title: Whole-genome sequencing overcomes pseudogene homology to diagnose autosomal dominant polycystic kidney disease.
abstract: Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic kidney disorder and is due to disease-causing variants in PKD1 or PKD2. Strong genotype-phenotype correlation exists although diagnostic sequencing is not part of routine clinical practice. This is because PKD1 bears 97.7% sequence similarity with six pseudogenes, requiring laborious and error-prone long-range PCR and Sanger sequencing to overcome. We hypothesised that whole-genome sequencing (WGS) would be able to overcome the problem of this sequence homology, because of 150 bp, paired-end reads and avoidance of capture bias that arises from targeted sequencing. We prospectively recruited a cohort of 28 unique pedigrees with ADPKD phenotype. Standard DNA extraction, library preparation and WGS were performed using Illumina HiSeq X and variants were classified following standard guidelines. Molecular diagnosis was made in 24 patients (86%), with 100% variant confirmation by current gold standard of long-range PCR and Sanger sequencing. We demonstrated unique alignment of sequencing reads over the pseudogene-homologous region. In addition to identifying function-affecting single-nucleotide variants and indels, we identified single- and multi-exon deletions affecting PKD1 and PKD2, which would have been challenging to identify using exome sequencing. We report the first use of WGS to diagnose ADPKD. This method overcomes pseudogene homology, provides uniform coverage, detects all variant types in a single test and is less labour-intensive than current techniques. This technique is translatable to a diagnostic setting, allows clinicians to make better-informed management decisions and has implications for other disease groups that are challenged by regions of confounding sequence homology.
authors.affiliation: Garvan Institute of Medical Research, Sydney, NSW, Australia.
authors.forename: Amali C
authors.initials: AC
authors.lastname: Mallawaarachchi
authors.affiliation: Garvan Institute of Medical Research, Sydney, NSW, Australia.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Hort
authors.affiliation: Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia.|St Vincent's Hospital Clinical School, University of New South Wales, Sydney, NSW, Australia.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Cowley
authors.affiliation: Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia.|St Vincent's Hospital Clinical School, University of New South Wales, Sydney, NSW, Australia.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: McCabe
authors.affiliation: Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia.
authors.forename: André
authors.initials: A
authors.lastname: Minoche
authors.affiliation: Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia.|St Vincent's Hospital Clinical School, University of New South Wales, Sydney, NSW, Australia.
authors.forename: Marcel E
authors.initials: ME
authors.lastname: Dinger
authors.affiliation: Garvan Institute of Medical Research, Sydney, NSW, Australia.
authors.forename: John
authors.initials: J
authors.lastname: Shine
authors.affiliation: Garvan Institute of Medical Research, Sydney, NSW, Australia.|Department of Renal Medicine, St Vincent's Hospital, Sydney, NSW, Australia.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Furlong
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D005260:Female; D017353:Gene Deletion; D005820:Genetic Testing / Q000379:methods*; D015894:Genome, Human; D006801:Humans; D008297:Male; D008875:Middle Aged; D010641:Phenotype; D016891:Polycystic Kidney, Autosomal Dominant / Q000175:diagnosis / Q000235:genetics*; D011544:Pseudogenes; D017422:Sequence Analysis, DNA / Q000379:methods; D017385:Sequence Homology; D050396:TRPP Cation Channels / Q000235:genetics
chemical_list: D050396:TRPP Cation Channels