The Role of the Choroid in Stargardt Disease.

Stargardt disease is the commonest juvenile macular dystrophy. It is caused by genetic mutations in the ABCA4 gene. Diagnosis is not always straightforward, and various phenocopies exist. Late-onset disease can be misdiagnosed with age-related macular disease. A correct diagnosis is particularly critical because of emergent gene therapies. Stargardt disease is known to affect retinal pigment epithelium and photoreceptors. Many studies have also highlighted the importance of the choroid in the diagnosis, pathophysiology, and progression of the disease. The choroid is in an integral relationship with the retinal pigment epithelium and photoreceptors, and its possible involvement during the disease should be considered. The purpose of this review is to analyze the current diagnostic tools for choroidal evaluation and the extrapolation of useful data for ophthalmologists and researchers studying the disease.

In vivo multimodal retinal imaging of disease-related pigmentary changes in retinal pigment epithelium.

Melanosomes, lipofuscin, and melanolipofuscin are the three principal types of pigmented granules found in retinal pigment epithelium (RPE) cells. Changes in the density of melanosomes and lipofuscin in RPE cells are considered hallmarks of various retinal diseases, including Stargardt disease and age-related macular degeneration (AMD). Herein, we report the potential of an in vivo multimodal imaging technique based on directional back-scattering and short-wavelength fundus autofluorescence (SW-FAF) to study disease-related changes in the density of melanosomes and lipofuscin granules in RPE cells. Changes in the concentration of these granules in Abca4-/- mice (a model of Stargardt disease) relative to age-matched wild-type (WT) controls were investigated. Directional optical coherence tomography (dOCT) was used to assess melanosome density in vivo, whereas the autofluorescence (AF) images and emission spectra acquired with a spectrometer-integrated scanning laser ophthalmoscope (SLO) were used to characterize lipofuscin and melanolipofuscin granules in the same RPE region. Subcellular-resolution ex vivo imaging using confocal fluorescence microscopy and electron microscopy was performed on the same tissue region to visualize and quantify melanosomes, lipofuscin, and melanolipofuscin granules. Comparisons between in vivo and ex vivo results confirmed an increased concentration of lipofuscin granules and decreased concentration of melanosomes in the RPE of Abca4-/- mice, and provided an explanation for the differences in fluorescence and directionality of RPE scattering observed in vivo between the two mouse strains.

CLASSIFYING ABCA4 MUTATION SEVERITY USING AGE-DEPENDENT ULTRA-WIDEFIELD FUNDUS AUTOFLUORESCENCE-DERIVED TOTAL LESION SIZE.

PURPOSE: To establish a mutation-specific age-dependent ultra-widefield fundus autofluorescence (UWF-FAF) trajectory in a large Stargardt disease (STGD1) cohort using total lesion size (TLS) and to develop a clinical method for variant classification. METHODS: A retrospective study of patients with biallelic ABCA4 mutations that were evaluated with UWF-FAF. Boundaries of TLS, defined by stippled hyper/hypoautofluorescence, were outlined manually. Pathogenicity was assessed according to ACMG/AMP criteria, and mutation severities were classified based on the current literature. Age-dependent trajectories in TLS were examined in patients with nullizygous, mild, and intermediate mutations. Mutations of uncertain severities were classified using a clinical criterion based on age of symptom onset and TLS. RESULTS: Eighty-one patients with STGD1 (mean age = 42 ± 20 years and mean visual acuity = 20/200) were recruited from 65 unrelated families. Patients with biallelic null/severe variants (n = 6) demonstrated an increase in TLS during their second decade reaching a mean ± SD of 796 ± 29 mm2 by age 40. Those harboring mild mutations c.5882G>A or c.5603A>T had lesions confined to the posterior pole with a mean ± SD TLS of 30 ± 39 mm2. Intermediate mutations c.6079C>T or c.[2588G>C;5603A>T] in trans with a null/severe mutation had a mean ± SD TLS of 397 ± 29 mm2. Thirty-two mutations were predicted to cause severe (n = 22), intermediate (n = 6), and mild (n = 5) impairment of ABCA4 function based on age of symptom onset and TLS. CONCLUSION: Age-dependent TLS showed unique ABCA4 mutation-specific trajectories. Our novel clinical criterion using age of symptom onset and TLS to segregate ABCA4 mutations into three severity groups requires further molecular studies to confirm its validity.

