PKD1 Duplicated regions limit clinical Utility of Whole Exome Sequencing for Genetic Diagnosis of Autosomal Dominant Polycystic Kidney Disease

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited monogenic renal disease characterised by the accumulation of clusters of fluid-filled cysts in the kidneys and is caused by mutations in PKD1 or PKD2 genes. ADPKD genetic diagnosis is complicated by PKD1 pseudogenes located proximal to the original gene with a high degree of homology. The next generation sequencing (NGS) technology including whole exome sequencing (WES) and whole genome sequencing (WGS), is becoming more affordable and its use in the detection of ADPKD mutations for diagnostic and research purposes more widespread. However, how well does NGS technology compare with the Gold standard (Sanger sequencing) in the detection of ADPKD mutations? Is a question that remains to be answered. We have evaluated the efficacy of WES, WGS and targeted enrichment methodologies in detecting ADPKD mutations in the PKD1 and PKD2 genes in patients who were clinically evaluated by ultrasonography and renal function tests. Our results showed that WES detected PKD1 mutations in ADPKD patients with 50% sensitivity, as the reading depth and sequencing quality were low in the duplicated regions of PKD1 (exons 1-32) compared with those of WGS and target enrichment arrays. Our investigation highlights major limitations of WES in ADPKD genetic diagnosis. Enhancing reading depth, quality and sensitivity of WES in the PKD1 duplicated regions (exons 1-32) is crucial for its potential diagnostic or research applications.

Figure 1

PKD1 mutations detected using targeted sequencing. A total of 6 mutations were found, 4 of which are novel.

Figure 2

Quality assessment of called variants in PKD1 and PKD2 using WES. Exons 1–32 of PKD1 had low reading depth, genotype quality and quality. ADPKD mutations shown in red.

Figure 3

WES and Target enrichment coverage map of PKD1. WES of exons 1 to 32 of PKD1 showed low coverage while PKD2 coverage showed proper depth of all exons. Target Enrichment showed proper coverage for coding regions of PKD1 and PKD2. SureSelect v6 improved the coverage of PKD1 in comparison to WES TruSeq v3 but exons 1 to 14 remained poorly covered. WGS showed proper covering of the entire PKD1.

Figure 4

Total number of reads per exon for PKD1 and PKD2 using WES. Upper image shows that PKD1 exons 1 to 32 have relatively lower number of total reads in comparison with PKD1 exons 33–46 while PKD2 exons “lower image” shows better distribution of readings per exon in comparison with PKD1.

Figure 5

WES, Target enrichment coverage map and WGS of PKD2. PKD2 was covered properly using all methods.