Clinical Wilson Disease Research Articles

Functional characterization of novel or yet uncharacterized ATP7B missense variants detected in patients with clinical Wilson's disease.

Wilson's disease (WD, MIM#277900) is an autosomal recessive disorder resulting in copper excess caused by biallelic variants in the ATP7B gene (MIM#606882) encoding a copper transporting P-type ATPase. ATP7B variants of unknown significance (VUS) are detected frequently, sometimes impeding a clear diagnosis. Functional analyses can help to classify these variants as benign or pathogenic. Additionally, variants already classified as (likely) pathogenic benefit from functional analyses to understand their pathomechanism, thus contribute to the development of personalized treatment approaches in the future. We described clinical features of six WD patients and functionally characterized five ATP7B missense variants (two VUS, three yet uncharacterized likely pathogenic variants), detected in these patients. We determined the protein level, copper export capacity, and cellular localization in an in vitro model and potential structural consequences using an ATP7B protein model based on AlphaFold. Our analyses give insight into the pathomechanism and allowed reclassification for the two VUS to likely pathogenic and for two of the three likely pathogenic variants to pathogenic.

Exogenous copper exposure causing clinical wilson disease in a patient with copper deficiency

BackgroundHuman Swayback is a disease characterized by acquired copper deficiency which primarily manifests as myeloneuropathy. Common causes include malabsorptive disorders, gastric surgery, total parenteral nutrition and excessive zinc intake. In contrast, copper supplementation should be closely monitored as excessive doses can lead to acute intoxication and in chronic cases, cirrhosis. Copper derangements are rare, however it is important to consider them due to potential severe complications.Case presentationWe present a middle-aged man who had been previously diagnosed with Human Swayback after presenting with various neurological symptoms. The patient was subsequently placed on copper supplementation. A decade later, he was referred to our hospital for liver transplant evaluation due to new diagnosis of decompensated end-stage liver disease after an abdominal surgery. His initial workup was suggestive of Wilson disease—subsequent ATP7B gene was negative. Ultimately, the patient underwent liver transplantation; liver explant was significant for a copper dry weight concentration of 5436 mcg/g.ConclusionsHuman Swayback is a very rare copper-related disease which deserves awareness due to its potential irreversible health effects in the human body. Additionally, in patients who require copper supplementation, serial levels should be monitored to ensure adequate copper levels.

MTF1 binds to metal-responsive element e within the ATP7B promoter and is a strong candidate in regulating the ATP7B expression.

Wilson's disease is an autosomal recessive disorder resulting from copper excess. Some patients with clinical Wilson's disease symptoms exhibit no or only heterozygous pathogenic variants in the coding region of the disease-causing ATP7B gene. Therefore, the ATP7B promoter region is of special interest. Metal-responsive elements (MREs) located in the ATP7B promoter are promising motifs in modulating the ATP7B expression. We studied protein interaction of MREe, MREc, and MREd by electrophoretic mobility shift assays and revealed specific interactions for all MREs. We further narrowed down the specific binding site. Proteins potentially binding to the three MREs were identified by MatInspector analyses. Metal regulatory transcription factor 1 (MTF1) could be validated to bind to MREe by electrophoretic mobility shift assays. ATP7B promoter-driven reporter gene expression was significantly increased because of this interaction. MTF1 is a strong candidate in regulating the ATP7B expression through MREe binding.

Laser Ablation Inductively Coupled Plasma Spectrometry: Metal Imaging in Experimental and Clinical Wilson Disease

Wilson disease is an inherited disorder caused by mutations in the ATP7B gene resulting in copper metabolism disturbances. As a consequence, copper accumulates in different organs with most common presentation in liver and brain. Chelating agents that nonspecifically chelate copper, and promote its urinary excretion, or zinc salts interfering with the absorption of copper from the gastrointestinal tract, are current medications. Also gene therapy, restoring ATP7B gene function or trials with bis-choline tetrathiomolybdate (WTX101) removing excess copper from intracellular hepatic copper stores and increasing biliary copper excretion, is promising in reducing body’s copper content. Therapy efficacy is mostly evaluated by testing for evidence of liver disease and neurological symptoms, hepatic synthetic functions, indices of copper metabolisms, urinary copper excretions, or direct copper measurements. However, several studies conducted in patients or Wilson disease models have shown that not only the absolute concentration of copper, but also its spatial distribution within the diseased tissue is relevant for disease severity and outcome. Here we discuss laser ablation inductively coupled plasma spectrometry imaging as a novel method for accurate determination of trace element concentrations with high diagnostic sensitivity, spatial resolution, specificity, and quantification ability in experimental and clinical Wilson disease specimens.

Presumed missense and synonymous mutations in ATP7B gene cause exon skipping in Wilson disease.