Total flow intensity, active flow intensity and volume related flow intensity as new quantitative metrics in optical coherence tomography angiography.

Optical coherence tomography (OCT) angiography (OCTA) is a non-invasive tool for the in-vivo study of the intraretinal vascular network. It is based on the analysis of motion particles within the retina to reconstruct the paths followed by the erythrocytes, i.e. retinal capillaries. To date, qualitative and quantitative information are based on the morphological features disclosed by retinal capillaries. In the present study, we proposed new quantitative functional metrics, named Total Flow Intensity (TFI), Active Flow Intensity (AFI), and Volume-related Flow Intensity (VFI), based on the processing of the blood flow signal detected by OCTA. We studied these metrics in a cohort of healthy subjects, and we assessed their clinical utility by including a cohort of age-matched patients affected by Stargardt disease. Moreover, we compared TFI, AFI, and VFI to the widely used vessel density (VD) parameter. TFI, AFI, and VFI were able to describe in detail the different properties of the retinal vascular compartment. In particular, TFI was intended as the overall amount of volumetric retinal blood flow. AFI represented a selective measure of voxels disclosing blood flow signal. VFI was developed to put in relationship the volumetric blood flow information with the not vascularized retinal volume. In conclusion, TFI, AFI, and VFI were proposed as feasible functional OCTA biomarkers based on the analysis of retinal blood flow signal.

Outer nuclear layer relevance in visual function correlated to quantitative enface OCT parameters in Stargardt disease.

PURPOSE: To evaluate the correlation between Best Corrected Visual Acuity (BCVA) and the following parameters in Stargardt Disease (STGD): Central Retinal Thickness (CR-T), Central Outer Nuclear Layer Thickness (C-ONL-T), Areas of macular Photoreceptor loss (PHRa), and Retinal Pigment Epithelium (RPE) loss (RPEa). METHODS: A total of 64 eyes of 32 STGD patients were included in the study. All patients received a comprehensive ophthalmological examination, color fundus photographs, fundus auto-fluorescence imaging, and Optical Coherence Tomography (OCT). The CR-T and C-ONL-T were evaluated from standard SD-OCT scans. The PHRa and RPEa were calculated from enface OCT scans (sub RPE slab and photoreceptor slab). The collected OCT parameters were evaluated for possible association with BCVA. RESULTS: The mean macular PHRa and RPEa was 16.16 ± 13.36 and 12.05 ± 12.57 mm2 respectively. The mean CR-T measured 120.78 ± 41.49 µm while the mean C-ONL-T was assessed at 4.60 ± 13.73 µm. BCVA showed the highest correlation with the C-ONL-T (r = -0.72; p < 0.001) while there was no correlation with the CR-T (r = -0.17; p = 1.00). CONCLUSIONS: Enface OCT permits a rapid and precise quantitative evaluation of the macular PHR and RPE atrophy area in STGD. Nonetheless, the OCT parameter that showed the highest correlation with visual acuity in STGD was the ONL thickness.

Genotypes Predispose Phenotypes-Clinical Features and Genetic Spectrum of ABCA4-Associated Retinal Dystrophies.

The ABCA4 gene is one of the most common disease-causing genes of inherited retinal degeneration. In this study, we report different phenotypes of ABCA4-associated retinal dystrophies in the Taiwanese population, its clinical progression, and its relationship with genetic characteristics. Thirty-seven subjects were recruited and all patients underwent serial ophthalmic examinations at a single medical center. Fundus autofluorescence (FAF) images were quantified for clinical evaluation, and panel-based next-generation sequencing testing was performed for genetic diagnosis. Visual preservation, disease progression, and genotype-phenotype correlation were analyzed. In this cohort, ABCA4-associated retinal degeneration presented as Stargardt disease 1 (STGD1, 62.16%), retinitis pigmentosa (32.43%), and cone-rod dystrophy (5.41%). STGD1 could be further divided into central and dispersed types. In each phenotype, the lesion areas quantified by FAF increased with age (p < 0.01) and correlated with poorer visual acuity. However, three patients had the foveal sparing phenotype and had relatively preserved visual acuity. Forty-two ABCA4 variants were identified as disease-causing, with c.1804C>T (p.Arg602Trp) the most frequent (37.84%). Patients with a combination of severe/null variants could have more extensive phenotypes, such as arRP and dispersed STGD1. This is the first cohort study of ABCA4-associated retinal degeneration in Taiwan with wide spectrums of both genotypic and phenotypic characteristics. An extremely high prevalence of c.1804C>T, which has not been reported in East Asia before, was noted. The extensiveness of retinal involvement might be regarded as a spectrum of ABCA4-associated retinal dystrophies. Different types of genetic variations could lead to distinctive phenotypes, according to the coding impact of variants.

