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.This work was supported by grants from the National Institute of Health Carlos III (Institute of Health Carlos III/ISCIII) (CP10/00572, PI13/02621 and RD16/0008/0027 to JRE, PI17/01413 to CI, and a Research Intensification Contract to ALdM); the Basque Government's Industry Department (SAIOTEK: SAIO11-PE11BN002; and SAIO12-PC12BN001 to JRE), a grant from the Mutua Madrilena Foundation and support from the Retinitis Pigmentosa Patients of Gipuzkoa Foundation (BEGISARE). JR-E is a Miguel Servet II Fellow, National Institute of Health Carlos III (ISCIII). MEI was supported by grants from the Basque Government's Department of Education (DEDUC14/309). OB is supported by funding from the Retinitis Pigmentosa Patients of Gipuzkoa Foundation (BEGISARE) and a grant from the Mutua Madrilena Foundation. AA was supported by grants from the Fundacion Jesus de Gangoiti Barrera and from the Basque Government's Departments of Industry and Education (SAIOTEK-11BN002/PC12BN001/DEPLC13/002). CI is partially supported by a Research Intensification Contract (INTBIO15/001). The authors are grateful to Xabier Elcoroaristizabal and Marta Fernandez-Mercado for their helpful advice on developing the base-calling setup. Maribel Gomez; Naiara Telletxea and Nahikari Pastoriza at the Basque Biobank for isolating DNA samples; and Dr. Carmen Ayuso for kindly providing control samples. We also give special thanks to all patients with IRD and their families involved in the study

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.Supported by the Fundacion Jesus de Gangoiti Barrera and from the Basque Government's Department of Industry and Education Grants SAIOTEK-PE11BN002, PC12BN001, and DEPLC13/002 (AA, JRE); funds from Foundation of Patients of Retinitis Pigmentosa of Gipuzkoa (Retinosis Gipuzkoa Begisare) (OB); a grant from the Fundacion Mutua Madrilena (OB); Basque Government's Department of Education grants DEDUC14/309 (MEI), Diputacion Foral de Gipuzkoa DFG15/006 (MM-C), and ELKARTEK 16/014 (MM-C); National Institute of Health Carlos III (Instituto de Salud Carlos III) Grants ISCIII: CP10/00572 (JRE), PI13/02621 (JRE); an Intensificacion Contract (ALdM) from the Basque Government's Department of Industry; and a grant from the Foundation of Patients of Retinitis Pigmentosa of Gipuzkoa (Retinosis Gipuzkoa Begisare) (JRE). JR-E is a Miguel Servet II Fellow, National Institute of Health Carlos III (Instituto de Salud Carlos III), ISCIII: CPII16/00012

Muscular and tendon degeneration after achilles rupture: new insights into future repair strategies

Achilles tendon rupture is a frequent injury with an increasing incidence. After clinical surgical repair, aimed at suturing the tendon stumps back into their original position, the repaired Achilles tendon is often plastically deformed and mechanically less strong than the pre-injured tissue, with muscle fatty degeneration contributing to function loss. Despite clinical outcomes, pre-clinical research has mainly focused on tendon structural repair, with a lack of knowledge regarding injury progression from tendon to muscle and its consequences on muscle degenerative/regenerative processes and function. Here, we characterize the morphological changes in the tendon, the myotendinous junction and muscle belly in a mouse model of Achilles tendon complete rupture, finding cellular and fatty infiltration, fibrotic tissue accumulation, muscle stem cell decline and collagen fiber disorganization. We use novel imaging technologies to accurately relate structural alterations in tendon fibers to pathological changes, which further explain the loss of muscle mechanical function after tendon rupture. The treatment of tendon injuries remains a challenge for orthopedics. Thus, the main goal of this study is to bridge the gap between clinicians'' knowledge and research to address the underlying pathophysiology of ruptured Achilles tendon and its consequences in the gastrocnemius. Such studies are necessary if current practices in regenerative medicine for Achilles tendon ruptures are to be improved

Evaluation of mammographic density patterns: reproducibility and concordance among scales

