Computational protocol: Exome Sequencing Reveals Novel and Recurrent Mutations with Clinical Significance in Inherited Retinal Dystrophies

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Protocol publication

[…] We performed exome sequencing using a modified Baylor College of Medicine protocol version 2.1 for sequencing on the SOLiD 5500xl platform as previously described , .Filtering and prioritization of variants were carried out with the BiERapp tool ( as follows. Variants which produce a synonymous change in the open reading frame and do not alter predicted splice sites were discarded. Then, variants present in NCBI dbSNP ( and 1000 Genomes project databases ( with a MAF higher than 0.01 were eliminated. An additional filtering step was performed by discarding variants observed in a group of 267 healthy controls of the Spanish population (The Medical Genome Project, Those genes presenting variants consistent with the inheritance pattern were considered as candidates. Finally, whole-exome data were further evaluated for second-site mutations acting as modifiers in other retinal disease genes (Retnet, [...] Each predicted disease-causing variant was confirmed by Sanger sequencing and co-segregation analysis was performed in the rest of family members DNA samples. We used Polyphen-2 (, SIFT ( and MutationTaster ( to evaluate the potential impact of novel missense substitutions on the function of the encoded protein. We used the DiANNA web server for disulfide connectivity prediction ( The correct name of the variation according to HGVS nomenclature guidelines was checked using Mutalyzer ( Furthermore, splice site tool Prediction by Neural Network , , were applied to estimate the pathogenic nature of intronic sequence variants that could affect the splicing process. The HOPE server ( was used to analyze and predict the structural variations in mutant C2orf71.In order to detect additional cases harboring novel mutations in RHO and C2orf71, we performed Sanger sequencing in those IRD families of our cohort without a molecular diagnosis. This study group was comprised of 282 families of which 92 were clinically diagnosed of arRP, 31 of adRP, 3 of cone rod dystrophies, 6 of Leber Congenital Amaurosis, 9 of Stargardt disease and 141 of sporadic RP. […]

Pipeline specifications

Software tools PolyPhen, SIFT, MutationTaster, DiANNA, Mutalyzer
Application Protein structure analysis
Organisms Homo sapiens
Diseases Retinitis Pigmentosa, Retinal Dystrophies