Extremely hypomorphic and severe deep intronic variants in the locus result in varying Stargardt disease phenotypes.

Autosomal recessive Stargardt disease (STGD1, MIM 248200) is caused by mutations in the gene. Complete sequencing of the locus in STGD1 patients identifies two expected disease-causing alleles in ∼75% of patients and only one mutation in ∼15% of patients. Recently, many possibly pathogenic variants in deep intronic sequences of have been identified in the latter group.

The Ultrastructure, Spatial Distribution, and Osmium Tetroxide Binding of Lipofuscin and Melanosomes in Aging Monkey Retinal Epithelium.

Purpose: To examine the ultrastructure of lipofuscin bodies and melanosomes in retinal epithelium of elderly rhesus monkeys and determines changes in their number and morphology as a function of retinal eccentricity.

Methods: Electron microscopy was used to describe and quantify two major organelles in elderly monkey retinal epithelium, lipofuscin bodies and melanosomes, at different retinal loci extending from the macula to the peri-macula, equator, periphery and ora serrata. Osmium tetroxide was used to distinguish lipofuscin bodies from melanosomes.

Novel organelles in primate retinal epithelium.

We are investigating age-related changes in organelles in monkey retinal epithelium using transmission and analytic electron microscopy. We previously described a circular organelle in retinal epithelium with a diameter of about 0.5μm.

Mitochondrial elongation in the macular RPE of aging monkeys, evidence of metabolic stress.

Purpose: This study was conducted to determine whether mitochondria of the macular retinal pigment epithelium (RPE) change with age in rhesus monkeys (Macaca mulatta). Mitochondria are the main instigators of oxidative stress, which has often been considered to play a role in the pathogenesis of age-related macular degeneration (AMD). Any pathological changes in the mitochondria of aging macular RPE, the main target of AMD, would be a clue to the pathogenesis of this common retinal degeneration afflicting both monkey and man.

Analysis of the ABCA4 genomic locus in Stargardt disease.

Autosomal recessive Stargardt disease (STGD1, MIM 248200) is caused by mutations in the ABCA4 gene. Complete sequencing of ABCA4 in STGD patients identifies compound heterozygous or homozygous disease-associated alleles in 65-70% of patients and only one mutation in 15-20% of patients. This study was designed to find the missing disease-causing ABCA4 variation by a combination of next-generation sequencing (NGS), array-Comparative Genome Hybridization (aCGH) screening, familial segregation and in silico analyses.

Transduction of photoreceptors with equine infectious anemia virus lentiviral vectors: safety and biodistribution of StarGen for Stargardt disease.

Purpose: StarGen is an equine infectious anemia virus (EIAV)-based lentiviral vector that expresses the photoreceptor-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) protein that is mutated in Stargardt disease (STGD1), a juvenile macular dystrophy. EIAV vectors are able to efficiently transduce rod and cone photoreceptors in addition to retinal pigment epithelium in the adult macaque and rabbit retina following subretinal delivery. The safety and biodistribution of StarGen following subretinal delivery in macaques and rabbits was assessed.

A novel melano-lysosome in the retinal epithelium of rhesus monkeys.

The large phagocytic load that confronts the retinal pigment epithelium (RPE) is thought to play a possible role in the pathogenesis of age related macular degeneration (AMD) that afflicts both humans and monkeys. Our knowledge of how RPE degrades phagosomes and other intra-cellular material by lysosomal action is still rudimentary. In this paper we examine organelles that play a role in this process, melanosome, lysosomes and phagosomes, in the RPE of young and old rhesus monkeys in order to better understand lysosomal autophagy and heterophagy in the RPE and its possible role in AMD.

Analysis of the ABCA4 gene by next-generation sequencing.

Purpose: To find all possible disease-associated variants in coding sequences of the ABCA4 gene in a large cohort of patients diagnosed with ABCA4-associated diseases.

