicine, have shown a preventive effect against Wilson's disease (WD)-induced neuronal damage in previous studies. However, the potential mechanisms need additional investigation. Combining metabonomics and network pharmacology revealed the GDL pathway against WD-induced neuronal damage. Methods: The WD rat model with a high copper load was developed, and nerve damage was assessed. Total metabonomics was used to identify distinct hippocampus metabolites and enriched metabolic pathways in MetaboAnalyst. The GDL's possible targets against WD neuron damage were then determined by network pharmacology. Cytoscape constructed compound metabonomics and pharmacology networks. Moreover, molecular docking and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) validated key targets. Results: GDL reduced WD-induced neuronal injury. Twenty-nine GDL-induced metabolites may protect against WD neuron injury. According to network pharmacology, we identified three essential gene clusters, of which genes in cluster 2 had the most significant impact on the metabolic pathway. A comprehensive investigation identified six crucial targets, including UGT1A1, CYP3A4, CYP2E1, CYP1A2, PIK3CB, and LPL, and their associated core metabolites and processes. Four targets reacted strongly with GDL active components. GDL therapy improved five targets' expression. Conclusion: This collaborative effort revealed the mechanisms of GDL against WD neuron damage and a way to investigate the potential pharmacological mechanisms of other Traditional Chinese Medicine (TCM).

Wilson disease complicated by Crohn disease: A case report and literature review.

RATIONAL: Wilson disease (WD), also known as hepatolenticular degeneration, is an autosomal-recessive hereditary disease with abnormal copper metabolism. Crohn disease (CD) is a chronic inflammatory gastrointestinal disease, which belongs to inflammatory bowel disease, all segments of the gastrointestinal tract can be affected, especially the terminal ileum and colon, accompanied by extraintestinal manifestations and related immune disorders. WD complicated by ulcerative colitis has been reported before, but WD complicated by CD has not been reported so far. PATIENT CONCERNS AND DIAGNOSIS: We presented the first report of a young patient with WD complicated by CD, who was admitted to the hospital because of repeated low fever, elevated C-reactive protein for 3 years, and anal fistula for 6 months. INTERVENTIONS AND OUTCOMES: In this complicated disease, Ustekinumab is safe and effective. LESSONS: We conclude that copper metabolism and oxidative stress play important roles in WD and CD.

Positron Emission Tomography Using 64-Copper as a Tracer for the Study of Copper-Related Disorders.

Copper is an essential trace element, functioning in catalysis and signaling in biological systems. Radiolabeled copper has been used for decades in studying basic human and animal copper metabolism and copper-related disorders, such as Wilson disease (WD) and Menke's disease. A recent addition to this toolkit is 64-copper (64Cu) positron emission tomography (PET), combining the accurate anatomical imaging of modern computed tomography (CT) or magnetic resonance imaging (MRI) scanners with the biodistribution of the 64Cu PET tracer signal. This allows the in vivo tracking of copper fluxes and kinetics, thereby directly visualizing human and animal copper organ traffic and metabolism. Consequently, 64Cu PET is well-suited for evaluating clinical and preclinical treatment effects and has already demonstrated the ability to diagnose WD accurately. Furthermore, 64Cu PET/CT studies have proven valuable in other scientific areas like cancer and stroke research. The present article shows how to perform 64Cu PET/CT or PET/MR in humans. Procedures for 64Cu handling, patient preparation, and scanner setup are demonstrated here.

Gandouling inhibits hepatic fibrosis in Wilson's disease through Wnt-1/ß-catenin signaling pathway.

ETHNOPHARMACOLOGIC SIGNIFICANCE: Wilson's disease (WD) hepatic fibrosis is the result of chronic liver injury induced by Cu2+ deposition in the liver. Gandouling (GDL) is a hospital preparation of the First Affiliated Hospital of Anhui University of Chinese Medicine. Previous studies have found that GDL can play an anti-inflammatory, anti-oxidation, and promote Cu2+ excretion, which has a clear anti-WD effect. AIM OF THE STUDY: We found that Wnt-1 was significantly up-regulated in the liver tissue of toxic-milk (TX) mouse in the WD gene mutant model, and the monomer components of GDL could combine well with Wnt-1. Therefore, in this work, we used RT-qPCR, Western blot, immunofluorescence, network pharmacology, molecular docking, and related methods to study the effects of GDL on hepatic stellate cell (HSC) activation and Wnt-1/ß-catenin pathway in TX mice to clarify the effect of GDL on WD hepatic fibrosis. RESULTS: GDL could alleviate hepatic fibrosis, improve liver function, and inhibit the activation of HSC in TX mice. Network pharmacology predicted that the Wnt-1/ß-catenin was the target of GDL, and molecular dynamics further revealed that GDL has a good binding ability with Wnt-1 and inhibits the Wnt/ß-catenin signaling pathway through Wnt-1. Furthermore, we found that GDL blocked the Wnt-1/ß-catenin signaling pathway in the liver of TX mice in vivo. In vitro, serum containing GDL blocked the Cu2+ ion-induced Wnt-1/ß-catenin signaling pathway in LX-2 cells. Therefore, GDL blocked the Wnt-1/ß-catenin signaling pathway, inhibited HSC activation, and improved WD hepatic fibrosis by binding to Wnt-1. CONCLUSION: GDL improves hepatic fibrosis in WD model mice by blocking the Wnt-1/ß-catenin signaling pathway, and Wnt-1 may be a new target for the diagnosis and treatment of WD. This reveals a new mechanism of GDL against WD, and promotes the clinical promotion of GDL.

