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Pseudoxanthoma elasticum (PXE) is a heritable disorder of the connective tissue. PXE patients frequently experience visual field loss and skin lesions, and occasionally cardiovascular complications1,2,3,4. Histopathological findings reveal calcification of the elastic fibres and abnormalities of the collagen fibrils5. Most PXE patients are sporadic, but autosomal recessive and dominant inheritance are also observed6,7. We previously localized the PXE gene to chromosome 16p13.1 (refs 8,9) and constructed a physical map10. Here we describe homozygosity mapping in five PXE families and the detection of deletions or mutations in ABCC6 (formerly MRP6) associated with all genetic forms of PXE in seven patients or families.

Retinitis pigmentosa (RP) denotes a group of hereditary retinal dystrophies, characterized by the early onset of night blindness followed by a progressive loss of the visual field. The primary defect underlying RP affects the function of the rod photoreceptor cell, and, subsequently, mostly unknown molecular and cellular mechanisms trigger the apoptotic degeneration of these photoreceptor cells. Retinitis pigmentosa is very heterogeneous, both phenotypically and genetically. In this review we propose a tentative classification of RP based on the functional systems affected by the mutated proteins. This classification connects the variety of phenotypes to the mutations and segregation patterns observed in RP. Current progress in the identification of the molecular defects underlying RP reveals that at least three distinct functional mechanisms may be affected: 1) the daily renewal and shedding of the photoreceptor outer segments, 2) the visual transduction cascade, and 3) the retinol (vitamin A) metabolism. The first group includes the rhodopsin and peripherin/RDS genes, and mutations in these genes often result in a dominant phenotype. The second group is predominantly associated with a recessive phenotype that results, as we argue, from continuous inactivation of the transduction pathway. Disturbances in the retinal metabolism seem to be associated with equal rod and cone involvement and the presence of deposits in the retinal pigment epithelium.

Best's macular dystrophy (BMD), also known as vitelliform macular degeneration type 2 (VMD2; OMIM 153700), is an autosomal dominant form of macular degeneration with mainly juvenile onset. BMD is characterized by the accumulation of lipofuscin within and beneath the retinal pigment epithelium. The gene causing the disease has been localized to 11q13 by recombination breakpoint mapping. Recently, we have identified the causative gene encoding a protein named bestrophin, and mutations have been found mainly to affect residues that are conserved from a family of genes in Caenorhabditis elegans. The function of bestrophin is so far unknown, and no reliable predictions can be made from sequence comparisons. We have investigated the bestrophin gene in 14 unrelated Swedish, Dutch, Danish, and Moroccan families affected with BMD and found eight new mutations. Including the previously published mutations, 15 different missense mutations have now been detected in 19 of the 22 families with BMD investigated by our laboratory. Interestingly, the mutations cluster in certain regions, and no nonsense mutations or mutations causing frame-shifts have been identified. Computer simulations of the structural elements in the bestrophin protein show that this protein is probably membrane bound, with four putative transmembrane regions.

We have identified a novel RPGR gene mutation in a large Dutch family with X-linked retinitis pigmentosa (RP3). In affected members, a G-->T transversion was found at position +1 of the 5' splice site of intron 5 of the RPGR (retinitis pigmentosa GTPase regulator) gene. Analysis of this mutation at the RNA level showed cryptic splicing upstream of the mutation in exon 5 leading to a frameshift and downstream termination codon. Identification of the causative mutation in this family has facilitated the detection of females at risk of having an affected son.

Dear editor We read with great interest the paper by Tsilis et al entitled “Systematic review of the association between Alzheimer’s disease and chronic glaucoma” published recently in this journal.1 The potential overlap in the pathobiological background of Alzheimer’s disease (AD) and primary open angle glaucoma (POAG) is currently a topic of intense discussion and could provide further insight into both of these complex diseases. Last year, we published an extensive review on POAG and the potential link between AD and POAG.2 Both AD and POAG consist of several clinical subtypes, and are genetically heterogeneous disorders. In our POAG disease model, we found that at least 65 candidate disease genes, together defining several molecular mechanisms (developmental dysfunction, lipid metabolism, and inflammatory processes), underlie the disease.2 For AD, at least 31 genes have been associated with the disease.3 We also showed that, on a molecular level, there is overlap between the molecular mechanisms of POAG and those of AD.4 When comparing two (genetically) heterogeneous disease entities, the conclusion that high heterogeneity and nonassociation will be found seems obvious. The study by Cumurcu et al5 cited by Tsilis et al, concerns a very distinct form of glaucoma, ie, pseudoexfoliation glaucoma, caused uniquely by mutations in the LOXL1 gene,6 and AD. We believe that this study should be considered separately, or pooled with data from other pseudoexfoliation glaucoma (and AD) studies. The data from this study cannot be extrapolated to others. We agree with Tsili et al that large and high-quality association studies, preferably with long follow-up, are needed to clarify the existence and nature of possible associations between POAG and AD. However, at the same time, these studies will only be useful if specific clinical or genetic subtypes of glaucoma and AD are considered. In this respect, we proposed to investigate the existence of a possible association between normal tension glaucoma and AD.2

X-linked retinitis pigmentosa (XLRP) results from mutations in at least two different loci, designated RP2 and RP3, located at Xp11.3 and Xp21.1, respectively. The RP3 gene was recently isolated by positional cloning, whereas the RP2 locus was mapped genetically to a 5-cM interval. We have screened this region for genomic rearrangements by the YAC representation hybridization (YRH) technique and detected a LINE1 (L1) insertion in one XLRP patient. The L1 retrotransposition occurred in an intron of a novel gene that consisted of five exons and encoded a polypeptide of 350 amino acids. Subsequently, nonsense, missense and frameshift mutations, as well as two small deletions, were identified in six additional patients. The predicted gene product shows homology with human cofactor C, a protein involved in the ultimate step of beta-tubulin folding. Our data provide evidence that mutations in this gene, designated RP2, are responsible for progressive retinal degeneration.

Best macular dystrophy (BMD), also known as vitelliform macular dystrophy (VMD2; OMIM 153700), is an autosomal dominant form of macular degeneration characterized by an abnormal accumulation of lipofuscin within and beneath the retinal pigment epithelium cells. In pursuit of the disease gene, we limited the minimum genetic region by recombination breakpoint analysis and mapped to this region a novel retina-specific gene (VMD2). Genetic mapping data, identification of five independent disease-specific mutations and expression studies provide evidence that mutations within the candidate gene are a cause of BMD. The 3′ UTR of the candidate gene contains a region of antisense complementarity to the 3′ UTR of the ferritin heavy-chain gene ( FTH1), indicating the possibility of antisense interaction between VMD2 and FTH1 transcripts.

Gamma-glutamyl carboxylase (GGCX) mutations have been reported in patients with a pseudoxanthoma elasticum (PXE)–like phenotype, loose redundant skin, and multiple vitamin K–dependent coagulation factor deficiencies. We report on the clinical findings and molecular results in 13 affected members of two families who had a uniform phenotype consisting of (PXE)-like skin manifestations in the neck and trunk, loose sagging skin of the trunk and upper limbs, and retinitis pigmentosa confirmed by electroretinographies in 10 affected individuals. There were no coagulation abnormalities. Molecular investigations of the ATP-binding cassette subfamily C member 6 did not yield causative mutations. All 13 affected family members were found to be homozygous for the splice-site mutation c.373+3G>T in the GGCX gene. All tested parents were heterozygous for the mutation, and healthy siblings were either heterozygous or had the wild type. We suggest that the present patients represent a hitherto unreported phenotype associated with GGCX mutations. Digenic inheritance has been suggested to explain the variability in phenotype in GGCX mutation carriers. Consequently, the present phenotype may not be explained only by the GGCX mutations only but may be influenced by variants in other genes or epigenetic and environmental factors.

Purpose Complete congenital stationary night blindness (CSNB1) is characterized by loss of night vision due to a defect in the retinal ON-bipolar cells (BCs). Mutations in GPR179, encoding the G-protein-coupled receptor 179, have been found in CSNB1 patients. In the mouse, GPR179 is localized to the tips of ON-BC dendrites. In this study we determined the ultrastructural localization of GPR179 in human retina and determined the functional consequences of mutations in GPR179 in patients and mice. Methods The localization of GRP179 was analyzed in postmortem human retinas with immunohistochemistry. The functional consequences of the loss of GPR179 were analyzed with standard and 15-Hz flicker ERG protocols. Results In the human retina, GPR179 is localized on the tips of ON-BC dendrites, which invaginate photoreceptors and terminate juxtaposed to the synaptic ribbon. The 15-Hz flicker ERG abnormalities found in patients with mutations in GPR179 more closely resemble those from patients with mutations in either TRPM1 or NYX than in GRM6. 15-Hz flicker ERG abnormalities of Gpr179(nob5) and Grm6(nob3) mice were comparable. Conclusions GRP179 is expressed on dendrites of ON-BCs, indicating that GRP179 is involved in the ON-BCs' signaling cascade. The similarities of 15-Hz flicker ERGs noted in GPR179 patients and NYX or TRPM1 patients suggest that the loss of GPR179 leads to the loss or closure of TRPM1 channels. The difference between the 15-Hz flicker ERGs of mice and humans indicates the presence of important species differences in the retinal activity that this signal represents.

