Dominant and Recessive Polycystic Kidney Disease: A Novel Molecular Diagnostics Approach Based on Next-Generation Sequencing

Polycystic Kidney Disease (PKD) is the most common genetic cause of kidney failure in children and adults and can be inherited as an autosomal dominant trait (ADPKD) or an autosomal recessive trait (ARPKD). ADPKD is the most common form, characterized by a late onset, caused by mutation in two causative gene PKD1 and PKD2; while ARPKD represents the rarest and most severe form, with early onset, caused by mutation in PKHD1 gene. The large size, the molecular complexity, the lack of mutational hotspot characterizing the causative genes and, in particular, the high homology of PKD1 with six pseudogenes make the molecular diagnostics challenging, time-consuming and expensive when using conventional sequencing methods. The aim of this work is to develop and validate a rapid and cost-saving genetic test for the molecular diagnosis of ADPKD and ARPKD based on Next-Generation Sequencing (NGS) using the Ion PGMTM platform. For PKD2 and PKHD1 screening the standard protocol for targeted-resequencing based on Ion Ampliseq technology was reliable; however for the PKD1 gene a strategy based on target-preselection using LR-PCR was applied in order to overcome the pseudogenes issue. The method validation, carried out on a cohort of patients with known molecular defects, showed 100% sensitivity and specificity. The prospective validation phase, carried out on different cohorts of patients with clinical suspect of ADPKD (n=125) or ARPKD (n=28), showed a detection rate of 90.4% and 85.7% respectively. Overall, 154 causative mutations were detected, 84 (54.5%) of which resulted previously undescribed, contributing the widening of the mutational spectrum of PKD. In conclusion, this NGS-based genetic approach is highly accurate and reliable for mutation analysis, achieves a high sensitivity, a faster turnaround time and lower cost in comparison to conventional Sanger sequencing. NGS would be an appropriate new standard for clinical genetic testing of PKD.