Analysis of retinal sublayer thicknesses and rates of change in ABCA4-associated Stargardt disease.

Stargardt disease, the most common inherited macular dystrophy, is characterized by vision loss due to central retinal atrophy. Although clinical trials for Stargardt are currently underway, the disease is typically slowly progressive, and objective, imaging-based biomarkers are critically needed. In this retrospective, observational study, we characterize the thicknesses of individual retinal sublayers by macular optical coherence tomography (OCT) in a large cohort of patients with molecularly-confirmed, ABCA4-associated Stargardt disease (STGD1) relative to normal controls. Automated segmentation of retinal sublayers was performed with manual correction as needed, and thicknesses in various macular regions were compared using mixed effects models. Relative to controls (42 eyes, 40 patients), STGD1 patients (107 eyes, 63 patients) had slight thickening of the nerve fiber layer and retinal pigment epithelium-Bruch's membrane, with thinning in other sublayers, especially the outer nuclear layer (ONL) (p < 0.0015). When comparing the rate of retinal sublayer thickness change over time (mean follow-up 3.9 years for STGD1, 2.5 years for controls), STGD1 retinas thinned faster than controls in the outer retina (ONL to photoreceptor outer segments). OCT-based retinal sublayer thickness measurements are feasible in STGD1 patients and may provide objective measures of disease progression or treatment response.

Clinical and genetic characteristics of Stargardt disease in a large Western China cohort: Report 1.

Stargardt disease 1 (STGD1) is the most prevalent retinal dystrophy caused by pathogenic biallelic ABCA4 variants. Forty-two unrelated patients mostly originating from Western China were recruited. Comprehensive ophthalmological examinations, including visual acuity measurements (subjective function), fundus autofluorescence (retinal imaging), and full-field electroretinography (objective function), were performed. Next-generation sequencing (target/whole exome) and direct sequencing were conducted. Genotype grouping was performed based on the presence of deleterious variants. The median age of onset/age was 10.0 (5-52)/29.5 (12-72) years, and the median visual acuity in the right/left eye was 1.30 (0.15-2.28)/1.30 (0.15-2.28) in the logarithm of the minimum angle of resolution unit. Ten patients (10/38, 27.0%) showed confined macular dysfunction, and 27 (27/37, 73.7%) had generalized retinal dysfunction. Fifty-eight pathogenic/likely pathogenic ABCA4 variants, including 14 novel variants, were identified. Eight patients (8/35, 22.8%) harbored multiple deleterious variants, and 17 (17/35, 48.6%) had a single deleterious variant. Significant associations were revealed between subjective functional, retinal imaging, and objective functional groups, identifying a significant genotype-phenotype association. This study illustrates a large phenotypic/genotypic spectrum in a large well-characterized STGD1 cohort. A distinct genetic background of the Chinese population from the Caucasian population was identified; meanwhile, a genotype-phenotype association was similarly represented.

Optical Gap Biomarker in Cone-Dominant Retinal Dystrophy.

PURPOSE: To characterize the progression of optical gaps and expand the known etiologies of this phenotype. DESIGN: Retrospective cohort study. METHODS: Thirty-six patients were selected based on the identification of an optical gap on spectral-domain optical coherence tomography (OCT) from a large cohort of patients (N = 746) with confirmed diagnoses of inherited retinal dystrophy. The width and height of the gaps in 70 eyes of 36 patients were measured by 2 independent graders using the caliper tool on Heidelberg Explorer. Measurements of outer and central retinal thickness were also evaluated and correlated with gap dimensions. RESULTS: Longitudinal analysis confirmed the progressive nature of optical gaps in patients with Stargardt disease, achromatopsia, occult macular dystrophy, and cone dystrophies (P < .003). Larger changes in gap width were noted in patients with Stargardt disease (78.1 µm/year) and cone dystrophies (31.9 µm/year) compared with patients with achromatopsia (16.2 µm/year) and occult macular dystrophy (15.4 µm/year). Gap height decreased in patients with Stargardt disease (6.5 µm/year; P = .02) but increased in patients with achromatopsia (3.3 µm/year) and occult macular dystrophy (1.2 µm/year). Gap height correlated with measurements of central retinal thickness at the fovea (r = 0.782, P = .00012). Interocular discordance of the gap was observed in 7 patients. Finally, a review of all currently described etiologies of optical gap was summarized. CONCLUSION: The optical gap is a progressive phenotype seen in an increasing number of etiologies. This progressive nature suggests a use as a biomarker in the understanding of disease progression. Interocular discordance of the phenotype may be a feature of Stargardt disease and cone dystrophies.

