Optimizing Next Generation Sequencing for Genetic Diagnosis in Autosomal Dominant Polycystic Kidney Disease.

PURPOSE Autosomal dominant polycystic kidney disease (ADPKD) affects 1:1000, causing 5-10% of kidney failure. The primary disease gene, PKD1, has six pseudogenes with 97-99% homology, a >12kb transcript, high GC content, and polypyrimidine tracts. While long-range PCR with Sanger sequencing has been the "gold-standard", next-generation sequencing (NGS) is increasingly used.

METHODS We performed exome sequencing (ES) on 203 ADPKD patients in 171 families from the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease (CRISP) cohort, with prior "gold standard" results: n=157 PKD1, n=27 PKD2, n=19 with no pathogenic variant detected. Clinical geneticists, blinded to genotype, reviewed ES data. We assessed effects of pipeline modifications, exome capture reagents, pseudogene alignment, and deeper ES or genome sequencing (GS) for unsolved cases.

RESULTS Optimized ES identified 95.5% of defined PKD1 pathogenic variants, all PKD2 variants, and at least 9 of 19 "unsolved" cases. Standard pipelines on research-grade ES missed at least 22 PKD1 variants due to GATK HardFiltering or alternative locus annotation. Higher-depth ES achieved 100% PKD1 variant detection. GS identified a balanced translocation t(1;16)(q31.1;p13.3).

CONCLUSION NGS matches "gold-standard" sensitivity, and considers additional disease genes. ES plus GS solve 96% of well-phenotyped ADPKD. We outline practical considerations for NGS on PKD1.