Metodologies d'alt rendiment per a la identificació de noves mutacions i gens causants de distròfies de retina: estudi funcional de nous candidats

Most diagnostic laboratories are confronted with the increasing demand for molecular diagnosis from patients and families and the ever-increasing genetic heterogeneity of visual disorders. Concerning Retinal Dystrophies (RD), almost 200 causative genes have been reported to date, and most families carry private mutations. We aimed to approach RD genetic diagnosis using all the available genetic information to prioritize candidates for mutational screening, and then restrict the number of cases to be analyzed by massive sequencing. We constructed and optimized a comprehensive cosegregation RDchip based on SNP genotyping and haplotype analysis. The RD-chip allows to genotype 768 selected SNPs (closely linked to 100 RD causative genes) in a single cost-, time-effective step. Full diagnosis was attained in 17/36 Spanish pedigrees, yielding 12 new and 12 previously reported mutations in 9 RD genes. The most frequently mutated genes were USH2A and CRB1. Notably, RD3–up to now only associated to Leber Congenital Amaurosis– was identified as causative of Retinitis Pigmentosa. The main assets of the RD-chip are: i) the robustness of the genetic information that underscores the most probable candidates, ii) the invaluable clues in cases of shared haplotypes, which are indicative of a common founder effect, and iii) the detection of extended haplotypes over closely mapping genes, which substantiates cosegregation, although the assumptions in which the genetic analysis is based could exceptionally lead astray. The combination of the genetic approach with whole exome sequencing (WES) greatly increases the diagnosis efficiency, and revealed novel mutations in USH2A and GUCY2D. Overall, the RD-chip diagnosis efficiency ranges from 16% in dominant, to 80% in consanguineous recessive pedigrees, with an average of 47%, well within the upper range of massive sequencing approaches, highlighting the validity of this timeand cost-effective approach whilst high-throughput methodologies become amenable for routine diagnosis in medium sized labs. Citation: de Castro-Miró M, Pomares E, Lorés-Motta L, Tonda R, Dopazo J, et al. (2014) Combined Genetic and High-Throughput Strategies for Molecular Diagnosis of Inherited Retinal Dystrophies. PLoS ONE 9(2): e88410. doi:10.1371/journal.pone. 0088410 Editor: Hector Escriva, Laboratoire Arago, France Received November 28, 2013; Accepted January 6, 2014; Published February 7, 2014 Copyright: ! 2014 de Castro-Miró et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: MdCM was a recipient of a FPI fellowship from the Spanish MICINN. This study was supported by grants SAF2009-08079 (RGD), BFU2010-15656 (GM), and BIO2011-27069 (JD) (Ministerio de Ciencia e Innovación), SGR2009-1427 (RGD)(Generalitat de Catalunya), CIBERER (U718 and U715), Retina Asturias (RGD) and ONCE (RGD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: No competing interests to be declared. * E-mail: rgonzalez@ub.edu ¤ Current address: Instituto de Microcirugı́a Ocular, Barcelona, Spain