Abnormal Visual Function Outside the Area of Atrophy Defined by Short-Wavelength Fundus Autofluorescence in Stargardt Disease.

Purpose: To examine the extent of visual function abnormality outside the dark lesion on short-wavelength fundus autofluorescence (SW-AF), and its correlation with background SW-AF features and optical coherence tomography (OCT) in recessive Stargardt disease (STGD1). Methods: Forty-nine eyes of 25 participants in the ProgStar (the Natural History of the Progression of Atrophy Secondary to Stargardt Disease) study at our center were included. Patients underwent microperimetry (both threshold and dense scotoma mapping), OCT, SW-AF, and visual acuity testing. The Fisher's exact test, the χ2 test, and unpaired t-tests were used to analyze the data. Results: Of 40 eyes without central fixation, 33 (82%) placed fixation remote (most ≥5°) from the dense scotoma edge, despite good intervening retinal sensitivity. OCT findings accounted for the remote fixation in 75%. Eighteen (37%) of all 49 eyes had dense scotoma extending past the dark lesion border. OCT was not adequate to define the edge of the scotoma. Of the 49 eyes, 28 (57%) had the mottled background pattern, 10 (20%) had the uniform pattern, and 11 (22%) had the other pattern, with >75% of eyes in each pattern having remote fixation. The dense scotoma exceeded the dark lesion primarily in the mottled pattern. The two eyes of each patient were concordant in all features. Conclusions: Functional abnormalities in STGD1 extend past the SW-AF dark lesion. The disruption of the ellipsoid zone shows that photoreceptor abnormality extends peripheral to the dark lesion, and it explains in part the remote fixation pattern and the dense scotoma exceeding the dark lesion. This has implications for clinical trials for STGD1.

Automated classification of normal and Stargardt disease optical coherence tomography images using deep learning.

PURPOSE: Recent advances in deep learning have seen an increase in its application to automated image analysis in ophthalmology for conditions with a high prevalence. We wanted to identify whether deep learning could be used for the automated classification of optical coherence tomography (OCT) images from patients with Stargardt disease (STGD) using a smaller dataset than traditionally used. METHODS: Sixty participants with STGD and 33 participants with a normal retinal OCT were selected, and a single OCT scan containing the centre of the fovea was selected as the input data. Two approaches were used: Model 1 - a pretrained convolutional neural network (CNN); Model 2 - a new CNN architecture. Both models were evaluated on their accuracy, sensitivity, specificity and Jaccard similarity score (JSS). RESULTS: About 102 OCT scans from participants with a normal retinal OCT and 647 OCT scans from participants with STGD were selected. The highest results were achieved when both models were implemented as a binary classifier: Model 1 - accuracy 99.6%, sensitivity 99.8%, specificity 98.0% and JSS 0.990; Model 2 - accuracy 97.9%, sensitivity 97.9%, specificity 98.0% and JSS 0.976. CONCLUSION: The deep learning classification models used in this study were able to achieve high accuracy despite using a smaller dataset than traditionally used and are effective in differentiating between normal OCT scans and those from patients with STGD. This preliminary study provides promising results for the application of deep learning to classify OCT images from patients with inherited retinal diseases.

Fundus autofluorescence, spectral-domain optical coherence tomography, and histology correlations in a Stargardt disease mouse model.

