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Predominance of hyperopia in autosomal dominant Best vitelliform macular dystrophy

Fri, 2020-11-27 05:00

Br J Ophthalmol. 2020 Nov 26:bjophthalmol-2020-317763. doi: 10.1136/bjophthalmol-2020-317763. Online ahead of print.


BACKGROUND/AIMS: Patients with BEST1-associated autosomal dominant Best vitelliform macular dystrophy (AD-BVMD) have been reported to be hyperopic, but the prevalence of refractive error has not been described. This study aimed to characterise the type and degree of refractive error in a large cohort of patients with AD-BVMD compared with an age-similar group with ABCA4-associated Stargardt disease.

METHODS: This was a retrospective chart review of consecutive patients with molecularly confirmed AD-BVMD and Stargardt macular dystrophy seen at a single academic centre. Demographic information, including age, gender and genotype were extracted from the chart. The best corrected visual acuity (BCVA), as well as type and degree of refractive error on manifest refraction for each eye on each visit, were recorded and compared.

RESULTS: A total of 178 eyes from 89 patients with AD-BVMD (35 women, 54 men; mean age 36.6 years) and 306 eyes from 153 patients (94 women, 59 men, mean age 30.2 years) with Stargardt disease were included in the study. Mean BCVA was excellent for both AD-BVMD and Stargardt eyes (logMAR 0.23 vs logMAR 0.31, respectively; p=0.55). At initial refraction, 73.0% of AD-BVMD eyes (130/178) were hyperopic, with mean spherical equivalent (SE) +1.38 dioptres (median +0.88) whereas 80.7% of Stargardt eyes (247/306) were myopic, with mean SE of -1.76 dioptres (median -1.19) (p<0.001).

CONCLUSION: Patients with AD-BVMD are predominantly hyperopic, whereas those with Stargardt disease are predominantly myopic. The findings provide further evidence of a role for BEST1 in ocular growth and development.

PMID:33243830 | DOI:10.1136/bjophthalmol-2020-317763

Patient derived stem cells for discovery and validation of novel pathogenic variants in inherited retinal disease

Sun, 2020-11-01 05:00

Prog Retin Eye Res. 2020 Oct 29:100918. doi: 10.1016/j.preteyeres.2020.100918. Online ahead of print.


Our understanding of inherited retinal disease has benefited immensely from molecular genetic analysis over the past several decades. New technologies that allow for increasingly detailed examination of a patient's DNA have expanded the catalog of genes and specific variants that cause retinal disease. In turn, the identification of pathogenic variants has allowed the development of gene therapies and low-cost, clinically focused genetic testing. Despite this progress, a relatively large fraction (at least 20%) of patients with clinical features suggestive of an inherited retinal disease still do not have a molecular diagnosis today. Variants that are not obviously disruptive to the codon sequence of exons can be difficult to distinguish from the background of benign human genetic variations. Some of these variants exert their pathogenic effect not by altering the primary amino acid sequence, but by modulating gene expression, isoform splicing, or other transcript-level mechanisms. While not discoverable by DNA sequencing methods alone, these variants are excellent targets for studies of the retinal transcriptome. In this review, we present an overview of the current state of pathogenic variant discovery in retinal disease and identify some of the remaining barriers. We also explore the utility of new technologies, specifically patient-derived induced pluripotent stem cell (iPSC)-based modeling, in further expanding the catalog of disease-causing variants using transcriptome-focused methods. Finally, we outline bioinformatic analysis techniques that will allow this new method of variant discovery in retinal disease. As the knowledge gleaned from previous technologies is informing targets for therapies today, we believe that integrating new technologies, such as iPSC-based modeling, into the molecular diagnosis pipeline will enable a new wave of variant discovery and expanded treatment of inherited retinal disease.

PMID:33130253 | DOI:10.1016/j.preteyeres.2020.100918

The Varying Optical Coherence Tomography Appearance of the Inner Choroid with Age: Possible Explanation and Histologic Correlate

Mon, 2020-10-26 05:00

Retina. 2020 Sep 21. doi: 10.1097/IAE.0000000000002985. Online ahead of print.


PURPOSE: To investigate the reflectivity of the structural optical coherence tomography images of the inner choroid as it relates to potential structural composition.

METHODS: The reflectivity of slab images 29 - 49 µm and 45 - 65 µm below the RPE, obtained with the Zeiss Plex Elite 9000, were evaluated. The mean and standard deviation of a group of subjects with no ocular disorders were determined. Binarization of the images was done a threshold level established at the mean plus one standard deviation for each slab depth. The proportion of area binarized was evaluated with generalized estimating equations. Representative histologic images obtained from autopsy donors were stained with Masson's trichrome, a staining method helpful in evaluating collagen and ground substance of tissue.

