Inhibition of MicroRNA 6937 Delays Photoreceptor and Vision Loss in a Mouse Model of Retinitis Pigmentosa.

Inherited retinal dystrophies (IRDs) are a group of rare retinal conditions, including retinitis pigmentosa (RP), caused by monogenic mutations in 1 out of more than 250 genes. Despite recent advancements in gene therapy, there is still a lack of an effective treatment for this group of retinal conditions. MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNAs that inhibit gene expression. Control of miRNAs-mediated protein expression has been described as a widely used mechanism for post-transcriptional regulation in many physiological and pathological processes in different organs, including the retina. Our main purpose was to test the hypothesis that modulation of a group of miRNAs can protect photoreceptor cells from death in the rd10 mouse model of retinitis pigmentosa. For this, we incorporated modulators of three miRNAs in adeno-associated viruses (AAVs), which were administered through sub-retinal injections. The results obtained indicate that inhibition of the miR-6937-5p slows down the visual deterioration of rd10 mice, reflected by an increased electroretinogram (ERG) wave response under scotopic conditions and significant preservation of the outer nuclear layer thickness. This work contributes to broadening our knowledge on the molecular mechanisms underlying retinitis pigmentosa and supports the development of novel therapeutic approaches for RP based on miRNA modulation.

Author Correction: A new approach based on targeted pooled DNA sequencing identifies novel mutations in patients with Inherited Retinal Dystrophies.

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A new approach based on targeted pooled DNA sequencing identifies novel mutations in patients with Inherited Retinal Dystrophies.

Inherited retinal diseases (IRD) are a heterogeneous group of diseases that mainly affect the retina; more than 250 genes have been linked to the disease and more than 20 different clinical phenotypes have been described. This heterogeneity both at the clinical and genetic levels complicates the identification of causative mutations. Therefore, a detailed genetic characterization is important for genetic counselling and decisions regarding treatment. In this study, we developed a method consisting on pooled targeted next generation sequencing (NGS) that we applied to 316 eye disease related genes, followed by High Resolution Melting and copy number variation analysis. DNA from 115 unrelated test samples was pooled and samples with known mutations were used as positive controls to assess the sensitivity of our approach. Causal mutations for IRDs were found in 36 patients achieving a detection rate of 31.3%. Overall, 49 likely causative mutations were identified in characterized patients, 14 of which were first described in this study (28.6%). Our study shows that this new approach is a cost-effective tool for detection of causative mutations in patients with inherited retinopathies.

Expression Profiling Analysis Reveals Key MicroRNA-mRNA Interactions in Early Retinal Degeneration in Retinitis Pigmentosa.

Purpose: The aim of this study was to identify differentially expressed microRNAs (miRNAs) that might play an important role in the etiology of retinal degeneration in a genetic mouse model of retinitis pigmentosa (rd10 mice) at initial stages of the disease. Methods: miRNAs-mRNA interaction networks were generated for analysis of biological pathways involved in retinal degeneration. Results: Of more than 1900 miRNAs analyzed, we selected 19 miRNAs on the basis of (1) a significant differential expression in rd10 retinas compared with control samples and (2) an inverse expression relationship with predicted mRNA targets involved in biological pathways relevant to retinal biology and/or degeneration. Seven of the selected miRNAs have been associated with retinal dystrophies, whereas, to our knowledge, nine have not been previously linked to any disease. Conclusions: This study contributes to our understanding of the etiology and progression of retinal degeneration.

SOX2 haploinsufficiency promotes impaired vision at advanced age.

Age-related vision loss has been associated with degeneration of the retina and decline in Müller glia cell activity. Sox2 is a critical transcription factor for the development and maintenance of the mammalian retina. Here we determined the role of Sox2 in retinal aging. We observed a decline in the number of Sox2-positive Müller, amacrine and ganglion cells with age. We also explored the impact of Sox2 haploinsufficiency (Sox2GFP ) on the activity of Müller glia cells and vision loss with age. Reduction of Sox2-positive cells promoted impaired Müller glia cell function at advanced age of Sox2GFP . These findings correlated with a significant decline in electroretinographic response in Sox2 haploinsufficient mice. Together, these results indicate that Sox2 is required for the maintenance of the transmission of visual information from cones and rods, and suggest that decline in Sox2 expression is responsible for retinal cell aging and age-related vision loss.

High prevalence of mutations affecting the splicing process in a Spanish cohort with autosomal dominant retinitis pigmentosa.

Retinitis pigmentosa is the most frequent group of inherited retinal dystrophies. It is highly heterogeneous, with more than 80 disease-causing genes 27 of which are known to cause autosomal dominant RP (adRP), having been identified. In this study a total of 29 index cases were ascertained based on a family tree compatible with adRP. A custom panel of 31 adRP genes was analysed by targeted next-generation sequencing using the Ion PGM platform in combination with Sanger sequencing. This allowed us to detect putative disease-causing mutations in 14 out of the 29 (48.28%) families analysed. Remarkably, around 38% of all adRP cases analysed showed mutations affecting the splicing process, mainly due to mutations in genes coding for spliceosome factors (SNRNP200 and PRPF8) but also due to splice-site mutations in RHO. Twelve of the 14 mutations found had been reported previously and two were novel mutations found in PRPF8 in two unrelated patients. In conclusion, our results will lead to more accurate genetic counselling and will contribute to a better characterisation of the disease. In addition, they may have a therapeutic impact in the future given the large number of studies currently underway based on targeted RNA splicing for therapeutic purposes.

Increased aquaporin 1 and 5 membrane expression in the lens epithelium of cataract patients.

