Mutation analyses by next-generation sequencing and multiplex ligation-dependent probe amplification in Japanese autosomal dominant polycystic kidney disease patients

  • Toshio MochizukiEmail author
  • Atsuko Teraoka
  • Hiroyuki Akagawa
  • Shiho Makabe
  • Taro Akihisa
  • Masayo Sato
  • Hiroshi Kataoka
  • Michihiro Mitobe
  • Toru Furukawa
  • Ken Tsuchiya
  • Kosaku Nitta
Original article



Autosomal dominant polycystic kidney disease (ADPKD), one of the most common hereditary kidney diseases, causes gradual growth of cysts in the kidneys, leading to renal failure. Owing to the advanced technology of next-generation sequencing (NGS), genetic analyses of the causative genes PKD1 and PKD2 have been improved.


We performed genetic analyses of 111 Japanese ADPKD patients using hybridization-based NGS and long-range (LR)-PCR-based NGS. Additionally, genetic analyses in exon 1 of PKD1 using Sanger sequencing because of an extremely low coverage of NGS and those using multiplex ligation-dependent probe amplification (MLPA) were performed.


The detection rate using NGS for 111 patients was 86.5%. One mutation in exon 1 of PKD1 and five deletions detected by MLPA were identified. When combined, the total detection rate was 91.9%.


Although NGS is useful, we propose the addition of Sanger sequencing for exon 1 of PKD1 and MLPA as indispensable for identifying mutations not detected by NGS.


Polycystic kidney disease ADPKD PKD1 PKD2 Next-generation sequencing MLPA 



We express our sincere appreciation to all the patients, collaborating physicians, and other medical staff for their important contributions to the study. This study was supported in part by JSPS KAKENHI Grant Number JP 15K09279 and by a Grant-in-Aid for Intractable Renal Diseases Research, Research on rare and intractable diseases, Health and Labour Sciences Research Grants from the Ministry of Health, Labour and Welfare of Japan.

Compliance with ethical standards

Conflict of interest

Toshio Mochizuki and Ken Tsuchiya received Travel fees and honoraria for lectures from Otsuka Pharmaceutical Co. Toshio Mochizuki and Hiroshi Kataoka belong to an endowed department sponsored by Otsuka Pharmaceutical Co, Chugai Pharmaceutical Co, Kyowa Hakko Kirin Co, MSD Co, and JMS Co.

Ethical approval

All procedures performed in this study were approved by the research ethics committee of Tokyo Women’s Medical University (No. 196B) in accordance with the 1964 Helsinki Declaration and its later amendments, or with comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

10157_2019_1736_MOESM1_ESM.pdf (96 kb)
Supplementary material 1 (PDF 95 kb)
10157_2019_1736_MOESM2_ESM.pdf (339 kb)
Supplementary material 2 (PDF 338 kb)


