Detection of PKD1 mutations in autosomal dominant polycystic kidney disease by Ion Torrent semiconductor sequencing

Abstract

Objective To develop and validate a method for detecting PKD1 mutations in autosomal dominant polycystic kidney disease by Ion Torrent semiconductor sequencing. Methods Molecular diagnosis of genetic diseases. Five Chinese patients with polycystic kidney disease were recruited from Nanjing Maternity and Child Health Care Hospital for genetic counseling from July 2013 to August 2014. Peripheral blood samples were collected for DNA analysis. The entire PKD1 coding region was amplified by nine long-range PCR reactions. Pooled amplicons from individual patients were sheared to short fragments using fragmentase. After template preparation, emulsion PCR, and ion sphere particles enrichment, the barcoded libraries were subsequently sequenced using the Ion Torrent sequencer. Candidate variants were validated by Sanger sequencing. Results By using the targeted Ion Torrent sequencing method, four PKD1 mutations were identified from 4 patients, including c.2966_2970delCGGCG in exon 12, c.5014_5015delAG in exon 15, c.8972-2A>G in intron 23, and c.10675G>A in exon 36. Conclusion The targeted Ion Torrent sequencing is an accurate and efficient method to detect PKD1 mutations in autosomal dominant polycystic kidney disease. (Chin J Lab Med, 2015, 38: 27-31) Key words: Polycystic kidney, autosomal dominant; TRPP cation channels; Mutation