A comprehensive clinical and biochemical functional study of a novel RPE65 hypomorphic mutation

PURPOSE. Later onset and progression of retinal dystrophy occur with some RPE65 missense mutations. The functional consequences of the novel P25L RPE65 mutation was correlated with its early-childhood phenotype and compared with other pathogenic missense mutations. METHODS. In addition to typical clinical tests, fundus autofluorescence (FAF), optical coherence tomography (OCT), and two-color threshold perimetry (2CTP) were measured. RPE65 mutations were screened by SSCP and direct sequencing. Isomerase activity of mutant RPE65 was assayed in 293F cells and quantified by HPLC analysis of retinoids. RESULTS. A very mild phenotype was detected in a now 7-yearold boy homozygous for the P25L mutation in RPE65. Although abnormal dark adaptation was noticed early, best corrected visual acuity was 20/20 at age 5 years and 20/30 at age 7 years. Nystagmus was absent. Cone electroretinogram (ERG) was measurable, rod ERG severely reduced, and FAF very low. 2CTP detected mainly cone-mediated responses in scotopic conditions, and light-adapted cone responses were approximately 1.5 log units below normal. High-resolution spectral domain OCT revealed morphologic changes. Isomerase activity in 293F cells transfected with RPE65/P25L was reduced to 7.7% of wild-type RPE65-transfected cells, whereas RPE65/ L22P-transfected cells had 13.5%. CONCLUSIONS. The mild clinical phenotype observed is consistent with the residual activity of a severely hypomorphic mutant RPE65. Reduction to Ͻ10% of wild-type RPE65 activity by homozygous P25L correlates with almost complete rod function loss and cone amplitude reduction. Functional survival of cones is possible in patients with residual RPE65 isomerase activity. This patient should profit most from gene therapy. (Invest Ophthalmol Vis Sci. 2008;49:5235-5242) DOI: 10.1167/iovs.07-1671 H uman mutations in the gene for the highly preferentially expressed RPE protein RPE65 are associated with a spectrum of retinal dystrophies ranging from more severe earlyonset conditions, variously described as Leber congenital amaurosis type 2/autosomal recessive childhood-onset severe retinal dystrophy or early-onset severe retinal dystrophy (LCA2/arCSR, EOSRD) to later onset conditions described as autosomal recessive retinitis pigmentosa (arRP). 1-7 Recently, RPE65 has been established as the isomerase central to the retinoid visual cycle. 8 -10 This cycle 11 is crucial for supply of the chromophore 11-cis retinal for visual pigment regeneration. Animal models have contributed greatly to our understanding of the role of RPE65 in the visual cycle, regeneration, and retinal dystrophy. Rpe65 knockout mice display a biochemical phenotype consisting of extreme chromophore starvation (no rhodopsin) in the photoreceptors concurrent with overaccumulation of all-trans retinyl esters in the RPE 10 and are extremely insensitive to light. This insensitivity to light protects Rpe65 Ϫ/Ϫ mice from light damage, establishing rhodopsin as the mediator of light-induced retinal damage. 12 There is also a natural mutation in mouse Rpe65 called rd12. 14,15 The utility of gene therapy was established by preclinical trials in these dogs. 21 This level appears to be more than enough to maintain near-normal function. In contrast, human RPE65 EOSRD displays a wide spectrum of severity, age of onset, and progression not seen in animal models. In this article, we present the mild phenotypic consequences of a homozygous P25L missense mutation in a young patient and correlate these with the biochemical effect of this mutation on RPE65 activity. We show that even though the isomerase activity of the mutant RPE65 was quite impaired, the patient had near-normal visual acuity. However, rod function was extremely impaired. In addition, short-wavelength cones appeared more impaired than long-wavelength cones, consistent with findings in other patients with RPE65 mutations that blue color vision is much more and earlier impaired than is red vision, opposite to the usual case in cone dystrophies. These From th

Real-world clinical experience with Idebenone in the treatment of Leber hereditary optic neuropathy

