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.

Figure 1

Flowchart illustrating the various steps of the DNA pooling and massive parallel resequencing approach, which was applied in order to perform mutation analysis for 18 genes in 120 patients with nephronophthisis associated ciliopathies.

Figure 2

Sequence chromatograms of 28 different novel mutations identified in the genes NPHP4 (4), IQCB1 (1), CEP290 (8), RPGRIP1L (2), TMEM67 (7), AHI1 (1), and CC2D2A (5) in individuals with a NPHP-AC. Gene name, patient identifier, nucleotide change, and inferred amino acid alteration are given above sequence traces. Wild type sequence chromatograms are shown below mutated sequences. All mutations were absent from at least 96 healthy control individuals. Note, that no second mutation has been identified in patients A394, A2420, and F787 in whom we identified a heterozygous truncating mutation in the genes IQCB1 (p.R364X), CEP290 (p.K484fsX8), and AHI1 (p.R891X), respectively. All mutations were found in the heterozygous state with the exception of a homozygous CEP290 missense mutation (p.L972P) in patient F335.

Figure 3

Obtained coverage depth along the entire concatenated sequence of all 376 amplicons (145 kb) derived from 18 different NPHP-AC genes. Shown as an example are the results from the MPR mutation analysis performed on 1 lane of a Solexa/Illumina flowcell in patient pool #3 (DNA pool of 24 patients). Note that exonic but also partial intronic regions of all amplicons are shown. The median coverage depth within exonic coding regions (total of 54 kb) is 4,186×. About 95% of all coding bases are covered at least 300-fold.

Figure 4

Sanger sequencing confirmation of “true mutations/variants” (filled diamonds) identified by MPR on Solexa/Illumina platform are plotted versus variants not confirmed “false positive” (open circles). The alignment of a total of about 83 million sequence reads (39 bp each) to the human reference sequence of targeted 376 exons using “CLC Genomics Workbench™” software revealed 114 variants/mutations fulfilling the following criteria: i) absent from SNP database SNPv130, ii) absent from a control pool of 96 DNAs of healthy individuals, and iii) damaging impact at protein level predicted by PolyPhen2 with scores above 0.7. The variant frequency (x-axis) is plotted against the variant counts (y-axis) for each of the 114 variants analyzed. Seventy four variants/mutations have been confirmed by Sanger sequencing (filled diamonds) and we identified the respective mutation carrier out of a pool of 24 patients. Note that Sanger sequencing confirmation (filled diamonds) has been successful for only 3 variants with allele frequencies below 1%. The expected frequency of a heterozygous change found in a pool of 24 patients DNA is about 2.1%. Changes with frequencies above 1% and high absolute counts (>50) are almost always confirmed by Sanger sequencing.