scholarly journals EYS mutations and implementation of minigene assay for variant classification in EYS-associated retinitis pigmentosa in northern Sweden

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ida Maria Westin ◽  
Frida Jonsson ◽  
Lennart Österman ◽  
Monica Holmberg ◽  
Marie Burstedt ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Exon Skipping ◽  
Molecular Testing ◽  
Northern Sweden ◽  
Human Embryonic Kidney Cells ◽  
Targeted Ngs ◽  
Minigene Assay

AbstractRetinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of inherited retinal degenerations. The ortholog of Drosophila eyes shut/spacemaker, EYS on chromosome 6q12 is a major genetic cause of recessive RP worldwide, with prevalence of 5 to 30%. In this study, by using targeted NGS, MLPA and Sanger sequencing we uncovered the EYS gene as one of the most common genetic cause of autosomal recessive RP in northern Sweden accounting for at least 16%. The most frequent pathogenic variant was c.8648_8655del that in some patients was identified in cis with c.1155T>A, indicating Finnish ancestry. We also showed that two novel EYS variants, c.2992_2992+6delinsTG and c.3877+1G>A caused exon skipping in human embryonic kidney cells, HEK293T and in retinal pigment epithelium cells, ARPE-19 demonstrating that in vitro minigene assay is a straightforward tool for the analysis of intronic variants. We conclude, that whenever it is possible, functional testing is of great value for classification of intronic EYS variants and the following molecular testing of family members, their genetic counselling, and inclusion of RP patients to future treatment studies.

scholarly journals EYS Mutations and Implementation of Minigene Assay for Variant Classification in EYS-Associated Retinitis Pigmentosa in Northern Sweden

2020 ◽  
Author(s):  
Ida Maria Westin ◽  
Frida Jonsson ◽  
Lennart Österman ◽  
Monica Holmberg ◽  
Marie Burstedt ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Exon Skipping ◽  
Molecular Testing ◽  
Northern Sweden ◽  
Human Embryonic Kidney Cells ◽  
Targeted Ngs ◽  
Minigene Assay

Abstract Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of inherited retinal degenerations. The ortholog of Drosophila eyes shut/spacemaker, EYS on chromosome 6q12 is a major genetic cause of recessive RP worldwide, with prevalence of 5 to 30%. In this study, by using targeted NGS, MLPA and Sanger sequencing we uncovered the EYS gene as the second most common genetic cause of RP in northern Sweden accounting for at least 18%. The most frequent pathogenic variant was c.8648_8655del that in some patients was identified in cis with c.1155T>A, indicating Finnish ancestry. We also showed that two novel EYS variants, c.2992_2992+6delinsTG and c.3877+1G>A caused exon skipping in human embryonic kidney cells, HEK293T and in retinal pigment epithelium cells, ARPE-19 demonstrating that in vitro minigene assay is a straightforward tool for the analysis of intronic variants. We conclude, that whenever it is possible, functional testing is of great value for classification of intronic EYS variants and the following molecular testing of family members, their genetic counselling, and inclusion of RP patients to future treatment studies.

scholarly journals Bestrophinopathies: perspectives on clinical disease, Bestrophin-1 function and developing therapies

2021 ◽  
Vol 13 ◽  
pp. 251584142199719
Author(s):  
Simranjeet Singh Grewal ◽  
Joseph J. Smith ◽  
Amanda-Jayne F. Carr
Keyword(s):  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Induced Pluripotent Stem Cell ◽  
Underlying Disease ◽  
Incomplete Penetrance ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
High Acuity ◽  
Induced Pluripotent

