DetectingPKD1variants in polycystic kidney disease patients by single-molecule long-read sequencing

Abstract

A genetic diagnosis of autosomal‐dominant polycystic kidney disease (ADPKD) is challenging due to allelic heterogeneity, high GC content, and homology of the PKD1 gene with six pseudogenes. Short‐read next‐generation sequencing approaches, such as whole‐genome sequencing and whole‐exome sequencing, often fail at reliably characterizing complex regions such as PKD1. However, long‐read single‐molecule sequencing has been shown to be an alternative strategy that could overcome PKD1 complexities and discriminate between homologous regions of PKD1 and its pseudogenes. In this study, we present the increased power of resolution for complex regions using long‐read sequencing to characterize a cohort of 19 patients with ADPKD. Our approach provided high sensitivity in identifying PKD1 pathogenic variants, diagnosing 94.7% of the patients. We show that reliable screening of ADPKD patients in a single test without interference of PKD1 homologous sequences, commonly introduced by residual amplification of PKD1 pseudogenes, by direct long‐read sequencing is now possible. This strategy can be implemented in diagnostics and is highly suitable to sequence and resolve complex genomic regions that are of clinical relevance.

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics

“…The easiest option to enrich for specific loci is amplifying the targets by doing a (multiplex) long-range PCR (up to 10 kb). To differentiate samples, barcodes can be added directly during PCR via primers ( 61 , 62 ), by a nested PCR approach ( 57 , 61 , 63 , 64 ), or by ligating hairpin adapters containing barcodes during library preparation (Pacific Biosciences Product Note: www.pacb.com/wp-content/uploads/2015/09/ProductNote-Barcoded-Adapters-Barcoded-Universal-Primers.pdf ). Therefore, for multiplexed long-amplicon tests only a single library preparation is needed after pooling the barcoded amplicons, as opposed to fragmentation and multiple barcoded library preparations for short-read platforms.…”

“…Overall, SMRT sequencing permits expanding from targeting specific CYP2D6 variants/exons, to identification of phased variants across the entire loci, including up/downstream and all introns, that will enhance identification of metabolizer phenotypes in tested individuals and enhance personalized medicine ( 61 ). Similar long-range PCR with PacBio applications have been used to genotype and discriminate other genes from pseudogenes (Table 2 ), including PKD1 for diagnosing autosomal-dominant polycystic kidney disease ( 64 ) and IKBKG for diagnosing primary immunodeficiency diseases in patients suffering from life-threatening invasive pyogenic bacterial infections ( 65 ).…”

A novel frameshift PKD1 mutation in a Chinese patient with autosomal dominant polycystic kidney disease and azoospermia: A case report

“…The sequencing of PKD1 is complex due to the six duplicated pseudogenes adjacent to it, which are highly homologous with exons 1-33 of PKD1 (23). Due to the long range and the six pseudogenes, single-molecule long-read sequencing is more suitable for the detection of genetic variants in patients with ADPKD (24)(25)(26). In the present study, long-range PCR (LR-PCR) amplification and targeted next-generation sequencing, as described previously (27), was used to detect the variants of PKD1 and PKD2 in the 33-year-old male patient.…”

Long-read sequencing in human genetics

“…Methylation analysis [20,22,38] Microbiome analysis [35,46,55] Pseudogene discrimination CYP2D6 [40], IKBKG [4], PKD1 [5], SMN1…”