Importance New funduscopic findings in patients with enhanced S-cone syndrome (ESCS) may help clinicians in diagnosing this rare autosomal recessive retinal dystrophy. Objective To expand the clinical spectrum of ESCS due to mutations in the NR2E3 gene. Design Retrospective, noncomparative case series of 31 patients examined between 1983 and 2012. Setting Academic and private ophthalmology practices specialized in retinal dystrophies. Participants A cohort of patients diagnosed with ESCS and harboring known NR2E3 mutations. Intervention Patients had ophthalmic examinations including visual function testing that led to the original diagnosis. Main Outcomes and Measures New fundus features captured with imaging modalities. Results New clinical observations in ESCS include (1) torpedo-like, deep atrophic lesions with a small hyperpigmented rim, variably sized and predominantly located along the arcades; (2) circumferential fibrotic scars in the posterior pole with a spared center and large fibrotic scars around the optic nerve head; and (3) yellow dots in areas of relatively normal-appearing retina. Conclusions and Relevance Enhanced S-cone syndrome has more pleiotropy than previously appreciated. While the nummular type of pigmentation at the level of the retinal pigment epithelium and cystoid or schisis-like maculopathy with typical functional findings remain classic hallmarks of the disease, changes such as circumferential fibrosis of the macula or peripapillary area and “torpedo-like” lesions along the vascular arcades may also direct the clinical diagnosis and focus on screening the NR2E3 gene for a molecular diagnosis.

Background: The choroid plexus epithelium (CPE) is a lobed neuro-epithelial structure that forms the outer blood-brain barrier. The CPE protrudes into the brain ventricles and produces the cerebrospinal fluid (CSF), which is crucial for brain homeostasis. Malfunction of the CPE is possibly implicated in disorders like Alzheimer disease, hydrocephalus or glaucoma. To study human genetic diseases and potential new therapies, mouse models are widely used. This requires a detailed knowledge of similarities and differences in gene expression and functional annotation between the species. The aim of this study is to analyze and compare gene expression and functional annotation of healthy human and mouse CPE.Methods: We performed 44k Agilent microarray hybridizations with RNA derived from laser dissected healthy human and mouse CPE cells. We functionally annotated and compared the gene expression data of human and mouse CPE using the knowledge database Ingenuity. We searched for common and species specific gene expression patterns and function between human and mouse CPE. We also made a comparison with previously published CPE human and mouse gene expression data.Results: Overall, the human and mouse CPE transcriptomes are very similar. Their major functionalities included epithelial junctions, transport, energy production, neuro-endocrine signaling, as well as immunological, neurological and hematological functions and disorders. The mouse CPE presented two additional functions not found in the human CPE: carbohydrate metabolism and a more extensive list of (neural) developmental functions. We found three genes specifically expressed in the mouse CPE compared to human CPE, being ACE, PON1 and TRIM3 and no human specifically expressed CPE genes compared to mouse CPE.Conclusion: Human and mouse CPE transcriptomes are very similar, and display many common functionalities. Nonetheless, we also identified a few genes and pathways which suggest that the CPE between mouse and man differ with respect to transport and metabolic functions.

Objective: To investigate the relative frequency of the genetic causes of the Schubert-Bornschein type of congenital stationary night blindness (CSNB) and to determine the genotype-phenotype correlations in CSNB1 and CSNB2. Design: Clinic-based, longitudinal, multicenter study. Participants: A total of 39 patients with CSNB1 from 29 families and 62 patients with CSNB2 from 43 families. Methods: Patients underwent full ophthalmologic and electrophysiologic examinations. On the basis of standard electroretinograms (ERGs), patients were diagnosed with CSNB1 or CSNB2. Molecular analysis was performed by direct Sanger sequencing of the entire coding regions in NYX, TRPM1, GRM6, and GPR179 in patients with CSNB1 and CACNA1F and CABP4 in patients with CSNB2. Main Outcome Measures: Data included genetic cause of CSNB, refractive error, visual acuity, nystagmus, strabismus, night blindness, photophobia, color vision, dark adaptation (DA) curve, and standard ERGs. Results: A diagnosis of CSNB1 or CSNB2 was based on standard ERGs. The photopic ERG was the most specific criterion to distinguish between CSNB1 and CSNB2 because it showed a "square-wave" appearance in CSNB1 and a decreased b-wave in CSNB2. Mutations causing CSNB1 were found in NYX (20 patients, 13 families), TRPM1 (10 patients, 9 families), GRM6 (4 patients, 3 families), and GPR179 (2 patients, 1 family). Congenital stationary night blindness 2 was primarily caused by mutations in CACNA1F (55 patients, 37 families). Only 3 patients had causative mutations in CABP4 (2 families). Patients with CSNB1 mainly had rod-related problems, and patients with CSNB2 had rod- and cone-related problems. The visual acuity on average was better in CSNB1 (0.30 logarithm of the minimum angle of resolution [logMAR]) than in CSNB2 (0.52 logMAR). All patients with CSNB1 and only 54% of the patients with CSNB2 reported night blindness. The dark-adapted threshold was on average more elevated in CSNB1 (3.0 log) than in CSNB2 (1.8 log). The 3 patients with CABP4 had a relative low visual acuity, were hyperopic, had severe nonspecific color vision defects, and had only 1.0 log elevated DA threshold. Conclusions: Congenital stationary night blindness 1, despite different causative mutations, shows 1 unique CSNB1 phenotype. Congenital stationary night blindness 2 caused by mutations in CABP4 merely shows cone-related problems and therefore appears to be distinct from CSNB2 caused by mutations in CACNA1F. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article. © 2013 by the American Academy of Ophthalmology.

BACKGROUND: The ciliary body epithelia (CBE) of the eye produce the aqueous humor (AH). The equilibrium between the AH production by the CBE and the outflow through the trabecular meshwork ultimately determines the intraocular pressure (IOP). An increased IOP is a major risk factor for primary open angle glaucoma (POAG). This study aims to elucidate the molecular machinery of the most important function of the CBE: the AH production and composition, and aims to find possible new molecular clues for POAG and AH production-lowering drugs.METHODS: We performed a gene expression analysis of the non-pigmented (NPE) and pigmented epithelia (PE) of the human CBE of post mortem eyes. We used 44 k Agilent microarrays against a common reference design. Functional annotations were performed with the Ingenuity knowledge database.RESULTS: We built a molecular model of AH production by combining previously published physiological data with our current genomic expression data. Next, we investigated molecular CBE transport features which might influence AH composition. These features included caveolin- and clathrin vesicle-mediated transport of large biomolecules, as well as a range of substrate specific transporters. The presence of these transporters implies that, for example, immunoglobins, thyroid hormone, prostaglandins, cholesterol and vitamins can be secreted by the CBE along with the AH. In silico, we predicted some of the molecular apical interactions between the NPE and PE, the side where the two folded epithelia face each other. Finally, we found high expression of seven POAG disease genes in the plasma membrane of extracellular space of the CBE, namely APOE, CAV1, COL8A2, EDNRA, FBN1, RFTN1 and TLR4 and we found possible new targets for AH lowering drugs in the AH.CONCLUSIONS: The CBE expresses many transporters, which account for AH production and/or composition. Some of these entries have also been associated with POAG. We hypothesize that the CBE may play a more prominent role than currently thought in the pathogenesis of POAG, for example by changing the composition of AH.

International audience; BACKGROUND: Pseudoxanthoma elasticum (PXE), caused by mutations in the ABCC6 gene, is a rare multiorgan disease characterized by the mineralization and fragmentation of elastic fibers in connective tissue. Cardiac complications reportedly associated with PXE are mainly based on case reports. METHODS: A cohort of 67 PXE patients was prospectively assessed. Patients underwent physical examination, electrocardiogram, transthoracic echocardiography, cardiac magnetic resonance imaging (CMR), treadmill testing, and perfusion myocardial scintigraphy (SPECT). Additionally, the hearts of a PXE mouse models (Abcc6(-/-)) and wild-type controls (WT) were analyzed. RESULTS: Three patients had a history of proven coronary artery disease. In total, 40 patients underwent exercise treadmill tests, and 28 SPECT. The treadmill tests were all negative. SPECT showed mild perfusion abnormalities in two patients. Mean left ventricular (LV) dimension and function values were within the normal range. LV hypertrophy was found in 7 (10.4%) patients, though the hypertrophy etiology was unknown for 3 of those patients. Echocardiography revealed frequent but insignificant mitral and tricuspid valvulopathies. Mitral valve prolapse was present in 3 patients (4.5%). Two patients exhibited significant aortic stenosis (3.0%). While none of the functional and histological parameters diverged significantly between the Abcc6(-/-) and WT mice groups at age of 6 and 12 months, the 24-month-old Abcc6(-/-) mice developed cardiac hypertrophy without contractile dysfunction. CONCLUSIONS: Despite sporadic cases, PXE does not appear to be associated with frequent cardiac complications. However, the development of cardiac hypertrophy in the 24-month-old Abcc6(-/-) mice suggests that old PXE patients might be prone to developing late cardiopathy.

