Clinical–Liver, Pancreas, and Biliary TractDefective cellular localization of mutant ATP7B in Wilson's disease patients and hepatoma cell lines☆
Abstract
Background & Aims: Wilson's disease, a hereditary disorder caused by mutations in the Wilson's disease gene (ATP7B), leads to hepatic and/or neurological pathology resulting from cellular copper overload. In vitro studies showed that ATP7B, located in the trans-Golgi network, traffics to a cytoplasmic vesicular compartment in response to increased copper concentration. Mislocalization and failed intracellular trafficking of ATP7B mutants are suggested to be among disease-causing mechanisms; however, the effect of mutations on ATP7B localization in human tissues has not been directly shown. Therefore, we characterized the subcellular localization of normal and mutant ATP7B in human livers and in hepatoma cell lines. Methods: Subcellular distribution of ATP7B in liver tissue from 3 control individuals and 3 Wilson's disease patients harboring a homozygous H1069Q-ATP7B mutation was analyzed by using immunogold electron microscopy. In addition, 14 ATP7B mutants tagged to green fluorescent protein were generated and expressed in HuH-7 and HepG2 cells; intracellular localization of these mutants was characterized by confocal microscopy. Results: In hepatocytes, ATP7B was localized in trans-Golgi vesicles, whereas H1069Q-ATP7B was trapped in the endoplasmic reticulum. Similar results were observed for wild-type ATP7B and H1069Q-ATP7B expressed in hepatoma cells. Most ATP7B proteins harboring missense mutations were distributed similarly to wild-type ATP7B. In contrast, truncated ATP7B mutants showed a diffuse, clustered, cytoplasmic pattern, distinct from the trans-Golgi network or endoplasmic reticulum. Conclusions: These results provide a detailed demonstration of the ATP7B distribution in control and diseased human livers and indicate that several Wilson's disease mutations lead to incorrect localization of ATP7B to distinct cell compartments.
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A simple TICT/ICT based molecular probe exhibiting ratiometric fluorescence Turn-On response in selective detection of Cu<sup>2+</sup>
2023, Journal of Photochemistry and Photobiology A: ChemistryThe work reported herein describes photophysical behavior of a new fluorescent probe APDA. The probe exhibited sensitivity for Cu2+ ion in 80% aqueous acetonitrile medium. Probe shows dual emission due to TICT and ICT states and upon interaction with different metal ions Cu2+ induces hydrolysis of imine bond to rejuvenate aldehyde derivative with ratiometric “turn–On” fluorescence response, through ICT state while emission due to TICT state get diminished. Similarly, the synthesized model compound ADA under similar experimental condition supported hydrolysis of imine bond in the presence of copper. Jobs plot analysis, based on emission titration data suggested a 1:1 binding stoichiometry between probe APDA and Cu2+ ion. The limit of detection (LOD) was found to be 4.4 × 10-8 M (44 nM). The probe has been applied on test paper strip to detect Cu2+ ion with naked-eye sensitive response. Also, the cell imaging studies revealed the potential applicability of probe to detect Cu2+ ion in live cells. The mechanism of sensing was confirmed by 1H, 13C NMR, FTIR and mass spectrometry data analysis.
ATP7B Genotype and Chronic Liver Disease Treatment Outcomes in Wilson Disease: Worse Survival With Loss-of-Function Variants
2023, Clinical Gastroenterology and HepatologyAlthough a good genotype-phenotype correlation has not been established in Wilson disease (WD), patients with loss-of-function (LOF) ATP7B variants demonstrate different clinical and biochemical characteristics. We aim to describe long-term treatment outcomes in the chronic liver disease (CLD) phenotype and evaluate an association with LOF variants.
This was a single-center retrospective review of WD patients with at least 1 variant in ATP7B. Demographic, biochemical, genetic, and clinical parameters were obtained. The composite clinical endpoint of liver transplantation or death was used for probands with CLD phenotype on chelators.
