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.

A potentially crucial role of the PKD1 C-terminal tail in renal prognosis

“…Sixty pathogenic mutations were newly described in 65 pedigrees, of which 57 were PKD1 , and 8 PKD2 mutations (Supplemental Table 1). These novel mutations have been described elsewhere [ 23 ].…”

“…The identification of pathogenic mutations by using a combination of NGS, LR-PCR, MLPA, and an analysis software package provided an overall PKD mutation detection rate of 93.1% (94/101) [ 23 ]. This detection rate was similar (84.5–93.8%) to that reported in recent large-scale PKD studies [ 5 , 7 , 8 , 10 , 12 ].…”

“…The detection and pathogenicity prediction of missense mutations were performed using next-generation sequencing (NGS), and long-range polymerase chain reaction (LR-PCR); data analysis was performed using software packages, as previously described [ 23 ]. Multiplex ligation-dependent probe amplification (MLPA) was used to analyze large genomic rearrangements, if pathogenic mutations were not detected by NGS.…”

Whole exome sequencing reveals a stop-gain mutation of PKD2 in an autosomal dominant polycystic kidney disease family complicated with aortic dissection

“…Conventional Sanger sequencing is hampered by coding sequence rich in GC nucleotides and six highly homogenous pseudogenes of PKD1 [ 17 ]. In recent years, next-generation sequencing has evolved as a robust method to detect mutations in ADPKD patients [ 18 ]. With an elaborate design of the capture strategy and improved sequencing depth, WES can achieve an excellent diagnostic effect.…”

Co‑segregation of candidate polymorphism rs201204878 of the PKD1 gene in a large Iranian family with autosomal dominant polycystic disease

“…To validate the variants predicted to be associated with ADPKD by computational analyses, the region of interest surrounding the variant was amplified by PCR using Taq DNA Polymerase Master Mix (Ampliqon A/S, Odense, Denmark). The variants were sequenced using Sanger sequencing methods and appropriate internal primers reported previously (Table I) (29).…”