An Efficient and Comprehensive Strategy for Genetic Diagnostics of Polycystic Kidney Disease
Fig 5
Comprehensive and complete testing strategy for ADPKD diagnostics.
Sequence capture based NGS for variant detection in PKD1 and all other targeted ciliopathy genes at standard filter criteria is the central and first step in the analysis which includes validation of putative mutations by conventional techniques (LR-PCR-Sanger, MLPA). By this, in more than 98% of our cohort the disease-causing mutation could already be identified. Further optional steps to minimize the risk of false negative results include deeper bioinformatic analysis of acquired NGS data and restricted use of conventional methods. Bioinformatic re-analysis by lowering the detection threshold (> 8% alternative reads) in mutation-negative samples might provide candidate sites in PKD1 where concurrent mapping of reads between the genuine gene and homologous regions impairs proper variant detection. Candidate sites are validated by conventional Sanger sequencing. If still no convincing mutation is present, restricted sequencing by LR-PCR-Sanger of insufficiently covered regions (exon 1) as well as those exons with empirically evaluated critical regions bearing mapping difficulties (in our study exons 5, 11, 17, 21, 26) is the next option and would have generated the correct result for patient 21 of our study. In parallel, MLPA analysis of PKD1 can be used to rule out false-negative CNV results. An optional final step might be ultra-deep sequencing with very high coverage of the PKD1 and PKD2 coding sequence (S9 Table) especially in those mutation-negative patients with a clear ADPKD phenotype and negative family history (*) to allow for detection of low-level mosaicism.