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Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy
  1. Edgar A Otto1,
  2. Gokul Ramaswami1,
  3. Sabine Janssen1,
  4. Moumita Chaki1,
  5. Susan J Allen1,
  6. Weibin Zhou1,
  7. Rannar Airik1,
  8. Toby W Hurd1,
  9. Amiya K Ghosh1,
  10. Matthias T Wolf2,
  11. Bernd Hoppe3,
  12. Thomas J Neuhaus4,
  13. Detlef Bockenhauer5,
  14. David V Milford6,
  15. Neveen A Soliman7,8,
  16. Corinne Antignac9,10,
  17. Sophie Saunier10,
  18. Colin A Johnson11,
  19. Friedhelm Hildebrandt1,12,13,
  20. the GPN Study Group
  1. 1Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA
  2. 2Pediatric Nephrology, Children's Medical Center, University of Texas Southwestern Medical Center, Dallas, Texas, USA
  3. 3Department of Pediatrics, Division of Pediatric Nephrology, University Hospital Cologne, Germany
  4. 4Department of Pediatrics, Children's Hospital Lucerne, Lucerne, Switzerland
  5. 5Department of Nephrology, Great Ormond Street Hospital for Children NHS Trust, London, UK
  6. 6Department of Pediatric Nephrology, Birmingham Children's Hospital, Birmingham, UK
  7. 7Center of Pediatric Nephrology & Transplantation, Cairo University, Cairo, Egypt
  8. 8Egyptian Group for Orphan Renal Diseases (EGORD), Cairo, Egypt
  9. 9Department of Genetics, Hopital Necker-Enfants Malades, Assistance Publique–Hopitaux de Paris, Paris, France
  10. 10INSERM U-983, Hopital Necker-Enfants Malades, Universite Paris Descartes, Paris, France
  11. 11Division of Molecular & Translational Medicine, Leeds Institute of Molecular Medicine, University of Leeds, Leeds, UK
  12. 12Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, USA
  13. 13Howard Hughes Medical Institute, Chevy Chase, Maryland, USA
  1. Correspondence to Professor Friedhelm Hildebrandt, University of Michigan Health System, 8220C MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5646, USA; fhilde{at}


Background Nephronophthisis associated ciliopathies (NPHP-AC) comprise a group of autosomal recessive cystic kidney diseases that includes nephronophthisis (NPHP), Senior-Loken syndrome (SLS), Joubert syndrome (JBTS), and Meckel-Gruber syndrome (MKS). To date, causative mutations in NPHP-AC have been described for 18 different genes, rendering mutation analysis tedious and expensive. To overcome the broad genetic locus heterogeneity, a strategy of DNA pooling with consecutive massively parallel resequencing (MPR) was devised.

Methods In 120 patients with severe NPHP-AC phenotypes, five pools of genomic DNA with 24 patients each were prepared which were used as templates in order to PCR amplify all 376 exons of 18 NPHP-AC genes (NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, TMEM67, INPP5E, TMEM216, AHI1, ARL13B, CC2D2A, TTC21B, MKS1, and XPNPEP3). PCR products were then subjected to MPR on an Illumina Genome-Analyser and mutations were subsequently assigned to their respective mutation carrier via CEL I endonuclease based heteroduplex screening and confirmed by Sanger sequencing.

Results For proof of principle, DNA from patients with known mutations was used and detection of 22 out of 24 different alleles (92% sensitivity) was demonstrated. MPR led to the molecular diagnosis in 30/120 patients (25%) and 54 pathogenic mutations (27 novel) were identified in seven different NPHP-AC genes. Additionally, in 24 patients only single heterozygous variants of unknown significance were found.

Conclusions The combined approach of DNA pooling followed by MPR strongly facilitates mutation analysis in broadly heterogeneous single gene disorders. The lack of mutations in 75% of patients in this cohort indicates further extensive heterogeneity in NPHP-AC.

