Melatonin inhibits liver ferroptosis in copper-laden rats: a potential therapy mechanism underlying Wilson’s disease

Wilson disease

Introduction Wilson’s disease (WD) is a potentially treatable, inherited disorder resulting from impaired copper metabolism. Pathological copper accumulation causes a range of symptoms, most commonly hepatic and a wide spectrum of neurological symptoms including tremor, dystonia, chorea, parkinsonism, dysphagia, dysarthria, gait and posture disturbances. To reduce copper overload, anti-copper drugs are used that improve liver function and neurological symptoms in up to 85% of patients. However, in some WD patients, treatment introduction leads to neurological deterioration, and in others, neurological symptoms persist with no improvement or improvement only after several years of treatment, severely affecting the patient’s quality of life. Areas covered This review appraises the evidence on various pharmacological and non-pharmacological therapies, neurosurgical procedures and liver transplantation for the management of neurological WD symptoms. The authors also discuss the neurological symptoms of WD, causes of deterioration and present symptomatic treatment options. Expert opinion Based on case and series reports, current recommendations and expert opinion, WD treatment is focused mainly on drugs leading to negative copper body metabolism (chelators or zinc salts) and copper-restricted diet. Treatment of WD neurological symptoms should follow general recommendations of symptomatic treatment. Patients should be always considered individually, especially in the case of severe, disabling neurological symptoms.

To determine the mutational characterization of P-type ATP7B gene and to explore the correlation of ATP7B genotype to phenotype in Chinese patients with Wilson disease (WD). Seventy-five patients with WD from 72 no-kinship families, 44 males and 31 females, were enrolled in this study. The age of onset ranged from 4 to 39 years, <=18 years in 72 patients. Some exons of ATP7B gene mutations were analyzed in patients with WD by using biochemical methods, polymerase chain reaction-single strand configuration polymorphism (PCR-SSCP) and DNA sequence analysis. A total of 778 coding regions were identified with restriction enzyme Msp I. The activity of Cu-ATPase was assessed by measuring inorganic phosphorus. Sixty-six of 75 patients (88%) had with hepatic manifestations, 39 of them had only hepatic manifestations, 27 patients had hepatic and neurological manifestations or other symptoms at the same time (16 patients had associated neurological manifestation, 3 patients had osteopathy, 8 patients had other symptoms). Eight of the 75 patients (10.7%) had only neurological symptoms, one patient (5 years old) had no symptom. Twelve changing patterns were detected in ATP7B gene by DNA sequencing, including seven mutations (R778L, C656X, G943D, V1140A, V1106I V1216M and 1384del17), six polymorphisms (IVS4-5t/c, A2495G, C2310G, IVS18+6c/t and IVS20+5a/g). R778L occurred in 49/66 patients (74%) with hepatic manifestations, homozygosis of R778L in 16 patients, heterozygosity of R778L in 33 patients. V1106I mutation of ATP7B gene occurred in 2 patients with delaying onset of clinical symptoms. Cu-ATPase activity of three patients with known mutations (R778L/V1106I/A2495G, R778L/V1216M and R778L/R778L) were determined, and the activity of Cu-ATPase was decreased by 44.55%, 88.23% and 69.49% respectively. 1384del17bp is a novel mutation found in WD patients. R778L is the most common mutation of ATP7B gene. There is a correlation between R778L and hepatic manifestations in WD patient.

