Abstract
PurposeRetinal 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.MethodsPatient 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.ResultsMolecular 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).ConclusionsTagging 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.
Highlights
Retinal dystrophies are to date the most genetically heterogeneous set of inherited conditions known to affect a single organ
This complicates genetic screening for conditions such as retinitis pigmentosa (RP), cone-rod dystrophy (CRD) and Leber congenital Amaurosis (LCA) since each can result from mutations in many genes which, with the exception of LCA, follow dominant, recessive or X-linked patterns of inheritance
A further incentive for seeking to improve this situation is the notable success of an increasing number of clinical trials for gene and other targeted therapies for retinal dystrophies [3,4,5,6,7]
Summary
Retinal dystrophies are to date the most genetically heterogeneous set of inherited conditions known to affect a single organ. A further incentive for seeking to improve this situation is the notable success of an increasing number of clinical trials for gene and other targeted therapies for retinal dystrophies [3,4,5,6,7]. These are gene-specific, meaning that only patients for whom mutations have been identified will benefit from these novel approaches to stratified medicine
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