RT Journal Article
SR Electronic
T1 Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy
JF Journal of Medical Genetics
JO J Med Genet
FD BMJ Publishing Group Ltd
SP 105
OP 116
DO 10.1136/jmg.2010.082552
VO 48
IS 2
A1 Edgar A Otto
A1 Gokul Ramaswami
A1 Sabine Janssen
A1 Moumita Chaki
A1 Susan J Allen
A1 Weibin Zhou
A1 Rannar Airik
A1 Toby W Hurd
A1 Amiya K Ghosh
A1 Matthias T Wolf
A1 Bernd Hoppe
A1 Thomas J Neuhaus
A1 Detlef Bockenhauer
A1 David V Milford
A1 Neveen A Soliman
A1 Corinne Antignac
A1 Sophie Saunier
A1 Colin A Johnson
A1 Friedhelm Hildebrandt
A1 the GPN Study Group
YR 2011
UL http://jmg.bmj.com/content/48/2/105.abstract
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.