Technical Evaluation: Identification of Pathogenic Mutations in PKD1 and PKD2 in Patients with Autosomal Dominant Polycystic Kidney Disease by Next-Generation Sequencing and Use of a Comprehensive New Classification System

Abstract

Genetic testing of PKD1 and PKD2 is expected to play an increasingly important role in determining allelic influences in autosomal dominant polycystic kidney disease (ADPKD) in the near future. However, to date, genetic testing is not commonly employed because it is expensive, complicated because of genetic heterogeneity, and does not easily identify pathogenic variants. In this study, we developed a genetic testing system based on next-generation sequencing (NGS), long-range polymerase chain reaction, and a new software package. The new software package integrated seven databases and provided access to five cloud-based computing systems. The database integrated 241 polymorphic nonpathogenic variants detected in 140 healthy Japanese volunteers aged >35 years, who were confirmed by ultrasonography as having no cysts in either kidney. Using this system, we identified 60 novel and 30 known pathogenic mutations in 101 Japanese patients with ADPKD, with an overall detection rate of 89.1% (90/101) [95% confidence interval (CI), 83.0%-95.2%]. The sensitivity of the system increased to 93.1% (94/101) (95% CI, 88.1%-98.0%) when combined with multiplex ligation-dependent probe amplification analysis, making it sufficient for use in a clinical setting. In 82 (87.2%) of the patients, pathogenic mutations were detected in PKD1 (95% CI, 79.0%-92.5%), whereas in 12 (12.8%) patients pathogenic mutations were detected in PKD2 (95% CI, 7.5%-21.0%); this is consistent with previously reported findings. In addition, we were able to reconfirm our pathogenic mutation identification results using Sanger sequencing. In conclusion, we developed a high-sensitivity NGS-based system and successfully employed it to identify pathogenic mutations in PKD1 and PKD2 in Japanese patients with ADPKD.

Conflict of interest statement

MK, RH, DK, KK, and KS are employees of Otsuka Pharmaceutical. TF and NG are employees of FALCO Biosystems. TO and KK are employees of World Fusion. KR and TH are employees of Omixon. MT is an employee of Samon-cho Clinic. There are no products in development or marketed products to declare. Otsuka Pharmaceutical has applied for patents of the functional capability of our genetic testing system; however, this does not alter our adherence to PLOS ONE policies on sharing data and materials as detailed online in the guide for authors. We have released all data, sequences of LR-PCR primers, reaction conditions, and the algorithm of analysis software. All relevant data, materials, and algorithm are within our article and its Supporting Information files. Although the algorithm is open source, the analysis software package is not a completely open source because it includes commercial software of OMIXON TARGET, which is nextgeneration-sequencing analysis software.

Figures

Fig 1. Schematic representation of the pipeline for identification of pathogenic mutations.

Pathogenic mutations were identified on the basis of seven annotated databases: PKDB, dbSNP, SnpEff, UCSC (for conservation probability), PubMed (article searches), Pseudogene.org, and a database of polymorphic variants in 140 healthy Japanese individuals. Novel missense mutations and potential splicing mutations were evaluated for pathogenicity using public cloud-based computing (SIFT, PolyPhen-2, Align-GVGD, MutationTaster, and NNSplice). PKDB; PKD mutation database, NCBI; National Center for Biotechnology Information, dbSNP; Single Nucleotide Polymorphism database, UCSC; University of California, Santa Cruz, SIFT; Sorting Intolerant from Tolerant, GVGD; Grantham Variation Grantham Deviation.

Fig 2. Schematic diagram of workflow in healthy volunteers.

140 healthy Japanese volunteers were recruited and they were age 35 or older and were confirmed as having no renal cysts by ultrasonography. Four nonsynonymous variants predicted to be likely pathogenic mutations in six subjects and other 134 subjects were predicted in likely neutral variants by the scoring protocol using cloud-based computing [31]. The specificity of the system was estimated to be 95.7%.

Fig 3. Schematic diagram of workflow in the patients with ADPKD.

52 definitely pathogenic mutations, 22 highly likely pathogenic mutations and 20 likely pathogenic mutations were identified in 101 Japanese patients with ADPKD. The sensitivity of the system was estimated to be 93.1% in combined with multiplex ligation-dependent probe amplification analysis (MLPA).