  • Next-generation sequencing
  • ciliopathy
  • nephronophthisis
  • genetics
  • renal medicine

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  • Contributing members of the GPN study group are: C Bergmann (Aachen, Germany); K Zerres (Aachen, Germany); J Gellermann (Berlin, Germany); A Münch (Berlin, Germany); L Neumann (Berlin, Germany); MJ Schürmann (Berlin, Germany); I Franke (Bonn, Germany); B Beck (Cologne, Germany); K Josefiak (Cologne, Germany); D Michalk (Cologne, Germany); Dr Stapenhorst (Cologne, Germany); T Ronda (Cologne, Germany); M Weber (Cologne, Germany); T Erler (Cottbus, Germany); B Weidner (Cottbus, Germany); KE Bonzel (Essen, Germany); A-M Wingen (Essen, Germany); J Dippell (Frankfurt, Germany); J Kirschner (Freiburg, Germany); R Korinthenberg (Freiburg, Germany); M Mall (Freiburg, Germany); H Omran (Freiburg, Germany); G Wolff, (Freiburg, Germany); S Fuchs (Hamburg, Germany); A Gal (Hamburg, Germany); M van Husen (Hamburg, Germany); S Lüttgen (Hamburg, Germany); DE Müller-Wiefel (Hamburg, Germany); J Drube (Hannover, Germany); JHH Ehrich (Hannover, Germany); S Fründ (Hannover, Germany); J Strehlau (Hannover, Germany); GF Hoffmann (Heidelberg, Germany); D Kiepe (Heidelberg, Germany); C Kneppo (Heidelberg, Germany); S Rieger (Heidelberg, Germany); B Tönshoff (Heidelberg, Germany); R Bambauer (Homburg, Germany); R Klüte (Ibbenbüren, Germany); M Heckel (Kronach, Germany); A Greiner (Leipzig, Germany); N Jeck (Marburg, Germany); R Roos (München, Germany); M, Bulla (Münster, Germany); S Fründ (Münster, Germany), B Frye (Münster, Germany); E Harms (Münster, Germany); E Kuwertz-Broeking (Münster, Germany); B Wittwer (Münster, Germany); R Sanwald (Pforzheim, Germany); H-J Stolpe (Rostock, Germany); J Höpfner (Schweinfurt, Germany); M Holder (Stuttgart, Germany); H-E Leichter (Stuttgart, Germany); G Baynam (Subiaco, Australia); C Edwards (Subiaco, Australia); H Peters (Victoria, Australia); C Jones (Victoria, Australia); A Janecke (Innsbruck, Austria); G Sunder-Plassmann (Vienna, Austria); K Devriendt, Leuven, Belgium); J Chow (Vancouver, Canada); P Trnka (Vancouver, Canada); K Õunap (Tartu, Estonia); T Apostolou (Athene, Greece); B Afroze (Kuala Lumpur, Malaysia); N Lock Hock (Kuala Lumpur, Malaysia); M Eccles (Otago, New Zealand); JW Dixon (Wellington, New Zealand; S Hashmi (Karachi, Pakistan); D Drozdz (Kraków, Poland); A Pogan (Kraków, Poland); A Peco-Antic (Belgrade, Serbia); B Milosevic (Novi Sad, Serbia); V Stojanovic (Novi Sad, Serbia); E Holmberg (Umea, Sweden); I Kern (Geneva, Switzerland); PH Axwijk (Amsterdam, The Netherlands); N Knoers (Nijmegen, The Netherlands); F Ozaltin (Ankara, Turkey); N Besbas (Ankara, Turkey); M Koyun (Antalya, Turkey); A Nayir (Istanbul, Turkey); H Kayserili (Istanbul, Turkey); S Ozturk (Istanbul, Turkey); D Pehlivan (Istanbul, Turkey); R Farrington (Cambridge, UK); FL Raymond (Cambridge, UK); R Sandford (Cambridge, UK); J Whittaker (Cambridge, UK); B Kerr (Manchester, UK); M Cadnapaphornchai (Denver, CO, USA); G Hidalgo (Detroit, MI, USA); S Andreoli (Indiananapolis, IN, USA); B Mills (Indiananapolis, IN, USA); M Bendel-Stenzel (Minneapolis, MN, USA); N Stover (Portland, OR, USA); R Weleber (Portland, OR, USA); M DeBeukelaer (Toledo, OH, USA); C Kozma (Washington, DC, USA); R Schonberg (Washington, DC, USA); M Bitzan (Winston-Salem, NC, USA).

  • Funding Other funders: NIH; Howard Hughes Medical Institute.

  • Competing interests None to declare.

  • Patient consent Obtained.

  • Ethics approval This study was conducted with the approval of the University of Michigan.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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