title: Mutation screening of retinal dystrophy patients by targeted capture from tagged pooled DNAs and next generation sequencing.
abstract: OBJECTIVE
  Retinal dystrophies are genetically heterogeneous, resulting from mutations in over 200 genes. Prior to the development of massively parallel sequencing, comprehensive genetic screening was unobtainable for most patients. Identifying the causative genetic mutation facilitates genetic counselling, carrier testing and prenatal/pre-implantation diagnosis, and often leads to a clearer prognosis. In addition, in a proportion of cases, when the mutation is known treatment can be optimised and patients are eligible for enrolment into clinical trials for gene-specific therapies.
  
  
  METHODS
  Patient genomic DNA was sheared, tagged and pooled in batches of four samples, prior to targeted capture and next generation sequencing. The enrichment reagent was designed against genes listed on the RetNet database (July 2010). Sequence data were aligned to the human genome and variants were filtered to identify potential pathogenic mutations. These were confirmed by Sanger sequencing.
  
  
  RESULTS
  Molecular analysis of 20 DNAs from retinal dystrophy patients identified likely pathogenic mutations in 12 cases, many of them known and/or confirmed by segregation. These included previously described mutations in ABCA4 (c.6088C>T,p.R2030*; c.5882G>A,p.G1961E), BBS2 (c.1895G>C,p.R632P), GUCY2D (c.2512C>T,p.R838C), PROM1 (c.1117C>T,p.R373C), RDH12 (c.601T>C,p.C201R; c.506G>A,p.R169Q), RPGRIP1 (c.3565C>T,p.R1189*) and SPATA7 (c.253C>T,p.R85*) and new mutations in ABCA4 (c.3328+1G>C), CRB1 (c.2832_2842+23del), RP2 (c.884-1G>T) and USH2A (c.12874A>G,p.N4292D).
  
  
  CONCLUSIONS
  Tagging and pooling DNA prior to targeted capture of known retinal dystrophy genes identified mutations in 60% of cases. This relatively high success rate may reflect enrichment for consanguineous cases in the local Yorkshire population, and the use of multiplex families. Nevertheless this is a promising high throughput approach to retinal dystrophy diagnostics.
authors.affiliation: Yorkshire Regional Genetics Service, St. James's University Hospital, Leeds, United Kingdom.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Watson
authors.affiliation: Section of Ophthalmology & Neuroscience, Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Mohammed
authors.initials: M
authors.lastname: El-Asrag
authors.affiliation: Section of Genetics, Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: David A
authors.initials: DA
authors.lastname: Parry
authors.affiliation: Section of Genetics, Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Joanne E
authors.initials: JE
authors.lastname: Morgan
authors.affiliation: Section of Ophthalmology & Neuroscience, Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Clare V
authors.initials: CV
authors.lastname: Logan
authors.affiliation: Section of Genetics, Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Carr
authors.affiliation: Section of Genetics, Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Eamonn
authors.initials: E
authors.lastname: Sheridan
authors.affiliation: Yorkshire Regional Genetics Service, St. James's University Hospital, Leeds, United Kingdom.
authors.forename: Ruth
authors.initials: R
authors.lastname: Charlton
authors.affiliation: Section of Ophthalmology & Neuroscience, Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Colin A
authors.initials: CA
authors.lastname: Johnson
authors.affiliation: Yorkshire Regional Genetics Service, St. James's University Hospital, Leeds, United Kingdom.
authors.forename: Graham
authors.initials: G
authors.lastname: Taylor
authors.affiliation: Section of Ophthalmology & Neuroscience, Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Carmel
authors.initials: C
authors.lastname: Toomes
authors.affiliation: Section of Ophthalmology & Neuroscience, Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, United Kingdom; Department of Ophthalmology, St. James's University Hospital, Leeds, United Kingdom.
authors.forename: Martin
authors.initials: M
authors.lastname: McKibbin
authors.affiliation: Section of Ophthalmology & Neuroscience, Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Chris F
authors.initials: CF
authors.lastname: Inglehearn
authors.affiliation: Section of Ophthalmology & Neuroscience, Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Manir
authors.initials: M
authors.lastname: Ali
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D056726:Genetic Association Studies; D020022:Genetic Predisposition to Disease; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D009154:Mutation; D010375:Pedigree; D058499:Retinal Dystrophies / Q000235:genetics*