ObjectivesTo explore the characteristics of myocardial involvement in Wilson Disease (WD) patients by cardiac magnetic resonance (CMR).MethodsWe prospectively included WD patients and age- and sex-matched healthy population. We applied CMR to analyze cardiac function, strain, T1 maps, T2 maps, extracellular volume fraction (ECV) maps, and LGE images. Subgroup analyzes were performed for patients with WD with predominantly neurologic manifestations (WD‐neuro +) or only hepatic manifestations (WD‐neuro −).ResultsForty-one WD patients (age 27.9 ± 8.0 years) and 40 healthy controls (age 25.4 ± 2.9 years) were included in this study. Compared to controls, the T1, T2, and ECV values were significantly increased in the WD group (T1 1085.1 ± 39.1 vs. 1046.5 ± 33.1 ms, T2 54.2 ± 3.3 ms vs. 51.5 ± 2.6 ms, ECV 31.8 ± 3.6% vs. 24.3 ± 3.7%) (all p < 0.001). LGE analysis revealed that LGE in WD patients was predominantly localized to the right ventricular insertion point and interventricular septum. Furthermore, the WD‐neuro + group showed more severe myocardial damage compared to WD‐neuro − group. The Unified Wilson Disease Rating Scale score was significantly correlated with ECV (Pearson’s r = 0.64, p < 0.001).ConclusionsCMR could detect early myocardial involvement in WD patients without overt cardiac function dysfunction. Furthermore, characteristics of myocardial involvement were different between WD‐neuro + and WD‐neuro − , and myocardial involvement might be more severe in WD‐neuro + patients.Critical relevance statementCardiac magnetic resonance enables early detection of myocardial involvement in Wilson disease patients, contributing to the understanding of distinct myocardial characteristics in different subgroups and potentially aiding in the assessment of disease severity.Key points• CMR detects WD myocardial involvement with increased T1, T2, ECV.• WD‐neuro + patients show more severe myocardial damage and correlation with ECV.• Differences of myocardial characteristics exist between WD‐neuro + and WD‐neuro − patients.Graphical

A High-Calorie Diet Aggravates Mitochondrial Dysfunction and Triggers Severe Liver Damage in Wilson Disease Rats.

Copper is essential for human physiology, but in excess it causes the severe metabolic disorder Wilson disease. Elevated copper is thought to induce pathological changes in tissues by stimulating the production of reactive oxygen species that damage multiple cell targets. To better understand the molecular basis of this disease, we performed genome-wide mRNA profiling as well as protein and metabolite analysis for Atp7b-/- mice, an animal model of Wilson disease. We found that at the presymptomatic stages of the disease, copper-induced changes are inconsistent with widespread radical-mediated damage, which is likely due to the sequestration of cytosolic copper by metallothioneins that are markedly up-regulated in Atp7b-/- livers. Instead, copper selectively up-regulates molecular machinery associated with the cell cycle and chromatin structure and down-regulates lipid metabolism, particularly cholesterol biosynthesis. Specific changes in the transcriptome are accompanied by distinct metabolic changes. Biochemical and mass spectroscopy measurements revealed a 3.6-fold decrease of very low density lipoprotein cholesterol in serum and a 33% decrease of liver cholesterol, indicative of a marked decrease in cholesterol biosynthesis. Consistent with low cholesterol levels, the amount of activated sterol regulatory-binding protein 2 (SREBP-2) is increased in Atp7b-/- nuclei. However, the SREBP-2 target genes are dysregulated suggesting that elevated copper alters SREBP-2 function rather than its processing or re-localization. Thus, in Atp7b-/- mice elevated copper affects specific cellular targets at the transcription and/or translation levels and has distinct effects on liver metabolic function, prior to appearance of histopathological changes. The identification of the network of specific copper-responsive targets facilitates further mechanistic analysis of human disorders of copper misbalance.