<p>Abstract</p> <p>Background</p> <p>Increased mammographic breast density is a moderate risk factor for breast cancer. Different scales have been proposed for classifying mammographic density. This study sought to assess intra-rater agreement for the most widely used scales (Wolfe, Tabár, BI-RADS and Boyd) and compare them in terms of classifying mammograms as high- or low-density.</p> <p>Methods</p> <p>The study covered 3572 mammograms drawn from women included in the DDM-Spain study, carried-out in seven Spanish Autonomous Regions. Each mammogram was read by an expert radiologist and classified using the Wolfe, Tabár, BI-RADS and Boyd scales. In addition, 375 mammograms randomly selected were read a second time to estimate intra-rater agreement for each scale using the kappa statistic. Owing to the ordinal nature of the scales, weighted kappa was computed. The entire set of mammograms (3572) was used to calculate agreement among the different scales in classifying high/low-density patterns, with the kappa statistic being computed on a pair-wise basis. High density was defined as follows: percentage of dense tissue greater than 50% for the Boyd, "heterogeneously dense and extremely dense" categories for the BI-RADS, categories P2 and DY for the Wolfe, and categories IV and V for the Tabár scales.</p> <p>Results</p> <p>There was good agreement between the first and second reading, with weighted kappa values of 0.84 for Wolfe, 0.71 for Tabár, 0.90 for BI-RADS, and 0.92 for Boyd scale. Furthermore, there was substantial agreement among the different scales in classifying high- versus low-density patterns. Agreement was almost perfect between the quantitative scales, Boyd and BI-RADS, and good for those based on the observed pattern, i.e., Tabár and Wolfe (kappa 0.81). Agreement was lower when comparing a pattern-based (Wolfe or Tabár) versus a quantitative-based (BI-RADS or Boyd) scale. Moreover, the Wolfe and Tabár scales classified more mammograms in the high-risk group, 46.61 and 37.32% respectively, while this percentage was lower for the quantitative scales (21.89% for BI-RADS and 21.86% for Boyd).</p> <p>Conclusions</p> <p>Visual scales of mammographic density show a high reproducibility when appropriate training is provided. Their ability to distinguish between high and low risk render them useful for routine use by breast cancer screening programs. Quantitative-based scales are more specific than pattern-based scales in classifying populations in the high-risk group.</p

ABCA4 c.6480-35A>G, a novel branchpoint variant associated with Stargardt disease

Introduction: Inherited retinal dystrophies (IRDs) can be caused by variants in more than 280 genes. The ATP-binding cassette transporter type A4 (ABCA4) gene is one of these genes and has been linked to Stargardt disease type 1 (STGD1), fundus flavimaculatus, cone–rod dystrophy (CRD), and pan-retinal CRD. Approximately 25% of the reported ABCA4 variants affect RNA splicing. In most cases, it is necessary to perform a functional assay to determine the effect of these variants.Methods: Whole genome sequencing (WGS) was performed in one Spanish proband with Stargardt disease. The putative pathogenicity of c.6480-35A&gt;G on splicing was investigated both in silico and in vitro. The in silico approach was based on the deep-learning tool SpliceAI. For the in vitro approach we used a midigene splice assay in HEK293T cells, based on a previously established wild-type midigene (BA29) containing ABCA4 exons 46 to 48. Through the analysis of WGS data, we identified two candidate variants in ABCA4 in one proband: a previously described deletion, c.699_768+342del (p.(Gln234Phefs*5)), and a novel branchpoint variant, c.6480-35A&gt;G. Segregation analysis confirmed that the variants were in trans. For the branchpoint variant, SpliceAI predicted an acceptor gain with a high score (0.47) at position c.6480-47. A midigene splice assay in HEK293T cells revealed the inclusion of the last 47 nucleotides of intron 47 creating a premature stop codon and allowed to categorize the variant as moderately severe. Subsequent analysis revealed the presence of this variant as a second allele besides c.1958G&gt;A p.(Arg653His) in an additional Spanish proband in a large cohort of IRD cases.Conclusion: A splice-altering effect of the branchpoint variant, confirmed by the midigene splice assay, along with the identification of this variant in a second unrelated individual affected with STGD, provides sufficient evidence to classify the variant as likely pathogenic. In addition, this research highlights the importance of studying non-coding regions and performing functional assays to provide a conclusive molecular diagnosis

High glucose-induced hyperosmolarity impacts proliferation, cytoskeleton remodeling and migration of human induced pluripotent stem cells via aquaporin-1

Background and objective: Hyperglycemia leads to adaptive cell responses in part due to hyperosmolarity. In endothelial and epithelial cells, hyperosmolarity induces aquaporin-1 (AQP1) which plays a role in cytoskeletal remodeling, cell proliferation and migration. Whether such impairments also occur in human induced pluripotent stem cells (iPS) is not known. We therefore investigated whether high glucose-induced hyperosmolarity impacts proliferation, migration, expression of pluripotency markers and actin skeleton remodeling in iPS cells in an AQP1-dependent manner. Methods and results: Human iPS cells were generated from skin fibroblasts by lentiviral transduction of four reprogramming factors (Oct4, Sox2, Klf4, c-Myc). After reprogramming, iPS cells were characterized by their adaptive responses to high glucose-induced hyperosmolarity by incubation with 5.5mmol/L glucose, high glucose (HG) at 30.5mM, or with the hyperosmolar control mannitol (HM). Exposure to either HG or HM increased the expression of AQP1. AQP1 co-immunoprecipitated with beta-catenin. HG and HM induced the expression of beta-catenin. Under these conditions, iPS cells showed increased ratios of F-actin to G-actin and formed increased tubing networks. Inhibition of AQP1 with small interfering RNA (siRNA) reverted the inducing effects of HG and HM. Conclusions: High glucose enhances human iPS cell proliferation and cytoskeletal remodeling due to hyperosmolarity-induced upregulation of AQP1

