Regulation of ABCA1 by AMD-Associated Genetic Variants and Hypoxia in iPSC-RPE

Age-related macular degeneration (AMD) is a progressive disease of the macula characterized by atrophy of the retinal pigment epithelium (RPE) and photoreceptor degeneration, leading to severe vision loss at advanced stages in the elderly population. Impaired reverse cholesterol transport (RCT) as well as intracellular lipid accumulation in the RPE are implicated in AMD pathogenesis. Here, we focus on ATP-binding cassette transporter A1 (ABCA1), a major cholesterol transport protein in the RPE, and analyze conditions that lead to ABCA1 dysregulation in induced pluripotent stem cell (iPSC)-derived RPE cells (iRPEs). Our results indicate that the risk-conferring alleles rs1883025 (C) and rs2740488 (A) in ABCA1 are associated with increased ABCA1 mRNA and protein levels and reduced efficiency of cholesterol efflux from the RPE. Hypoxia, an environmental risk factor for AMD, reduced expression of ABCA1 and increased intracellular lipid accumulation. Treatment with a liver X receptor (LXR) agonist led to an increase in ABCA1 expression and reduced lipid accumulation. Our data strengthen the homeostatic role of cholesterol efflux in the RPE and suggest that increasing cellular cholesterol export by stimulating ABCA1 expression might lessen lipid load, improving RPE survival and reducing the risk of developing AMD

Disruption of the basal body protein POC1B results in autosomal-recessive cone-rod dystrophy

Exome sequencing revealed a homozygous missense mutation (c.317C>G [p.Arg106Pro]) in POC1B, encoding POC1 centriolar protein B, in three siblings with autosomal-recessive cone dystrophy or cone-rod dystrophy and compound-heterozygous POC1B mutations (c.199_201del [p.G1n67del] and c.810+1G>T) in an unrelated person with cone-rod dystrophy. Upon overexpression of POC1B in human TERT-immortalized retinal pigment epithelium 1 cells, the encoded wild-type protein localized to the basal body of the primary cilium, whereas this localization was lost for p.Arg106Pro and p.G1n67del variant forms of POC1B. Morpholino-oligonucleotide-induced knockdown of poc1b translation in zebrafish resulted in a dose-dependent small-eye phenotype, impaired optokinetic responses, and decreased length of photoreceptor outer segments. These ocular phenotypes could partially be rescued by wild-type human POC1B mRNA, but not by c.199_201del and c.317C>G mutant human POC1B mRNAs. Yeast two-hybrid screening of a human retinal cDNA library revealed FAM161A as a binary interaction partner of POC1B. This was confirmed in coimmunoprecipitation and colocalization assays, which both showed loss of FAM161A interaction with p.Arg106Pro and p.G1n67del variant forms of POC1B. FAM161A was previously implicated in autosomal-recessive retinitis pigmentosa and shown to be located at the base of the photoreceptor connecting cilium, where it interacts with several other ciliopathy-associated proteins. Altogether, this study demonstrates that POC1B mutations result in a defect of the photoreceptor sensory cilium and thus affect cone and rod photoreceptors

The p53 codon 72 PRO/PRO genotype may be associated with initial central visual field defects in Caucasians with primary open angle glaucoma

Background: Loss of vision in glaucoma is due to apoptotic retinal ganglion cell loss. While p53 modulates apoptosis, gene association studies between p53 variants and glaucoma have been inconsistent. In this study we evaluate the association between a p53 variant functionally known to influence apoptosis (codon 72 Pro/Arg) and the subset of primary open angle glaucoma (POAG) patients with early loss of central visual field. Methods: Genotypes for the p53 codon 72 polymorphism (Pro/Arg) were obtained for 264 POAG patients and 400 controls from the U.S. and in replication studies for 308 POAG patients and 178 controls from Australia (GIST). The glaucoma patients were divided into two groups according to location of initial visual field defect (either paracentral or peripheral). All cases and controls were Caucasian with European ancestry. Results: The p53-PRO/PRO genotype was more frequent in the U.S. POAG patients with early visual field defects in the paracentral regions compared with those in the peripheral regions or control group (p = 2.761025). We replicated this finding in the GIST cohort (p = 7.361023, and in the pooled sample (p = 6.661027) and in a meta-analysis of both the US and GIST datasets (1.361026, OR 2.17 (1.58–2.98 for the PRO allele). Conclusions: These results suggest that the p53 codon 72 PRO/PRO genotype is potentially associated with early paracentral visual field defects in primary open-angle glaucoma patients.NHMRC: This study was supported by National Institutes of Health/National Eye Institute grants: R01EY015872 (Wiggs), R01EY015473 (Pasquale), P30EY014104 (Wiggs), Research to Prevent Blindness (Wiggs, Pasquale, Realini), the Harvard Glaucoma Center of Excellence (Wiggs, Pasquale), The Massachusetts Lions Eye Research Fund (Wiggs, Pasquale), National Health & Medical Research Council Project grant 229960, the Ophthalmic Research Institute of Australia, and Glaucoma Australia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Clinical characteristics of rod and cone photoreceptor dystrophies in patients with mutations