Methods: One hundred sixty-eight patients who had been clinically diagnosed with Stargardt disease, cone-rod dystrophy, and other ABCA4-associated phenotypes were prescreened for mutations in ABCA4 with the ABCA4 microarray, resulting in finding 1 of 2 expected mutations in 111 patients and 0 of 2 mutations in 57 patients. The next-generation sequencing (NGS) strategy was applied to these patients to sequence the entire coding region and the splice sites of the ABCA4 gene.

Loss of peripapillary sparing in non-group I Stargardt disease.

The aim of this study was to assess peripapillary sparing in patients with non-group I Stargardt disease. We suggest this as a useful clinical sign for formulating disease severity. Patients with a diagnosis of Stargardt disease were grouped by electroretinogram (ERG).

Topographic and age-related changes of the retinal epithelium and Bruch's membrane of rhesus monkeys.

Purpose: To examine structural differences in the retinal pigmented epithelium (RPE) and Bruch's membrane of rhesus monkeys (Macaca mulatta) as a function of topography and age.

Methods: The retinas of two old (24 and 26 years old) and two young (1 and 6 years old) female monkeys were examined by light fluorescence and electron microscopy at the macula, equator, and ora serrata.

Results: All monkeys lacked fluorescence and lipofuscin granules in the RPE at the ora serrata where photoreceptors are absent.

Bestrophin detected in the basal membrane of the retinal epithelium and drusen of monkeys with drusenoid maculopathy.

Purpose: To determine if bestrophin is present in the basal membrane of macular retinal pigment epithelium (RPE) and in drusen of rhesus monkeys with age-related drusenoid maculopathy.

Methods: The macular region of three rhesus monkeys (Macaca mulatta), 23-24 years of age, with drusenoid maculopathy was dissected from eyes fixed with 4% paraformaldehyde. The macula was sectioned into rectangular pieces.

Phenotypic features of patients with NR2E3 mutations.

Objective: To describe the phenotypes of 5 patients with NR2E3 mutations.

Methods: Two patients with familial and 3 with sporadic early-onset nyctalopia and retinal pigment abnormalities were screened for mutations in the NR2E3 gene (OMIM 604485). The clinical course, fundus features, visual field test results, and fluorescein angiographic and electrophysiologic findings were compared.

Cone inputs to murine striate cortex.

Background: We have recorded responses from single neurons in murine visual cortex to determine the effectiveness of the input from the two murine cone photoreceptor mechanisms and whether there is any unique selectivity for cone inputs at this higher region of the visual system that would support the possibility of colour vision in mice. Each eye was stimulated by diffuse light, either 370 (strong stimulus for the ultra-violet (UV) cone opsin) or 505 nm (exclusively stimulating the middle wavelength sensitive (M) cone opsin), obtained from light emitting diodes (LEDs) in the presence of a strong adapting light that suppressed the responses of rods.

Results: Single cells responded to these diffuse stimuli in all areas of striate cortex.

Mutations in NR2E3 can cause dominant or recessive retinal degenerations in the same family.

NR2E3, a photoreceptor-specific nuclear receptor (PNR), represses cone-specific genes and activates several rod-specific genes. In humans, mutations in NR2E3 have been associated with the recessively-inherited enhanced short-wavelength sensitive S-cone syndrome (ESCS) and, recently, with autosomal dominant (ad) retinitis pigmentosa (RP) (adRP). In the present work, we describe two additional families affected by adRP that carry a heterozygous c.

Drusenoid maculopathy in rhesus monkeys (Macaca mulatta): effects of age and gender.

Purpose: To compare drusenoid maculopathy in monkeys with human age-related macular degeneration, and evaluate the influence of age, gender and caloric restriction.

Methods: Examination by indirect ophthalmoscopy, slit-lamp biomicroscopy and fundus photography, including in some cases fluorescein angiography, was performed on 61 male and 60 female rhesus macaques of ages 10-39 years. Fifty-four of the monkeys were maintained on a calorically restricted diet (approximately 30% lower than control levels) and 67 on an approximately ad libitum diet for 2-19 years, with all other environmental factors held constant.

