Functional Characterization of Novel ATP7B Variants for Diagnosis of Wilson Disease

Abstract

Background: Diagnosis of rare Wilson disease (WD) in pediatric patients is difficult, in particular when hepatic manifestation is absent. Genetic analysis of ATP7B represents the single major determinant of the diagnostic scoring system in WD children having mild symptoms.Objectives: To assess the impact of molecularly expressed ATP7B gene products in order to assist diagnosis of Wilson disease in pediatric patients having a novel mutation and subtle neuropsychiatric disease.Methods: The medical history, clinical presentation, biochemical parameters, and the genetic analysis of ATP7B were determined. Due to ambiguous clinical and biochemical findings and identification of a novel compound ATP7B mutation with unknown disease-causing status, a molecular analysis of the ATP7B gene products in a previously well characterized cell model was performed.Results: The ATP7B variants were transgenically expressed and the respective gene function molecularly characterized. Despite normal mRNA expression, low ATP7B protein expression of the mutants p.L168P and p.S1423N was observed (34.3 ± 8% and 66.0 ± 8%, respectively). Copper exposure did not result in decreased viability of transgenic cells as compared to wild type. Intracellular copper accumulation was reduced (≤47.9 ± 8%) and intracellular protein trafficking was impaired.Conclusion: Our report suggests that functional characterization of novel ATP7B mutants can assist diagnosis; however mild functional impairments of ATP7B variants may hamper the value of such approaches.

Polarized trafficking and copper transport activity of ATP7B: A mutational approach to establish genotype–phenotype correlation in Wilson disease

“…A third mutation located at end of C‐terminal selected for functional characterization is ATP7B‐Ser1423Asn. This disease‐causing variant was reported as compound heterozygous with Leu168Pro in a child with mild neuropsychiatric symptoms without hepatic manifestation (Guttmann et al, 2018). The mutation Ser1423Asn was modeled using PyMOL and the predicted changes in local protein interaction is shown in Figure 6i.…”

“…We selected four nonsynonymous point mutations spanning the N-terminal domain, Gly85Val, Leu168Pro, Leu492Ser, and Gly591Asp (de Bie et al, 2007;Guttmann et al, 2018;Hamza et al, 1999;Huster et al, 2012;Van Den Berghe et al, 2009;Vanderwerf et al, 2001) for characterization on the basis of copper transport in the biosynthetic pathway and copper-induced polarized trafficking.…”

ATP7B variant penetrance explains differences between genetic and clinical prevalence estimates for Wilson disease

“…The functional characterisation of these variants would be useful for confirming that they are indeed low penetrant or non-causative. However, recent research suggests that current cell-based systems may not be accurate at measuring mild impairments in ATP7B function (Guttmann et al 2018).…”

Complex ATP7B mutation patterns in Wilson disease and evaluation of a yeast model for functional analysis of variants

“…Although in recent years several outstanding software programs (Adzhubei et al, ; Ng & Henikoff, ; Schwarz, Cooper, Schuelke, & Seelow, ) have become available for predicting the functional effect of mutations, direct experimental evidence is still considered the gold standard when researching variant function (Sunyaev, ). Several cell lines (e.g., CHO cell line, HepG2 cell line) and animal models (e.g., toxic milk mouse) have been used to investigate the function of ATP7B variants (Chandhok et al, ; Guttmann et al, ; Luoma, Deeb, Macintyre, & Cox, ; Lv et al, ; Parisi et al, ). The present study developed and evaluated a yeast model, which is more simple and convenient and may provide another feasible method for rapid evaluation of functional consequence of the individual ATP7B variants (J. R. Forbes & Cox, ).…”