  1. 1.
    Mochizuki T, Tsuchiya K, Nitta K. Autosomal dominant polycystic kidney disease: recent advances in pathogenesis and potential therapies. Clin Exp Nephrol. 2013;17:317–26.CrossRefGoogle Scholar
  2. 2.
    Cornec-Le Gall E, Audrezet MP, Chen JM, Hourmant M, Morin MP, Perrichot R, Charasse C, Whebe B, Renaudineau E, Jousset P, Guillodo MP, Grall-Jezequel A, Saliou P, Ferec C, Le Meur Y. Type of PKD1 mutation influences renal outcome in ADPKD. J Am Soc Nephrol JASN. 2013;24:1006–13.CrossRefGoogle Scholar
  3. 3.
    Torres VE, Chapman AB, Devuyst O, Gansevoort RT, Grantham JJ, Higashihara E, Perrone RD, Krasa HB, Ouyang J, Czerwiec FS, Investigators TT. Tolvaptan in patients with autosomal dominant polycystic kidney disease. N Engl J Med. 2012;367:2407–18.CrossRefGoogle Scholar
  4. 4.
    Torres VE, Chapman AB, Devuyst O, Gansevoort RT, Perrone RD, Dandurand A, Ouyang J, Czerwiec FS, Blais JD, Investigators TT. Multicenter, open-label, extension trial to evaluate the long-term efficacy and safety of early versus delayed treatment with tolvaptan in autosomal dominant polycystic kidney disease: the TEMPO 4:4 Trial. Nephrol Dialysis Transplant. 2017. Scholar
  5. 5.
    Phakdeekitcharoen B, Watnick TJ, Germino GG. Mutation analysis of the entire replicated portion of PKD1 using genomic DNA samples. J Am Soc Nephrol JASN. 2001;12:955–63.Google Scholar
  6. 6.
    Watnick TJ, Piontek KB, Cordal TM, Weber H, Gandolph MA, Qian F, Lens XM, Neumann HP, Germino GG. An unusual pattern of mutation in the duplicated portion of PKD1 is revealed by use of a novel strategy for mutation detection. Hum Mol Genet. 1997;6:1473–81.CrossRefGoogle Scholar
  7. 7.
    Watnick T, Phakdeekitcharoen B, Johnson A, Gandolph M, Wang M, Briefel G, Klinger KW, Kimberling W, Gabow P, Germino GG. Mutation detection of PKD1 identifies a novel mutation common to three families with aneurysms and/or very-early-onset disease. Am J Hum Genet. 1999;65:1561–71.CrossRefGoogle Scholar
  8. 8.
    Rossetti S, Hopp K, Sikkink RA, Sundsbak JL, Lee YK, Kubly V, Eckloff BW, Ward CJ, Winearls CG, Torres VE, Harris PC. Identification of gene mutations in autosomal dominant polycystic kidney disease through targeted resequencing. J Am Soc Nephrol JASN. 2012;23:915–33.CrossRefGoogle Scholar
  9. 9.
    Qi XP, Du ZF, Ma JM, Chen XL, Zhang Q, Fei J, Wei XM, Chen D, Ke HP, Liu XZ, Li F, Chen ZG, Su Z, Jin HY, Liu WT, Zhao Y, Jiang HL, Lan ZZ, Li PF, Fang MY, Dong W, Zhang XN. Genetic diagnosis of autosomal dominant polycystic kidney disease by targeted capture and next-generation sequencing: utility and limitations. Gene. 2013;516:93–100.CrossRefGoogle Scholar
  10. 10.
    Kinoshita M, Higashihara E, Kawano H, Higashiyama R, Koga D, Fukui T, Gondo N, Oka T, Kawahara K, Rigo K, Hague T, Katsuragi K, Sudo K, Takeshi M, Horie S, Nutahara K. 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;11:e0166288.CrossRefGoogle Scholar
  11. 11.
    Kozlowski P, Bissler J, Pei Y, Kwiatkowski DJ. Analysis of PKD1 for genomic deletion by multiplex ligation-dependent probe assay: absence of hot spots. Genomics. 2008;91:203–8.CrossRefGoogle Scholar
  12. 12.
    Yu G, Qian X, Wu Y, Li X, Chen J, Xu J, Qi J. Analysis of gene mutations in PKD1/PKD2 by multiplex ligation-dependent probe amplification: some new findings. Ren Fail. 2015;37:366–71.CrossRefGoogle Scholar
  13. 13.
    Pei Y, Obaji J, Dupuis A, Paterson AD, Magistroni R, Dicks E, Parfrey P, Cramer B, Coto E, Torra R, San Millan JL, Gibson R, Breuning M, Peters D, Ravine D. Unified criteria for ultrasonographic diagnosis of ADPKD. J Am Soc Nephrol JASN. 2009;20:205–12.CrossRefGoogle Scholar
  14. 14.