Drusenoid maculopathy in rhesus monkeys: autofluorescence, lipofuscin and drusen pathogenesis.

Purpose: To examine patterns of retinal pigment epithelial autofluorescence and lipofuscin accumulation in relation to drusen and to explore the pathogenesis of drusen in rhesus monkeys.

Methods: The macular areas of six rhesus monkeys, euthanized at 19 to 28 years of age, were studied by bright field and fluorescence light microscopy and transmission electron microscopy.

Results: There was strong autofluorescence in the retinal epithelium that tended to diminish over drusen.

Correction of the disease phenotype in the mouse model of Stargardt disease by lentiviral gene therapy.

Autosomal recessive Stargardt disease (STGD1) is a macular dystrophy caused by mutations in the ABCA4 (ABCR) gene. The disease phenotype that is most recognized in STGD1 patients, and also in the Abca4-/- mouse (a disease model), is lipofuscin accumulation in retinal pigment epithelium. Here, we tested whether delivery of the normal (wt) human ABCA4 gene to the subretinal space of the Abca4 -/- mice via lentiviral vectors would correct the disease phenotype; that is, reduce accumulation of the lipofuscin pigment A2E.

Development of photoreceptor-specific promoters and their utility to investigate EIAV lentiviral vector mediated gene transfer to photoreceptors.

Background: We wanted to investigate the ability of recombinant equine infectious anemia virus (EIAV) vectors to transduce photoreceptor cells by developing a series of photoreceptor-specific promoters that drive strong gene expression in photoreceptor cells.

Methods: Promoter fragments derived from the rhodopsin (RHO), the beta phosphodiesterase (PDE) and the retinitis pigmentosa (RP1) genes were cloned in combination with an enhancer element, derived from the interphotoreceptor retinoid-binding protein gene (IRBP), into luciferase reporter plasmids. An in vitro transient reporter assay was carried out in the human Y-79 retinoblastoma cell line.

Behavior of retinal epithelium to bleb detachment versus retinectomy.

Purpose: To compare the behavior of the retinal pigment epithelium to a bleb detachment versus removal of a segment of neural retina.

Methods: A bleb detachment was performed on 14 adult pigmented rabbits. In seven rabbits, the neural retina was removed within the bleb detachment.

Alteration in choroidal blood flow produced by local pressure.

Background: To investigate how transient pressure applied to the retinal pigment epithelium (RPE) layer and choroid affects choroidal blood flow in rabbits.

Methods: Twelve rabbits underwent vitrectomy and local retinectomy. In nine of the rabbits a glass rod was used to exert brief pressure on the RPE layer and choroid.

Cone and rod inputs to murine retinal ganglion cells: evidence of cone opsin specific channels.

To identify ultraviolet (UV) and middle- (M) wavelength-sensitive cone and rod signals in murine retinal ganglion cells, single ganglion cell responses were studied in anesthetized, light-adapted C57/BL6 mice with tungsten microelectrodes driven through the sclera and vitreous to the neural retina. One hundred fifty-four ganglion cells were examined in 43 retinas of 34 mice. The retina was stimulated with diffuse flashes and/or pulses of ultraviolet (360 nm) or green (520 nm) light in the presence and absence of a strong steady orange adapting light.

Retinal degeneration associated with RDH12 mutations results from decreased 11-cis retinal synthesis due to disruption of the visual cycle.

Retinoid dehydrogenases/reductases catalyze key oxidation-reduction reactions in the visual cycle that converts vitamin A to 11-cis retinal, the chromophore of the rod and cone photoreceptors. It has recently been shown that mutations in RDH12, encoding a retinol dehydrogenase, result in severe and early-onset autosomal recessive retinal dystrophy (arRD). In a cohort of 1011 individuals diagnosed with arRD, we have now identified 20 different disease-associated RDH12 mutations, of which 16 are novel, in a total of 22 individuals (2.