Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy

Edgar A. Otto, Gokul Ramaswami, Sabine Janssen, Moumita Chaki, Susan J. Allen, Weibin Zhou, Rannar Airik, Toby W. Hurd, Amiya K. Ghosh, Matthias T. Wolf, Bernd Hoppe, Thomas J. Neuhaus, Detlef Bockenhauer, David V. Milford, Neveen A. Soliman, Corinne Antignac, Sophie Saunier, Colin A. Johnson, Friedhelm Hildebrandt, C. BergmannK. Zerres, J. Gellermann, A. Münch, L. Neumann, M. J. Schürmann, I. Franke, B. Beck, K. Josefiak, D. Michalk, Stapenhorst, T. Ronda, M. Weber, T. Erler, B. Weidner, K. E. Bonzel, A. M. Wingen, J. Dippell, J. Kirschner, R. Korinthenberg, M. Mall, H. Omran, G. Wolff, S. Fuchs, A. Gal, M. Van Husen, S. Lüttgen, D. E. Müller-Wiefel, J. Drube, J. H H Ehrich, S. Fründ, J. Strehlau, G. F. Hoffmann, D. Kiepe, C. Kneppo, S. Rieger, B. Tönshoff, R. Bambauer, R. Klüte, M. Heckel, A. Greiner, N. Jeck, R. Roos, M. Bulla, S. Fründ, B. Frye, E. Harms, E. Kuwertz-Broeking, B. Wittwer, R. Sanwald, H. J. Stolpe, J. Höpfner, M. Holder, H. E. Leichter, G. Baynam, C. Edwards, H. Peters, C. Jones, A. Janecke, G. Sunder-Plassmann, K. Devriendt, J. Chow, P. Tmka, K. Õunap, T. Apostolou, B. Afroze, N. Lock Hock, M. Eccles, J. W. Dixon, S. Hashmi, D. Drozdz, A. Pogan, A. Peco-Antic, B. Milosevic, V. Stojanovic, E. Holmberg, I. Kern, P. H. Axwijk, N. Knoers, F. Ozaltin, N. Besbas, M. Koyun, A. Nayir, H. Kayserili, S. Ozturk, D. Pehlivan, R. Farrington, F. L. Raymond, R. Sandford, J. Whittaker, B. Kerr, M. Cadnapaphornchai, G. Hidalgo, S. Andreoli, B. Mills, M. Bendel-Stenzel, N. Stover, R. Weleber, M. DeBeukelaer, C. Kozma, R. Schonberg, M. Bitzan

Research output: Contribution to journalArticle

90 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)105-116
Number of pages12
JournalJournal of Medical Genetics
Volume48
Issue number2
DOIs
StatePublished - Feb 2011

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Mutation
DNA
Genes
Cystic Kidney Diseases
Polymerase Chain Reaction
Genetic Loci
Genetic Heterogeneity
Ciliopathies
Deoxyribonuclease I
Exons
Alleles
Genome
Phenotype

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

Cite this

Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy. / Otto, Edgar A.; Ramaswami, Gokul; Janssen, Sabine; Chaki, Moumita; Allen, Susan J.; Zhou, Weibin; Airik, Rannar; Hurd, Toby W.; Ghosh, Amiya K.; Wolf, Matthias T.; Hoppe, Bernd; Neuhaus, Thomas J.; Bockenhauer, Detlef; Milford, David V.; Soliman, Neveen A.; Antignac, Corinne; Saunier, Sophie; Johnson, Colin A.; Hildebrandt, Friedhelm; Bergmann, C.; Zerres, K.; Gellermann, J.; Münch, A.; Neumann, L.; Schürmann, M. J.; Franke, I.; Beck, B.; Josefiak, K.; Michalk, D.; Stapenhorst; Ronda, T.; Weber, M.; Erler, T.; Weidner, B.; Bonzel, K. E.; Wingen, A. M.; Dippell, J.; Kirschner, J.; Korinthenberg, R.; Mall, M.; Omran, H.; Wolff, G.; Fuchs, S.; Gal, A.; Van Husen, M.; Lüttgen, S.; Müller-Wiefel, D. E.; Drube, J.; Ehrich, J. H H; Fründ, S.; Strehlau, J.; Hoffmann, G. F.; Kiepe, D.; Kneppo, C.; Rieger, S.; Tönshoff, B.; Bambauer, R.; Klüte, R.; Heckel, M.; Greiner, A.; Jeck, N.; Roos, R.; Bulla, M.; Fründ, S.; Frye, B.; Harms, E.; Kuwertz-Broeking, E.; Wittwer, B.; Sanwald, R.; Stolpe, H. J.; Höpfner, J.; Holder, M.; Leichter, H. E.; Baynam, G.; Edwards, C.; Peters, H.; Jones, C.; Janecke, A.; Sunder-Plassmann, G.; Devriendt, K.; Chow, J.; Tmka, P.; Õunap, K.; Apostolou, T.; Afroze, B.; Lock Hock, N.; Eccles, M.; Dixon, J. W.; Hashmi, S.; Drozdz, D.; Pogan, A.; Peco-Antic, A.; Milosevic, B.; Stojanovic, V.; Holmberg, E.; Kern, I.; Axwijk, P. H.; Knoers, N.; Ozaltin, F.; Besbas, N.; Koyun, M.; Nayir, A.; Kayserili, H.; Ozturk, S.; Pehlivan, D.; Farrington, R.; Raymond, F. L.; Sandford, R.; Whittaker, J.; Kerr, B.; Cadnapaphornchai, M.; Hidalgo, G.; Andreoli, S.; Mills, B.; Bendel-Stenzel, M.; Stover, N.; Weleber, R.; DeBeukelaer, M.; Kozma, C.; Schonberg, R.; Bitzan, M.