    Irazabal MV, Rangel LJ, Bergstralh EJ, Osborn SL, Harmon AJ, Sundsbak JL, Bae KT, Chapman AB, Grantham JJ, Mrug M, Hogan MC, El-Zoghby ZM, Harris PC, Erickson BJ, King BF, Torres VE. Imaging classification of autosomal dominant polycystic kidney disease: a simple model for selecting patients for clinical trials. J Am Soc Nephrol JASN. 2015;26:160–72.CrossRefGoogle Scholar
  15. 15.
    Tsuchiya K, Komeda M, Takahashi M, Yamashita N, Cigira M, Suzuki T, Suzuki K, Nihei H, Mochizuki T. Mutational analysis within the 3′ region of the PKD1 gene in Japanese families. Mutat Res. 2001;458:77–84.Google Scholar
  16. 16.
    Hayashi T, Mochizuki T, Reynolds DM, Wu G, Cai Y, Somlo S. Characterization of the exon structure of the polycystic kidney disease 2 gene (PKD2). Genomics. 1997;44:131–6.CrossRefGoogle Scholar
  17. 17.
    Audrezet MP, Cornec-Le Gall E, Chen JM, Redon S, Quere I, Creff J, Benech C, Maestri S, Le Meur Y, Ferec C. Autosomal dominant polycystic kidney disease: comprehensive mutation analysis of PKD1 and PKD2 in 700 unrelated patients. Hum Mutat. 2012;33:1239–50.CrossRefGoogle Scholar
  18. 18.
    Grantham R. Amino acid difference formula to help explain protein evolution. Science. 1974;185:862–4.CrossRefGoogle Scholar
  19. 19.
    Tavtigian SV, Greenblatt MS, Lesueur F, Byrnes GB, Group IUGVW. In silico analysis of missense substitutions using sequence-alignment based methods. Hum Mutat. 2008;29:1327–36.CrossRefGoogle Scholar
  20. 20.
    Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7:248–9.CrossRefGoogle Scholar
  21. 21.
    Ng PC, Henikoff S. Accounting for human polymorphisms predicted to affect protein function. Genome Res. 2002;12:436–46.CrossRefGoogle Scholar
  22. 22.
    Ng PC, Henikoff S. Predicting deleterious amino acid substitutions. Genome Res. 2001;11:863–74.CrossRefGoogle Scholar
  23. 23.
    Schwarz JM, Rodelsperger C, Schuelke M, Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods. 2010;7:575–6.CrossRefGoogle Scholar
  24. 24.
    Genomes Project C, Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, Handsaker RE, Kang HM, Marth GT, McVean GA. An integrated map of genetic variation from 1,092 human genomes. Nature. 2012;491:56–65.CrossRefGoogle Scholar
  25. 25.
    Higasa K, Miyake N, Yoshimura J, Okamura K, Niihori T, Saitsu H, Doi K, Shimizu M, Nakabayashi K, Aoki Y, Tsurusaki Y, Morishita S, Kawaguchi T, Migita O, Nakayama K, Nakashima M, Mitsui J, Narahara M, Hayashi K, Funayama R, Yamaguchi D, Ishiura H, Ko WY, Hata K, Nagashima T, Yamada R, Matsubara Y, Umezawa A, Tsuji S, Matsumoto N, Matsuda F. Human genetic variation database, a reference database of genetic variations in the Japanese population. J Hum Genet. 2016;61:547–53.CrossRefGoogle Scholar
  26. 26.
    Kurashige M, Hanaoka K, Imamura M, Udagawa T, Kawaguchi Y, Hasegawa T, Hosoya T, Yokoo T, Maeda S. A comprehensive search for mutations in the PKD1 and PKD2 in Japanese subjects with autosomal dominant polycystic kidney disease. Clin Genet. 2015;87:266–72.CrossRefGoogle Scholar

Copyright information

© Japanese Society of Nephrology 2019

Authors and Affiliations

  • Toshio Mochizuki
    • 1
    • 2
    Email author
  • Atsuko Teraoka
    • 1
  • Hiroyuki Akagawa
    • 3
  • Shiho Makabe
    • 1
  • Taro Akihisa
    • 1
  • Masayo Sato
    • 1
  • Hiroshi Kataoka
    • 1
    • 2
  • Michihiro Mitobe
    • 1
  • Toru Furukawa
    • 3
    • 4
  • Ken Tsuchiya
    • 5
  • Kosaku Nitta
    • 1
  1. 1.Department of Medicine, Kidney CenterTokyo Women’s Medical UniversityTokyoJapan
  2. 2.Clinical Research Division for Polycystic Kidney Disease, Department of Medicine, Kidney CenterTokyo Women’s Medical UniversityTokyoJapan
  3. 3.Tokyo Women’s Medical University Institute for Integrated Medical Sciences (TIIMS)TokyoJapan
  4. 4.Department of HistopathologyTohoku University Graduate School of MedicineSendaiJapan
  5. 5.Department of Blood Purification, Kidney CenterTokyo Women’s Medical UniversityTokyoJapan

Personalised recommendations