METHODS. Eight patients-four diagnosed with retinitis pigmentosa (RP) and four with conerod dystrophy (CRD), carrying causal C8orf37 mutations-were clinically evaluated, including extensive medical history taking, slit-lamp biomicroscopy, ophthalmoscopy, kinetic perimetry, electroretinography (ERG), spectral-domain optical coherence tomography (SD-OCT), autofluorescence (AF) imaging, and fundus photography. RESULTS. In families A and D, respectively, one and three patients showed a classic RP phenotype with night blindness followed by concentric loss of visual field. Severe visual loss to light perception occurred early in the course of the disease. The symptoms initiated during infancy (family A) or adolescence (family D). Ophthalmoscopy revealed macular atrophy, bone spicules, attenuated vessels, and waxy pale optic discs. SD-OCT showed profound photoreceptor degeneration and AF demonstrated atrophy of the retinal pigment epithelium (RPE). ERG responses were nonrecordable in these patients. In families B and C, the patients were diagnosed with CRD. Initial symptoms were photophobia or loss of visual acuity and occurred during infancy (family B) or adolescence (family C). Ophthalmoscopy and AF revealed profound macular RPE atrophy and SD-OCT demonstrated macular photoreceptor degeneration. ERG responses were severely reduced in a cone-rod pattern or were nonrecordable. Interestingly, both patients in family B demonstrated polydactyly. CONCLUSIONS. Mutations in C8orf37 give rise to an early or adolescent-onset autosomal recessive CRD or RP phenotype with early macular atrophy. The occurrence of postaxial polydactyly in one family suggests a syndromic phenotype, which may indicate C8orf37 has a ciliary function

Significance Analysis of Tourist Sports Events

Import 04/11/2015Cílem této bakalářské práce je popsání významu sportovních akcí v cestovním ruchu. Práce je rozdělena do dvou částí. První část představuje teoretická východiska zaměřená na cestovní ruch, sportovní událost, sportovní marketing a metody použité v práci. Druhá část práce obsahuje analýzu mezinárodních sportovních událostí v podobě olympijských her, Mistrovství světa ve fotbale. Podrobněji analyzuji sportovní události v České republice v roce 2015, Mistrovství Evropy ve fotbale do 21 let, Světový pohár v Biatlonu, Halové Mistrovství Evropy v Atletice a nejdůkladněji zkoumám Mistrovství světa v ledním hokeji 2015. Dotazník směřovaný pro návštěvníky hokejové sportovní události zkoumá dopady na turismus a cestovní ruch.The aim of this bachelor thesis is to describe significance analysis of tourist sports events. This thesis is divided in two parts. The first part presents the theoretical basis of tourist, sports events and sports marketing and methods used in work. The second part contains an analysis of international sporting events as the Olympic games and FIFA World Cup. I explain the influence of significant sport events in Czech republic in 2015, European Football Championship under 21 years, Biathlon World Cup and European Athletic Indoor Championships. I analyze in detail IIHF Hockey World Championship 2015. Questionnaire was made with questions focused on impact of tourism destinations and tourism.115 - Katedra managementuvýborn

Association of Risk Variants in the <i>CFH </i>Gene With Elevated Levels of Coagulation and Complement Factors in Idiopathic Multifocal Choroiditis