PURPOSE: Complement activation plays an unequivocal role in the pathogenesis of age-related macular degeneration (AMD). More recent evidence suggests an additional role in AMD for the ubiquitin proteasome pathway (UPP), a protein-degradation nanomachinery present in all types of eukaryotic cells. The purpose of this study was to elaborate on these findings and investigate whether the complement system directly contributes to derangements in the UPP through the activated complement components C3a and C5a. METHODS: In the retinal pigment epithelial cells (RPE) of monocyte chemoattractant protein-1-deficient CCL2(-/-) mice, a mouse model that may serve as a model for age-related atrophic degeneration of the RPE, proteasome function was investigated by immunohistochemistry of household (β5) and immuno (β5i) subunit expression. Subsequently, proteasome overall activity was determined using the BodipyFl-Ahx3L3VS probe in primary-cultured human retinal pigment epithelial cells (HRPE) cells that were exposed to different stimuli including C3a and C5a, using confocal laser scanning microscopy and flow cytometry. Gene expression and protein levels of proteasome subunits α7, PA28α, β5, and β5i were also studied in RPE cells after exposure to IFN-γ, C3a, and C5a by real-time PCR and Western blotting. RESULTS: Retinal pigment epithelial cells of CCL2(-/-) mice showed immunoproteasome upregulation. C3a, but not C5a supplementation, induced a decreased proteasome overall activity in HRPE cells, whereas mRNA and protein levels of household proteasome and immunoproteasome subunits were unaffected. CONCLUSIONS: In HRPE cells, C3a induces decreased proteasome-mediated proteolytic activity, whereas in a mouse model of age-related RPE atrophy, the immunoproteasome was upregulated, indicating a possible role for complement-driven posttranslational alterations in proteasome activity in the cascade of pathologic events that result in AMD.

Contains fulltext : 117915.pdf (Publisher’s version ) (Open Access) Leber congenital amaurosis (LCA) is the earliest and most severe retinal degeneration (RD), and the most common cause of incurable blindness diagnosed in children. It is occasionally the presenting symptom of multisystemic ciliopathies which diagnosis will require a specific care of patients. Nineteen LCA genes are currently identified and three of them account for both non-syndromic and syndromic forms of the disease. RD3 (LCA12) was implicated as a LCA gene based on the identification of homozygous truncating mutations in two LCA families despite the screening of large cohorts of patients. Here we provide a comprehensive survey of RD3 mutations and of their clinical expression through the screening of a cohort of 852 patients originating worldwide affected with LCA or early-onset and severe RD. We identified three RD3 mutations in seven unrelated consanguineous LCA families - i.e., a 2 bp deletion and two nonsense mutations - predicted to cause complete loss of function. Five families originating from the Southern Shores of the Mediterranean segregated a similar mutation (c.112C>T, p.R38*) suggesting that this change may have resulted from an ancient founder effect. Considering the low frequency of RD3 carriers, the recurrence risk for LCA in non-consanguineous unions is negligible for both heterozygote and homozygote RD3 individuals. The LCA12 phenotype in our patients is highly similar to those of patients with mutant photoreceptor-specific guanylate cyclase (GUCY2D/LCA1). This observation is consistent with the report of the role of RD3 in trafficking of GUCYs and gives further support to a common mechanism of photoreceptor degeneration in LCA12 and LCA1, i.e., inability to increase cytoplasmic cGMP concentration in outer segments and thus to recover the dark-state. Similar to LCA1, LCA12 patients have no extraocular symptoms despite complete inactivation of both RD3 alleles, supporting the view that extraocular investigations in LCA infants with RD3 mutations should be avoided.

Purpose: The ciliary body (CB) of the human eye consists of the non-pigmented (NPE) and pigmented (PE) neuro-epithelia. We investigated the gene expression of NPE and PE, to shed light on the molecular mechanisms underlying the most important functions of the CB. We also developed molecular signatures for the NPE and PE and studied possible new clues for glaucoma.Methods: We isolated NPE and PE cells from seven healthy human donor eyes using laser dissection microscopy. Next, we performed RNA isolation, amplification, labeling and hybridization against 44xk Agilent microarrays. For microarray conformations, we used a literature study, RT-PCRs, and immunohistochemical stainings. We analyzed the gene expression data with R and with the knowledge database Ingenuity.Results: The gene expression profiles and functional annotations of the NPE and PE were highly similar. We found that the most important functionalities of the NPE and PE were related to developmental processes, neural nature of the tissue, endocrine and metabolic signaling, and immunological functions. In total 1576 genes differed statistically significantly between NPE and PE. From these genes, at least 3 were cell-specific for the NPE and 143 for the PE. Finally, we observed high expression in the (N)PE of 35 genes previously implicated in molecular mechanisms related to glaucoma.Conclusion: Our gene expression analysis suggested that the NPE and PE of the CB were quite similar. Nonetheless, cell-type specific differences were found. The molecular machineries of the human NPE and PE are involved in a range of neuro-endocrinological, developmental and immunological functions, and perhaps glaucoma.

Background: variation in the complement factor H gene (CFH) is associated with risk of late age-related macular degeneration (AMD). Previous studies have been case–control studies in populations of European ancestry with little differentiation in AMD subtype, and insufficient power to confirm or refute effect modification by smoking. Methods: to precisely quantify the association of the single nucleotide polymorphism (SNP rs1061170, ‘Y402H’) with risk of AMD among studies with differing study designs, participant ancestry and AMD grade and to investigate effect modification by smoking, we report two unpublished genetic association studies (n?=?2759) combined with data from 24 published studies (26 studies, 26?494 individuals, including 14?174 cases of AMD) of European ancestry, 10 of which provided individual-level data used to test gene–smoking interaction; and 16 published studies from non-European ancestry. Results: in individuals of European ancestry, there was a significant association between Y402H and late-AMD with a per-allele odds ratio (OR) of 2.27 [95% confidence interval (CI) 2.10–2.45; P?=?1.1?x?10?161]. There was no evidence of effect modification by smoking (P?=?0.75). The frequency of Y402H varied by ancestral origin and the association with AMD in non-Europeans was less clear, limited by paucity of studies. Conclusion: the Y402H variant confers a 2-fold higher risk of late-AMD per copy in individuals of European descent. This was stable to stratification by study design and AMD classification and not modified by smoking. The lack of association in non-Europeans requires further verification. These findings are of direct relevance for disease prediction. New research is needed to ascertain if differences in circulating levels, expression or activity of factor H protein explain the genetic association

Objective: To evaluate the clinical course, genetic etiology, and visual prognosis in patients with cone dystrophy (CD) and cone-rod dystrophy (CRD). Design: Clinic-based, longitudinal, multicenter study. Participants: Consecutive probands with CD (N = 98), CRD (N = 83), and affected relatives (N = 41 and N = 17, respectively) from various ophthalmogenetic clinics in The Netherlands, Belgium, and the United Kingdom. Methods: Data on best-corrected Snellen visual acuity, color vision, ophthalmoscopy, fundus photography, Goldmann perimetry, and full-field standard electroretinogram (ERG) from all patients were registered from medical charts over a mean follow-up of 19 years. The ABCA4, CNGB3, KCNV2, PDE6C, and RPGR genes were analyzed by direct sequencing in autosomal recessive (AR) and X-linked (XL), respectively. Genotyping was not undertaken for autosomal-dominant cases. Main Outcome Measures: The 10-year progression of all clinical parameters and cumulative lifetime risk of low vision and legal blindness were assessed. Results: The mean age onset for CD was 16 years (standard deviation, 11), and of CRD 12 years (standard deviation, 11; P = 0.02). The pattern of inheritance was AR in 92% of CD and 90% of CRD. Ten years after diagnosis, 35% of CD and 51% of CRD had a bull's eye maculopathy; 70% of CRD showed absolute peripheral visual field defects and 37% of CD developed rod involvement on ERG. The mean age of legal blindness was 48 (standard error [SE], 3.1) years in CD, and 35 (SE, 1.1; P

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by mineralization of connective tissue, which leads to pathology in eye, skin and blood vessels. The disease is caused by mutations in ABCC6. To learn more about PXE eye pathology, we analyzed Bruch's membrane (BM) of the eye of an Abcc6 knockout mouse. With age, BM differences between Abcc6-/- and wild type mice became apparent. At two years of age, von Kossa staining indicated clear calcification of BM in Abcc6-/- mice, and not in healthy controls. Electron microscopy revealed BM changes as early as at 10 months of age: Fibrous structures with abnormal high electron-density were present in the central layers of BM of Abcc6-/- mice. EDX (Energy Dispersive X-ray) analysis demonstrated that these structures contained elevated levels of Ca, P and O. Since some of these electron-dense structures showed a banding pattern with periodicity of about 50 nm, they most likely represent calcified collagen fibers. Immunoelectron microscopy showed that the calcified structures were positive for collagen III. Remarkably, the elastic layer of BM appeared to have a normal ultrastructure, even in 2.5 year old Abcc6-/- mice. Our results suggest that Abcc6 deficiency in the mouse causes calcification of BM. While PXE is considered to affect primarily the elastic fibers, we found predominantly mineralization of collagen fibers. (C) 2012 Elsevier Ltd. All rights reserved

Background— Pseudoxanthoma elasticum (PXE) is an inborn disorder of the connective tissue with specific skin, ocular, and cardiovascular disease (CVD) manifestations. Recently, we and others have identified mutations in the gene coding for the ABCC6 transporter in PXE patients with ocular and skin involvement. In the Netherlands, as in the rest of Europe, a particular premature truncation variant ABCC6 (R1141X) was found in a large cohort of PXE patients. Given the association between CVD and PXE, we hypothesized that heterozygosity of this ABCC6 mutation could also confer an increased risk for CVD. Methods and Results— To assess the relationship between the frequent R1141X mutation in the ABCC6 gene and the prevalence of premature coronary artery disease (CAD), we conducted a case-control study of 441 patients under the age of 50 years who had definite CAD and 1057 age- and sex-matched population-based controls who were free of coronary disease. Strikingly, the prevalence of the R1141X mutation was 4.2 times higher among patients than among controls (3.2% versus 0.8%; P P = 0.001). Conclusion— The presence of the R1141X mutation in the ABCC6 gene is associated with a sharply increased risk of premature CAD.