1H NMR-based metabolomic study of striatal injury in rats with copper-loaded Wilson's disease by Chinese and Western medicine intervention.

OBJECTIVE: To investigate the metabolic mechanisms of Chinese and Western medicines on the metabolic network of striatal injury in a copper-loaded rat model of Wilson disease (WD) from a metabolomic perspective. METHODS: We divided 60 rats into 4 groups of 15 rats each according to a random number table, namely the control group, the model group, the Bushen Huoxue Huazhuo Recipe group, and the penicillamine group, and subsequently replicated the WD copper-loaded rat model according to the literature method for a total of 12 weeks. From the 7th week onwards, each intervention group was given an equivalent dose of the corresponding drug, and the control and model groups were given an equal volume of saline gavage until the end of the model replication. We used 1H NMR metabolomics techniques combined with multivariate statistical methods to describe the changes in the striatal metabolic profile of nerve injury in Wilson's disease and to analyze the effect of different treatments on their biomarker interventions. RESULTS: Nerve cell damage was evident in the WD copper-loaded rat model and could be reduced to varying degrees by different methods of intervention in the striatal nerve cells. The content of glycine, serine metabolism, and valine metabolism decreased in WD copper-loaded rat model; aspartate content increased after penicillamine intervention; glycolytic metabolism, valine metabolism, taurine metabolism, and tyrosine metabolism increased in the group of Bushen Huoxue Huazhuo Recipe. CONCLUSION: Different intervention methods of Chinese and Western medicine affect aspartate, glycolysis, taurine, tyrosine, valine, and carbon metabolism in striatal tissues of WD copper-loaded rats, and can regulate the metabolism of small molecules, which in turn have certain repairing effects on nerve damage in WD copper-loaded rats.

Structures of the human Wilson disease copper transporter ATP7B.

The P-type ATPase ATP7B exports cytosolic copper and plays an essential role in the regulation of cellular copper homeostasis. Mutants of ATP7B cause Wilson disease (WD), an autosomal recessive disorder of copper metabolism. Here, we present cryoelectron microscopy (cryo-EM) structures of human ATP7B in the E1 state in the apo, the putative copper-bound, and the putative cisplatin-bound forms. In ATP7B, the N-terminal sixth metal-binding domain (MBD6) binds at the cytosolic copper entry site of the transmembrane domain (TMD), facilitating the delivery of copper from the MBD6 to the TMD. The sulfur-containing residues in the TMD of ATP7B mark the copper transport pathway. By comparing structures of the E1 state human ATP7B and E2-Pi state frog ATP7B, we propose the ATP-driving copper transport model of ATP7B. These structures not only advance our understanding of the mechanisms of ATP7B-mediated copper export but can also guide the development of therapeutics for the treatment of WD.

Atp7b deficiency induces zebrafish eye developmental defects.

As a copper (Cu) transport ATPase, ATP7B plays an important role in maintaining Cu homeostasis in the body and its dysfunction is associated with retinal disease. How ATP7B dysfunction and the subsequent Cu overload induce retinal damage, however, are unknown. Here, we show that atp7b-/- homozygous zebrafish larvae are insensitive to light stimulation, with a reduction in retinal cells but normal like morphological phenotypes. Additionally, a series of differentially expressed genes are unveiled in atp7b-/- mutated larvae, which enrich in photo-transduction, structural constituent of eye lens, sensory perception of light stimulus, oxidative phosphorylation, and ATPase activity. Moreover, we show the Cu accumulation in retinal cells in atp7b-/- mutated larvae, which results in endoplasmic reticulum (ER) stress and retinal cell apoptosis and subsequent retinal defects. The integral data in this study demonstrate that atp7b mutation leads to Cu accumulation in zebrafish retinal cells and the consequence ER stress and retinal cell death. These data may give some possible hints to explain retinal disease occurred in the Cu dysregulation syndromes Wilson's disease with ATP7B mutation.

