Abstract
Purpose: :
Genetic disorders of the retina are extremely heterogeneous, and can be caused by multiple genes (for overview, see:(http://www.sph.uth.tmc.edu/Retnet/home.htm). Screening of all those genes in patients by traditional genetic screening methods (dHPLC, SSCP etc.) or sequencing is neither cost-effective nor efficient. In this study, we present a novel strategy for cost-effective, large scale mutation analysis.
Methods: :
Up to 1600 amplicons from 87 disease genes (per chip) were amplified, and hybridized against custom designed 300 kb affymetrix resequencing chips, using a new strategy. Mutation analysis was carried out with Affymetrix standard software, and the novel, superior, computerprogram SEQC.
Results: :
At least eight chips were hybridized with (subsets of) amplicons from 87 disease genes, amplified from DNA of patients with a variety of retinal disorders. These genes included, for example, the ABCC6, OPA1, OCA1, OCA2, ABCR, CEP290, and CFH402 genes. Mutation and SNP detection call rates were between 90-100% of hybridized fragments, the average of which will improve further since the SEQC program contains a "learning module". Amongst others, we confirmed and found the following mutations: ABBC6 p.Arg1141X, ABCC6 p.Arg1221Cys, OPA1 p.Trp89X, OCA1 p.Val183Met, OCA1 p.Thr373Lys, OCA2 p.Trp61X, OCA2 p.Cys793Phe, and many more.
Conclusions: :
We implemented a new, cost-effective, mutation detection strategy for mutation analysis in 87 retinal disease genes for different patients and diseases, which include autosomal recessive retinitis pigmentosa (ARRP), autosomal dominant retinitis pigmentosa (ADRP), cone-rod dystrophies, usher syndrome, congenital stationary nightblindness, Bardet-Biedle syndrome, and others.
Keywords: mutations • gene screening • retinal degenerations: hereditary