Objective— ABCC6 genetic deficiency underlies pseudoxanthoma elasticum (PXE) in humans, characterized by ectopic calcification, and early cardiac disease. The spectrum of PXE has been noted in Abcc6 -deficient mice, including dystrophic cardiac calcification. We tested the role of Abcc6 in response to cardiac ischemia-reperfusion (I/R) injury. Methods and Results— To determine the role of Abcc6 in cardioprotection, we induced ischemic injury in mice in vivo by occluding the left anterior descending artery (30 minutes) followed by reperfusion (48 hours). Infarct size was increased in Abcc6 -deficient mice compared with wild-type controls. Additionally, an Abcc6 transgene significantly reduced infarct size on the background of a naturally occurring Abcc6 deficiency. There were no differences in cardiac calcification following I/R, but increased cardiac apoptosis was noted in Abcc6 -deficient mice. Previous studies have implicated the bone morphogenetic protein (BMP) signaling pathway in directing calcification, and here we showed that the BMP responsive transcription factors pSmad1/5/8 were increased in hearts of Abcc6 mice. Consistent with this finding, BMP4 and BMP9 were increased and activin receptor-like kinase-2 and endoglin were downregulated in cardiac extracts from Abcc6 -deficient mice versus controls. Conclusion— These data identify Abcc6 as a novel modulator of cardiac myocyte survival after I/R. This cardioprotective mechanism may involve inhibition of the BMP signaling pathway, which modulates apoptosis.

PURPOSE. Transient receptor potential subfamily melastatin (TRPM)1 cation channels of retinal ON-bipolar cells are modulated via a mGluR6 (GMR6) signaling cascade. While lightmicroscopy shows these channels are located on the tips of ON-bipolar cells dendrites, near rod and cone synaptic ribbons, TRPM1 localization at the electron-microscope level is currently not described. The authors report here the ultrastructural localization of TRPM1 in the human retina. METHODS. TRPM1 was localized in postmortem human retinas by immunohistochemistry at both the light and electron microscope levels. Additionally, TRPM1 expression was studied using in situ hybridization, laser dissection microscopy, and PCR techniques. RESULTS. TRPM1-immunoreactivity was located on the dendrites and soma of ON-bipolar cells at the light microscope level. At the electron microscope level TRPM1-immunoreactivity was located on the tips of ON-bipolar cell dendrites that were invaginating cone pedicles and rod spherules. In addition, TRPM1-immunoreactivity was occasionally found on the rod spherules ribbons, suggesting that at least a proportion of rods may also express TRPM1. In situ hybridization showed TRPM1 encoding RNA in inner nuclear layer somata and in some photoreceptors. The presence of TRPM1-RNA in photoreceptors was confirmed by PCR in pure photoreceptor material obtained with a laser dissection microscope. CONCLUSIONS. In the human retina TRPM1 is expressed on ON-bipolar cell dendrites that invaginate photoreceptor terminals. TRPM1 is also expressed on the synaptic ribbons of a subclass of rods, suggesting a dual function for TRPM1 in the ON-pathway. (Invest Ophthalmol Vis Sci. 2011;52:8356‐8362) DOI:10.1167/iovs.11-7575

Open-angle glaucoma (glaucoma) is a major eye disorder characterized by optic disc pathology. Recent genome-wide association studies identified new loci associated with clinically relevant optic disc parameters, such as the optic disc area and vertical cup-disc ratio (VCDR). We examined to what extent these loci are involved in glaucoma. The loci studied include ATOH7, CDC7/TGFBR3 and SALL1 for optic disc area, and CDKN2B, SIX1, SCYL1/LTBP3, CHEK2, ATOH7 and DCLK1 for VCDR. We performed a meta-analysis using data from six independent studies including: the Rotterdam Study (n = 5736), Genetic Research in Isolated Populations combined with Erasmus Rucphen Family study (n = 1750), Amsterdam Glaucoma Study (n = 296) and cohorts from Erlangen and Tubingen (n = 1363), Southampton (n = 702) and deCODE (n = 36 151) resulting in a total of 3161 glaucoma cases and 42 837 controls. Of the eight loci, we found significant evidence (P = 1.41 x 10(-8)) for the association of CDKN2B with glaucoma [odds ratio (OR) for those homozygous for the risk allele: 0.76; 95% confidence interval (CI): 0.70-0.84], for the role of ATOH7 (OR: 1.28; 95% CI: 1.12-1.47) and for SIX1 (OR: 1.20; 95% CI: 1.10-1.31) when adjusting for the number of tested loci. Furthermore, there was a borderline significant association of CDC7/TGFBR3 and SALL1 (both P = 0.04) with glaucoma. In conclusion, we found consistent evidence for three common variants (CDKN2B, ATOH7 and SIX1) significantly associated with glaucoma. These findings may shed new light on the pathophysiological protein pathways leading to glaucoma, and point to pathways involved in the growth and development of the optic nerve.

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

Differential Alu PCR fingerprint cloning was used to isolate a DNA probe from the Xp 11.4→p11.21 region of the human X chromosome. This novel sequence, cpXr318 (DXS742), detects a new submicroscopic deletion interval at the Norrie disease locus (NDP). Combining our data with the consensus genetic map of the proximal short arm of the X chromosome, we propose the physical order Xcen – DXS14 – DXS255 – (DXS426, TIMP) – (DXS742 – ([MAOB – MAOA -DXS7], NDP) – DXS77 – DXS228) – DXS209 – DXS148 -DXS196 – Xpter. The cpXr318 probe and a subclone from a cosmid corresponding to the DXS7 locus were converted into sequence-tagged sites. Finally, DXS742, DXS7, DXS77, and MAOA were integrated into a physical map spanning the Norrie disease locus.

The optic nerve head is involved in many ophthalmic disorders, including common diseases such as myopia and open-angle glaucoma. Two of the most important parameters are the size of the optic disc area and the vertical cup-disc ratio (VCDR). Both are highly heritable but genetically largely undetermined. We performed a meta-analysis of genome-wide association (GWA) data to identify genetic variants associated with optic disc area and VCDR. The gene discovery included 7,360 unrelated individuals from the population-based Rotterdam Study I and Rotterdam Study II cohorts. These cohorts revealed two genome-wide significant loci for optic disc area, rs1192415 on chromosome 1p22 (p = 6.72×10−19) within 117 kb of the CDC7 gene and rs1900004 on chromosome 10q21.3-q22.1 (p = 2.67×10−33) within 10 kb of the ATOH7 gene. They revealed two genome-wide significant loci for VCDR, rs1063192 on chromosome 9p21 (p = 6.15×10−11) in the CDKN2B gene and rs10483727 on chromosome 14q22.3-q23 (p = 2.93×10−10) within 40 kbp of the SIX1 gene. Findings were replicated in two independent Dutch cohorts (Rotterdam Study III and Erasmus Rucphen Family study; N = 3,612), and the TwinsUK cohort (N = 843). Meta-analysis with the replication cohorts confirmed the four loci and revealed a third locus at 16q12.1 associated with optic disc area, and four other loci at 11q13, 13q13, 17q23 (borderline significant), and 22q12.1 for VCDR. ATOH7 was also associated with VCDR independent of optic disc area. Three of the loci were marginally associated with open-angle glaucoma. The protein pathways in which the loci of optic disc area are involved overlap with those identified for VCDR, suggesting a common genetic origin., Author Summary Morphologic characteristics of the optic nerve head are involved in many ophthalmic diseases. Its size, called the optic disc area, is an important measure and has been associated with e.g. myopia and open-angle glaucoma (OAG). Another important and clinical parameter of the optic disc is the vertical cup-disc ratio (VCDR). Although studies have shown a high heritability of optic disc area and VCDR, its genetic determinants are still undetermined. We therefore conducted a genome-wide association (GWA) study on these quantitative traits, using data of over 11,000 Caucasian participants, and related the findings to myopia and OAG. We found evidence for association of three loci with optic disc area: CDC7/TGFBR3 region, ATOH7, and SALL1; and six with VCDR: CDKN2B, SIX1, SCYL1, CHEK2, ATOH7, and DCLK1; and additionally one borderline significant locus: BCAS3. None of the loci could be related to myopia. There was marginal evidence for association of ATOH7, CDKN2B, and SIX1 with OAG, which remains to be confirmed. The present study reveals new insights into the physiological development of the optic nerve and may shed light on the pathophysiological protein pathways leading to (neuro-) ophthalmologic diseases such as OAG.

