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 we devised a strategy of DNA pooling with consecutive massively parallel resequencing (MPR). Methods In 120 patients with severe NPHP-AC phenotypes we prepared 5 pools of genomic DNA with 24 patients each 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 a Illumina Genome-Analyzer 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 we used DNA from patients with known mutations and demonstrated the detection of 22 out of 24 different alleles (92% sensitivity). MPR led to the molecular diagnosis in 30/120 patients (25%) and we identified 54 pathogenic mutations (27 novel) in 7 different NPHP-AC genes. Additionally, in 24 patients we only found single heterozygous variants of unknown significance. 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 our cohort indicates further extensive heterogeneity in NPHP-AC.

Identification of Gene Mutations in Autosomal Dominant Polycystic Kidney Disease through Targeted Resequencing

“…Pooling of DNA before amplification would further simplify the upstream work by greatly reducing the number of amplicons to generate, check, normalize, and pool. 45 However, in our experiments, this did not seem ideal and led to loss of sensitivity and a higher number of false positives. This may be due to variability of DNA quality and errors in DNA concentration measurements due to innate DNA viscosity, as previously suggested.…”

Missense variants in TMEM67 in a patient with Joubert syndrome

“…This results in a change from the amino acid serine at position 312 to proline: c.934T>C; p.(Ser312Pro). Although this variant is not in a known functional domain, other patients with Joubert syndrome have been found to carry missense mutations at nearby nucleotide positions 903 and 986 (c.903C>G (p.Asp301Glu)) and (c.986A>C (p.Lys329Thr)) 12, 16. This variant is a novel missense change that is not present in any population databases (ACMG PM2).…”

Rapid Detection of Monogenic Causes of Childhood-Onset Steroid-Resistant Nephrotic Syndrome

“…Mutations were examined whether they segregate with the affected status. The protocol for the PCR for confirmation was performed using a touchdown protocol described previously (40). Sanger sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit on an ABI 3730 XL sequencer (Applied Biosystems).…”