ARBM101 (Methanobactin SB2) Drains Excess Liver Copper via Biliary Excretion in Wilson's Disease Rats.

BACKGROUND & AIMS: Excess copper causes hepatocyte death in hereditary Wilson's disease (WD). Current WD treatments by copper-binding chelators may gradually reduce copper overload; they fail, however, to bring hepatic copper close to normal physiological levels. Consequently, lifelong daily dose regimens are required to hinder disease progression. This may result in severe issues due to nonadherence or unwanted adverse drug reactions and also due to drug switching and ultimate treatment failures. This study comparatively tested bacteria-derived copper binding agents-methanobactins (MBs)-for efficient liver copper depletion in WD rats as well as their safety and effect duration. METHODS: Copper chelators were tested in vitro and in vivo in WD rats. Metabolic cage housing allowed the accurate assessment of animal copper balances and long-term experiments related to the determination of minimal treatment phases. RESULTS: We found that copper-binding ARBM101 (previously known as MB-SB2) depletes WD rat liver copper dose dependently via fecal excretion down to normal physiological levels within 8 days, superseding the need for continuous treatment. Consequently, we developed a new treatment consisting of repetitive cycles, each of ∼1 week of ARBM101 applications, followed by months of in-between treatment pauses to ensure a healthy long-term survival in WD rats. CONCLUSIONS: ARBM101 safely and efficiently depletes excess liver copper from WD rats, thus allowing for short treatment periods as well as prolonged in-between rest periods.

A Study of Dopaminergic Pathway in Neurologic Wilson Disease with Movement Disorder.

Movement disorder (MD) is an important manifestation of neurologic Wilson disease (NWD), but there is a paucity of information on dopaminergic pathways. We evaluate dopamine and its receptors in patients with NWD and correlate the changes with MD and MRI changes. Twenty patients with NWD having MD were included. The severity of dystonia was assessed using BFM (Burke-Fahn-Marsden) score. The neurological severity of NWD was categorized as grades I to III based on the sum score of 5 neurological signs and activity of daily living. Dopamine concentration in plasma and CSF was measured using liquid chromatography-mass spectrometry, and D1 and D2 receptor expression at mRNA by reverse transcriptase polymerase chain reaction in patients and 20 matched controls. The median age of the patients was 15 years and 7 (35%) were females. Eighteen (90%) patients had dystonia and 2 (10%) had chorea. The CSF dopamine concentration (0.08 ± 0.02 vs 0.09 ± 0.017 pg/ml; p = 0.42) in the patients and controls was comparable, but D2 receptor expression was reduced in the patients (0.41 ± 0.13 vs 1.39 ± 1.04; p = 0.01). Plasma dopamine level correlated with BFM score (r = 0.592, p < 0.01) and D2 receptor expression with the severity of chorea (r = 0.447, p < 0.05). The neurological severity of WD correlated with plasma dopamine concentration (p = 0.006). Dopamine and its receptors were not related to MRI changes. The central nervous system dopaminergic pathway is not enhanced in NWD, which may be due to structural damage to the corpus striatum and/or substantia nigra.

[Wilson's disease: overview]. / Enfermedad de Wilson.

Wilson's disease (WD) is an uncommon hereditary disorder caused by a deficiency in the ATP7B transporter. The protein codified by this gene facilitates the incorporation of the copper into ceruloplasmin. Therefore, WD accumulates copper primary in the liver and secondary in other organs, such as the central nervous system. It represents a wide spectrum of disease, ranging from being asymptomatic in some patients to promote an acute liver failure in others. The diagnosis requires a combination of clinical signs and symptoms, as well as some diagnostic tests such as the measurement of serum ceruloplasmin, the urinary excretion of copper, the liver biopsy or the genetic testing. The treatment must be maintained lifelong and includes some drugs such as chelating agents (penicillamine and trientine) and inhibitors of the copper absorption (zinc salts). Lastly, the liver transplant should be an option for patients with end-stage liver disease.

Pathogenicity of Intronic and Synonymous Variants of ATP7B in Wilson Disease.

