Molecular diagnosis of autosomal dominant polycystic kidney disease using next-generation sequencing.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2. However, genetic analysis is complicated by six PKD1 pseudogenes, large gene sizes, and allelic heterogeneity. We developed a new clinical assay for PKD gene analysis using paired-end next-generation sequencing (NGS) by multiplexing individually bar-coded long-range PCR libraries and analyzing them in one Illumina MiSeq flow cell. The data analysis pipeline has been optimized and automated with Unix shell scripts to accommodate variant calls. This approach was validated using a cohort of 25 patients with ADPKD previously analyzed by Sanger sequencing. A total of 250 genetic variants were identified by NGS, spanning the entire exonic and adjacent intronic regions of PKD1 and PKD2, including all 16 pathogenic mutations. In addition, we identified three novel mutations in a mutation-negative cohort of 24 patients with ADPKD previously analyzed by Sanger sequencing. This NGS method achieved sensitivity of 99.2% (95% CI, 96.8%-99.9%) and specificity of 99.9% (95% CI, 99.7%-100.0%), with cost and turnaround time reduced by as much as 70%. Prospective NGS analysis of 25 patients with ADPKD demonstrated a detection rate comparable with Sanger standards. In conclusion, the NGS method was superior to Sanger sequencing for detecting PKD gene mutations, achieving high sensitivity and improved gene coverage. These characteristics suggest that NGS would be an appropriate new standard for clinical genetic testing of ADPKD.