Background: Abnormal nutritional status is frequently seen in patients with chronic diseases. To date, no study has investigated the detailed characteristics of abnormal nutritional status among Wilson's disease (WD) patients in the Chinese cohort. This study aimed to describe the nutritional status of WD patients, with a particular focus on the differences between patients with different phenotypes.Methods: The study subjects comprised 119 healthy controls, 129 inpatients (hepatic subtype, n = 34; neurological subtype, n = 95) who were being treated at the affiliated hospital of the Institute of Neurology, Anhui University of Chinese Medicine. All of the subjects were assessed for body composition by using bioelectrical impedance analysis. All WD patients received anthropometry, nutritional risk screening 2002 (NRS2002), and laboratory test (hemocyte and serum biomarkers) additionally.Results: Compared with healthy controls, the fat mass and rate of total body and trunk were significantly higher in WD patients (P < 0.001), the muscle and skeletal muscle mass of total body and trunk were significantly lower in WD patients (P < 0.001). Compared with hepatic subtype patients, the fat mass and rate of total body, trunk, and limbs were significantly lower in neurological subtype patients (P<0.01); while there were no significant differences in muscle and skeletal muscle between these two subtypes. The overall prevalence of abnormal nutritional status in WD patients was 43.41% (56/129). The prevalence of high-nutritional risk and overweight in WD patients was 17.83% (23 of 129) and 25.58% (33 of 129), respectively. Compare with patients with high nutritional risk, macro platelet ratio, alkaline phosphatase, the basal metabolic rate (p < 0.05), creatinine, trunk fat rate (p < 0.01) and appendicular skeletal muscle mass (p < 0.001) were significantly higher in patients without nutritional risk (p < 0.001). Patients with a high nutritional risk tend to have a lower cholinesterase concentration (x2 = 4.227, p < 0.05).Conclusion: Both patients with H-subtype and N-subtype are prone to have an abnormal nutritional status. Longitudinal studies are required to investigate if nutritional status and body composition could reflect prognosis in WD patients, and which of these body composition indexes contribute to malnutrition and worse prognosis.

Wilson disease (WD) is the most common genetic metabolic liver disease. Some studies have shown that comorbidities may have important effects on WD. Data on hepatitis B virus (HBV) infection in patients with WD are limited. To investigate the prevalence and clinical impact of HBV infection in patients with WD. The clinical data of patients with WD were analyzed retrospectively, and the data of patients with concurrent WD and HBV infection were compared with those of patients with isolated WD. Among a total of 915 WD patients recruited, the total prevalence of current and previous HBV infection was 2.1% [95% confidence interval (CI): 1.2%-3.0%] and 9.2% (95%CI: 7.3%-11.1%), respectively. The main finding of this study was the identification of 19 patients with concurrent WD and chronic hepatitis B (CHB) infection. The diagnosis of WD was missed in all but two patients with CHB infection. The mean delay in the diagnosis of WD in patients with concurrent WD and CHB infection was 32.5 mo, which was significantly longer than that in patients with isolated WD (10.5 mo). The rates of severe liver disease and mortality in patients with concurrent WD and CHB infection were significantly higher than those in patients with isolated WD (63.1% vs 19.3%, P = 0.000 and 36.8% vs 4.1%, P < 0.001, respectively). Binary logistic regression analysis revealed a significantly higher risk of severe liver disease at the diagnosis of WD in patients with current HBV infection [odds ratio (OR) = 7.748; 95%CI: 2.890-20.774; P = 0.000)] or previous HBV infection (OR = 5.525; 95%CI: 3.159-8.739; P = 0.000) than in patients with isolated WD. The total prevalence of current HBV infection in patients with WD was 2.1%. The diagnosis of WD in CHB patients is usually missed. HBV infection is an independent risk factor for severe liver disease in WD patients. The diagnosis of WD should be ruled out in some patients with CHB infection.