Wilson disease (WD) is a hereditary disorder of copper metabolism, resulting from mutations within ATP7B. Early diagnosis is essential for affected individuals. However, there are still patients with clinically suspected WD who do not have detectable pathogenic variants, which makes diagnosis difficult and delays treatment. This study included such patients from the authors' center and screened for the full-length sequence of ATP7B by next-generation sequencing. Newly identified synonymous and intronic variants were then analyzed with in silico tools. A minigene system was constructed to determine the pathogenicity of these variants in terms of splicing and blood RNA extraction, and RT-PCR experiments were performed on several patients to verify the splicing alterations. The phenotypes of the patients were also analyzed. Fourteen suspected pathogenic variants, including nine synonymous and five intronic variants, were detected in 12 patients with clinically suspected WD. Among them, four synonymous variants (c.1050G>A, c.1122C>G, c.3243G>A, and c.4014T>A) and four intronic variants (c.1543 +40G>A, c.1707+6_1707+16del, c.1870-49A>G, and c.2731-67A>G) resulted in splicing changes in ATP7B. After the above analysis, the diagnosis of WD could be confirmed in eight clinically suspected patients with WD who showed a late age of onset.

Glutathione improves testicular spermatogenesis through inhibiting oxidative stress, mitochondrial damage, and apoptosis induced by copper deposition in mice with Wilson disease.

BACKGROUND AND OBJECTIVE: There are considerable evidence of reproductive impairment in male organisms with Wilson disease (WD). The purpose of this study was to observe spermatogenesis, mitochondrial damage, apoptosis, and the level of oxidative stress in the testes of Wilson disease model TX mice, and to observe the effect and mechanism of glutathione on testicular spermatogenesis. METHODS: Mice were divided into a normal control group (control group), Wilson disease model TX mice group (WD group), penicillamine-treated TX mice group (penicillamine group) and glutathione-treated TX mice group (glutathione group). Testicular coefficient, histomorphology of testis and epididymis, number of spermatozoa, apoptosis of spermatogenic cells and expression of apoptosis-related proteins were observed. Ultrastructural analysis of mitochondria and mitochondrial membrane potential (MMP) monitored using JC-1 dye were used to detect mitochondrial damage. The levels of malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and reactive oxygen species (ROS) in testicular cells were measured to assess oxidative stress. RESULTS: Testicular coefficient did not change in mice with Wilson disease. However, the tissue structure of the testicular seminiferous tubules was damaged, and the number of spermatozoa in the epididymal lumen was significantly reduced in WD group. The apoptosis rate in the testes was significantly increased. The protein expression of the pro-apoptotic proteins Bax and Caspase-3 significantly increased, and the expressions of the anti-apoptotic protein Bcl-2 significantly decreased. The levels of ROS and MDA significantly increased, and the levels of CAT and GSH significantly decreased. Mitochondria with abnormal ultrastructure and the rate of JC-1 positive cells were significantly increased in the WD group. After copper chelation by penicillamine, the structure of the testicular seminiferous tubules and the number of spermatozoa in the epididymal lumen were significantly improved. The number of apoptotic cells was significantly reduced. The levels of Bax and Caspase-3 decreased, and the expression of Bcl-2 increased. The contents of CAT and GSH increased, and the levels of ROS and MDA decreased significantly. The abnormal mitochondria and JC-1 positive cells was significantly decreased. The histomorphology of seminiferous tubules, spermatogenic function, apoptosis rate, apoptosis-related proteins, mitochondrial damage, and oxidative stress in Wilson disease TX mice significantly improved after glutathione treatment. CONCLUSION: Copper deposition in Wilson disease can lead to oxidative stress injury, mitochondrial damage, and apoptosis in the testis, leading to the impairment of spermatogenesis. Glutathione may improve testicular spermatogenesis in male Wilson disease TX mice by inhibiting copper deposition-induced oxidative stress, mitochondrial damage, and apoptosis.

The Role of Zinc in the Treatment of Wilson's Disease.

Wilson's disease (WD) is a hereditary disorder of copper metabolism, producing abnormally high levels of non-ceruloplasmin-bound copper, the determinant of the pathogenic process causing brain and hepatic damage and dysfunction. Although the disease is invariably fatal without medication, it is treatable and many of its adverse effects are reversible. Diagnosis is difficult due to the large range and severity of symptoms. A high index of suspicion is required as patients may have only a few of the many possible biomarkers. The genetic prevalence of ATP7B variants indicates higher rates in the population than are currently diagnosed. Treatments have evolved from chelators that reduce stored copper to zinc, which reduces the toxic levels of circulating non-ceruloplasmin-bound copper. Zinc induces intestinal metallothionein, which blocks copper absorption and increases excretion in the stools, resulting in an improvement in symptoms. Two meta-analyses and several large retrospective studies indicate that zinc is equally effective as chelators for the treatment of WD, with the advantages of a very low level of toxicity and only the minor side effect of gastric disturbance. Zinc is recommended as a first-line treatment for neurological presentations and is gaining acceptance for hepatic presentations. It is universally recommended for lifelong maintenance therapy and for presymptomatic WD.