Diagnosis of autosomal dominant polycystic kidney disease using efficient PKD1 and PKD2 targeted next-generation sequencing
Trujillano, Daniel (Centro de Investigación Biomédica en Red de Epidemiología y Salud Pública)
Bullich Vilanova, Gemma (Institut d'Investigació Biomèdica Sant Pau)
Ossowski, Stephan (Universitat Pompeu Fabra)
Ballarín Castan, José Aurelio (Institut d'Investigació Biomèdica Sant Pau)
Torra Balcells, Roser (Institut d'Investigació Biomèdica Sant Pau)
Estivill, Xavier (Grup Quirónsalud (Barcelona, Catalunya))
Ars, Elisabet (Institut d'Investigació Biomèdica Sant Pau)
Universitat Autònoma de Barcelona

Date:	2014
Abstract:	Molecular diagnostics of autosomal dominant polycystic kidney disease (ADPKD) relies on mutation screening of PKD1 and PKD2, which is complicated by extensive allelic heterogeneity and the presence of six highly homologous sequences of PKD1. To date, specific sequencing of PKD1 requires laborious long-range amplifications. The high cost and long turnaround time of PKD1 and PKD2 mutation analysis using conventional techniques limits its widespread application in clinical settings. We performed targeted next-generation sequencing (NGS) of PKD1 and PKD2. Pooled barcoded DNA patient libraries were enriched by in-solution hybridization with PKD1 and PKD2 capture probes. Bioinformatics analysis was performed using an in-house developed pipeline. We validated the assay in a cohort of 36 patients with previously known PKD1 and PKD2 mutations and five control individuals. Then, we used the same assay and bioinformatics analysis in a discovery cohort of 12 uncharacterized patients. We detected 35 out of 36 known definitely, highly likely, and likely pathogenic mutations in the validation cohort, including two large deletions. In the discovery cohort, we detected 11 different pathogenic mutations in 10 out of 12 patients. This study demonstrates that laborious long-range PCRs of the repeated PKD1 region can be avoided by in-solution enrichment of PKD1 and PKD2 and NGS. This strategy significantly reduces the cost and time for simultaneous PKD1 and PKD2 sequence analysis, facilitating routine genetic diagnostics of ADPKD.
Grants:	Instituto de Salud Carlos III FIS/FEDER/PI11/00733 Instituto de Salud Carlos III FIS/FEDER/PI12/01523 Instituto de Salud Carlos III FIS/FEDER/PI13/01731 European Commission 261123 European Commission 262055 Instituto de Salud Carlos III REDinREN/RD06/0016 Instituto de Salud Carlos III FEDER/RD012/0021 Agència de Gestió d'Ajuts Universitaris i de Recerca 2009/SGR-1116
Note:	Altres ajuts: Spanish Renal Network for Research 16/06; Fundación Renal Íñigo Álvarez de Toledo
Note:	Altres ajuts: SAF/2008-00357
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Subject:	Autosomal dominant polycystic kidney disease ; Genetic counseling ; Molecular diagnostics ; Targeted NGS
Published in:	Molecular genetics & genomic medicine, Vol. 2 (may 2014) , p. 412-421, ISSN 2324-9269

DOI: 10.1002/mgg3.82
PMID: 25333066

10 p, 715.2 KB

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Research literature > UAB research groups literature > Research Centres and Groups (research output) > Health sciences and biosciences > Institut de Recerca Sant Pau
Articles > Research articles
Articles > Published articles