Of 117 patients with hepatic WD: 71 had CLD, 27 had fulminant hepatic failure requiring urgent liver transplantation, and 19 were diagnosed through family screening. Median age at diagnosis was 13.1 (interquartile range, 9.7–17.6) years. In total, 91 variants in ATP7B were identified in the study population. At least 1 LOF variant was present in 60 (51.3%) patients. During median follow-up of 10.7 (interquartile range, 6.7–18.9) years, 10 (14.1%) of the probands with CLD reached the composite endpoint. There was a worse transplant-free survival for patients prescribed chelation therapy in patients with at least 1 LOF variant (P = .03).
Patients with WD and CLD phenotype on chelators, who have at least 1 LOF variant in ATP7B, have a worse prognosis during long-term follow up. This subgroup of patients requires close monitoring for signs of progressive liver disease. Sequencing of ATP7B may be used in the diagnosis of WD, and in addition, it may provide useful prognostic information for patients with hepatic WD.
Developmental and Inherited Liver Disease
2023, MacSween's Pathology of the Liver, Eighth EditionA section on the approach to diagnostic histological interpretation is the overture to this chapter on inherited and developmental disorders. This initial section is split chronologically into the early neonatal and infantile period and later childhood and adulthood, with the intention of reflecting clinical practice as closely and succinctly as possible. Disorders of the biliary tree, bile formation and secretion and hepatocyte metabolism are the core of this chapter, a merger of Chapters 3 and 4 of previous editions. Considerations on the pathogenetic and/or clinical overlap among developmental, genetic and metabolic disorders were the rationale behind this change. The complexity of hepatocyte metabolism is reflected into the myriad of related pathological conditions. Two short new paragraphs on disorders of manganese metabolism and DNA repair and nuclear envelope have been added. Recent technological advances, particularly in genomics in the last 5 years, have resulted in a plethora of new entities and changes in terminology, challenging the authors to balance detail and application to clinical practice. Tables and figures from the previous edition have been largely kept due to their quality and contemporary relevance, and updated where necessary. Liver involvement in immunodeficiency and miscellaneous disorders precede the final section on anatomical anomalies. Vascular anomalies are now included in the chapters on vascular disorders.
Mutation spectrum of ATP7B gene in pediatric patients with Wilson disease in Vietnam
2022, Molecular Genetics and Metabolism ReportsWilson disease (WD) is caused by mutations in the copper-transporting P-type adenosine triphosphatase encoded by the ATP7B gene. In this study, we screened and identified the ATP7B mutations among unrelated Vietnamese pediatric patients.
One-hundred-thirteen pediatric patients with clinically diagnosed WD were recruited. DNA samples were extracted from peripheral blood. Mutations in the ATP7B gene were identified by Sanger sequencing.
Approximately 98% of the clinically diagnosed WD patients carried ATP7B mutations. A total of 35 different ATP7B variants were detected, including five novel mutations (L658P, L792P, T977K, IVS4 + 1G > A and IVS20 + 4A > G). Remarkably, this study revealed that S105* was the most prevalent variant (32.27%), followed by L1371P (9.09%), I1148T (7.27%), R778L (6.36%), T850I (5.45%), V176Sfs*28 and IVS14-2A > G (4.55%). Most ATP7B mutations were located in the exon 2 (37.73%), exon 16 (10.00%), exon 8 (9.55%), exon 20 (9.09%), exon 10 and exon 18 (5.45%), exon 14 (5.00%), exon 13 and intron 14 (4.55%). We developed a streamlined procedure to quickly characterize mutations in the ATP7B gene in the Vietnamese children, starting with sequencing exon 2 and subsequently to exons 8,10,13-16,18, and 20 to allow quick diagnosis of clinically suspected patients.
The mutational spectrum and hotspots of ATP7B gene in the Vietnamese population were fairly different from other East Asian populations. A streamlined procedure was developed to screen exon 2 in ATP7B gene among suspected WD patients to reduce genetically diagnostic cost, to facilitate early detection and intervention in countries with limited resources.
Copper
2021, Handbook on the Toxicology of Metals: Fifth EditionCopper is an essential trace element found in all organs and cells. The redox chemistry of this element makes copper highly suitable as a catalytic cofactor in oxidative enzymes. Copper is involved in numerous biological processes, primarily as an integral part of enzymes, such as those involved in cellular respiration (cytochrome c oxidase), antioxidant defense (superoxide dismutase), connective tissue formation (lysyl oxidase and related proteins), neurotransmitter biosynthesis (dopamine beta-hydroxylase), peptide hormone maturation (peptidyl-glycine alpha-amidating monooxygenase), pigmentation (tyrosinase), keratinization (sulfhydryl oxidase), and iron homeostasis (ceruloplasmin and hephaestin).