Background: Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in the developed countries and is caused by both environmental and genetic factors. A recent study (Tuo et al., PNAS) reported an association between AMD and a single nucleotide polymorphism (SNP) (rs3793784) in the ERCC6 (NM_000124) gene. The risk allele also increased ERCC6 expression. ERCC6 is involved in DNA repair and mutations in ERCC6 cause Cockayne syndrome (CS). Amongst others, photosensitivity and pigmentary retinopathy are hallmarks of CS. Methodology/Principal Findings: Separate and combined data from three large AMD case-control studies and a prospective population-based study (The Rotterdam Study) were used to analyse the genetic association between ERCC6 and AMD (2682 AMD cases and 3152 controls). We also measured ERCC6 mRNA levels in retinal pigment epithelium (RPE) cells of healthy and early AMD affected human donor eyes. Rs3793784 conferred a small increase in risk for late AMD in the Dutch population (The Rotterdam and AMRO-NL study), but this was not replicated in two non-European studies (AREDS, Columbia University). In addition, the AMRO-NL study revealed no significant association for 9 other variants spanning ERCC6. Finally, we determined that ERCC6 expression in the human RPE did not depend on rs3793784 genotype, but, interestingly, on AMD status: Early AMD-affected donor eyes had a 50% lower ERCC6 expression than healthy donor eyes (P = 0.018). Conclusions/Significance: Our meta-analysis of four Caucasian cohorts does not replicate the reported association between SNPs in ERCC6 and AMD. Nevertheless, our findings on ERCC6 expression in the RPE suggest that ERCC6 may be functionally involved in AMD. Combining our data with those of the literature, we hypothesize that the AMD-related reduced transcriptional activity of ERCC6 may be caused by diverse, small and heterogeneous genetic and/or environmental determinants.

Background: To identify and functionally annotate cell type-specific gene expression in the human retinal pigment epithelium (RPE), a key tissue involved in age-related macular degeneration and retinitis pigmentosa. Methodology: RPE, photoreceptor and choroidal cells were isolated from selected freshly frozen healthy human donor eyes using laser microdissection. RNA isolation, amplification and hybridization to 44 k microarrays was carried out according to Agilent specifications. Bioinformatics was carried out using Rosetta Resolver, David and Ingenuity software. Principal Findings: Our previous 22 k analysis of the RPE transcriptome showed that the RPE has high levels of protein synthesis, strong energy demands, is exposed to high levels of oxidative stress and a variable degree of inflammation. We currently use a complementary new strategy aimed at the identification and functional annotation of RPE-specific expressed transcripts. This strategy takes advantage of the multilayered cellular structure of the retina and overcomes a number of limitations of previous studies. In triplicate, we compared the transcriptomes of RPE, photoreceptor and choroidal cells and we deduced RPE specific expression. We identified at least 114 entries with RPE-specific gene expression. Thirty-nine of these 114 genes also show high expression in the RPE, comparison with the literature showed that 85% of these 39 were previously identified to be expressed in the RPE. In the group of 114 RPE specific genes there was an overrepresentation of genes involved in (membrane) transport, vision and ophthalmic disease. More fundamentally, we found RPE-specific involvement in the RAR-activation, retinol metabolism and GABA receptor signaling pathways. Conclusions: In this study we provide a further specification and understanding of the RPE transcriptome by identifying and analyzing genes that are specifically expressed in the RPE.

Objective: To investigate the association between variants in the complement component 5 (C5) gene and age-related macular degeneration (AMD). Design: Separate and combined data from 3 large AMD case-control studies and a prospective population-based study (The Rotterdam Study). Participants: A total of 2599 AMD cases and 3458 ethnically matched controls. Methods: Fifteen single nucleotide polymorphisms (SNPs) spanning the C5 gene were initially genotyped in 375 cases and 199 controls from The Netherlands (The Amsterdam/Rotterdam-Netherlands [AMRO-NL] study population). Replication testing of selected SNPs was performed in the Rotterdam Study (NL) and study populations from Southampton, United Kingdom (UK), and New York, United States (US). Main Outcome Measures: Early and late stages of prevalent and incident AMD, graded according to (a modification of) the international grading and classification system of AMD. Results: Significant allelic or genotypic associations between 8 C5 SNPs and AMD were found in the AMRO-NL study and this risk seemed to be independent of CFH Y402H, LOC387715 A69S, age, and gender. None of these findings could be confirmed consistently in 3 replication populations. Conclusions: Although the complement pathway, including C5, plays a crucial role in AMD, and the C5 protein is present in drusen, no consistent significant associations between C5 SNPs and AMD were found in any of these studies. The implications for genetic screening of AMD are discussed. Financial Disclosure(s): The authors have no proprietary or commercial interest in any of the materials discussed in this article. Ophthalmology 2010; 117: 500-511 (C) 2010 by the American Academy of Ophthalmology.

Eleven infant boys presented with chin-up head posture, tonic downgaze and, on attempted upgaze, large-amplitude upward saccades with deceleration during the slow phase downward. The gaze-evoked upward saccades disappeared at the age of 2 or 3 years. In addition, they had high-frequency, small-amplitude horizontal pendular nystagmus that remained. Among these infant boys were 2 pairs of maternally related half-brothers, 2 cousins, and 2 siblings. Visual acuity ranged from 0.1 to 0.6, ERG-amplitudes (both A- and B-wave) were reduced, and severe myopia was found in 5 cases. Eight boys had CACNA1F mutations, and 1 boy had a NYX mutation, compatible with incomplete or complete congenital stationary nightblindness (iCSNB or cCSNB), respectively. This points to a defective synapse between the rod and the ON-bipolar cell causing the motility disorder: CACNA1F is located on the rod side of this synapse, whereas NYX is located on the side of the ON-bipolar cell. The coexistence of horizontal and vertical nystagmus has been previously described in dark-reared cats.

Contains fulltext : 87254.pdf (Publisher’s version ) (Closed access) PURPOSE: To describe the disease course in patients with vitelliform macular dystrophy (VMD) with a Best1 mutation and to determine the association between Best1 genotype and visual prognosis. DESIGN: Consecutive case series. PARTICIPANTS: Fifty-three patients with VMD with Best1 mutations from 27 Dutch families, aged 11 to 87 years. METHODS: Best-corrected visual acuity (VA), fundus appearance, and Arden ratio on the electro-oculogram (EOG) during clinical follow-up were assessed from medical records. Mutation analysis of the Best1 gene was performed on DNA samples using denaturing high-pressure liquid chromatography and direct sequencing. MAIN OUTCOME MEASURES: Cumulative lifetime risk of visual decline below 0.5, 0.3, and 0.1 for the entire group and stratified for genotype. RESULTS: Median age of onset of visual symptoms was 33 years (range: 2-78). The cumulative risk of VA below 0.5 (20/40) was 50% at 55 years and 75% at 66 years. The cumulative risk of decline less than 0.3 (20/63) was 50% by age 66 years and 75% by age 74 years. Two patients progressed to VA less than 0.1 (20/200). Fourteen different mutations were found. Most patients (96%) had missense mutations; the Thr6Pro, Ala10Val, and Tyr227Asn mutations were most common. Visual decline was significantly faster in patients with an Ala10Val mutation than either the Thr6Pro or the Tyr227Asn mutation (P=0.001). CONCLUSIONS: Age of onset of visual symptoms varies greatly among patients with VMD. All patients show a gradual decrease in VA, and most progress to visual impairment at a relatively late age. Our data suggest a phenotype-genotype correlation, because the Ala10Val mutation has a more rapid disease progression than other common mutations. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article. 01 juli 2010

Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impaired night vision and variable decreased visual acuity. We report here that six out of eight female probands with autosomal-recessive complete CSNB (cCSNB) had mutations in TRPM1, a retinal transient receptor potential (TRP) cation channel gene. These data suggest that TRMP1 mutations are a major cause of autosomal-recessive CSNB in individuals of European ancestry. We localized TRPM I in human retina to the ON bipolar cell dendrites in the outer plexifom layer. Our results suggest that in humans, TRPM1 is the channel gated by the mGluR6 (GRM6) signaling cascade, which results in the light-evoked response of ON bipolar cells. Finally, we showed that detailed electroretinography is an effective way to discriminate among patients with mutations in either TRPM1 or GRM6, another autosomal-recessive cCSNB disease gene. These results add to the growing importance of the diverse group of TRP channels in human disease and also provide new insights into retinal circuitry