In: Journal of Medical Genetics, Vol. 48, No. 2, 02.2011, p. 105-116.

Research output: Contribution to journalArticle

Otto, EA, Ramaswami, G, Janssen, S, Chaki, M, Allen, SJ, Zhou, W, Airik, R, Hurd, TW, Ghosh, AK, Wolf, MT, Hoppe, B, Neuhaus, TJ, Bockenhauer, D, Milford, DV, Soliman, NA, Antignac, C, Saunier, S, Johnson, CA, Hildebrandt, F, Bergmann, C, Zerres, K, Gellermann, J, Münch, A, Neumann, L, Schürmann, MJ, Franke, I, Beck, B, Josefiak, K, Michalk, D, Stapenhorst, Ronda, T, Weber, M, Erler, T, Weidner, B, Bonzel, KE, Wingen, AM, Dippell, J, Kirschner, J, Korinthenberg, R, Mall, M, Omran, H, Wolff, G, Fuchs, S, Gal, A, Van Husen, M, Lüttgen, S, Müller-Wiefel, DE, Drube, J, Ehrich, JHH, Fründ, S, Strehlau, J, Hoffmann, GF, Kiepe, D, Kneppo, C, Rieger, S, Tönshoff, B, Bambauer, R, Klüte, R, Heckel, M, Greiner, A, Jeck, N, Roos, R, Bulla, M, Fründ, S, Frye, B, Harms, E, Kuwertz-Broeking, E, Wittwer, B, Sanwald, R, Stolpe, HJ, Höpfner, J, Holder, M, Leichter, HE, Baynam, G, Edwards, C, Peters, H, Jones, C, Janecke, A, Sunder-Plassmann, G, Devriendt, K, Chow, J, Tmka, P, Õunap, K, Apostolou, T, Afroze, B, Lock Hock, N, Eccles, M, Dixon, JW, Hashmi, S, Drozdz, D, Pogan, A, Peco-Antic, A, Milosevic, B, Stojanovic, V, Holmberg, E, Kern, I, Axwijk, PH, Knoers, N, Ozaltin, F, Besbas, N, Koyun, M, Nayir, A, Kayserili, H, Ozturk, S, Pehlivan, D, Farrington, R, Raymond, FL, Sandford, R, Whittaker, J, Kerr, B, Cadnapaphornchai, M, Hidalgo, G, Andreoli, S, Mills, B, Bendel-Stenzel, M, Stover, N, Weleber, R, DeBeukelaer, M, Kozma, C, Schonberg, R & Bitzan, M 2011, 'Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy', Journal of Medical Genetics, vol. 48, no. 2, pp. 105-116. https://doi.org/10.1136/jmg.2010.082552
Otto, Edgar A. ; Ramaswami, Gokul ; Janssen, Sabine ; Chaki, Moumita ; Allen, Susan J. ; Zhou, Weibin ; Airik, Rannar ; Hurd, Toby W. ; Ghosh, Amiya K. ; Wolf, Matthias T. ; Hoppe, Bernd ; Neuhaus, Thomas J. ; Bockenhauer, Detlef ; Milford, David V. ; Soliman, Neveen A. ; Antignac, Corinne ; Saunier, Sophie ; Johnson, Colin A. ; Hildebrandt, Friedhelm ; Bergmann, C. ; Zerres, K. ; Gellermann, J. ; Münch, A. ; Neumann, L. ; Schürmann, M. J. ; Franke, I. ; Beck, B. ; Josefiak, K. ; Michalk, D. ; Stapenhorst ; Ronda, T. ; Weber, M. ; Erler, T. ; Weidner, B. ; Bonzel, K. E. ; Wingen, A. M. ; Dippell, J. ; Kirschner, J. ; Korinthenberg, R. ; Mall, M. ; Omran, H. ; Wolff, G. ; Fuchs, S. ; Gal, A. ; Van Husen, M. ; Lüttgen, S. ; Müller-Wiefel, D. E. ; Drube, J. ; Ehrich, J. H H ; Fründ, S. ; Strehlau, J. ; Hoffmann, G. F. ; Kiepe, D. ; Kneppo, C. ; Rieger, S. ; Tönshoff, B. ; Bambauer, R. ; Klüte, R. ; Heckel, M. ; Greiner, A. ; Jeck, N. ; Roos, R. ; Bulla, M. ; Fründ, S. ; Frye, B. ; Harms, E. ; Kuwertz-Broeking, E. ; Wittwer, B. ; Sanwald, R. ; Stolpe, H. J. ; Höpfner, J. ; Holder, M. ; Leichter, H. E. ; Baynam, G. ; Edwards, C. ; Peters, H. ; Jones, C. ; Janecke, A. ; Sunder-Plassmann, G. ; Devriendt, K. ; Chow, J. ; Tmka, P. ; Õunap, K. ; Apostolou, T. ; Afroze, B. ; Lock