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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.

SUBMITTER: Tan AY

PROVIDER: S-EPMC3937536 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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

Tan Adrian Y AY Michaeel Alber A Liu Genyan G Elemento Olivier O Blumenfeld Jon J Donahue Stephanie S Parker Tom T Levine Daniel D Rennert Hanna H

The Journal of molecular diagnostics : JMD 20131227 2

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 she ...[more]

PMID: 24374109

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Project description:ImportanceMost studies of autosomal dominant polycystic kidney disease (ADPKD) genetics have used kidney specialty cohorts, focusing on PKD1 and PKD2. These can lead to biased estimates of population prevalence of ADPKD-associated gene variants and their phenotypic expression.ObjectiveTo determine the prevalence of ADPKD and contributions of PKD1, PKD2, and other genes related to cystic kidney disease in a large, unselected cohort.Design, setting, and participantsThis retrospective observational study used an unselected health system-based cohort in central and northeast Pennsylvania with exome sequencing (enrolled from 2004 to 2020) and electronic health record data (up to October 2021). The genotype-first approach included the entire cohort and the phenotype-first approach focused on patients with ADPKD diagnosis codes, confirmed by chart and imaging review.ExposuresLoss-of-function (LOF) variants in PKD1, PKD2, and other genes associated with cystic kidney disease (ie, ALG8, ALG9, DNAJB11, GANAB, HNF1B, IFT140, SEC61B, PKHD1, PRKCSH, SEC63); likely pathogenic missense variants in PKD1 and PKD2.Main outcomes and measuresGenotype-first analysis: ADPKD diagnosis code (Q61.2, Q61.3, 753.13, 753.12); phenotype-first analysis: presence of a rare variant in PKD1, PKD2, or other genes associated with cystic kidney disease.ResultsOf 174 172 patients (median age, 60 years; 60.6% female; 93% of European ancestry), 303 patients had ADPKD diagnosis codes, including 235 with sufficient chart review data for confirmation. In addition to PKD1 and PKD2, LOF variants in IFT140, GANAB, and HNF1B were associated with ADPKD diagnosis after correction for multiple comparisons. Among patients with LOF variants in PKD1, 66 of 68 (97%) had ADPKD; 43 of 43 patients (100%) with LOF variants in PKD2 had ADPKD. In contrast, only 24 of 77 patients (31.2%) with a PKD1 missense variant previously classified as "likely pathogenic" had ADPKD, suggesting misclassification or variable penetrance. Among patients with ADPKD diagnosis confirmed by chart review, 180 of 235 (76.6%) had a potential genetic cause, with the majority being rare variants in PKD1 (127 patients) or PKD2 (34 patients); 19 of 235 (8.1%) had variants in other genes associated with cystic kidney disease. Of these 235 patients with confirmed ADPKD, 150 (63.8%) had a family history of ADPKD. The yield for a genetic determinant of ADPKD was higher for those with a family history of ADPKD compared with those without family history (91.3% [137/150] vs 50.6% [43/85]; difference, 40.7% [95% CI, 29.2%-52.3%]; P < .001). Previously unreported PKD1, PKD2, and GANAB variants were identified with pedigree data suggesting pathogenicity, and several PKD1 missense variants previously reported as likely pathogenic appeared to be benign.Conclusions and relevanceThis study demonstrates substantial genetic and phenotypic variability in ADPKD among patients within a regional health system in the US.

Dataset Information

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

Publications

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

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