OP-2 WILSON DISEASE (WD) DIAGNOSIS WITH NEXT-GENERATION SEQUENCING (NGS) IN CLINICAL PRACTICE: A PILOT STUDY

Abstract

Conflict of interest: Yes, Ultragenyx has provided 25 kits for copper panel genotyping by NGS at Mendelics laboratory Introduction and Objectives: Wilson disease (WD) is an autosomal recessive disorder caused by a defect in the ATP7B protein, leading to copper overload. ATP7B genotyping has been performed by Sanger method, but NGS techniques have recently become available. Our aim was to analyze the impact of Sanger direct sequencing and NGS in the diagnosis of WD. Patients / Materials and Methods: A series of 287 WD patients included 160 individuals who provided DNA after informed consent. All patients met ≥4 points of the European WD scoring system. DNA for Sanger sequencing was extracted from peripheral leukocytes and for NGS from oral cells using a buccal swab. ATP7B mutations were identified in 135 patients by Sanger sequencing only, in 17 by NGS and in 8 by both methods. Sanger sequencing was performed as previously published (Deguti et al, 2004). In targeted NGS using a ''copper panel'' (Laboratório Mendelics), libraries were prepared with Illumina DNA with enrichment, target regions were captured using specific probes (Twist Biosciences v.3) for all exons/intronic regions, and variants and indels were identified using GATL v4 program and CNVs using ExomeDepth. Results and Discussion: Of the 320 alleles analyzed, the most frequent mutations were p.A1135fs (26.7%), L708P (15.8%), p.H1069Q (5.3%) and p.M645R (3.1%).The remaining 49.1% of alleles had 38 distinct disease-causing mutations, each with a frequency of <3.0%. Sanger sequencing detected a mutation in 264/286 alleles (92.3%), while the NGS method detected a mutation in 50/50 (100%). The table shows the results of the 8 patients genotyped with both methods. Conclusions: The Sanger direct sequencing methods were laborious and detected mutations in 92.3% of ATP7B alleles, while NGS techniques yielded 100%, impacting the WD score by up to 4 points. This can be crucial in difficult and potentially severe cases.