Bestrophinopathies are a group of clinically distinct inherited retinal dystrophies that typically affect the macular region, an area synonymous with central high acuity vision. This spectrum of disorders is caused by mutations in bestrophin1 ( BEST1), a protein thought to act as a Ca2+-activated Cl- channel in the retinal pigment epithelium (RPE) of the eye. Although bestrophinopathies are rare, over 250 individual pathological mutations have been identified in the BEST1 gene, with many reported to have various clinical expressivity and incomplete penetrance. With no current clinical treatments available for patients with bestrophinopathies, understanding the role of BEST1 in cells and the pathological pathways underlying disease has become a priority. Induced pluripotent stem cell (iPSC) technology is helping to uncover disease mechanisms and develop treatments for RPE diseases, like bestrophinopathies. Here, we provide a comprehensive review of the pathophysiology of bestrophinopathies and highlight how patient-derived iPSC-RPE are being used to test new genomic therapies in vitro.

scholarly journals Ocular Toxoplasmosis: Mechanisms of Retinal Infection and Experimental Models

Parasitologia ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 50-60
Author(s):  
Veronica Rodriguez Fernandez ◽  
Giovanni Casini ◽  
Fabrizio Bruschi
Keyword(s):  
Ex Vivo ◽  
Cell Damage ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Treatment Strategies ◽  
Experimental Models ◽  
Ocular Toxoplasmosis ◽  
Cell Infection

Ocular toxoplasmosis (OT) is caused by the parasite Toxoplasma gondii and affects many individuals throughout the world. Infection may occur through congenital or acquired routes. The parasites enter the blood circulation and reach both the retina and the retinal pigment epithelium, where they may cause cell damage and cell death. Different routes of access are used by T. gondii to reach the retina through the retinal endothelium: by transmission inside leukocytes, as free parasites through a paracellular route, or after endothelial cell infection. A main feature of OT is the induction of an important inflammatory state, and the course of infection has been shown to be influenced by the host immunogenetics. On the other hand, there is evidence that the T. gondii phenotype also has an impact on the distribution of the pathology in different areas. Although considerable knowledge has been acquired on OT, a deeper knowledge of its mechanisms is necessary to provide new, more targeted treatment strategies. In particular, in addition to in vitro and in vivo experimental models, organotypic, ex vivo retinal explants may be useful in this direction.

scholarly journals Bacillus cereus Induces Permeability of an In Vitro Blood-Retina Barrier

2008 ◽  
Vol 76 (4) ◽  
pp. 1358-1367 ◽  
Author(s):  
A. L. Moyer ◽  
R. T. Ramadan ◽  
J. Thurman ◽  
A. Burroughs ◽  
M. C. Callegan
Keyword(s):  
Quorum Sensing ◽  
Bacillus Cereus ◽  
Barrier Function ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Toxin Production ◽  
Global Regulator ◽  
Barrier Permeability ◽  
Cell Monolayers

ABSTRACT Most Bacillus cereus toxin production is controlled by the quorum-sensing-dependent, pleiotropic global regulator plcR, which contributes to the organism's virulence in the eye. The purpose of this study was to analyze the effects of B. cereus infection and plcR-regulated toxins on the barrier function of retinal pigment epithelium (RPE) cells, the primary cells of the blood-retina barrier. Human ARPE-19 cells were apically inoculated with wild-type or quorum-sensing-deficient B. cereus, and cytotoxicity was analyzed. plcR-regulated toxins were not required for B. cereus-induced RPE cytotoxicity, but these toxins did increase the rate of cell death, primarily by necrosis. B. cereus infection of polarized RPE cell monolayers resulted in increased barrier permeability, independent of plcR-regulated toxins. Loss of both occludin and ZO-1 expression occurred by 8 h postinfection, but alterations in tight junctions appeared to precede cytotoxicity. Of the several proinflammatory cytokines analyzed, only interleukin-6 was produced in response to B. cereus infection. These results demonstrate the deleterious effects of B. cereus infection on RPE barrier function and suggest that plcR-regulated toxins may not contribute significantly to RPE barrier permeability during infection.

