Mutation screening of retinal dystrophy patients by targeted capture from tagged pooled DNAs and next generation sequencing

• Published in: PloS one Vol. 9; no. 8; p. e104281
• Main Authors: Watson, Christopher M, El-Asrag, Mohammed, Parry, David A, Morgan, Joanne E, Logan, Clare V, Carr, Ian M, Sheridan, Eamonn, Charlton, Ruth, Johnson, Colin A, Taylor, Graham, Toomes, Carmel, McKibbin, Martin, Inglehearn, Chris F, Ali, Manir
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 18.08.2014; Public Library of Science (PLoS)
• Subjects: Analysis; Biology and Life Sciences; Clinical trials; Congenital diseases; Deoxyribonucleic acid; Disease; DNA; DNA Mutational Analysis - methods; DNA sequencing; Dystrophy; Enrichment; Female; Gene mutation; Gene sequencing; Genes; Genetic aspects; Genetic Association Studies; Genetic counseling; Genetic counselling; Genetic Predisposition to Disease; Genetic screening; Genetic testing; Genetics; Genomes; Genomics; Guanylate cyclase 1; High-Throughput Nucleotide Sequencing; Hospitals; Humans; Implantation; Male; Medical genetics; Medical research; Medicine and Health Sciences; Multiprocessing; Mutation; Neurosciences; Nucleotide sequence; Patients; Pedigree; Prognosis; Retina; Retinal degeneration; Retinal Dystrophies - genetics; Screening; Tagging; USH2A protein; United Kingdom > UK
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0104281

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. 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. 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). 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.