Background To determine level, variability and functional annotation of gene expression of the human retinal pigment epithelium (RPE), the key tissue involved in retinal diseases like age-related macular degeneration and retinitis pigmentosa. Macular RPE cells from six selected healthy human donor eyes (aged 63–78 years) were laser dissected and used for 22k microarray studies (Agilent technologies). Data were analyzed with Rosetta Resolver, the web tool DAVID and Ingenuity software. Results In total, we identified 19,746 array entries with significant expression in the RPE. Gene expression was analyzed according to expression levels, interindividual variability and functionality. A group of highly (n = 2,194) expressed RPE genes showed an overrepresentation of genes of the oxidative phosphorylation, ATP synthesis and ribosome pathways. In the group of moderately expressed genes (n = 8,776) genes of the phosphatidylinositol signaling system and aminosugars metabolism were overrepresented. As expected, the top 10 percent (n = 2,194) of genes with the highest interindividual differences in expression showed functional overrepresentation of the complement cascade, essential in inflammation in age-related macular degeneration, and other signaling pathways. Surprisingly, this same category also includes the genes involved in Bruch's membrane (BM) composition. Among the top 10 percent of genes with low interindividual differences, there was an overrepresentation of genes involved in local glycosaminoglycan turnover. Conclusion Our study expands current knowledge of the RPE transcriptome by assigning new genes, and adding data about expression level and interindividual variation. Functional annotation suggests that the RPE has high levels of protein synthesis, strong energy demands, and is exposed to high levels of oxidative stress and a variable degree of inflammation. Our data sheds new light on the molecular composition of BM, adjacent to the RPE, and is useful for candidate retinal disease gene identification or gene dose-dependent therapeutic studies.

Two recent studies have provided conflicting evidence about the association between the gene encoding serpin peptidase inhibitor, clade G, member 1 (SERPING1) and age-related macular degeneration (AMD). An Article in The Lancet1 suggested that a variant (rs2511989) in intron 6 of SERPING1 conferred protection against the development of AMD in case–control studies from the UK and USA. A follow-up study2 showed the absence of any association in two US case–control studies, including the AREDS cohort.3 As a member of the classical complement pathway, SERPING1 is a plausible candidate gene for AMD since several genes encoding proteins involved in the innate immune response (CFH,4–7 C2/CFB,8 C39,10), are robustly associated with the development of this disorder. To test this hypothesis further, we genotyped rs2511989 in seven large case–control studies involving individuals of European descent (4881 patients with AMD and 2842 matched controls; see webappendix for details). Samples from all cohorts in the seven centres were collected and genotyped independently to minimise data selection, analysis, or interpretation bias. Data from the seven studies are summarised in the table and figure. They show—both independently and collectively—that there is no significant association between the rs2511989 variant in the SERPING1 gene and AMD. In fact, data from no single study suggested even a trend towards protective association (p>0·3 in all cohorts; table); one cohort (AREDS) suggested an opposite, marginally significant (p=0·03), trend towards increased susceptibility. However, the study by Park and colleagues2 found no association after genotyping more AREDS samples, thereby suggesting that the marginal association detected in our study is due to a limited sample size. The minor allele frequency (MAF) for the rs2511989 single nucleotide polymorphism (SNP) varied substantially between studies, from 0·35 and 0·45 both in AMD patients (average 0·40) and in controls matched for age and ethnic group (average 0·40). However, there was almost no difference in the MAF between patients and controls within all, except for the AREDS, studies. When summarising the data across cohorts, the MAF of the rs2511989 SNP was practically identical in 2842 controls and in 4881 patients with AMD (0·40), clearly showing no association (p=0·99; odds ratio 1, table). The same lack of significance was seen for all AMD subphenotypes, early AMD, geographic atrophy, and choroidal neovascularisation, when analysed separately. Since the other SNPs (eg, rs2509879, rs2511988, etc) reported in the study by Ennis and colleagues are in high linkage disequilibrium with the rs2511989 variant,1 the MAFs for those confirmed the rs2511989 data (not shown),2 as did a combined analysis of all three studies by the Mantel-Haenszel fixed effects model (figure). Figure Forest plot of the combined analysis of ten case–control cohorts from the three studies Table Genotype counts and association analysis of the SERPING1 rs2511989 variant in seven studies Excluding experimental errors discussed by Park and colleagues,2 the difference between our collective findings and those published by Ennis and colleagues could be explained by the two following scenarios: Whereas most methods used in our study closely match those used by Ennis and colleagues (genotyping methods, selection criteria for AMD patients, etc), the selection and matching of controls in the seven centres in this study were more rigorous by age and, in some centres (Germany, the Netherlands), also by ethnicity (see webappendix). The significantly higher MAFs in controls in both cohorts used in Ennis and colleagues’ study (average 0·46 vs 0·40 in this study and the one by Park and colleagues2, p=8×10−6) could be explained by the fact that controls were younger than AMD patients.1 Other possible sources for the difference in results could be non-random sampling of study populations or population substructure (stratification). For example, although there was no significant difference in MAFs between any of the AMD and control cohorts within any one study, the same numbers varied significantly between some studies (MAF 0·35 in AMD cases from New York vs >0·41 in all other cohorts), probably originating from varied ethnic composition within populations of European descent. The New York (Columbia University) study population originates mainly from eastern Europe, whereas that from Iowa from western Europe and Scandinavia. At the same time it is of interest that one of the two cohorts in Ennis and colleagues’ study (248 AMD cases) was collected at exactly the same location, the University of Iowa, as was one of the seven cohorts in our study (368 AMD cases). However, whereas the frequencies of the minor allele in Iowa controls were very similar in the two studies (0·44 vs 0·42), they differed significantly in patients with AMD (0·35 vs 0·44; p=0·001). In summary, analysis of the rs2511989 variant in SERPING1 in seven large, well characterised case–control studies did not confirm an association between this variant and AMD. Since there is also no evidence of a functional role for this variant, it is unlikely that the SERPING1 gene has a significant, if any, role in the aetiology of AMD.

Accumulating evidence demonstrates that disregulation of the local inflammatory and immunologic response is an important causal pathway in age-related macular degeneration. Initial proof of this insight was provided by histopathology studies that showed that drusen contain complement components, complement regulators, immunoglobulins, and anaphylatoxins.1 Recently, the role of inflammation in AMD was further established by multiple genetic studies. Genes involved in the complement pathway, such as the complement factor H (CFH) gene, the complement factor B (FB) gene, and the complement component 2 (C2) gene have repetitively been associated with AMD.2–8 The general hypothesis is that dys-function of these genes may lead to an increase in complement activation and a high release of proinflammatory proteins, which results in an augmentation of the local inflammatory response. It is currently unclear whether inflammatory pathways other than complement regulation are involved in AMD pathogenesis. The immune system detects and responds to infection mainly through a family of pattern recognition receptors called toll-like receptors (TLRs).9 These receptors recognize a wide range of microbial molecules (e.g., lipopolysaccharide, peptidoglycan, lipopeptide) and induce phagocytosis after binding. They are expressed by many immune cells, as well as by corneal cells and retinal pigment epithelium (RPE) cells.10 TLRs trigger a signal transduction cascade that results in activation of transcription factor NF-κB, which leads to increased expression of proinflammatory genes.11,12 A significant association between a common single nucleotide polymorphism (SNP; rs4986790, D299G) in the TLR4 gene and AMD was reported by Zareparsi et al.13 This genetic variant alters the extracellular domain of the receptor, which interrupts the signaling transduction cascade14 and interferes with the expression of genes such as TNF-a, IL-1, IL-6, monocyte chemo-attractant protein-1 (MCP-1 or CCL2), and its cognate receptor C-C chemokine receptor-2 (CCR2).15 Although biologically plausible, the reported association between TLR4 and AMD awaits confirmation.16 CCL2 and CCR2 are key mediators in the infiltration of monocytes from blood into foci of inflammation. The CCL2 protein is ubiquitously expressed and exerts its effect after binding to its receptor CCR2, which leads to actin rearrangement, shape change, and movement of monocytes.17 Evidence of a potential role of CCL2 and CCR2 in AMD was provided by Ambati et al.,18 who showed that aging mice deficient in these genes develop hallmarks of AMD (i.e., accumulation of drusen and lipofuscin, photoreceptor atrophy, and choroidal neovascularization). Similar to human AMD, complement-associated proteins such as C5, IgG, vitronectin, CD46, and serum amyloid P component were also present in the RPE of these mice. The occurrence of AMD-like disease in these knockout mice raises the question of whether CCL2 and CCR2 play a role in human AMD. In this study, we assessed the association with the D299G allele of TLR4 in independent case— control studies from the Netherlands and the United States. Furthermore, we performed a comprehensive genetic analysis of the CCL2 and CCR2 genes in the Dutch study and validated common variants of these genes in the U.S. study. We also performed quantitative (q)PCR experiments to investigate whether mRNA expression of these genes in the retinal pigment epithelium was different between individuals with AMD and healthy control subjects.