Hock, N. ; Eccles, M. ; Dixon, J. W. ; Hashmi, S. ; Drozdz, D. ; Pogan, A. ; Peco-Antic, A. ; Milosevic, B. ; Stojanovic, V. ; Holmberg, E. ; Kern, I. ; Axwijk, P. H. ; Knoers, N. ; Ozaltin, F. ; Besbas, N. ; Koyun, M. ; Nayir, A. ; Kayserili, H. ; Ozturk, S. ; Pehlivan, D. ; Farrington, R. ; Raymond, F. L. ; Sandford, R. ; Whittaker, J. ; Kerr, B. ; Cadnapaphornchai, M. ; Hidalgo, G. ; Andreoli, S. ; Mills, B. ; Bendel-Stenzel, M. ; Stover, N. ; Weleber, R. ; DeBeukelaer, M. ; Kozma, C. ; Schonberg, R. ; Bitzan, M. / Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy. In: Journal of Medical Genetics. 2011 ; Vol. 48, No. 2. pp. 105-116.
@article{ce8f829eb4e04661b9fdd78a7b6bd502,
title = "Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy",
abstract = "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.",
author = "Otto, {Edgar A.} and Gokul Ramaswami and Sabine Janssen and Moumita Chaki and Allen, {Susan J.} and Weibin Zhou and Rannar Airik and Hurd, {Toby W.} and Ghosh, {Amiya K.} and Wolf, {Matthias T.} and Bernd Hoppe and Neuhaus, {Thomas J.} and Detlef Bockenhauer and Milford, {David V.} and Soliman, {Neveen A.} and Corinne Antignac and Sophie Saunier and Johnson, {Colin A.} and Friedhelm Hildebrandt and C. Bergmann and K. Zerres and J. Gellermann and A. M{\"u}nch and L. Neumann and Sch{\"u}rmann, {M. J.} and I. Franke and B. Beck and K. Josefiak and D. Michalk and Stapenhorst and T. Ronda and M. Weber and T. Erler and B. Weidner and Bonzel, {K. E.} and Wingen, {A. M.} and J. Dippell and J. Kirschner and R. Korinthenberg and M. Mall and H. Omran and G. Wolff and S. Fuchs and A. Gal and {Van Husen}, M. and S. L{\"u}ttgen and M{\"u}ller-Wiefel, {D. E.} and J. Drube and Ehrich, {J. H H} and S. Fr{\"u}nd and J. Strehlau and Hoffmann, {G. F.} and D. Kiepe and C. Kneppo and S. Rieger and B. T{\"o}nshoff and R. Bambauer and R. Kl{\"u}te and M. Heckel and A. Greiner and N. Jeck and R. Roos and M. Bulla and S. Fr{\"u}nd and B. Frye and E. Harms and E. Kuwertz-Broeking and B. Wittwer and R. Sanwald and Stolpe, {H. J.} and J. H{\"o}pfner and M. Holder and Leichter, {H. E.} and G. Baynam and C. Edwards and H. Peters and C. Jones and A. Janecke and G. Sunder-Plassmann and K. Devriendt and J. Chow and P. Tmka and K. {\~O}unap and T. Apostolou and B. Afroze and {Lock Hock}, N. and M. Eccles and Dixon, {J. W.} and S. Hashmi and D. Drozdz and A. Pogan and A. Peco-Antic and B. Milosevic and V. Stojanovic and E. Holmberg and I. Kern and Axwijk, {P. H.} and N. Knoers and F. Ozaltin and N. Besbas and M. Koyun and A. Nayir and H. Kayserili and S. Ozturk and D. Pehlivan and R. Farrington and Raymond, {F. L.} and R. Sandford and J. Whittaker and B. Kerr and M. Cadnapaphornchai and G. Hidalgo and S. Andreoli and B. Mills and M. Bendel-Stenzel and N. Stover and R. Weleber and M. DeBeukelaer and C. Kozma and R. Schonberg and M. Bitzan",
year = "2011",
month = "2",
doi = "10.1136/jmg.2010.082552",
language = "English (US)",
volume = "48",
pages = "105--116",
journal = "Journal of Medical Genetics",
issn = "0022-2593",
publisher = "BMJ Publishing Group",
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TY - JOUR

