Challenges in molecular diagnosis of Wilson disease: viewpoint from the clinical laboratory

Correspondence to Dr Karen Tan, Laboratory Medicine, National University Hospital Singapore, Singapore 119704, Singapore; karen_ml_tan@nuhs.edu.sg Wilson disease Wilson disease (WD), a disorder of copper metabolism resulting in copper accumulation in the liver and other organs, is an inherited autosomal recessive genetic condition.1 Diagnosis is challenging and is based on clinical symptoms (neuropsychiatric or hepatic dysfunction), clinical signs (presence of Kayser-Fleischer (KF) rings) and laboratory test results (abnormal liver function tests, low serum caeruloplasmin and elevated urinary copper excretion).2 However, the biochemical findings may be inconclusive as concentrations of serum caeruloplasmin and urinary copper in patients with WD can overlap with those of healthy individuals.2 To try to overcome such issues, laboratory-derived indices have been suggested, such as non-caeruloplasmin bound copper, percentage non-caeruloplasmin bound copper, copper caeruloplasmin ratio and copper adjusted for caeruloplasmin, but the reference ranges and cut-offs differ between laboratories.3–6 In addition, scoring systems exist,7 some of which may incorporate genetic testing.8 Molecular diagnostic testing is performed to confirm or exclude the diagnosis in patients with symptoms and biochemical findings suggestive of WD.2 7 Sequencing the ATP7B gene Genetic testing of WD is performed by screening for loss-of-function variants in the ATP7B gene. More than 500 WD-causing variants in the ATP7B gene including single nucleotide variants, small deletions and insertions have been reported in literature and public databases such as ClinVar, Leiden Open Variation Database, Human Genetic Variation Database and the Wilson Disease Mutation Database.9 Although the gene harbours several population-specific variants, many pathogenic variants throughout the entire gene have been found to be rare and only familial-specific.10 Therefore, screening tests that contain only specific, most frequent variants, although not as labour-intensive and time-consuming as whole gene screening, may miss rare pathogenic variants. Recently, we developed an amplicon-based next-generation sequencing (NGS) assay to overcome the limitations of Sanger sequencing.11 We showed 100% concordance in detection of pathogenic variants with an additional advantage of the NGS assay being able to detect variants within the non-coding regions of the gene (deep intronic variants).11 Although the NGS assay reduced