Analysis of Wilson disease mutations revealed that interactions between different ATP7B mutants modify their properties

Abstract

Wilson disease (WD) is an autosomal-recessive disorder caused by mutations in the copper (cu)transporter ATP7B. Thus far, studies of WD mutations have been limited to analysis of ATP7B mutants in the homozygous states. However, the majority of WD patients are compound-heterozygous, and how different mutations on two alleles impact ATP7B properties is unclear. We characterized five mutations identified in Indian WD patients, first by expressing each alone and then by co-expressing two mutants with dissimilar properties. Mutations located in the regulatory domains of ATP7B-A595T, S1362A, and S1426I-do not affect ATP7B targeting to the trans-Golgi network (TGN) but reduce its Cu-transport activity. The S1362A mutation also inhibits Cu-dependent trafficking from the TGN. The G1061E and G1101R mutations, which are located within the ATP-binding domain, cause ATP7B retention in the endoplasmic reticulum, inhibit Cu-transport, and lower ATP7B protein abundance. Co-expression of the A595T and G1061E mutations, which mimics the compoundheterozygous state of some WD patients, revealed an interaction between these mutants that altered their intracellular localization and trafficking under both low and high Cu conditions. These findings highlight the need to study WD variants in both the homozygous and compound-heterozygous states to better understand the genotype-phenotype correlations and incomplete penetrance observed in WD. Copper (Cu) is a redox-active metal that is indispensable for human growth and development. Cu is required for the activity of several enzymes, including cytochrome c oxidase, Cu/Zn-dependent superoxide dismutases, tyrosinase, dopamine-β-hydroxylase, ceruloplasmin, and peptidyl-α-monooxygenase 1. In order to ensure an adequate supply of Cu without toxic accumulation, Cu homeostatic mechanisms are tightly regulated. Either Cu deficiency or overabundance is associated with pathologic changes and disease, such as, Menkes disease or Wilson disease (WD), respectively 2,3. WD is a genetic disorder caused by mutations in the Cu-transporter ATP7B that causes abnormal deposition of Cu in the liver and the brain 4. WD has a spectrum of hepatic and neurological manifestations, along with a broad range of disease onsets 5. The mechanisms behind this variability remain poorly understood. Studies of WD patients identified over 600 ATP7B mutations 6 and revealed the diverse effects of mutations on the functional and cellular properties of the ATP7B protein. Efforts to link different phenotypic presentations of WD to specific ATP7B mutations have not produced strong correlations and have sometimes led to conflicting results 7. High prevalence of compound-heterozygous mutations further complicates the task of genotype-phenotype correlation 8 .

Estimating the clinical prevalence of Wilson’s disease in the UK

“… 18 , 25 Several reasons are likely to contribute to this difference including the presence of more than 600 pathogenic or likely pathogenic variants in ATP7B, 10 difference in the severity of certain variants e.g. truncating variants that lead to a significant decrease in the protein vs. missense changes, and also the combination of variants in either homozygous or compound-heterozygous states, 26 new classification criteria for assessing pathogenicity of variants, 27 variation in penetrance of certain variants, 11 the potential for epigenetic mechanisms of gene expression regulation causing clinical disease 28 and the likeliness of undiagnosed cases being missed in clinical prevalence studies. 29 …”

Investigation of the Wilson gene ATP7B transcriptional start site and the effect of core promoter alterations

“…Therefore, the two frequent core promoter polymorphisms are candidate genetic modifiers of Wilson disease phenotype. The marked variability of the Wilson disease clinical phenotype that is even observed in carriers of the same mutation(s) has not yet been explained; several modifying influences have been suggested to contribute: besides environmental factors, epigenetic and transgenetic effectors and protein interactions of different missense variants are in the list 40 , 43 , 44 .…”

Wilson’s disease: update on pathogenesis, biomarkers and treatments

“…In a similar study, Roy et al characterised the functional properties of further variants demonstrating differential effects on intracellular copper concentrations. 18 Intriguingly, they also found that co-expressing the G1061E variant, which is usually retained in the ER, with the A595T variant restored localisation of ATP7B to the TGN. ATP7B has previously been shown to form stable dimers in vitro and so genetic and functional studies may need to consider interactions between heterozygous variants in the future.…”