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

PLOS ONE, Dec 2019

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.

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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

November 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 Moritoshi Kinoshita 0 1 2 3 Eiji Higashihara 0 1 3 Haruna Kawano 0 1 3 Ryo Higashiyama 0 1 2 3 Daisuke Koga 0 1 2 3 Takafumi Fukui 0 1 3 Nobuhisa Gondo 0 1 3 Takehiko Oka 0 1 3 Kozo Kawahara 0 1 3 Krisztina Rigo 0 1 3 4 Tim Hague 0 1 3 4 Kiyonori Katsuragi 0 1 2 3 Kimiyoshi Sudo 0 1 2 3 Masahiko Takeshi 0 1 3 Shigeo Horie 0 1 3 Kikuo Nutahara 0 1 3 0 Current address: Shinanozaka Clinic, Medical Corporation Shinanokai , Tokyo , Japan 1 Funding: This study was funded by Otsuka Pharmaceutical Co., Ltd. The funder only provided 2 Diagnostic Division, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan, 2 Department of ADPKD Research, School of Medicine, Kyorin University , Tokyo , Japan , 3 Department of Urology, School of Medicine, Kyorin University , Tokyo , Japan , 4 Department of Urology, Graduate School of Medicine, Juntendo University , Tokyo , Japan , 5 FALCO biosystems Ltd., Kyoto, Japan, 6 World Fusion Co., Ltd. , Tokyo , Japan 3 Editor: Hao Deng, Central South University , CHINA 4 Omixon Ltd., Budapest, Hungary, 8 Samon-cho Clinic, Medical Corporation Shinanokai , Tokyo , Japan 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- - OPEN ACCESS financial support in the form of salaries for MK, RH, DK, KK, and KS and did not have any additional role in the study design, data collection and analysis, decision to publish, or manuscript preparation. Similarly, FALCO Biosystems, World Fusion, Omixon, and Samon-cho clinic provided support in the form of salaries for TF, NG, TO, KK, KR, TH, and MT but did not have any additional role in the study design, data collection and analysis, decision to publish, or manuscript preparation. EH, SH, and KN have received research funding from Otsuka Pharmaceutical. HK and MT have no conflict of interest directly relevant to the content of this article. Competing Interests: 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. sensitivity NGS-based system and successfully employed it to identify pathogenic mutations in PKD1 and PKD2 in Japanese patients with ADPKD. Introduction Polycystic kidney disease (PKD) is one of the most common inherited disorders affecting the renal tubules. It comprises autosomal dominant PKD (ADPKD) and autosomal recessive PKD [1±3]. Aro (...truncated)


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Moritoshi Kinoshita, Eiji Higashihara, Haruna Kawano, Ryo Higashiyama, Daisuke Koga, Takafumi Fukui, Nobuhisa Gondo, Takehiko Oka, Kozo Kawahara, Krisztina Rigo, Tim Hague, Kiyonori Katsuragi, Kimiyoshi Sudo, Masahiko Takeshi, Shigeo Horie, Kikuo Nutahara. 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, PLOS ONE, 2016, Volume 11, Issue 11, DOI: 10.1371/journal.pone.0166288