Background: Leber hereditary optic neuropathy (LHON) leads to bilateral central vision loss. In a clinical trial setting, idebenone has been shown to be safe and to provide a trend toward improved visual acuity, but long-term evidence of effectiveness in real-world clinical practice is sparse. Methods: Open-label, multicenter, retrospective, noncontrolled analysis of long-term visual acuity and safety in 111 LHON patients treated with idebenone (900 mg/day) in an expanded access program. Eligible patients had a confirmed mitochondrial DNA mutation and had experienced the onset of symptoms (most recent eye) within 1 year before enrollment. Data on visual acuity and adverse events were collected as per normal clinical practice. Efficacy was assessed as the proportion of patients with either a clinically relevant recovery (CRR) or a clinically relevant stabilization (CRS) of visual acuity. In the case of CRR, time to and magnitude of recovery over the course of time were also assessed. Results: At time of analysis, 87 patients had provided longitudinal efficacy data. Average treatment duration was 25.6 months. CRR was observed in 46.0% of patients. Analysis of treatment effect by duration showed that the proportion of patients with recovery and the magnitude of recovery increased with treatment duration. Average gain in best-corrected visual acuity for responders was 0.72 logarithm of the minimal angle of resolution (logMAR), equivalent to more than 7 lines on the Early Treatment Diabetic Retinopathy Study (ETDRS) chart. Furthermore, 50% of patients who had a visual acuity below 1.0 logMAR in at least one eye at initiation of treatment successfully maintained their vision below this threshold by last observation. Idebenone was well tolerated, with most adverse events classified as minor. Conclusions: These data demonstrate the benefit of idebenone treatment in recovering lost vision and maintaining good residual vision in a real-world setting. Together, these findings indicate that idebenone treatment should be initiated early and be maintained more than 24 months to maximize efficacy. Safety results were consistent with the known safety profile of idebenone

Mutations in RD3

PURPOSE. To identify the underlying mutation and describe the phenotype in a consanguineous Kurdish family with Leber's congenital amaurosis (LCA)/early onset severe retinal dystrophy (EOSRD). METHODS. Members of the index family were followed up to 22 years by ophthalmological examinations, including best corrected visual acuity (BCVA), Goldmann visual field (GVF), two-color-threshold perimetry (2CTP) and Ganzfeld electroretinogram (ERG), fundus photographs, fundus autofluorescence (FAF), and optical coherence tomography (OCT). After excluding seven of nine known LCA/EOSRD genes in the index patient, linkage analysis was performed in the family using a microarray followed by microsatellite fine mapping and direct sequencing of candidate genes. RD3 was screened by direct sequencing of 85 independent patients with LCA/EOSRD presenting with a BCVA ≥ 1.0 LogMAR before the age of 2 years to assess the prevalence of RD3 mutations in LCA/EOSRD. Since RD3 and RetGC1 have a functional relation, study authors screened for a modifying effect of RD3 mutations in 17 independent patients with mutations in GUCY2D. RESULTS. BCVA was severely reduced from the earliest examinations (as early as 3 months), never exceeding 1.3 LogMAR. The disease presented as cone-rod dystrophy with dystrophic changes in the macula and bone spicules in the periphery on progression. Linkage analysis narrowed the region of interest towards the LCA12 locus. Direct sequencing of RD3 revealed a homozygous nonsense mutation (c.180C > A) in all affected members tested. Screening of additional unrelated LCA/EOSRD patients revealed only polymorphisms in RD3. CONCLUSIONS. This is the second family reported so far with mutations in RD3. Mutations in RD3 are a very rare cause of LCA associated with an extremely severe form of retinal dystrophy

Nachnutzung der gemeinsamen JOIN2\mathrm{JOIN^2} –Repository-Infrastruktur für den KDSF-Objektbereich Publikation?