Copper in our body is absorbed from the diet. The absorption is dependent on the amount ingested, its chemical form, and the composition of other dietary components such as zinc. Drinking water may contribute significantly to the daily copper intake due to the widespread use of copper piping in water supplies.
Because copper is a highly reactive metal and thus harmful to cells if present as free ions, intracellular copper levels are strictly controlled by a substantial number of integral transmembrane transporters, including copper-transporting ATPases (ATP7A and ATP7B, respectively), and metallochaperones. ATP7B transports copper into the hepatocyte secretory pathway for incorporation into ceruloplasmin and excretion into the bile. ATP7A transports copper across the gastrointestinal tract, blood–brain barrier, and placenta. ATP7A is also responsible for the copper loading of a large number of the other copper-requiring enzymes.
Wilson disease and Menkes disease are inherited disorders of copper transport, caused by mutations in the ATP7B and ATP7A genes, respectively. The clinical features of patients with Wilson disease can be attributed to impaired biliary copper excretion, whereas Menkes disease arises from an altered distribution of copper in the body, with some tissues showing a deficiency and others showing excess accumulation.
Ingestion of a large amount of copper salts causes gastrointestinal disturbances. The first symptom to occur is nausea, with increased reporting starting at about 4 mg/L copper in drinking water. In severe cases, systemic effects, especially hemolysis, liver and kidney damage, can also occur. In contrast to data obtained after ingestion, comparatively little is known about health effects related to the inhalation of copper and copper fumes. Copper may cause irritation of the respiratory tract and metal fume fever. For reviews on copper, see Linder and Hazegh-Azam (1996), WHO (1998), Barceloux (1999), Gaetke and Chow (2003), Harris (2003), Tapiero et al. (2003), Stern et al. (2007), Romaňa et al. (2011), Nevitt et al. (2012), Skjørringe et al. (2012), Scheiber et al. (2014), Kaplan and Maryon (2016), Lenartowicz et al. (2016), and Michniewicz et al. (2021).
Direct Measurement of ATP7B Peptides Is Highly Effective in the Diagnosis of Wilson Disease
2021, GastroenterologyBoth existing clinical criteria and genetic testing have significant limitations for the diagnosis of Wilson disease (WD), often creating ambiguities in patient identification and leading to delayed diagnosis and ineffective management. ATP7B protein concentration, indicated by direct measurement of surrogate peptides from patient dried blood spot samples, could provide primary evidence of WD. ATP7B concentrations were measured in patient samples from diverse backgrounds, diagnostic potential is determined, and results are compared with biochemical and genetic results from individual patients.
Two hundred and sixty-four samples from biorepositories at 3 international and 2 domestic academic centers and 150 normal controls were obtained after Institutional Review Board approval. Genetically or clinically confirmed WD patients with a Leipzig score >3 and obligate heterozygote (carriers) from affected family members were included. ATP7B peptide measurements were made by immunoaffinity enrichment mass spectrometry.
Two ATP7B peptides were used to measure ATP7B protein concentration. Receiver operating characteristics curve analysis generates an area under the curve of 0.98. ATP7B peptide analysis of the sequence ATP7B 887 was found to have a sensitivity of 91.2%, specificity of 98.1%, positive predictive value of 98.0%, and a negative predictive value of 91.5%. In patients with normal ceruloplasmin concentrations (>20 mg/dL), 14 of 16 (87.5%) were ATP7B-deficient. In patients without clear genetic results, 94% were ATP7B-deficient.
Quantification of ATP7B peptide effectively identified WD patients in 92.1% of presented cases and reduced ambiguities resulting from ceruloplasmin and genetic analysis. Clarity is brought to patients with ambiguous genetic results, significantly aiding in noninvasive diagnosis. A proposed diagnostic score and algorithm incorporating ATP7B peptide concentrations can be rapidly diagnostic and supplemental to current Leipzig scoring systems.
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Supported in part by the Deutsche Forschungsgemeinschaft (DH: Hu 932/1-1).