Maintenance of adult porcine retina and retinal pigment epithelium in perfusion culture: Characterisation of an organotypic in vitro model

2008 ◽  
Vol 86 (4) ◽  
pp. 661-668 ◽  
Author(s):  
Karin Kobuch ◽  
Wolfgang A. Herrmann ◽  
Carsten Framme ◽  
Helmut G. Sachs ◽  
Veit-Peter Gabel ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
In Vitro Model ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Perfusion Culture ◽  
Vitro Model

scholarly journals USH2A-Related Retinitis Pigmentosa: Staging of Disease Severity and Morpho-Functional Studies

Diagnostics ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 213
Author(s):  
Benedetto Falsini ◽  
Giorgio Placidi ◽  
Elisa De Siena ◽  
Maria Cristina Savastano ◽  
Angelo Maria Minnella ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Usher Syndrome ◽  
Collaborative Study ◽  
Staging System ◽  
International Standards ◽  
Full Field ◽  
Functional Studies ◽  
Cumulative Score

Usher syndrome type 2A (USH2A) is a genetic disease characterized by bilateral neuro-sensory hypoacusia and retinitis pigmentosa (RP). While several methods, including electroretinogram (ERG), describe retinal function in USH2A patients, structural alterations can be assessed by optical coherence tomography (OCT). According to a recent collaborative study, RP can be staged considering visual acuity, visual field area and ellipsoid zone (EZ) width. The aim of this study was to retrospectively determine RP stage in a cohort of patients with USH2A gene variants and to correlate the results with age, as well as additional functional and morphological parameters. In 26 patients with established USH2A genotype, RP was staged according to recent international standards. The cumulative staging score was correlated with patients’ age, amplitude of full-field and focal flicker ERGs, and the OCT-measured area of sub-Retinal Pigment Epithelium (RPE) illumination (SRI). RP cumulative score (CS) was positively correlated (r = 0.6) with age. CS was also negatively correlated (rho = −0.7) with log10 ERG amplitudes and positively correlated (r = 0.5) with SRI. In USH2A patients, RP severity score is correlated with age and additional morpho-functional parameters not included in the international staging system and can reliably predict their abnormality at different stages of disease.

The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo

1988 ◽  
Vol 91 (2) ◽  
pp. 303-312
Author(s):  
N.M. McKechnie ◽  
M. Boulton ◽  
H.L. Robey ◽  
F.J. Savage ◽  
I. Grierson
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cytokeratin 18 ◽  
Rpe Cells ◽  
Human Retinal Pigment Epithelium ◽  
Cytoskeletal Elements

The cytoskeletal elements of normal (in situ) and cultured human retinal pigment epithelium (RPE) were studied by a variety of immunocytochemical techniques. Primary antibodies to vimentin and cytokeratins were used. Positive immunoreactivity for vimentin was obtained with in situ and cultured material. The pattern of reactivity obtained with antisera and monoclonals to cytokeratins was more complex. Cytokeratin immunoreactivity could be demonstrated in situ and in cultured cells. The pattern of cytokeratin expression was similar to that of simple or glandular epithelia. A monoclonal antibody that specifically recognizes cytokeratin 18 identified a population of cultured RPE cells that had particularly well-defined filamentous networks within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, but upon culture cytokeratin 18 positive cells were identifiable. Cytokeratin 18 positive cells were identified in all RPE cultures (other than early primaries), regardless of passage number, age or sex of the donor. In post-confluent cultures cytokeratin 18 cells were identified growing over cytokeratin 18 negative cells, suggesting an association of cytokeratin 18 immunoreactivity with cell proliferation. Immunofluorescence studies of retinal scar tissue from two individuals revealed the presence of numerous cytokeratin 18 positive cells. These findings indicate that RPE cells can be identified by their cytokeratin immunoreactivity and that the overt expression of cytokeratin 18 may be associated with proliferation of human RPE both in vitro and in vivo.

Sign in / Sign up

Export Citation Format

Share Document