Mutations in the CSB gene cause Cockayne syndrome (CS), a DNA repair disorder characterized by UV sensitivity and severe physical and neurological impairment. CSB functions in the transcription-coupled repair subpathway of nucleotide excision repair. This function may explain the UV sensitivity but hardly clarifies the other CS symptoms. Many of these, including retinopathy, are associated with premature aging. We studied eye pathology in a mouse model for CS. Csb(m/m) mice were hypersensitive to UV light and developed epithelial hyperplasia and squamous cell carcinomas in the cornea, which underscores the importance of transcription-coupled repair of photolesions in the mouse. In addition, we observed a spontaneous loss of retinal photoreceptor cells with age in the Csb(m/m) retina, resulting in a 60% decrease in the number of rods by the age of 18 months. Importantly, when Csb(m/m) mice (as well as Csa(-/-) mice) were exposed to 10 Gy of ionizing radiation, we noticed an increase in apoptotic photoreceptor cells, which was not observed in wild-type animals. This finding, together with our observation that the expression of established oxidative stress marker genes is upregulated in the Csb(m/m) retina, suggests that (endogenous) oxidative DNA lesions play a role in this CS-specific premature-aging feature and supports the oxidative DNA damage theory of aging.

Beside calcification, the impact of ABCC6 deficiency on the vasculature remains unclear. We investigated arterial structure and function in Abcc6-/- mice, a model of the human Pseudoxanthoma Elasticum (PXE). Arterial calcium accumulation determined by atomic absorption spectrometry was 1.5 – to 2-fold higher in Abcc6-/- than in wild-type mice. Calcium also accumulated locally leading to a specific punctuated pattern. Abcc6-/- mesenteric arteries mounted on a wire myograph displayed slight increase in arterial vasoconstrictor tone in response to phenylephrine and thromboxane A2. Interestingly, myogenic tone (Bayliss effect) determined using a pressure myograph was significantly elevated in Abcc6-/- compared to wild type arteries. Arterial blood pressure was not significantly modified in Abcc6-/- animals, despite higher variability. These changes were accompanied with deregulated gene expression (RTqPCR) in both liver and resistance arteries. Old Abcc6-/- mouse mesenteric arteries expressed markers of both osteogenic (Runx2, opn) and chondrogenic lineage (Sox9, col2a1). Surprisingly Enpp1 and Alpl genes encoding ectonucleotide pyrophosphatase/phosphodiesterase 1 and alkaline phosphatase were deregulated within Abcc6-/- liver and this was corroborated with reduced alkaline phosphatase circulating levels in PXE patients. As a conclusion, scattered calcium depositions result from osteochondrogenic transdifferentiation of vascular cells. The lower elasticity and increased myogenic tone evidenced in aged Abcc6-/- mice suggest a reduced control of local blood flow, which in turn may alter vascular homeostasis. Our findings argue in favor of a deregulated arterial function and may help to decipher consequences of ABCC6 deficiency since PXE is a significant risk factor for small vessel disease and particularly ischemic stroke.

In response to injury, the adult mammalian retina shows signs of structural remodeling, possibly in an attempt to preserve or regain some of its functional neural connections. In order to study the mechanisms involved in injury-induced plasticity, we have studied changes in growth associated protein 43 (GAP-43) after retinal ischemia/reperfusion in the rat. GAP-43 is a marker for neuronal remodeling and is involved in synapse formation. Ischemic injury of the rat retina was induced by 60 min of ischemia followed by reperfusion times varying from 2 h up to 4 weeks. GAP-43 mRNA levels were significantly increased between 12 h and 72 h reperfusion with a peak around 24 h. GAP-43 specific antibodies showed that the total amount of GAP-43 labeling in the inner plexiform layer was diminished after 12 h of reperfusion by approximately 35% and remained at this level up to 1 week postischemia despite the reduction in thickness of this layer during this period resulting from the ischemia-induced cell loss. At 2 and 4 weeks reperfusion, the amount of labeling was reduced by 70%, simultaneously with a decrease of GAP-43 transcript level. Between 72 h up to 2 weeks postischemia, the induction of intense GAP-43 labeling was observed in NeuN- and β-tubulin-positive ganglion cell somata and in horizontally and vertically oriented processes in the inner plexiform layer. Ischemia also induced GAP-43 expression in some GFAP-positive Muller cells. Double-labeling showed that in controls and after ischemia GAP-43 was expressed by some amacrine cells of the glycinergic (glycine transporter 1), calretinin-positive, and dopaminergic (tyrosine hydroxylase) subpopulations. No increase of GAP-43 expression levels was found in these amacrine cells. The results demonstrate that ganglion cells show an elevated expression of GAP-43 after ischemia-inflicted damage. These findings suggest a temporal window during which ganglion cells may remodel their neuronal network in the damaged retina.

CONTEXT: The evidence that inflammation is an important pathway in age-related macular degeneration (AMD) is growing. Recent case-control studies demonstrated an association between the complement factor H (CFH) gene, a regulator of complement, and AMD. OBJECTIVES: To assess the associations between the CFH gene and AMD in the general population and to investigate the modifying effect of smoking, serum inflammatory markers, and genetic variation of C-reactive protein (CRP). DESIGN, SETTING, AND PARTICIPANTS: Population-based, prospective cohort study of individuals aged 55 years or older (enrollment between March 20, 1990, and July 31, 1993, and 3 follow-up examinations that were performed between September 1, 1993, and December 31, 2004) in Rotterdam, the Netherlands. The CFH Y402H polymorphism was determined in a total of 5681 individuals. Information on smoking, erythrocyte sedimentation rate, CRP serum levels, and haplotypes of the CRP gene were assessed at baseline. MAIN OUTCOME MEASURES: All severity stages of prevalent and incident AMD, graded according to the international classification and grading system for AMD. RESULTS: The frequency of CFH Y402H was 36.2% (4116/11,362 alleles). At baseline, there were 2062 persons (36.3%) with any type of AMD (prevalent cases), including 78 (1.4%) with late AMD (stage 4). During follow-up (mean, 8 years; median, 10 years), 1649 (35.5%) of 4642 participants progressed to a higher stage of AMD (incident cases), including 93 (5.6%) who developed late AMD. The odds ratio (OR) of AMD increased in an allele-dose manner with 2.00 (95% confidence interval [CI], 1.56-2.55) for stage 2 AMD, 4.58 (95% CI, 2.82-7.44) for stage 3 AMD, and 11.02 (95% CI, 6.82-11.81) for stage 4 (late, vision threatening) AMD for homozygous persons. Cumulative risks calculated by Kaplan-Meier analysis of late AMD by age 95 years were 48.3% for homozygotes, 42.6% for heterozygotes, and 21.9% for noncarriers. The population-attributable risk for CFH Y402H was 54.0%. Elevated erythrocyte sedimentation rates further increased the OR to 20.2 (95% CI, 9.5-43.0), elevated serum CRP levels to 27.7 (95% CI, 10.7-72.0), and smoking to 34.0 (95% CI, 13.0-88.6) for homozygotes compared with noncarriers without these determinants. The CRP haplotypes conferring high levels of CRP significantly increased the effect of CFH Y402H (P

Age-related macular degeneration (ARMD) is the leading cause of irreversible visual loss in the Western world, affecting approximately 25 million people worldwide. The pathogenesis is complex and missense mutations in FBLN5 have been reported in association with ARMD. We have investigated the role of fibulin 5 in ARMD by completing the first European study of the gene FBLN5 in ARMD (using 2 European cohorts of 805 ARMD patients and 279 controls) and by determining the functional effects of the missense mutations on fibulin 5 expression. We also correlated the FBLN5 genotype with the ARMD phenotype. We found two novel sequence changes in ARMD patients that were absent in controls and expressed these and the other nine reported FBLN5 mutations associated with ARMD and two associated with the autosomal recessive disease cutis laxa. Fibulin 5 secretion was significantly reduced (P

Context: The evidence that inflammation is an important pathway in age-related macular degeneration (AMD) is growing. Recent case-control studies demonstrated an association between the complement factor H (CFH) gene, a regulator of complement, and AMD. Objectives: To assess the associations between the CFH gene and AMD in the general population and to investigate the modifying effect of smoking, serum inflammatory markers, and genetic variation of C-reactive protein (CRP). Design, Setting, and Participants: Population-based, prospective cohort study of individuals aged 55 years or older (enrollment between March 20, 1990, and July 31, 1993, and 3 follow-up examinations that were performed between September 1, 1993, and December 31, 2004) in Rotterdam, the Netherlands. The CFH Y402H polymorphism was determined in a total of 5681 individuals. Information on smoking, erythrocyte sedimentation rate, CRP serum levels, and haplotypes of the CRP gene were assessed at baseline. Main Outcome Measures: All severity stages of prevalent and incident AMD, graded according to the international classification and grading system for AMD.

X-linked juvenile retinoschisis (RS) is a progressive vitreoretinal degeneration localised in Xp22.1-p22.2. A human homologue of the retinal degeneration gene C (rdgC), a gene that in Drosophila melanogaster prevents light-induced retinal degeneration, was localised in the RS obligate gene region. We have tested the gene, designated PPEF in humans, as a candidate gene in RS patients using RT-PCR and the protein truncation test on RNA and SSCP on DNA. No mutations were identified, making it highly unlikely that PPEF is the gene implicated in RS. The data presented facilitate mutation analysis of the PPEF gene in other diseases which have been or will be localised to this region.