RESULTS: There were 67 eyes of 38 subjects with a mean age of 44.5 (range 22 - 82) years. Using generalized estimating equations, age was found to be a significant predictor for the proportion of binarized pixels in both the 29 - 49 µm (P=.034) and the 45 - 65 µm (P<.001) slabs. The histologic specimens illustrated the loss of ground substance with increasing compaction of collagen fibers in the choroidal stroma with advancing age.

CONCLUSIONS: The reflectivity from the inner choroid is not uniform and changes with age. As suggested by the histologic specimens, we propose the OCT reflectance from the inner choroid is related, in part, to the packing density of collagen fibers present there.

PMID:33104324 | DOI:10.1097/IAE.0000000000002985


Wed, 2020-10-21 05:00

Retina. 2020 Nov;40(11):e68-e69. doi: 10.1097/IAE.0000000000002958.


PMID:33086372 | DOI:10.1097/IAE.0000000000002958

Stepwise differentiation and functional characterization of human induced pluripotent stem cell-derived choroidal endothelial cells

Thu, 2020-09-24 05:00

Stem Cell Res Ther. 2020 Sep 23;11(1):409. doi: 10.1186/s13287-020-01903-4.


BACKGROUND: Endothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds. While all ECs share a number of structural and molecular features, heterogeneity exists depending on their resident tissue. ECs lining the choriocapillaris in the human eye are lost early in the pathogenesis of age-related macular degeneration (AMD), a common and devastating form of vision loss. In order to study the mechanisms leading to choroidal endothelial cell (CEC) loss and to develop reagents for repairing the choroid, a reproducible in vitro model, which closely mimic CECs, is needed. While a number of protocols have been published to direct induced pluripotent stem cells (iPSCs) into ECs, the goal of this study was to develop methods to differentiate iPSCs into ECs resembling those found in the human choriocapillaris specifically.

METHODS: We transduced human iPSCs with a CDH5p-GFP-ZEO lentiviral vector and selected for transduced iPSCs using blasticidin. We generated embryoid bodies (EBs) from expanded iPSC colonies and transitioned from mTESR™1 to EC media. One day post-EB formation, we induced mesoderm fate commitment via addition of BMP-4, activin A, and FGF-2. On day 5, EBs were adhered to Matrigel-coated plates in EC media containing vascular endothelial cell growth factor (VEGF) and connective tissue growth factor (CTGF) to promote CEC differentiation. On day 14, we selected for CECs using either zeocin resistance or anti-CD31 MACS beads. We expanded CECs post-selection and performed immunocytochemical analysis of CD31, carbonic anhydrase IV (CA4), and RGCC; tube formation assays; and transmission electron microscopy to access vascular function.

RESULTS: We report a detailed protocol whereby we direct iPSC differentiation toward mesoderm and utilize CTGF to specify CECs. The CDH5p-GFP-ZEO lentiviral vector facilitated the selection of iPSC-derived ECs that label with antibodies directed against CD31, CA4, and RGCC; form vascular tubes in vitro; and migrate into empty choroidal vessels. CECs selected using either antibiotic selection or CD31 MACS beads showed similar characteristics, thereby making this protocol easily reproducible with or without lentiviral vectors.

CONCLUSION: ECs generated following this protocol exhibit functional and biochemical characteristics of CECs. This protocol will be useful for developing in vitro models toward understanding the mechanisms of CEC loss early in AMD.

PMID:32967716 | DOI:10.1186/s13287-020-01903-4

Retinal Tropism and Transduction of Adeno-Associated Virus (AAV) Varies by Serotype and Route of Delivery (Intravitreal, Subretinal or Suprachoroidal) in Rats

Sat, 2020-09-19 05:00

Hum Gene Ther. 2020 Sep 18. doi: 10.1089/hum.2020.043. Online ahead of print.