Importance: Idiopathic multifocal choroiditis (MFC) is poorly understood, thereby hindering optimal treatment and monitoring of patients. Objective: To identify the genes and pathways associated with idiopathic MFC. Design, Setting, and Participants: This was a case-control genome-wide association study (GWAS) and protein study of blood plasma samples conducted from March 2006 to February 2022. This was a multicenter study involving 6 Dutch universities. Participants were grouped into 2 cohorts: cohort 1 consisted of Dutch patients with idiopathic MFC and controls, and cohort 2 consisted of patients with MFC and controls. Plasma samples from patients with idiopathic MFC who had not received treatment were subjected to targeted proteomics. Idiopathic MFC was diagnosed according to the Standardization of Uveitis Nomenclature (SUN) Working Group guidelines for punctate inner choroidopathy and multifocal choroiditis with panuveitis. Data were analyzed from July 2021 to October 2022. Main outcomes and measures: Genetic variants associated with idiopathic MFC and risk variants associated with plasma protein concentrations in patients. Results: This study included a total of 4437 participants in cohort 1 (170 [3.8%] Dutch patients with idiopathic MFC and 4267 [96.2%] controls; mean [SD] age, 55 [18] years; 2443 female [55%]) and 1344 participants in cohort 2 (52 [3.9%] patients with MFC and 1292 [96.1%] controls; 737 male [55%]). The primary GWAS association mapped to the CFH gene with genome-wide significance (lead variant the A allele of rs7535263; odds ratio [OR], 0.52; 95% CI, 0.41-0.64; P = 9.3 × 10-9). There was no genome-wide significant association with classical human leukocyte antigen (HLA) alleles (lead classical allele, HLA-A*31:01; P = .002). The association with rs7535263 showed consistent direction of effect in an independent cohort of 52 cases and 1292 control samples (combined meta-analysis OR, 0.58; 95% CI, 0.38-0.77; P = 3.0 × 10-8). In proteomic analysis of 87 patients, the risk allele G of rs7535263 in the CFH gene was strongly associated with increased plasma concentrations of factor H-related (FHR) proteins (eg, FHR-2, likelihood ratio test, adjusted P = 1.1 × 10-3) and proteins involved in platelet activation and the complement cascade. Conclusions and relevance: Results suggest that CFH gene variants increase systemic concentrations of key factors of the complement and coagulation cascades, thereby conferring susceptibility to idiopathic MFC. These findings suggest that the complement and coagulation pathways may be key targets for the treatment of idiopathic MFC.</p

Mutation screening of retinal dystrophy patients by targeted capture from tagged pooled DNAs and next generation sequencing.

Purpose: Retinal dystrophies are genetically heterogeneous, resulting from mutations in over 200 genes. Prior to the development of massively parallel sequencing, comprehensive genetic screening was unobtainable for most patients. Identifying the causative genetic mutation facilitates genetic counselling, carrier testing and prenatal/pre-implantation diagnosis, and often leads to a clearer prognosis. In addition, in a proportion of cases, when the mutation is known treatment can be optimised and patients are eligible for enrolment into clinical trials for gene-specific therapies. Methods: Patient genomic DNA was sheared, tagged and pooled in batches of four samples, prior to targeted capture and next generation sequencing. The enrichment reagent was designed against genes listed on the RetNet database (July 2010). Sequence data were aligned to the human genome and variants were filtered to identify potential pathogenic mutations. These were confirmed by Sanger sequencing. Results: Molecular analysis of 20 DNAs from retinal dystrophy patients identified likely pathogenic mutations in 12 cases, many of them known and/or confirmed by segregation. These included previously described mutations in ABCA4 (c.6088C>T,p.R2030*; c.5882G>A,p.G1961E), BBS2 (c.1895G>C,p.R632P), GUCY2D (c.2512C>T,p.R838C), PROM1 (c.1117C>T,p.R373C), RDH12 (c.601T>C,p.C201R; c.506G>A,p.R169Q), RPGRIP1 (c.3565C>T,p.R1189*) and SPATA7 (c.253C>T,p.R85*) and new mutations in ABCA4 (c.3328+1G>C), CRB1 (c.2832_2842+23del), RP2 (c.884-1G>T) and USH2A (c.12874A>G,p.N4292D). Conclusions: Tagging and pooling DNA prior to targeted capture of known retinal dystrophy genes identified mutations in 60% of cases. This relatively high success rate may reflect enrichment for consanguineous cases in the local Yorkshire population, and the use of multiplex families. Nevertheless this is a promising high throughput approach to retinal dystrophy diagnostics