Pseudoxanthoma elasticum (PXE) is a hereditary disorder of connective tissue with skin, cardiovascular, and visual involvement. In familial cases, PXE usually segregates in an autosomal recessive fashion. The aim of this manuscript is to describe an efficient strategy for DNA diagnosis of PXE. The two most frequent mutations, R1141X and an ABCC6 del exons 23-29, as well as a core set of mutations, were identified by restriction enzyme digestion and size separation on agarose gels. Next, in the remaining patient group in which only one or no mutant allele was found, the complete coding sequence was analyzed using denaturing high-performance liquid chromatography (dHPLC). All variations found were confirmed by direct DNA sequencing. Finally, Southern blot was used to investigate the potential presence of small or large deletions. Twenty different mutations, including two novel mutations in the ABCC6 gene, were identified in 80.3% of the 76 patients, and 58.6% of the 152 ABCC6 alleles analyzed. With this strategy, 70 (78.7%) out of 89 mutant alleles could be detected within a week. We conclude that this strategy leads to both reliable and time-saving screening for mutations in the ABCC6 gene in sporadic cases and in families with PXE.

Background: Pseudoxanthoma elasticurn (PXE) is an inherited disorder of elastic tissue. We recently found that heterozygosity for the frequent (0.8% prevalence in Dutch population) R 1141 X mutation in the PXE gene coding for the ABCC6 transporter, is associated with a fourfold risk of premature coronary artery disease. Yet, it is not clear whether or not heterozygosity for this mutation results in a mild PXE phenotype. The objective of our study was to determine if skin and/or eye abnormalities related to a PXE phenotype could be found in patients with premature coronary artery disease, with and without the R 1141 X mutation. Methods: R1141X mutation carriers with premature coronary artery disease (cases) and patients with premature coronary artery disease with no-or not known-mutation (controls) were studied. Cases and controls were examined for PXE-like skin changes and retinal angioid streaks, peau d'orange or pigment epithelium changes. Results: 7 cases and 31 controls were analysed. In both the mutation-positive and the control group, skin inspection and eye fundus examination did not reveal any dermatological or ocular signs of PXE. Conclusions: Carriers for the ABCC6 R 114 1 X mutation, which is frequent and confers a high risk of premature coronary artery disease, do not commonly have skin or eye abnormalities consistent with a mild PXE phenotype. (c) 2004 Elsevier Ireland Ltd. All rights reserved

Skin biopsies in patients with pseudoxanthoma elasticum (PXE) show elastic fiber fragmentation and calcium and proteoglycans accumulation. Assuming such changes to be present in the artery wall as well, we studied the influence of such alterations on function and structure of the human common carotid artery (CCA). Indeed, elastin fragmentation and increased calcium and proteoglycans content were present in the arteries of the two PXE patients examined. Internal diameter, distension and intima-media thickness (IMT) in the CCA of PXE patients (n = 19) and controls (n = 39) were determined by ultrasound (US). Pulse pressure was assessed in the brachial artery. The distensibility and compliance coefficients as well as the Young's modulus were calculated. Diameter and pulse pressure were not significantly different in PXE patients and controls. The distensibility and compliance coefficients were significantly greater in older PXE patients than in older controls. The distensibility coefficient decreased with age in both PXE patients and in controls. Unlike in controls, the compliance coefficient did not decrease and the Young's modulus barely increased with age in PXE patients. IMT was significantly greater at both younger and older ages and the Young's modulus was significantly smaller at older ages in PXE patients than in controls. The carotid artery is thicker and more elastic in PXE patients than in control subjects; differences are most pronounced at older ages. These alterations might be explained by the elastin fragmentation and proteoglycans accumulation as observed in these patients.

Purpose: To investigate the prevalence of myocilin (MYOC) mutations in a population-based sample of open-angle glaucoma (OAG) cases and to describe a family with both juvenile and adult onset OAG caused by a mutation in MYOC. Methods: MYOC was screened in cases derived from the Rotterdam Study in the Netherlands. Definite OAG was defined as a glaucomatous optic neuropathy together with a glaucomatous visual field defect. Upon the identification of the Asn480Lys mutation in one case, seven additional family members were studied. To test for a founder effect with earlier reported families with this mutation, the haplotypes of MYOC flanking markers D1S2851, D1S242, D1S218, and D I S 1165 were compared. Results: Seven sequence alterations in MYOC were found in 14 of 47 OAG cases; six of these were also found in controls. In one case, an Asn480Lys mutation was found. In relatives of the latter patient, the phenotype ranged from a glaucomatous optic neuropathy without visual field defect in a 70-year-old patient to severely affected optic discs and a remaining temporal remnant in a 34-year-old patient; those without the mutation had no signs of OAG. Haplotype analysis suggested a different origin of the mutation. Conclusions: The prevalence of MYOC mutations (2.2%) was similar to that found in hospital-based studies. Although mutations in MYOC are rare, relatives carrying this mutation run a high risk of developing the disease. Instead of submitting all members of a family with the Asn480Lys mutation to frequent follow-up, medical care can be restricted to those carrying the mutation

BACKGROUND: The neuroepithelia of the choroid plexus (CP) in the brain and the ciliary body (CB) of the eye have common embryological origins and share similar micro-structure and functions. The CP epithelium (CPE) and the non-pigmented epithelium (NPE) of the CB produce the cerebrospinal fluid (CSF) and the aqueous humor (AH) respectively. Production and outflow of the CSF determine the intracranial pressure (ICP); production and outflow of the AH determine the intraocular pressure (IOP). Together, the IOP and ICP determine the translaminar pressure on the optic disc which may be involved in the pathophysiology of primary open angle glaucoma (POAG). The aim of this study was to compare the molecular machinery of the secretory neuroepithelia of the CP and CB (CPE versus NPE) and to determine their potential role in POAG.METHODS: We compared the transcriptomes and functional annotations of healthy human CPE and NPE. Microarray and bioinformatic studies were performed using an Agilent platform and the Ingenuity Knowledge Database (IPA).RESULTS: Based on gene expression profiles, we found many similar functions for the CPE and NPE including molecular transport, neurological disease processes, and immunological functions. With commonly-used selection criteria (fold-change > 2.5, p-value 5, p-value CONCLUSIONS: The transcriptomes of the CPE and NPE were less similar than we previously anticipated. High expression of CSF/AH production genes and candidate POAG disease genes in the CPE and NPE suggest that both might be involved in POAG.

Incomplete X-linked congenital stationary night blindness (CSNB) is a recessive, non-progressive eye disorder characterized by abnormal electroretinogram and psychophysical testing and can include impaired night vision, decreased visual acuity, myopia, nystagmus, and strabismus. Including the 20 families previously reported (Bech-Hansen et al. 1998b), we have now analyzed patients from a total of 36 families with incomplete CSNB and identified 20 different mutations in the calcium channel gene CACNA1F. Three of the mutations account for incomplete CSNB in two or more families, and a founder effect is clearly demonstrable for one of these mutations. Of the 20 mutations identified, 14 (70%) are predicted to cause premature protein truncation and six (30%) to cause amino acid substitutions or deletions at conserved positions in the alpha1F protein. In characterizing transcripts of CACNA1F we have identified several splice variants and defined a prototypical sequence based on the location of mutations in splice variants and comparison with the mouse orthologue, Cacnalf.

X-linked retinoschisis (XLRS) is the most common cause of juvenile macular degeneration in males, resulting in vision loss early in life. The gene involved in XLRS was identified recently. It encodes a protein with a disoidin domain, suggested to be involved in cell-cell interactions. We have screened the gene for mutations in 234 familial and sporadic retinoschisis cases and identified 82 different mutations in 214 (91%). Thirty one mutations were found more than once, i.e. 2-10 times, with the exception of the 214G→A mutation which was found in 34 apparently unrelated cases. The origin of the patients, the linkage data and the site of the mutations (mainly CG dinucleotides) indicate that most recurrent mutations had independent origins and thus suggest the existence of a significant new mutation rate in XLRS1. The mutations identified cover the entire spectrum, from small intra-genic deletions (7%), to nonsense (6%), missense (75%), small frameshifting insertions/deletions (6%) and splice site mutations (6%). Since, regardless of the mutation type, no females with a typical RS phenotype were identified, RS seems to be caused by loss-of-function mutations only. Mutations occurred non-randomly, with hotspots at several CG dinudeotides and a C6 stretch. Exons 1-3 contained few, mainly translation-truncating mutations, arguing against an important functional role for this segment of the protein. Exons 4-6, encoding the discoidin domain, contained most, mainly missense mutations. An alignment of 32 discoidin domain proteins was constructed to reveal the consensus sequence and to deduce the functional importance of the missense mutations identified. The mutation analysis revealed a high preponderance of mutations involving or creating cysteine residues, pointing to sites important for the tertiary folding and/or protein function, and highlights several amino acids which may be involved in XLRS1-specific protein-protein interactions. Despite the enormous mutation heterogeneity, patients have relatively uniform clinical manifestations although with great intra-familial variation in age at onset and progression.