Long-term Outcome for Wilson Disease: 85% Good

Introduction Liver transplantation (LT) is an effective option for Wilson's disease (WD) patients with neurologic symptoms, acute liver failure and with advance decompansated liver disease. In this retrospective study we aimed to review our LT results for WD patients. Materials and Methods Between December 1988 and October 2017 we performed 580 LT procedures at our center (age range, 6 months-64 years). We performed 53 LT in WD (45 living donor LT, 8 deceased donor LT) and after discharge we followed them for 12-350 months. We evaluated the regression of neurologic symptoms and reported survival, graft function of WD patients after LT. Results We performed LT for acute liver failure in 7 WD patients and for end stage liver failure in 41 WD patients. Five patients had LT for neurological WD. One of these neurological WD patients had auxiliary partial orthotopic LT (APOLT) due to neurological WD without hepatic failure (child A). After APOLT he is doing well with normal liver functions and ceruloplasmin levels. All neurologic symptoms were regressed in all of these 5 neurological WD patients. Four of these 53 WD patients had retransplantation (due to 1 primary non-function and 3 chronic rejections) and 3 retransplant patients are doing well with normal liver functions. Thirteen of 53 WD patients died; 6 patients died in early postoperative period, 7 patients died during their follow up due to complications unconnected to WD. The remaining 40 patients have normal liver functions in their follow up (range 12-350 months). Our long term survival is 85%. Conclusion Liver transplantation for WD with end stage liver failure and neurological WD has good outcomes. We may also consider auxiliary partial orthotopic LT for progressive neurological WD patients with no hepatic insufficiency.

Late-Onset Wilson’s Disease

Visualization of Copper Metabolism by 64CuCl2-PET

N-terminal domains of the Wilson's and Menkes disease proteins (N-WND and N-MNK) were overexpressed in a soluble form in Escherichia coli as fusions with maltose-binding protein, purified, and their metal-binding properties were characterized. Both N-MNK and N-WND bind copper specifically as indicated by the results of metal-chelate chromatography, direct copper-binding measurements, and chemical modification of Cys residues in the presence of different heavy metals. When E. coli cells are grown in the presence of copper, N-MNK and N-WND bind copper in vivo with stoichiometry of 5-6 nmol of copper/nmol of protein. Copper released from the copper-N-MNK and copper-N-WND complexes reacts with the Cu(I)-selective chelator bicinchoninic acid in the absence of reducing agents. This suggests that in proteins, it is bound in reduced Cu(I) form, in agreement with the spectroscopic properties of the copper-bound domains. Copper bound to the domains in vivo or in vitro specifically protects the N-MNK and N-WND against labeling with the cysteine-directed probe; this indicates that Cys residues in the repetitive motifs GMTCXXCXXXIE are involved in coordination of copper. Direct involvement of the N-terminal domains in the binding of copper suggests their important role in copper-dependent functions of human copper-transporting adenosine triphosphatases (Wilson's and Menkes disease proteins).

Background: A number of polymerase chain reaction (PCR) based techniques like single-strand conformational polymorphism (SSCP), amplification refractory mutation system (ARMS)-PCR, semi-nested PCR and dinucleotide-repeat marker analysis have been used in the diagnosis of asymptomatic Wilson disease (WD) patients and the carrier status of WD families. In the present study, we explore the utility of mutation analysis in combination with restriction fragment length polymorphism (RFLP) in the genetic diagnosis of WD.Aim: The study was planned to provide a molecular diagnostic tool for the diagnosis of asymptomatic WD patients as well as assessment of the carrier status of WD families.Subjects and methods: Four WD families were analyzed in which parents and siblings showed no clinical manifestations or biochemical abnormalities. The parents of the WD patients were not consanguineous and had no family history of WD. Mutations in ATP7B were characterized using SSCP and DNA sequencing. Further, RFLP was developed for the analysis of characterized mutations in ATP7B from the WD patients, their parents and siblings.Results: Three mutations, Q1256R, A1003T and I1102T, were characterized in WD patients, using SSCP and DNA sequencing. These mutations created/deleted restriction sites for AccII, Bsh1236I and EcoRI restriction enzymes respectively. Despite having no clinical manifestations nor any significant alteration in biochemical investigations, eight carriers and one asymptomatic WD patient were diagnosed in 13 members of the patients families by restriction digestion analysis.Conclusion: The report demonstrates that mutation analysis in combination with RFLP is useful for diagnosis of asymptomatic WD patients as well as for the elucidation of the carrier status of the patients’ family members. It is noteworthy that this combinational methodology provides a positive diagnosis in siblings/parents where biochemical parameters are ambiguous.

Liver pathology in Wilson's disease: From copper overload to cirrhosis