Molecular diagnosis of autosomal dominant polycystic kidney disease using next-generation sequencing

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2. However, genetic analysis is complicated by six PKD1 pseudogenes, large gene sizes, and allelic heterogeneity. We developed a new clinical assay for PKD gene analysis using paired-end next-generation sequencing (NGS) by multiplexing individually bar-coded long-range PCR libraries and analyzing them in one Illumina MiSeq flow cell. The data analysis pipeline has been optimized and automated with Unix shell scripts to accommodate variant calls. This approach was validated using a cohort of 25 patients with ADPKD previously analyzed by Sanger sequencing. A total of 250 genetic variants were identified by NGS, spanning the entire exonic and adjacent intronic regions of PKD1 and PKD2, including all 16 pathogenic mutations. In addition, we identified three novel mutations in a mutation-negative cohort of 24 patients with ADPKD previously analyzed by Sanger sequencing. This NGS method achieved sensitivity of 99.2% (95% CI, 96.8%-99.9%) and specificity of 99.9% (95% CI, 99.7%-100.0%), with cost and turnaround time reduced by as much as 70%. Prospective NGS analysis of 25 patients with ADPKD demonstrated a detection rate comparable with Sanger standards. In conclusion, the NGS method was superior to Sanger sequencing for detecting PKD gene mutations, achieving high sensitivity and improved gene coverage. These characteristics suggest that NGS would be an appropriate new standard for clinical genetic testing of ADPKD.

Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Visualization of the NGS workflow. PKD1 and PKD2 genes were individually amplified as 10 locus-specific LR-PCR products (1.4 to 10.9 kb in size), with all coding regions and most intronic regions covered, in total, an approximately 68.0 kb genomic region. A: Map of the PKD1 and PKD2 genes showing the position of the 10 pairs of primers used for LR-PCR amplification of the coding regions. The highlighted green and yellow regions correspond to the duplicated and single-copy sequences of PKD1, respectively. B: Amplification quality was verified using agarose gel electrophoresis. C: LR-PCR products from each patient were pooled together at equimolar ratio, followed by fragmentation and library preparation. The finished libraries were quantified and batched together at equimolar amounts in groups of 25 patient samples and were assessed for quality by a high-sensitivity chip using an Agilent Bioanalyzer instrument. D: The pooled libraries were sequenced on an Illumina MiSeq platform. The raw sequencing reads were sorted by bar code first and then were subjected to quality control analysis before proceeding with the mutation analysis. The quality score (Phred-like score) is shown at each position of the reads. E: Reads were then mapped back to the PKD1/PKD2 loci of human genome assembly hg19 using the BWA program. In this example, PKD1 sequencing coverage is shown using the Integrative Genomics Viewer (Broad Institute, Cambridge, MA). Red areas, reads from the plus DNA strands; blue areas, reads from the minus strands. F: Variant callings were made by the GATK software package and were visualized using the Integrative Genomics Viewer. Ex, exon; FU, fluorescence unit.

Figure 2

Illustration of the data analysis pipelines. Listed are the analysis steps (A) and the corresponding software/application programs involved (B).

Figure 3

Read depth and coverage analysis results. Plot shows the base coverage (y axis) of each LR-PCR amplicon of the PKD1 and PKD2 genes of one patient. The x axis represents the genomic interval. The average read depth for each fragment is indicated under each amplicon. The plots were generated using the Integrative Genomics Viewer. Ex, exon.

Figure 4

Visualization of typical PKD1 NGS gene variation calls. A nonsense mutation (A) and a 10-bp deletion variant (B) are shown. NGS reads were piled up and are shown on the Integrative Genomics Viewer on top; Sanger sequencing confirmations are shown below.

Figure 5

Distribution of the ALT allele frequencies. The analysis was performed using R software version 3.0.1 (http://www.r-project.org). The results are shown as a function of the ALT allele frequency percentage.

Figure 6

Coverage plot illustrating the identification of a large-sized deletion in PKD2. The patient had an approximately 6-kb deletion that included PKD2 exon (Ex) 5 and adjacent intronic regions compared with the control sample.