Development and validation of a whole genome amplification long-range PCR sequencing method for ADPKD genotyping of...

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in two large genes, PKD1 and PKD2, but genetic testing is complicated by the large transcript sizes and the duplication of PKD1 exons 1-33 as six pseudogenes on chromosome 16. Long-range PCR (LR-PCR) represents the gold standard approach for PKD1 genetic analysis. However, a major issue with this approach is that it requires large quantities of genomic DNA (gDNA) material limiting its application primarily to DNA extracted from blood. In this study, we have developed a whole genome amplification (WGA)-based genotyping assay for PKD1 and PKD2, and examined whether this approach can be applied to biosamples with low DNA yield, including blood, buccal cells and urine. DNA samples were amplified by multiple displacement amplification (MDA) and a high-fidelity DNA polymerase followed by LR-PCR and exon-specific amplifications of PKD1 and PKD2 respectively, and Sanger sequencing. This method has generated large amounts of DNA with high average product length (>10 kb), which were uniformly amplified across all sequences assessed. When compared to the gDNA direct sequencing method for six ADPKD samples, a total of 89 variants were detected including all 86 variations previously reported, in addition to three new variations, including one pathogenic mutation not previously detected by the standard gDNA-based analysis. We have further applied WGA to ADPKD mutation analysis of low DNA-yield specimens, successfully detecting all 63 gene variations. Compared to the gDNA method the WGA-based assay had a sensitivity and specificity of 100%. In conclusion, WGA-based LR-PCR represents a major technical improvement for PKD genotyping from trace amounts of DNA.