Wilson disease is an inborn error of metabolism caused by abnormalities of the copper-transporting protein-encoding gene ATP7B. Recently, the phenomenon of exon skipping, in which exonic mutations result in abnormal splicing, has been associated with various diseases. The present study investigated the splicing defects of the ATP7B exonic variants identified in a cohort of 44 patients with Wilson disease. All patients were analysed for ATP7B gene by direct sequencing or multiplex ligation-dependent probe amplification analysis. To identify the potential pathogenicity of the candidate mutations that may induce exon skipping, both in vivo RT-PCR analysis using RNA from peripheral leukocytes and in vitro functional splicing by minigene construction were conducted. The patterns of inheritance of the mutations in ATP7B identified in 44 patients exhibited homozygotes (7 patients), compound heterozygotes (32 patients) and heterozygotes (5 patients). In all patients, we detected 25 different ATP7B mutations, including 17 missenses, 1 frameshift, 3 nonsenses, 2 exonic deletions and 2 splicing alteration. In these mutations, 4 mutations have not been previously described in the literature or entered in human genome mutation database. Furthermore, we identified synonymous mutation c.4014T>A and missense mutation R919G caused exon skipping in the ATP7B mRNA transcript. Our results suggest that aberrant exon skipping associated to putative splicing enhancer disruption and silencer creation is one previously unrecognized mechanism in Wilson disease. What is more, the multiplex ligation-dependent probe amplification assay for the detection of exon deletions may be valuable in individuals with clinical Wilson disease diagnosis where one or no mutation has been identified by sequencing.

Laser ablation inductively coupled plasma mass spectrometry imaging of metals in experimental and clinical Wilson's disease.

Wilson's disease is an autosomal recessive disorder in which the liver does not properly release copper into bile, resulting in prominent copper accumulation in various tissues. Affected patients suffer from hepatic disorders and severe neurological defects. Experimental studies in mutant mice in which the copper-transporting ATPase gene (Atp7b) is disrupted revealed a drastic, time-dependent accumulation of hepatic copper that is accompanied by formation of regenerative nodes resembling cirrhosis. Therefore, these mice represent an excellent exploratory model for Wilson's disease. However, the precise time course in hepatic copper accumulation and its impact on other trace metals within the liver is yet poorly understood. We have recently established novel laser ablation inductively coupled plasma mass spectrometry protocols allowing quantitative metal imaging in human and murine liver tissue with high sensitivity, spatial resolution, specificity and quantification ability. By use of these techniques, we here aimed to comparatively analyse hepatic metal content in wild-type and Atp7b deficient mice during ageing. We demonstrate that the age-dependent accumulation of hepatic copper is strictly associated with a simultaneous increase in iron and zinc, while the intrahepatic concentration and distribution of other metals or metalloids is not affected. The same findings were obtained in well-defined human liver samples that were obtained from patients suffering from Wilson's disease. We conclude that in Wilson's disease the imbalances of hepatic copper during ageing are closely correlated with alterations in intrahepatic iron and zinc content.

Prion protein gene codon 129 modulates clinical course of neurological Wilson disease

The polymorphism in the human prion protein gene at codon 129 (PRNP 129) determines susceptibility to prion disease, and has been associated with early onset and a more severe course of other neurodegenerative disorders. Here, we tested the hypothesis that PRNP is a disease-modifying gene in clinical Wilson disease with a neurological phenotype. Allele frequencies in patients with clinical Wilson disease were not different from those of a healthy German control population, and PRNP 129 genotypes did not result in different serum copper, serum ceruloplasmin, or copper in 24-h urine concentrations. PRNP 129 methionine homozygosity, however, led to significantly more severe neurological symptoms in elderly patients, particularly tremor, supporting the notion that PRNP 129 homozygosity contributes to neuronal vulnerability.

Metabolic studies in Wilson's disease: Evaluation of efficacy of chelation therapy in respect to copper balance

Long-term copper balance studies were performed in fifteen patients with Wilson's disease, six of their parents and siblings, and one normal control subject. Patients with Wilson's disease required a daily dietary copper intake of 1.1 mg or less to attain negative copper balance whereas their family members were in balance at 1.4 mg/day. Penicillamine therapy elicited a markedly negative copper balance initially by increasing the urinary excretion of copper, but as therapy continued, both urinary and fecal copper excretion diminished. The effect of oral ehelators on fecal copper varied: DTPA was of no value; carbacrylamine resin and potassium sulfide increased the fecal copper content. Most patients with Wilson's disease could be maintained in negative copper balance with a daily dietary copper intake of 1.5 mg or less with these latter agents. The addition of these drugs offered no advantage over the use of penicillamine alone. The combination of a low copper diet and the oral chelating agent, carbacrylamine resin, should be considered for interim periods to treat presymptomatic Wilson's disease or in patients with clinical Wilson's disease whose tissue copper stores have been reduced by penicillamine therapy.

PRESYMPTOMATIC WILSON'S DISEASE

Presymptomatic Wilson's disease must be diagnosed because the severe consequences of the disease may be prevented by adequate chelation therapy. Seventeen children from five families having members with Wilson's disease have been studied to detect presymptomatic subjects. Seven had clinical Wilson's disease, five were diagnosed as presymptomatic (homozygotes), and five were believed normal or heterozygotes. Kayser-Fleischer rings must be looked for with a slit lamp. They were present in symptomatic but absent in presymptomatic patients. All presymptomatic homozygotes had low serum copper and cæruloplasmin levels, but these estimations could not distinguish them with certainty from heterozygotes. A liver-copper concentration, performed on needle-biopsy samples, over 25 mg. per 100 g. dry weight, confirmed the presymptomatic state. The liver histology may be normal or abnormal, depending on the stage of the disease. Two heterozygotes