Molecular Diagnosis of Wilson's Disease Using Next Generation Sequencing Platform

Background and Aim: In patients with hepatic Wilson's disease (WD), confirmatory diagnosis is often elusive. Currently available genetic tools for WD diagnosis are time consuming and expensive. Next generation sequencing (NGS) offers high throughput sample processing at a relatively lower cost. Therefore, we aim to investigate the feasibility of the NGS-based approach to sequence ATP7B gene in patients with hepatic WD. Method: Patients diagnosed with hepatic WD (low serum Ceruloplasmin, high urinary copper excretion and presence of Kayser-Fleischer ring on eye examination) were included after informed consent and blood was collected for DNA extraction. All 21 exons and flanking 5′ and 3′ UTR regions of ATP7B gene were amplified in four multiplex PCRs. Libraries were generated from the pooled amplicons and subjected to sequencing on Ion-Torrent platform. Data analysis was performed using IonTorrent Suite™ Software v.5.0 and genetic variants were then identified using Torrent Suite Variant Caller pipeline v.5.0. Bioinformatic tools (SIFT, Polyphen, Mutation taster) were used to predict the effect of the observed variants. Sanger sequencing was subsequently performed in the corresponding exon to confirm the identified mutations. Results: Thirteen patients with unequivocal hepatic WD were included for the study (Table 1). Coverage analysis showed >99% of the target was sequenced with a mean coverage of 20x. Overall we identified 9 mutations (missense: 7 nonsense: 1, deletion: 1) in 11 patients (84.6%). Four patients were compound heterozygous, six were homozygous and one was heterozygous for mutations (Table 1). Among the identified mutations, three were novel and all others have been previously reported. We failed to identify point mutations in two patients. Sanger sequencing results were 100% concordant with NGS data.Table 1Demographic Characteristics and Mutation Details in Patients With Wilson's Disease.UPN NoAgeSexLocationATP7B mutationAminoacid changeExonSIFTPolyphenMTWD116MJHc[3895C>T]c.3182G>A]p.L1299FaNovel mutation.G1061E18,4DamagingProbably damagingDisease causingWD224FTNc.[813C>A]p.C271x2––Disease-causingWD326FTNc.[3971A>G]p.N1324S19ToleratedProbably damagingDisease-causingWD424FTNc[3971A>G]p.N1324S19ToleratedProbably damagingDisease-causingWD523MWBc[3182G>A]c[2131G>T]p.G1061EG711W14,8DamagingProbably damagingDisease-causingWD616FAPc[2131G>T]p.G711W8DamagingProbably damagingDisease-causingWD79MWBc[3662G>T]c[3053C>T]p.G1221VaNovel mutation.p.A1018V17,13Tolerated DamagingProbably damagingDisease-causingWD836MBGDNEGATIVE–––––WD927MWBNEGATIVE–––––WD1017MWBc.[3567delT]p.C1189fsaNovel mutation.17––Disease-causingWD1115MUPC[813C>A[=]p.C271X[=]2––Disease-causingWD1222MWBc.[3182G>A]p.G1061E14DamagingProbably damagingDisease-causingWD1317MWBc.[3802G>A]c.[813C>A]p.G1268R, p.C271x18,2DamagingProbably damagingDisease-causingGeographic Location: JH-Jharkhand, WB-West Bengal, TN-Tamilnadu, UP-Uttar Pradesh, AP-Andhra Pradesh, BGD-Bangladesh.a Novel mutation. Open table in a new tab Geographic Location: JH-Jharkhand, WB-West Bengal, TN-Tamilnadu, UP-Uttar Pradesh, AP-Andhra Pradesh, BGD-Bangladesh. Conclusion: In this pilot study, we report the use of NGS platform for ATP7B gene mutation analysis for molecular diagnosis of WD. Our data suggests NGS is a reliable method for the diagnosis of Wilson's disease. The authors have none to declare.