A common allele in RPGRIP1L is a modifier of retinal degeneration in ciliopathies

Despite rapid advances in the identification of genes involved in disease, the predictive power of the genotype remains limited, in part owing to poorly understood effects of second-site modifiers. Here we demonstrate that a polymorphic coding variant of RPGRIP1L (retinitis pigmentosa GTPase regulator-interacting protein-1 like), a ciliary gene mutated in Meckel-Gruber (MKS) and Joubert (JBTS) syndromes, is associated with the development of retinal degeneration in individuals with ciliopathies caused by mutations in other genes. As part of our resequencing efforts of the ciliary proteome, we identified several putative loss-of-function RPGRIP1L mutations, including one common variant, A229T. Multiple genetic lines of evidence showed this allele to be associated with photoreceptor loss in ciliopathies. Moreover, we show that RPGRIP1L interacts biochemically with RPGR, loss of which causes retinal degeneration, and that the Thr229-encoded protein significantly compromises this interaction. Our data represent an example of modification of a discrete phenotype of syndromic disease and highlight the importance of a multifaceted approach for the discovery of modifier alleles of intermediate frequency and effect.This work was supported by grants R01EY007961 from the National Eye Institute (H.K. and A.S.), R01HD04260 from the National Institute of Child Health and Development (N.K.), R01DK072301, R01DK075972 (N.K.), R01DK068306, R01DK064614, R01DK069274 (F.H.), NRSA fellowship F32 DK079541 (E.E.D.) from the National Institute of Diabetes, Digestive and Kidney disorders, Intramural program of NEI (A.S.), the Macular Vision Research Foundation (N.K.), the Foundation for Fighting Blindness (H.K., S.S.B., A.S. and N.K.), the Foundation for Fighting Blindness Canada (R.K.K.), Le Fonds de la recherche en sante du Québec (FRSQ) (R.K.K.), Research to Prevent Blindness (A.S.), Harold Falls Collegiate Professorship (A.S.), the Midwest Eye Banks and Transplantation Center (H.K.), the Searle Scholars Program (M.A.B.), the Deutsche Forschungsgemeinschaft (DFG grant BE 3910/4-1; C.B.) the UK Medical Research Council (grant number G0700073; C.A.J.), NIHR Biomedical Research Centre for Ophthalmology (S.S.B.) and EU-GENORET Grant LSHG-CT-2005-512036 (S.S.B.). F.H. is an investigator of the Howard Hughes Medical Institute (HHMI) and a Doris Duke Distinguished Clinical Scientist (DDCF)

Clinical course of cone dystrophy caused by mutations in the RPGR gene

Contains fulltext : 97720.pdf (publisher's version ) (Closed access)BACKGROUND: Mutations in the RPGR gene predominantly cause rod photoreceptor disorders with a large variability in clinical course. In this report, we describe two families with mutations in this gene and cone involvement. METHODS: We investigated an X-linked cone dystrophy family (1) with 25 affected males, 25 female carriers, and 21 non-carriers, as well as a small family (2) with one affected and one unaffected male. The RPGR gene was analyzed by direct sequencing. All medical records were evaluated, and all available data on visual acuity, color vision testing, ophthalmoscopy, fundus photography, fundus autofluorescence, Goldmann perimetry, SD-OCT, dark adaptation, and full-field electroretinography (ERG) were registered. Cumulative risks of visual loss were studied with Kaplan-Meier product-limit survival analysis. RESULTS: Both families had a frameshift mutation in ORF15 of the RPGR gene; family 1 had p.Ser1107ValfsX4, and family 2 had p.His1100GlnfsX10. Mean follow up was 13 years (SD 10). Virtually all affected males showed reduced photopic and normal scotopic responses on ERG. Fifty percent of the patients had a visual acuity of <0.5 at age 35 years (SE 2.2), and 75% of the patients was legally blind at age 60 years (SE 2.3). Female carriers showed no signs of ocular involvement. CONCLUSIONS: This report describes the clinical course and visual prognosis in two families with cone dystrophy due to RPGR mutations in the 3' terminal region of ORF15. Remarkable features were the consistent, late-onset phenotype, the severe visual outcome, and the non-expression in female carriers. Expression of RPGR mutations in this particular region appears to be relatively homogeneous and predisposed to cones