Diverse Functional Properties of Wilson Disease ATP7B Variants

Abstract

BACKGROUND & AIMS Wilson disease is a severe disorder of copper metabolism caused by mutations in ATP7B, which encodes a copper-transporting adenosine triphosphatase. The disease presents with a variable phenotype that complicates the diagnostic process and treatment. Little is known about the mechanisms that contribute to the different phenotypes of the disease. METHODS We analyzed 28 variants of ATP7B from patients with Wilson disease that affected different functional domains; the gene products were expressed using the baculovirus expression system in Sf9 cells. Protein function was analyzed by measuring catalytic activity and copper (64Cu) transport into vesicles. We studied intracellular localization of variants of ATP7B that had measurable transport activities and were tagged with green fluorescent protein in mammalian cells using confocal laser scanning microscopy. RESULTS Properties of ATP7B variants with pathogenic amino-acid substitution varied greatly even if substitutions were in the same functional domain. Some variants had complete loss of catalytic and transport activity, whereas others lost transport activity but retained phosphor-intermediate formation or had partial losses of activity. In mammalian cells, transport-competent variants differed in stability and subcellular localization. CONCLUSIONS Variants in ATP7B associated with Wilson disease disrupt the protein’s transport activity, result in its mislocalization, and reduce its stability. Single assays are insufficient to accurately predict the effects of ATP7B variants the function of its product and development of Wilson disease. These findings will contribute to our understanding of genotype–phenotype correlation and mechanisms of disease pathogenesis.

Distinct phenotype of a Wilson disease mutation reveals a novel trafficking determinant in the copper transporter ATP7B

“…In addition to p.S653Y, mutations p.G626A and p.M645R were reported with p.H1069Q as the second allele, and one patient was homozygous for p.D642H (Table S1). The biochemical properties of p.G626A and p.M645R mutants were previously studied in vitro, where both variants showed diminished but significant copper transport activity (21). We reasoned that mutations in this region might disrupt Cu-specific functions of ATP7B characteristic of vertebrates, such as Cu-dependent trafficking.…”

“…Review of WD patient mutations revealed two more common patient mutations, ATP7B G710S in TM2 and ATP7B G943S in TM5, which may behave similarly to ATP7B S653Y . Both mutants were reported to localize to the Golgi in basal medium and exhibit some Cu(I) transport activity (5,18,21). Trafficking studies in fibroblasts showed that ATP7B G943S failed to exit the TGN in elevated copper.…”

Linkage Disequilibrium and Haplotype Analysis of theATP7BGene in Alzheimer's Disease

“…A number of studies have investigated whether these variants are really disease-causing mutations rather than rare normal variants, highlighting that ATP7B mutations in the same protein domain can have diverse functional effects. [29][30][31][32] Nevertheless, it is possible to evaluate the pathogenetic potential of our LD blocks using the Wilson Disease Mutation Database to calculate the density of For the descriptive characteristics p < 0.05 (*) and p < 0.01 (**) are considered significant, whereas for ATP7B genotypes p < 0.017 (*) and p < 0.003 (**) are significant according multiple test correction.…”

Establishment of hepatic and neural differentiation platforms of Wilson’s disease specific induced pluripotent stem cells

“…It is located in the frequently mutated exon 8 and is one of the hotspot mutations of WD in the Caucasian population (Curtis et al, 1999;Huster et al, 2004;Lepori et al, 2007). Biochemical analysis has revealed a significantly impaired transport activity of the M769V mutant ATP7B (Huster et al, 2012). We firstly reprogrammed the patient fibroblasts to iPSC by retroviruses encoding Oct4, Sox2, Klf4, c-Myc and green fluorescent protein (GFP) (Takahashi et al, 2007).…”

“…The disease gene ATP7B encodes for a copper transporting P-type ATPase which is critical in the distribution and elimination of excess copper from cells. Various WD associated mutations of ATP7B disrupt the transport activity of the protein, which also results in protein mislocalization and reduced stability (Huster et al, 2012). WD is characterized by the toxic accumulation of copper in the liver and subsequently in the central nervous system (CNS) and other organs, thereby causing severe liver damage and/or neurological abnormality.…”