Im Rahmen des JOIN2-Projekts haben Bibliotheks- & Dokumentationseinheiten (Deutsches Elektronensynchrotron DESY Hamburg/Zeuten, Deutsches Krebsforschungszentrum DKFZ Heidelberg, Forschungszentrum Jülich, GSI Helmholtzzentrum für Schwerionenforschung Darmstadt, Maier-Leibnitz-Zentrum Garching, Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen, KIT Institut für experimentelle Kernphysik Karlsruhe) eine gemeinsame Repository-Infrastruktur für ihre Wissenschaftler und Wissenschaftlerinnen geschaffen. Das Poster dokumentiert Überlegungen, welche Anforderungen des Kerndatensatzes Forschung im Objektbereich abgebildet werden können, wo Probleme und fehlende Normierungen in der Praxis auftauchen könnten und vor allem, an welchen Stellen Kompromisse in Hinblick auf die JOIN2-Serviceorientierung für den Wissenschaftsbereich eingegangen werden müssen.Schlagwörter: Repositorium; VeröffentlichungsdatenbankSchwerpunktbereich: Identifikatoren & Anbindung von Drittsystemen, z.B. von Repositorie

Inactivation of the murine X-linked juvenile retinoschisis gene, Rs1h, suggests a role of retinoschisin in retinal cell layer organization and synaptic structure

Deleterious mutations in RS1 encoding retinoschisin are associated with X-linked juvenile retinoschisis (RS), a common form of macular degeneration in males. The disorder is characterized by a negative electroretinogram pattern and by a splitting of the inner retina. To gain further insight into the function of the retinoschisin protein and its role in the cellular pathology of RS, we have generated knockout mice deficient in Rs1h, the murine ortholog of the human RS1 gene. We show that pathologic changes in hemizygous Rs1h(−/Y) male mice are evenly distributed across the retina, apparently contrasting with the macula-dominated features in human. Similar functional anomalies in human and Rs1h(−/Y) mice, however, suggest that both conditions are a disease of the entire retina affecting the organization of the retinal cell layers as well as structural properties of the retinal synapse

JOIN2^2 empowering a bibliographic infrastructure to support scientists

Most scientific organizations have established bibliographic databases to collect and present the scholarly output generated by their researchers and research projects. In many cases, an institutional repository is included to enable and promote open access. Besides publications lists on the Web, the data is even more valuable to support internal procedures such as reuse in citation management software for new publications or grant applications and in particular to alleviate the burden of administrative reporting according to complex sorting or filtering criteria.In 2010 the libraries and documentation departments of DESY (Hamburg), GSI (Darmstadt), Forschungszentrum Jülich (Jülich), Heinz Maier-Leibnitz Zentrum (MLZ) (Garching) and RWTH Aachen University (Aachen) founded join² to establish such an infrastructure based on Invenio. Main focuses were a solution, which is easy to use and accepted by scientists, to incorporate specific and precise data sources of an organization on for example, people and projects, as well as (inter-)nationally accepted authority records -e.g. on journals- and sources for data import. By merging bibliographic database and repository the addition of full texts is encouraged, furthering OpenAccess. Local protected working areas for publication exchange serve as an additional value to the scientists. The project addresses that each organizations has its specifics and thus the infrastructure must be suitable configured and tailored while still profiting from the shared development resources.As a result six independent repositories are currently in operation. As an open project, we recently also welcomed Deutsches Krebsforschungszentrum (Heidelberg) as a new partner.join² repositories today serve more than 20.000 staff members and more than 5.000 local visitors annually on campus. They provide more than 300.000 records as well as nearly 90.000 high quality authority records freely accessible on the Internet. This makes join² one of the larger Invenio users world wide.We present the flexible structure of our project based on shared content, its various use cases and give an overview of its internal work flows

Increasing the Yield in Targeted Next-Generation Sequencing by Implicating CNV Analysis, Non-Coding Exons and the Overall Variant Load: The Example of Retinal Dystrophies

<div><p>Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover “hidden mutations” such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5′ exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of <i>EYS</i> revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in <i>RP1</i>, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5′-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.</p></div