Determining the causes of recessive retinal dystrophy

Inherited retinal dystrophies (RDs) are a clinically heterogeneous group of eye diseases that result from mutations in more than 250 genes. Genetic diagnosis of these diseases has, until recently, been hampered by the lack of suitable technologies to perform high throughput screening. This thesis describes two different strategies for using next generation sequencing (NGS) in RD patients to find the pathogenic mutation(s) involved. In the first results chapter, a customised capture reagent (called Retinome) designed against the known retinal dystrophy genes (RetNet, June 2010) was used in NGS analysis of 20 RD families. The disease-causing mutations were identified in 12 of 20 cases (60%). These included previously reported mutations in ABCA4 (c.6088C>T, p.R2030*; c.5882G>A, p.G1961E), RDH12 (c.601T>C, p.C201R; c.506G>A, p.R169Q), PROM1 (c.1117C>T, p.R373C), GUCY2D (c.2512C>T, p.R838C), RPGRIP1 (c.3565C>T, p.R1189*), BBS2 (c.1895G>C, p.R632P) and SPATA7 (c.253C>T, p.R85*) and new mutations in CRB1 (c.2832_2842+23del), USH2A (c.12874A>G, p.N4292D), RP2 (c.884-1G>T) and ABCA4 (c.3328+1G>C). In eight cases the causative mutation could not be unambiguously identified. In the second results chapter, whole-exome NGS was performed on five RD families that had been pre-screened with the Retinome reagent. This identified mutations in three known RD genes, MFSD8 (c.1006G>C, p.E336Q; c.1394G>A, p.R465Q), C8orf37 (c.555G>A, p.W185*) and TTLL5 (c.1627G>A, p.E543K), and mutations in two potentially new RD genes, LARGE (c.2089G>T, p.V697L) and FDFT1 (c.930C>G, p.F310L). In the third results chapter, whole-exome NGS was performed, without pre-screening of known genes, in a family with atypical adult-onset RD with early macular involvement. NGS identified a mutation in a novel RD gene, DRAM2 (c.140delG, p.G47Vfs*3). Further DRAM2 screening in DNA panels identified a compound heterozygote case (c.494G>A, p.W165*; c.131G>A, p.S44N). DRAM2 was localised to the photoreceptor inner segment and retinal pigment epithelium. The relative merits of each approach are discussed. Identifying the pathogenic mutation facilitates counselling, carrier testing and may lead to a clearer prognosis. It may also influence future prospects for these families as new treatments become available