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Ass. Lect. Mohammed Elsayed Mohammed Elasrag :: Publications:

Title:
Mutation Screening of Retinal Dystrophy Patients by Targeted Capture from Tagged Pooled DNAs and Next Generation Sequencing
Authors: Christopher M. Watson*, Mohammed El-Asrag*, David A. Parry, Joanne E. Morgan, Clare V. Logan, Ian M. Carr, Eamonn Sheridan, Ruth Charlton, Colin A. Johnson, Graham Taylor, Carmel Toomes, Martin McKibbin, Chris F. Inglehearn, Manir Ali*
Year: 2014
Keywords: Human Genetics, Retinal dystrophies, Targeted capture and next generation sequencing, Gene.
Journal: PLOS ONE
Volume: 9
Issue: 8
Pages: e104281
Publisher: PLOS ONE
Local/International: International
Paper Link:
Full paper Mohammed Elsayed Mohammed Elasrag_PLOS ONE_2014.pdf
Supplementary materials Not Available
Abstract:

Purpose: 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.

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