Targeted next-generation sequencing of the ATP7B gene for molecular diagnosis of Wilson disease

Objectives: In recent years, next-generation sequencing (NGS) technologies, which enable high throughput sample processing at relatively lower costs, are adopted in both research and clinical settings. A multiplex PCR-based NGS assay to identify mutations in the ATP7B gene for routine molecular diagnosis of Wilson disease was evaluated in comparison with the gold standard direct Sanger sequencing.

Design and methods: Five multiplex PCRs to amplify the partial promoter, 5' untranslated and the entire coding regions of the ATP7B gene were designed. Indexed paired-end libraries were generated from the pooled amplicons using Nextera XT DNA Sample Preparation Kit and subjected to NGS on the MiSeq platform. DNA from the peripheral blood of 12 patients with Wilson disease, 2 B-lymphocyte cell lines and 3 external quality assurance samples were sequenced by the MiSeq and Sanger sequencing.

Results: Complete coverage was achieved across the targeted bases without any drop-out sequences. The observed read depth in a single run with 20 samples was >100X. Comparison of the NGS results against Sanger sequencing data on a panel of clinical specimens, cell lines and European Molecular Genetics Quality Networks (EMQN) quality assurance samples showed 100% concordance in identifying pathogenic mutations.

Conclusion: With the capability of generating relatively higher throughput in a short time period, the NGS assay is a viable alternative to Sanger sequencing for detecting ATP7B mutations causally linked to Wilson disease in the clinical diagnostic laboratory.