T1 - Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy

AU - Otto, Edgar A.

AU - Ramaswami, Gokul

AU - Janssen, Sabine

AU - Chaki, Moumita

AU - Allen, Susan J.

AU - Zhou, Weibin

AU - Airik, Rannar

AU - Hurd, Toby W.

AU - Ghosh, Amiya K.

AU - Wolf, Matthias T.

AU - Hoppe, Bernd

AU - Neuhaus, Thomas J.

AU - Bockenhauer, Detlef

AU - Milford, David V.

AU - Soliman, Neveen A.

AU - Antignac, Corinne

AU - Saunier, Sophie

AU - Johnson, Colin A.

AU - Hildebrandt, Friedhelm

AU - Bergmann, C.

AU - Zerres, K.

AU - Gellermann, J.

AU - Münch, A.

AU - Neumann, L.

AU - Schürmann, M. J.

AU - Franke, I.

AU - Beck, B.

AU - Josefiak, K.

AU - Michalk, D.

AU - Stapenhorst,

AU - Ronda, T.

AU - Weber, M.

AU - Erler, T.

AU - Weidner, B.

AU - Bonzel, K. E.

AU - Wingen, A. M.

AU - Dippell, J.

AU - Kirschner, J.

AU - Korinthenberg, R.

AU - Mall, M.

AU - Omran, H.

AU - Wolff, G.

AU - Fuchs, S.

AU - Gal, A.

AU - Van Husen, M.

AU - Lüttgen, S.

AU - Müller-Wiefel, D. E.

AU - Drube, J.

AU - Ehrich, J. H H

AU - Fründ, S.

AU - Strehlau, J.

AU - Hoffmann, G. F.

AU - Kiepe, D.

AU - Kneppo, C.

AU - Rieger, S.

AU - Tönshoff, B.

AU - Bambauer, R.

AU - Klüte, R.

AU - Heckel, M.

AU - Greiner, A.

AU - Jeck, N.

AU - Roos, R.

AU - Bulla, M.

AU - Fründ, S.

AU - Frye, B.

AU - Harms, E.

AU - Kuwertz-Broeking, E.

AU - Wittwer, B.

AU - Sanwald, R.

AU - Stolpe, H. J.

AU - Höpfner, J.

AU - Holder, M.

AU - Leichter, H. E.

AU - Baynam, G.

AU - Edwards, C.

AU - Peters, H.

AU - Jones, C.

AU - Janecke, A.

AU - Sunder-Plassmann, G.

AU - Devriendt, K.

AU - Chow, J.

AU - Tmka, P.

AU - Õunap, K.

AU - Apostolou, T.

AU - Afroze, B.

AU - Lock Hock, N.

AU - Eccles, M.

AU - Dixon, J. W.

AU - Hashmi, S.

AU - Drozdz, D.

AU - Pogan, A.

AU - Peco-Antic, A.

AU - Milosevic, B.

AU - Stojanovic, V.

AU - Holmberg, E.

AU - Kern, I.

AU - Axwijk, P. H.

AU - Knoers, N.

AU - Ozaltin, F.

AU - Besbas, N.

AU - Koyun, M.

AU - Nayir, A.

AU - Kayserili, H.

AU - Ozturk, S.

AU - Pehlivan, D.

AU - Farrington, R.

AU - Raymond, F. L.

AU - Sandford, R.

AU - Whittaker, J.

AU - Kerr, B.

AU - Cadnapaphornchai, M.

AU - Hidalgo, G.

AU - Andreoli, S.

AU - Mills, B.

AU - Bendel-Stenzel, M.

AU - Stover, N.

AU - Weleber, R.

AU - DeBeukelaer, M.

AU - Kozma, C.

AU - Schonberg, R.

AU - Bitzan, M.

PY - 2011/2

Y1 - 2011/2

N2 - 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.

AB - 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.

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