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J Med Genet doi:10.1136/jmedgenet-2013-101602
  • Methods
  • Original article

Next generation diagnostics of cystic fibrosis and CFTR-related disorders by targeted multiplex high-coverage resequencing of CFTR

  1. X Estivill4,1,2,3
  1. 1Genetic Causes of Disease Group, Centre for Genomic Regulation (CRG), Barcelona, Catalonia, Spain
  2. 2Universitat Pompeu Fabra (UPF), Barcelona, Catalonia, Spain
  3. 3Hospital del Mar Medical Research Institute (IMIM), Barcelona, Catalonia, Spain
  4. 4CIBER in Epidemiology and Public Health (CIBERESP), Barcelona, Catalonia, Spain
  5. 5Human Molecular Genetics Group, IDIBELL, L'Hospitalet de Llobregat, Barcelona, Catalonia, Spain
  6. 6Genomic and Epigenomic Variation in Disease Group, Centre for Genomic Regulation (CRG), Barcelona, Catalonia, Spain
  7. 7qGENOMICS, Quantitative Genomic Medicine Laboratories SL, Barcelona, Catalonia, Spain
  1. Correspondence to Dr X Estivill, Genetic Causes of Disease Group, Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG), Doctor Aiguader 88, Barcelona, Catalonia 08003, Spain; xavier.estivill{at}crg.cat
  • Received 13 February 2013
  • Revised 28 March 2013
  • Accepted 16 April 2013
  • Published Online First 17 May 2013

Abstract

Background Here we have developed a novel and much more efficient strategy for the complete molecular characterisation of the cystic fibrosis (CF) transmembrane regulator (CFTR) gene, based on multiplexed targeted resequencing. We have tested this approach in a cohort of 92 samples with previously characterised CFTR mutations and polymorphisms.

Methods After enrichment of the pooled barcoded DNA libraries with a custom NimbleGen SeqCap EZ Choice array (Roche) and sequencing with a HiSeq2000 (Illumina) sequencer, we applied several bioinformatics tools to call mutations and polymorphisms in CFTR.

Results The combination of several bioinformatics tools allowed us to detect all known pathogenic variants (point mutations, short insertions/deletions, and large genomic rearrangements) and polymorphisms (including the poly-T and poly-thymidine-guanine polymorphic tracts) in the 92 samples. In addition, we report the precise characterisation of the breakpoints of seven genomic rearrangements in CFTR, including those of a novel deletion of exon 22 and a complex 85 kb inversion which includes two large deletions affecting exons 4–8 and 12–21, respectively.

Conclusions This work is a proof-of-principle that targeted resequencing is an accurate and cost-effective approach for the genetic testing of CF and CFTR-related disorders (ie, male infertility) amenable to the routine clinical practice, and ready to substitute classical molecular methods in medical genetics.