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.
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.
Diagnostic Division, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan.
Moritoshi
http://orcid.org/0000-0002-8944-9052
M
Kinoshita
Department of ADPKD Research, School of Medicine, Kyorin University, Tokyo, Japan.|Department of Urology, School of Medicine, Kyorin University, Tokyo, Japan.
Eiji
E
Higashihara
Department of Urology, Graduate School of Medicine, Juntendo University, Tokyo, Japan.
Haruna
H
Kawano
Diagnostic Division, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan.
Ryo
R
Higashiyama
Diagnostic Division, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan.
Daisuke
D
Koga
FALCO biosystems Ltd., Kyoto, Japan.
Takafumi
T
Fukui
FALCO biosystems Ltd., Kyoto, Japan.
Nobuhisa
N
Gondo
World Fusion Co., Ltd., Tokyo, Japan.
Takehiko
T
Oka
World Fusion Co., Ltd., Tokyo, Japan.
Kozo
K
Kawahara
Omixon Ltd., Budapest, Hungary.
Krisztina
K
Rigo
Omixon Ltd., Budapest, Hungary.
Tim
T
Hague
Diagnostic Division, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan.
Kiyonori
K
Katsuragi
Diagnostic Division, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan.
Kimiyoshi
K
Sudo
Samon-cho Clinic, Medical Corporation Shinanokai, Tokyo, Japan.
Masahiko
M
Takeshi
Department of Urology, Graduate School of Medicine, Juntendo University, Tokyo, Japan.
Shigeo
S
Horie
Department of Urology, School of Medicine, Kyorin University, Tokyo, Japan.
Kikuo
K
Nutahara
D000328:Adult; D018389:Codon, Nonsense; D004252:DNA Mutational Analysis / Q000379:methods; D016368:Frameshift Mutation; D015321:Gene Rearrangement; D005820:Genetic Testing / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D009154:Mutation; D020125:Mutation, Missense; D016891:Polycystic Kidney, Autosomal Dominant / Q000175:diagnosis / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D050396:TRPP Cation Channels / Q000235:genetics*
D018389:Codon, Nonsense; D022821:RNA Splice Sites; D050396:TRPP Cation Channels