Validation of autosomal dominant polycystic kidney disease variants using long-range pcr and third generation sequencing

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary disorder which is characterized by the formation and progressive enlargement of multiple bilateral kidney cysts with an estimated prevalence of 1 in 1000 to 1 in 2500 individuals. ADPKD is also associated with a high phenotypic variability even between affected members of the same family such that the age of onset and the severity of the disease differ significantly between affected individuals. With molecular testing, it is known that 80-85 % of the affected individuals possess a pathogenic variant in PKD1 and almost all the rest are characterized by a pathogenic variant in PKD2. This study involved 3 objectives. The first objective was to confirm the presence, location and pathogenicity of the PKD1 p.C508R variant. Long range (LR) PCR followed by long-read 3rd generation amplicon sequencing were performed which successfully determined that the p.C508R variant is present on PKD1P3 in both ADPKD patients and in non-PKD individuals and thus should be reclassified as a likely benign polymorphism. The second objective was to confirm previously identified variants which were shortlisted by 2nd generation high-throughput sequencing (HTS) in the Malta NGS Project. LR-PCR and/or nested PCR followed by Sanger sequencing were used, which successfully confirmed 7 out of 11 potential causative variants. These variants were re-evaluated to determine pathogenicity. The third and final objective of this project was to carry out further testing on the patients from the 2 pedigrees (P12 and P13) in which no potential causative variant had been identified. PacBio whole genome sequencing (WGS) was performed on a single Sequel IIe SMRT flowcell however, unsatisfactory results were obtained, with insufficient coverage across several parts of the genome and almost no coverage across PKD1. Additional sequencing runs on other Sequel IIe SMRT flowcells per genome or higher throughput HiFi systems such as Revio or Vega are necessary. This study highlights the differences between short-read and long-read sequencing techniques and encourages the integration of LR-PCR with long-read 3rd generation sequencing especially for complex genomic regions or genes with homologous pseudogenes such as PKD1.