Viral-mediated gene augmentation offers tremendous promise for the treatment of inherited retinal diseases. The development of effective gene therapy requires an understanding of the vector's tissue-specific behavior, which may vary depending on serotype, route of delivery, or target species. Using an ex vivo organotypic explant system, we previously demonstrated that retinal tropism and transduction of adeno-associated virus type 2 (AAV2) varies significantly depending on serotype in human eyes. However, the ex vivo system has limited ability to assess route of ocular delivery, and relatively little literature exists on tropic differences between serotypes and routes of delivery in vivo. In this study, we demonstrate that retinal tropism and transduction efficiency of five different AAV2 serotypes (AAV2/1, AAV2/2, AAV2/6, AAV2/8, AAV2/9) expressing eGFP driven by a cytomegalovirus promoter vary greatly depending on serotype and route of delivery (intravitreal, subretinal, or suprachoroidal) in rats. With subretinal delivery, all serotypes successfully transduced the retinal pigmented epithelium and outer nuclear layer (ONL), with AAV2/1 displaying the highest transduction efficiency and AAV2/2 and AAV2/6 showing lower ONL transduction. There was minimal transduction of the inner retina via subretinal delivery for any serotype. Tropism by suprachoroidal delivery mirrored that of subretinal delivery for all AAV serotypes but resulted in a wider distribution and greater ONL transduction. With intravitreal delivery, retinal transduction was seen primarily in the inner retina (retinal nerve fiber, ganglion cell, and inner nuclear layers) for AAV2/1 and AAV2/6, with AAV2/6 showing the highest transduction. When compared to data from human explant models, there are substantial differences in tropism and transduction that are important to consider when using rats as preclinical models for the development of ocular gene therapies for humans.

PMID:32948113 | DOI:10.1089/hum.2020.043

Spectacle: An interactive resource for ocular single-cell RNA sequencing data analysis

Thu, 2020-09-10 05:00

Exp Eye Res. 2020 Sep 7:108204. doi: 10.1016/j.exer.2020.108204. Online ahead of print.


Single-cell RNA sequencing has revolutionized ocular gene expression studies. This technology has enabled researchers to identify expression signatures for rare cell types and characterize how gene expression changes across biological conditions, such as topographic region or disease status. However, sharing single-cell RNA sequencing results remains a major obstacle, particular for individuals without a computational background. To address these limitations, we developed Spectacle, an interactive web-based resource for exploring previously published single-cell RNA sequencing data from ocular studies. Spectacle is powered by a locally developed R package, cellcuratoR, which utilizes the Shiny framework in R to generate interactive visualizations for single-cell expression data. Spectacle contains five pre-processed ocular single-cell RNA sequencing data sets and is accessible via the web at With Spectacle, users can interactively identify which cell types express a gene of interest, detect transcriptomic subpopulations within a cell type, and perform highly flexible differential expression analyses. The freely-available Spectacle system reduces the bioinformatic barrier for interacting with rich single-cell RNA sequencing studies from ocular tissues, making it easy to quickly identify cell types that express a gene of interest.

PMID:32910939 | DOI:10.1016/j.exer.2020.108204

Response to letter to the editor by Dhananjay Shukla.

Thu, 2020-08-27 04:56
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Response to letter to the editor by Dhananjay Shukla.

Retina. 2020 Aug 18;:

Authors: Sohn EH, Mullins RF, Eliott D

PMID: 32826793 [PubMed - as supplied by publisher]

Prospective, Single-center, Open-label, Pilot Study Using Cryopreserved Umbilical Tissue Containing Viable Cells in the Treatment of Complex Acute and Chronic Wounds.

Thu, 2020-08-27 04:56
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Prospective, Single-center, Open-label, Pilot Study Using Cryopreserved Umbilical Tissue Containing Viable Cells in the Treatment of Complex Acute and Chronic Wounds.

Wounds. 2020 Jun 21;:

Authors: Mullins RF, Hassan Z, Homsombath B, Fagan S, Craft-Coffman B, Wilson J, Rumbaugh JG, Saunders M, Danilkovitch A

INTRODUCTION: Complex wounds with exposed bone, muscle, tendon, or hardware continue to be a therapeutic challenge for wound care providers. Wounds with exposed structures are more susceptible to infection, necrosis, and amputation. As such, rapid granulation to cover exposed deep tissue structures is essential for patient recovery.
OBJECTIVE: In this prospective, pilot study, the authors evaluate the clinical outcomes of a cryopreserved umbilical tissue graft containing viable cells (vCUT) in the treatment of complex wounds.
MATERIALS AND METHODS: Ten patients with 12 wounds each received 1 application of vCUT. Two patients did not complete the study and were removed from the per-protocol population. Data analyses were performed on the remaining 8 patients with 10 wounds. The average wound area was 16.5 cm2 with an average duration of 10 months. Post-application, patients were followed for an additional 4 weeks for granulation, closure, and safety outcomes.
RESULTS: By the end of the study, 8 of 10 (80.0%) vCUT-treated wounds achieved 100% granulation, and 3 wounds (30.0%) went on to achieve complete closure. The median area reduction was 40.5% and the median volume reduction was 59.4%.
CONCLUSIONS: The results of this study suggest vCUT in conjunction with standard of care can be a viable treatment option for acute and chronic lower extremity complex wounds.

PMID: 32813668 [PubMed - as supplied by publisher]