Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy.
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.
Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA.
Edgar A
EA
Otto
Gokul
G
Ramaswami
Sabine
S
Janssen
Moumita
M
Chaki
Susan J
SJ
Allen
Weibin
W
Zhou
Rannar
R
Airik
Toby W
TW
Hurd
Amiya K
AK
Ghosh
Matthias T
MT
Wolf
Bernd
B
Hoppe
Thomas J
TJ
Neuhaus
Detlef
D
Bockenhauer
David V
DV
Milford
Neveen A
NA
Soliman
Corinne
C
Antignac
Sophie
S
Saunier
Colin A
CA
Johnson
Friedhelm
F
Hildebrandt
D002923:Cilia / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods*; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D052177:Kidney Diseases, Cystic / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction