Background

A pediatric patient presented with rapidly progressive vision loss, nyctalopia and retinal dystrophy. This is the first report of homozygosity for the p.Arg602Trp mutation in the ABCA4 gene. The child became legally blind within a period of 2 years.

Case presentation

An eight year-old Hispanic female presented with bilateral decreased vision following a febrile gastrointestinal illness with nausea and vomiting. Extensive workup involved pediatric infectious disease and rheumatology consultations.Initial visual acuity was 20/60 at distance and 20/30 at near in both eyes. Rapidly progressive vision loss occurred during a 2-year period resulting in visual acuities of 20/200 at distance in both eyes. Fundus exam disclosed attenuated vessels and multiple subretinal blister-like elevations. Optical coherence tomography showed far more lesions than were clinically evident with different levels of elevation. Autofluorescence imagery showed dramatic and widespread geographic areas of atrophy. The deposits that appeared drusen-like on clinical exam were hyperfluorescent, consistent with lipofuscin deposits containing A2e (N-retinylidene-N-retinylethanolamine) indicative of RPE cell dysfunction. Electroretinography was consistent with cone dystrophy, with relative preservation of rod function. Blood analysis and rheumatology evaluation found no evidence of a diffuse post-infectious/inflammatory process. The unique and rapid progression of her subretinal blister-like lesions was documented by fluorescein angiography, optical coherence tomography, autofluorescence imagery, and fundus photography. Family pedigree history disclosed consanguinity, her parents being first cousins. DNA analysis by whole exomic sequencing revealed homozygosity of p.Arg602Trp in the ABCA4 gene.

Conclusion

The pediatric patient presented with a striking clinical appearance and dramatic rate of progression that was clinically more characteristic of an infectious or inflammatory process. This case expands the diverse range of phenotypes attributed to ABCA4 mutations and further supports the role of whole exome sequencing as a powerful new tool available to aid clinicians in establishing diagnosis for challenging cases.

The aim of this study is to evaluate the retinal safety and toxicity of a novel synthetic biopolymer to be used as a patch to treat rhegmatogenous retinal detachment. Thirty one adult wild type albino mice were divided in 2 groups. In Group A (n=9) 0.2 μl balanced salt solution (BSS) and in Group B (n=22), 0.2 μl biopolymer was injected in the subretinal space. Trans-scleral subretinal injection was performed in one eye and the fellow eye was used as control. In both groups, in vivo color fundus photography, electroretinogram (ERG), spectral domain optical coherence tomography (SD-OCT) were performed before injection and at days 7 and 14 post-intervention. Histological analysis was performed following euthanization at days 1, 7 and 21 post-injection. The biopolymer was visualized in the subretinal space in vivo by SD-OCT and post-life by histology up to 1 week after the injection. There were no significant differences in ERG parameters between the two groups at 1 and 2 weeks post-injection. Minimal inflammatory response and loss of photoreceptor cells was only observed in the immediate proximity of the site of scleral perforation, which was similar in both groups. Overall integrity of the outer, inner retina and retinal pigment epithelial (RPE) layers was unaffected by the presence of the biopolymer in the subretinal space. Functional and histological evaluation suggests that the synthetic biopolymer is non-inflammatory and non-toxic to the eye. It may represent a safe therapeutic agent in the future, for the treatment of rhegmatogenous retinal detachment.

Lipid peroxidation from oxidative stress is considered a major contributor to age-related macular degeneration (AMD). The retina is abundant with circulating low-density lipoproteins (LDL), which are taken up by LDL receptor (LDLR) in the RPE and Müller cells. The purpose of this study is to investigate the role of LDLR in the NaIO₃-induced model of dry AMD. Confluent primary human RPE (hRPE) and LDLR-silenced ARPE-19 cells were stressed with 150 µM tert-butyl hydroperoxide (tBH) and caspase 3/7 activation was determined. WT and Ldlr^-/- mice were administered NaIO₃ (20 mg/kg) intravenously. On day 7, fundus imaging, OCT, ERG, and retinal thickness were measured. Histology, TUNEL, cleaved caspase 3 and lipid accumulation were assessed. Treatment of hRPE with tBH markedly decreased LDLR expression. Caspase 3/7 activation was significantly increased in LDLR-silenced ARPE-19 cells treated with tBH. In Ldlr^-/- mice, NaIO₃ administration resulted in significant (a) retinal thinning, (b) compromised photoreceptor function, (c) increased percentage of cleaved caspase 3 positive and apoptotic cells, and (d) increased lipid droplet accumulation in the RPE, Bruch membrane, choroid, and sclera, compared to WT mice. Our findings imply that LDLR loss leads to lipid accumulation and impaired retinal function, which may contribute to the development of AMD.

IMPORTANCE: Binocular summation (BiS) is defined as the superiority of visual function for binocular over monocular viewing. Binocular summation decreases with age and large interocular differences in visual acuity. To our knowledge, BiS has not heretofore been well studied as a functional measure of binocularity in strabismus. OBJECTIVE: To evaluate the effect of strabismus on BiS using a battery of psychophysical tasks that are clinically relevant and easy to use and to determine whether strabismus is associated with binocular inhibition in extreme cases. DESIGN: Case-control study. SETTING: University-based eye institute. PARTICIPANTS: Strabismic patients recruited during 2010 to 2012 from a preoperative clinic and control participants with no history of eye disease other than refractive error. INTERVENTION: A battery of psychophysical and electrophysiological tests including Early Treatment Diabetic Retinopathy Study visual acuity, Sloan low-contrast acuity (LCA) (2.5% and 1.25%), Pelli-Robson contrast sensitivity, and sweep visual evoked potential contrast sensitivity. MAIN OUTCOME AND MEASURE: Binocular summation was calculated as the ratio between binocular and better-eye individual scores. RESULTS: Sixty strabismic and 80 control participants were prospectively examined (age range, 8-60 years). Mean BiS was significantly lower in the strabismic patients than controls for LCA (2.5% and 1.25%, P = .005 and <.001, respectively). For 1.25% LCA, strabismic patients had a mean BiS score less than 1, indicating binocular inhibition (ie, the binocular score was less than that of the better eyes monocular score). There was no significant difference in BiS for contrast thresholds on Early Treatment Diabetic Retinopathy Study visual acuity, Pelli-Robson contrast sensitivity, or sweep visual evoked potential contrast sensitivity. Regression analysis revealed a significant worsening of BiS with strabismus for 2.5% (P = .009) and 1.25% (P = .002) LCA, after accounting for age. CONCLUSIONS AND RELEVANCE: Strabismic patients demonstrate subnormal BiS and even binocular inhibition for LCA, suggesting that strabismus impairs visual function more than previously appreciated. This may explain why strabismic patients who are not diplopic close 1 eye in visually demanding situations. This finding clarifies the visual deficits impacting quality of life in strabismic patients and may represent a novel measure by which to evaluate and monitor function in strabismus.

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

The key to reducing the individual and societal burden of age-related macular degeneration (AMD)-related vision loss, is to be able to initiate therapies that slow or halt the progression at a point that will yield the maximum benefit while minimizing personal risk and cost. There is a critical need to find clinical markers that, when combined with the specificity of genetic testing, will identify individuals at the earliest stages of AMD who would benefit from preventive therapies. These clinical markers are endophenotypes for AMD, present in those who are likely to develop AMD, as well as in those who have clinical evidence of AMD. Clinical characteristics associated with AMD may also be possible endophenotypes if they can be detected before or at the earliest stages of the condition, but we and others have shown that this may not always be valid. Several studies have suggested that dynamic changes in rhodopsin regeneration (dark adaptation kinetics and/or critical flicker fusion frequencies) may be more subtle indicators of AMD-associated early retinal dysfunction. One can test for the relevance of these measures using genetic risk profiles based on known genetic risk variants. These functional measures may improve the sensitivity and specificity of predictive models for AMD and may also serve to delineate clinical subtypes of AMD that may differ with respect to prognosis and treatment.

Although AMD is a complex disease, oxidative stress is a crucial contributor to its development, especially in view of the higher oxygen demand of the retina. Paraoxonase 2 (PON2) is a ubiquitously and constitutively expressed antioxidant protein that is found intracellularly associated with mitochondrial membranes and modulates mitochondrial ROS production and function. The contribution of PON2 to AMD has not been studied to date. In this study, we examined the role of PON2 in AMD utilizing both in vitro and in vivo models of AMD with emphasis on mitochondrial function. Mitochondrial localization and regulation of PON2 following oxidative stress were determined in human primary cultured retinal pigment epithelium (hRPE) cells. PON2 was knocked down in RPE cells using siRNA and mitochondrial bioenergetics were measured. To investigate the function of PON2 in the retina, WT and PON2-deficient mice were administered NaIO₃ (20 mg/kg) intravenously; fundus imaging, optical coherence tomography (OCT), electroretinography (ERG) were conducted; and retinal thickness and cell death were measured and quantified. In hRPE, mitochondrial localization of PON2 increased markedly with stress. Moreover, a time-dependent regulation of PON2 was observed following oxidative stress, with an initial significant increase in expression followed by a significant decrease. Mitochondrial bioenergetic parameters (basal respiration, ATP production, spare respiratory capacity, and maximal respiration) showed a significant decrease with oxidative stress, which was further exacerbated in the absence of PON2. NaIO₃ treatment caused significant retinal degeneration, retinal thinning, and reduced rod and cone function in PON2-deficient mice when compared to WT mice. The apoptotic cells and active caspase 3 significantly increased in PON2-deficient mice treated with NaIO_3, when compared to WT mice. Our investigation demonstrates that deficiency of PON2 results in RPE mitochondrial dysfunction and a decline in retinal function. These findings imply that PON2 may have a beneficial role in retinal pathophysiology and is worthy of further investigation.

Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world. The retinal pigment epithelium (RPE) is a critical site of pathology in AMD. Oxidative stress plays a key role in the development of AMD. We generated a chimeric high-density lipoprotein (HDL), mimetic peptide named HM-10/10, with anti-oxidant properties and investigated its potential for the treatment of retinal disease using cell culture and animal models of RPE and photoreceptor (PR) degeneration. Treatment with HM-10/10 peptide prevented human fetal RPE cell death caused by tert-Butyl hydroperoxide (tBH)-induced oxidative stress and sodium iodate (NaIO₃), which causes RPE atrophy and is a model of geographic atrophy in mice. We also show that HM-10/10 peptide ameliorated photoreceptor cell death and significantly improved retinal function in a mouse model of N-methyl-N-nitrosourea (MNU)-induced PR degeneration. Our results demonstrate that HM-10/10 protects RPE and retina from oxidant injury and can serve as a potential therapeutic agent for the treatment of retinal degeneration.

AbstractBackgroundThe commonest genetic form of juvenile or early adult onset macular degeneration is Stargardt Disease (STGD) caused by recessive mutations in the gene ABCA4. However, high phenotypic and allelic heterogeneity and a small but non-trivial amount of locus heterogeneity currently impede conclusive molecular diagnosis in a significant proportion of cases.MethodsWe performed whole exome sequencing (WES) of nine putative Stargardt Disease probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Follow-up dideoxy sequencing was performed for confirmation and to screen for mutations in an additional set of affected individuals lacking a definitive molecular diagnosis.ResultsWhole exome sequencing revealed seven likely disease-causing variants across four genes, providing a confident genetic diagnosis in six previously uncharacterized participants. We identified four previously missed mutations in ABCA4 across three individuals. Likely disease-causing mutations in RDS/PRPH2, ELOVL, and CRB1 were also identified.ConclusionsOur findings highlight the enormous potential of whole exome sequencing in Stargardt Disease molecular diagnosis and research. WES adequately assayed all coding sequences and canonical splice sites of ABCA4 in this study. Additionally, WES enables the identification of disease-related alleles in other genes. This work highlights the importance of collecting parental genetic material for WES testing as the current knowledge of human genome variation limits the determination of causality between identified variants and disease. While larger sample sizes are required to establish the precision and accuracy of this type of testing, this study supports WES for inherited early onset macular degeneration disorders as an alternative to standard mutation screening techniques.

Effective treatments and animal models for the most prevalent neurodegenerative form of blindness in elderly people, called age-related macular degeneration (AMD), are lacking. Genome-wide association studies have identified lipid metabolism and inflammation as AMD-associated pathogenic pathways. Given liver X receptors (LXRs), encoded by the nuclear receptor subfamily 1 group H members 2 and 3 (NR1H3 and NR1H2), are master regulators of these pathways, herein we investigated the role of LXR in human and mouse eyes as a function of age and disease and tested the therapeutic potential of targeting LXR. We identified immunopositive LXR fragments in human extracellular early dry AMD lesions and a decrease in LXR expression within the retinal pigment epithelium (RPE) as a function of age. Aged mice lacking LXR presented with isoform-dependent ocular pathologies. Specifically, loss of the Nr1h3 isoform resulted in pathobiologies aligned with AMD, supported by compromised visual function, accumulation of native and oxidized lipids in the outer retina, and upregulation of ocular inflammatory cytokines, while absence of Nr1h2 was associated with ocular lipoidal degeneration. LXR activation not only ameliorated lipid accumulation and oxidant-induced injury in RPE cells but also decreased ocular inflammatory markers and lipid deposition in a mouse model, thereby providing translational support for pursuing LXR-active pharmaceuticals as potential therapies for dry AMD.