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Inherited syndromic retinopathies are a highly heterogeneous group of diseases that involve retinal anomalies and systemic manifestations. They include retinal ciliopathies, other well-defined clinical syndromes presenting with retinal alterations and cases of non-specific multisystemic diseases. The heterogeneity of these conditions makes molecular and clinical characterization of patients challenging in daily clinical practice. We explored the capacity of targeted resequencing and copy-number variation analysis to improve diagnosis of a heterogeneous cohort of 47 patients mainly comprising atypical cases that did not clearly fit a specific clinical diagnosis. Thirty-three likely pathogenic variants were identified in 18 genes (ABCC6, ALMS1, BBS1, BBS2, BBS12, CEP41, CEP290, IFT172, IFT27, MKKS, MYO7A, OTX2, PDZD7, PEX1, RPGRIP1, USH2A, VPS13B, and WDPCP). Molecular findings and additional clinical reassessments made it possible to accurately characterize 14 probands (30% of the total). Notably, clinical refinement of complex phenotypes was achieved in 4 cases, including 2 de novo OTX2-related syndromes, a novel phenotypic association for the ciliary CEP41 gene, and the co-existence of biallelic USH2A variants and a Koolen-de-Vries syndrome-related 17q21.31 microdeletion. We demonstrate that combining next-generation sequencing and CNV analysis is a comprehensive and useful approach to unravel the extensive phenotypic and genotypic complexity of inherited syndromic retinopathies, This study was funded by several grants from the Instituto de Salud Carlos III (ISCIII) from the Spanish Ministry of Health, including CIBERER (06/07/0036), FIS - FEDER (European Regional Development Fund) (PI016/00425), IIS-FJD Biobank PT13/0010/0012. In addition, sponsored chair HU-FJD-UAM “Cátedra de Patrocinio Medicina Genómica”, the Spanish National Organization of the Blind (ONCE) and the Spanish Fighting Blindness Foundation (FUNDALUCE) also supported our work. ISN and RSA are sponsored by Sara Borrell Postdoctoral Program (CD13-00085 and CD12-00676) from ISCIII/FEDER. MC is supported by the Miguel Servet Program (CP12/03256) from ISCIII/FEDER. RPC is supported by Fundación Conchita Rábago and LRJS is supported by CAPES Foundation, Ministry of Education of Brazil

Retinitis pigmentosa (RP) is a group of inherited progressive retinal dystrophies (RD) characterized by photoreceptor degeneration. RP is highly heterogeneous both clinically and genetically, which complicates the identification of causative genes and mutations. Targeted next-generation sequencing (NGS) has been demonstrated to be an effective strategy for the detection of mutations in RP. In our study, an in-house gene panel comprising 75 known RP genes was used to analyze a cohort of 47 unrelated Spanish families pre-classified as autosomal recessive or isolated RP. Disease-causing mutations were found in 27 out of 47 cases achieving a mutation detection rate of 57.4%. In total, 33 pathogenic mutations were identified, 20 of which were novel mutations (60.6%). Furthermore, not only single nucleotide variations but also copy-number variations, including three large deletions in the USH2A and EYS genes, were identified. Finally seven out of 27 families, displaying mutations in the ABCA4, RP1, RP2 and USH2A genes, could be genetically or clinically reclassified. These results demonstrate the potential of our panel-based NGS strategy in RP diagnosis.

Choroideremia (CHM) is a rare X-linked disease leading to progressive retinal degeneration resulting in blindness. The disorder is caused by mutations in the CHM gene encoding REP-1 protein, an essential component of the Rab geranylgeranyltransferase (GGTase) complex. In the present study, we evaluated a multi-technique analysis algorithm to describe the mutational spectrum identified in a large cohort of cases and further correlate CHM variants with phenotypic characteristics and biochemical defects of choroideremia patients. Molecular genetic testing led to the characterization of 36 out of 45 unrelated CHM families (80%), allowing the clinical reclassification of four CHM families. Haplotype reconstruction showed independent origins for the recurrent p.Arg293* and p.Lys178Argfs*5 mutations, suggesting the presence of hotspots in CHM, as well as the identification of two different unrelated events involving exon 9 deletion. No certain genotype-phenotype correlation could be established. Furthermore, all the patients´ fibroblasts analyzed presented significantly increased levels of unprenylated Rabs proteins compared to control cells; however, this was not related to the genotype. This research demonstrates the major potential of the algorithm proposed for diagnosis. Our data enhance the importance of establish a differential diagnosis with other retinal dystrophies, supporting the idea of an underestimated prevalence of choroideremia. Moreover, they suggested that the severity of the disorder cannot be exclusively explained by the genotype.

Retinitis pigmentosa (RP) is a group of inherited progressive retinal dystrophies (RD) characterized by photoreceptor degeneration. RP is highly heterogeneous both clinically and genetically, which complicates the identification of causative genes and mutations. Targeted next-generation sequencing (NGS) has been demonstrated to be an effective strategy for the detection of mutations in RP. In our study, an in-house gene panel comprising 75 known RP genes was used to analyze a cohort of 47 unrelated Spanish families pre-classified as autosomal recessive or isolated RP. Disease-causing mutations were found in 27 out of 47 cases achieving a mutation detection rate of 57.4%. In total, 33 pathogenic mutations were identified, 20 of which were novel mutations (60.6%). Furthermore, not only single nucleotide variations but also copy-number variations, including three large deletions in the USH2A and EYS genes, were identified. Finally seven out of 27 families, displaying mutations in the ABCA4, RP1, RP2 and USH2A genes, could be genetically or clinically reclassified. These results demonstrate the potential of our panel-based NGS strategy in RP diagnosis.

PURPOSE Next-generation sequencing (NGS) has been demonstrated to be an effective strategy for the detection of mutations in retinal dystrophies, a group of inherited diseases that are highly heterogeneous. Therefore, the aim of this study is the application of an NGS-based approach in a Spanish cohort of autosomal dominant retinitis pigmentosa (RP) patients to find out causative mutations. METHODS Index cases of 59 Spanish families with initial diagnosis of autosomal dominant RP and unsuccessfully studied for mutations in the most common RP causal genes, were selected for application of a NGS-based approach with a custom panel for 73 genes related to retinal dystrophies. Candidate variants were select based on frequency, pathogenicity, inherited model, and phenotype. Subsequently, confirmation by Sanger sequencing, cosegregation analysis, and population studies, was applied for determining the implication of those variants in the pathology. RESULTS Overall 31 candidate variants were selected. From them, 17 variants were considered as mutations causative of the disease, 64% (11/17) of them were novel and 36% (6/17) were known RP-related mutations. Therefore, applying this technology16 families were characterized rendering a mutation detection rate of 27% (16/59). Of them, 5% (3/59) of cases displayed mutations in recessive or X-linked genes (ABCA4, RPGR, and RP2) allowing a genetic and clinical reclassification of those families. Furthermore, seven novel variants with uncertain significance and seven novel variants probably not causative of disease were also found. CONCLUSIONS This NGS strategy is a fast, effective, and reliable tool to detect known and novel mutations in autosomal dominant RP patients allowing genetic reclassification in some cases and increasing the knowledge of pathogenesis in retinal dystrophies.