Potassium Dependent Regulation of Astrocyte Water Permeability Is Mediated by cAMP Signaling

Astrocytes express potassium and water channels to support dynamic regulation of potassium homeostasis. Potassium kinetics can be modulated by aquaporin-4 (AQP4), the essential water channel for astrocyte water permeability regulation. We investigated whether extracellular potassium ([K+]o) can regulate astrocyte water permeability and the mechanisms of such an effect. Studies were performed on rat primary astrocytes and a rat astrocyte cell line transfected with AQP4. We found that 10mM [K+]o caused an immediate, more than 40%, increase in astrocyte water permeability which was sustained in 5min. The water channel AQP4 was a target for this regulation. Potassium induced a significant increase in intracellular cAMP as measured with a FRET based method and with enzyme immunoassay. We found that protein kinase A (PKA) could phosphorylate AQP4 in vitro. Further elevation of [K+]o to 35mM induced a global intracellular calcium response and a transient water permeability increase that was abolished in 5min. When inwardly rectifying potassium (Kir)-channels were blocked, 10mM [K+]o also induced a calcium increase and the water permeability increase no longer persisted. In conclusion, we find that elevation of extracellular potassium regulates AQP4 and astrocyte water permeability via intracellular signaling involving cAMP. A prolonged increase of astrocyte water permeability is Kir-channel dependent and this response can be impeded by intracellular calcium signaling. Our results support the concept of coupling between AQP4 and potassium handling in astrocytes

AAV-mediated photoreceptor transduction of the pig cone-enriched retina

Recent success in clinical trials supports the use of adeno-associated viral (AAV) vectors for gene therapy of retinal diseases caused by defects in the retinal pigment epithelium (RPE). In contrast, evidence of the efficacy of AAV-mediated gene transfer to retinal photoreceptors, the major site of inherited retinal diseases, is less robust. In addition, although AAV-mediated RPE transduction appears efficient, independently of the serotype used and species treated, AAV-mediated photoreceptor gene transfer has not been systematically investigated thus so far in large animal models, which also may allow identifying relevant species-specific differences in AAV-mediated retinal transduction. In the present study, we used the porcine retina, which has a high cone/rod ratio. This feature allows to properly evaluate both cone and rod photoreceptors transduction and compare the transduction characteristics of AAV2/5 and 2/8, the two most efficient AAV vector serotypes for photoreceptor targeting. Here we show that AAV2/5 and 2/8 transduces both RPE and photoreceptors. AAV2/8 infects and transduces photoreceptor more efficiently than AAV2/5, similarly to what we have observed in the murine retina. The use of the photoreceptor-specific rhodopsin promoter restricts transgene expression to porcine rods and cones, and results in photoreceptor transduction levels similar to those obtained with the ubiquitous promoters tested. Finally, immunological, toxicological and biodistribution studies support the safety of AAV subretinal administration to the large porcine retina. The data presented here on AAV-mediated transduction of the cone-enriched porcine retina may affect the development of gene-based therapies for rare and common severe photoreceptor diseases

Astrocytic Mechanisms Explaining Neural-Activity-Induced Shrinkage of Extraneuronal Space

Neuronal stimulation causes ∼30% shrinkage of the extracellular space (ECS) between neurons and surrounding astrocytes in grey and white matter under experimental conditions. Despite its possible implications for a proper understanding of basic aspects of potassium clearance and astrocyte function, the phenomenon remains unexplained. Here we present a dynamic model that accounts for current experimental data related to the shrinkage phenomenon in wild-type as well as in gene knockout individuals. We find that neuronal release of potassium and uptake of sodium during stimulation, astrocyte uptake of potassium, sodium, and chloride in passive channels, action of the Na/K/ATPase pump, and osmotically driven transport of water through the astrocyte membrane together seem sufficient for generating ECS shrinkage as such. However, when taking into account ECS and astrocyte ion concentrations observed in connection with neuronal stimulation, the actions of the Na+/K+/Cl− (NKCC1) and the Na+/HCO3− (NBC) cotransporters appear to be critical determinants for achieving observed quantitative levels of ECS shrinkage. Considering the current state of knowledge, the model framework appears sufficiently detailed and constrained to guide future key experiments and pave the way for more comprehensive astroglia–neuron interaction models for normal as well as pathophysiological situations