In this work we have analyzed the expression levels of the main aquaporins (AQPs) expressed in human lens epithelial cells (HLECs) using 112 samples from patients treated with cataract surgery and 36 samples from individuals treated with refractive surgery, with transparent lenses as controls. Aquaporin-1 (AQP1) is the main AQP, representing 64.1% of total AQPs in HLECs, with aquaporin-5 (AQP5) representing 35.9% in controls. A similar proportion of each AQP in cataract was found. Although no differences were found at the mRNA level compared to controls, a significant 1.65-fold increase (p=0.001) in AQP1protein expression was observed in HLECs from cataract patients, with the highest differences being found for nuclear cataracts (2.1-fold increase; p<0.001). A similar trend was found for AQP5 (1.47-fold increase), although the difference was not significant (p=0.161). Moreover we have shown increased membrane AQP5 protein expression in HLECs of patients with cataracts. No association of AQP1 or AQP5 expression levels with age or sex was observed in either group. Our results suggest regulation of AQP1 and AQP5 at the post-translational level and support previous observations on the implication of AQP1 and 5 in maintenance of lens transparency in animal models. Our results likely reflect a compensatory response of the crystalline lens to delay cataract formation by increasing the water removal rate.

A Cost-Effective Mutation Screening Strategy for Inherited Retinal Dystrophies.

OBJECTIVE: We developed a simple, time- and cost-effective Excel-based genetic screening strategy for the diagnosis of inherited retinal dystrophies (IRD). DESIGN: 76 patients diagnosed with IRD and 112 nonaffected family members, from 55 unrelated families, were included. DNA samples were analyzed using Axiom Exome Genotyping Array Plates (Affymetrix) that contain over 300,000 genetic variants, including more than 5,000 variants present in 181 genes involved in IRD. We used a simple Excel-based data mining strategy in order to screen IRD variants likely involved in the development of IRD. RESULTS: A total of 5 relevant genetic variants were found in 5 IRD genes. Four variants were reported either as pathogenic or with a prediction of probably damaging, and 1 variant was reported to affect a regulatory region. These variants were present in 14 patients and in 11 carriers, in 10 unrelated families. CONCLUSION: Using our Excel-based data screening strategy, we were able to assign likely genetic diagnoses in a fast and cost-effective manner to over 18% of patients analyzed, with a comparable ratio of genetic findings to that reported with retina-specific arrays for about 1/5 of the cost. Our approach proved efficient in reducing costs and time for IRD diagnosis as a first tier genetic screening method.

Genetic high throughput screening in Retinitis Pigmentosa based on high resolution melting (HRM) analysis.

Retinitis Pigmentosa (RP) involves a group of genetically determined retinal diseases caused by a large number of mutations that result in rod photoreceptor cell death followed by gradual death of cone cells. Most cases of RP are monogenic, with more than 80 associated genes identified so far. The high number of genes and variants involved in RP, among other factors, is making the molecular characterization of RP a real challenge for many patients. Although HRM has been used for the analysis of isolated variants or single RP genes, as far as we are concerned, this is the first study that uses HRM analysis for a high-throughput screening of several RP genes. Our main goal was to test the suitability of HRM analysis as a genetic screening technique in RP, and to compare its performance with two of the most widely used NGS platforms, Illumina and PGM-Ion Torrent technologies. RP patients (n = 96) were clinically diagnosed at the Ophthalmology Department of Donostia University Hospital, Spain. We analyzed a total of 16 RP genes that meet the following inclusion criteria: 1) size: genes with transcripts of less than 4 kb; 2) number of exons: genes with up to 22 exons; and 3) prevalence: genes reported to account for, at least, 0.4% of total RP cases worldwide. For comparison purposes, RHO gene was also sequenced with Illumina (GAII; Illumina), Ion semiconductor technologies (PGM; Life Technologies) and Sanger sequencing (ABI 3130xl platform; Applied Biosystems). Detected variants were confirmed in all cases by Sanger sequencing and tested for co-segregation in the family of affected probands. We identified a total of 65 genetic variants, 15 of which (23%) were novel, in 49 out of 96 patients. Among them, 14 (4 novel) are probable disease-causing genetic variants in 7 RP genes, affecting 15 patients. Our HRM analysis-based study, proved to be a cost-effective and rapid method that provides an accurate identification of genetic RP variants. This approach is effective for medium sized (<4 kb transcript) RP genes, which constitute over 80% of the total of known RP genes.

Current mutation discovery approaches in Retinitis Pigmentosa.

With a worldwide prevalence of about 1 in 3500-5000 individuals, Retinitis Pigmentosa (RP) is the most common form of hereditary retinal degeneration. It is an extremely heterogeneous group of genetically determined retinal diseases leading to progressive loss of vision due to impairment of rod and cone photoreceptors. RP can be inherited as an autosomal-recessive, autosomal-dominant, or X-linked trait. Non-Mendelian inheritance patterns such as digenic, maternal (mitochondrial) or compound heterozygosity have also been reported. To date, more than 65 genes have been implicated in syndromic and non-syndromic forms of RP, which account for only about 60% of all RP cases. Due to this high heterogeneity and diversity of inheritance patterns, the molecular diagnosis of syndromic and non-syndromic RP is very challenging, and the heritability of 40% of total RP cases worldwide remains unknown. However new sequencing methodologies, boosted by the human genome project, have contributed to exponential plummeting in sequencing costs, thereby making it feasible to include molecular testing for RP patients in routine clinical practice within the coming years. Here, we summarize the most widely used state-of-the-art technologies currently applied for the molecular diagnosis of RP, and address their strengths and weaknesses for the molecular diagnosis of such a complex genetic disease.