Stargardt disease (STGD1), known as inherited retinal dystrophy, is caused by ABCA4 mutations. The pigmented Abca4-/- mouse strain only reflects the early stage of STGD1 since it is devoid of retinal degeneration. This blue light-illuminated pigmented Abca4-/- mouse model presented retinal pigment epithelium (RPE) and photoreceptor degeneration which was similar to the advanced STGD1 phenotype. In contrast, wild-type mice showed no RPE degeneration after blue light illumination. In Abca4-/- mice, the acute blue light diminished the mean autofluorescence (AF) intensity in both fundus short-wavelength autofluorescence (SW-AF) and near-infrared autofluorescence (NIR-AF) modalities correlating with reduced levels of bisretinoid-fluorophores. Blue light-induced RPE cellular damage preceded the photoreceptors loss. In late-stage STGD1-like patient and blue light-illuminated Abca4-/- mice, lipofuscin and melanolipofuscin granules were found to contribute to NIR-AF, indicated by the colocalization of lipofuscin-AF and NIR-AF under the fluorescence microscope. In this mouse model, the correlation between in vivo and ex vivo assessments revealed histological characteristics of fundus AF abnormalities. The flecks which are hyper AF in both SW-AF and NIR-AF corresponded to the subretinal macrophages fully packed with pigment granules (lipofuscin, melanin, and melanolipofuscin). This mouse model, which has the phenotype of advanced STGD1, is important to understand the histopathology of Stargardt disease.

Prospective Cohort Study of Childhood-Onset Stargardt Disease: Fundus Autofluorescence Imaging, Progression, Comparison with Adult-Onset Disease, and Disease Symmetry.

PURPOSE: To determine the reliability and repeatability of quantitative evaluation of areas of decreased autofluorescence (DAF) from fundus autofluorescence (FAF) images and track disease progression in children with Stargardt disease (STGD1), and to investigate clinical and genotype correlations, disease symmetry, and intrafamilial variability. DESIGN: Prospective cohort study. METHODS: Children and adults with molecularly confirmed STGD1 (n = 90) underwent longitudinal FAF imaging with subsequent semiautomated measurement of the area of DAF and calculation of the annual rate of progression. The age of disease onset was recorded for all subjects, as well as the electroretinography (ERG) group at baseline (n = 86). Patients were grouped for analysis based on the age at baseline and age of onset, into children (n = 56), adults with childhood-onset STGD1 (n = 15), and adults with adult-onset (n = 19). Fifty FAF images were selected randomly and analyzed by 2 observers to evaluate repeatability and reproducibility. Differences between groups, interocular symmetry, genotype-phenotype correlations, and intrafamilial variability were also investigated both for baseline measurements as well as progression rates. We measured visual acuity, molecular genetics, ERG group, FAF metrics, and their correlations. RESULTS: The mean age of onset ± SD was 9.6 ± 3.4 years for childhood-onset (n = 71) and 28.3 ± 7.8 years for adult-onset STGD1 (n = 19). The intra- and interobserver reliability of DAF quantification was excellent (intraclass correlation coefficients 0.995 and 0.987, respectively). DAF area was symmetric between eyes and the mean rate of progression (SD) was 0.69 (0.72), 0.78 (0.48), and 0.40 (0.36) mm2/year for children, adults with childhood-onset, and adults with adult-onset disease, respectively. Patients belonging to a group 3 ERG phenotype (generalized cone and rod dysfunction) had a significantly greater progression rate. Limited intrafamilial variability was observed. CONCLUSIONS: This is the first large prospective study of FAF in a cohort of molecularly confirmed children with STGD1. DAF area quantification was highly reliable and may thereby serve as a robust structural endpoint. A high rate of progression was observed in childhood-onset disease, making this subtype of STGD1 ideally suited to be considered for prioritization in clinical trials.

Choroidal hyper-reflective foci and vascularity in retinal dystrophy.

Purpose: To investigate choroidal hyper-reflective foci (HRF) in subjects with retinal dystrophy [Stargardt's disease (SGD) and retinitis pigmentosa (RP)] and their association with demographics, visual acuity, choroidal thickness (CT), and choroidal vascularity index (CVI). Methods: Single center retrospective study of subjects with previously diagnosed SGD or RP. Swept-source optical coherence tomography images were analyzed for the presence of choroidal HRFs and CVI using previously validated automated algorithm. A Spearman's rank correlation coefficient was used to evaluate the correlation between the number of HRF and various baseline parameters including age, visual acuity, intraocular pressure, and other optical coherence tomography (OCT) parameters (CT, choroidal area, and CVI) were evaluated in these subjects. Results: This study included 46 eyes (23 subjects) and 55 eyes (28 subjects) with previously diagnosed RP and SGD, respectively. In the RP group, the mean number of HRFs was 247.9 ± 57.1 and mean CVI was 0.56 ± 0.04. In SGD group, mean HRF was 192.5 ± 44.3 and mean CVI was 0.41 ± 0.04. Mean HRF was significantly greater in the RP group (0.02), however, the mean CVI was not statistically different. In RP, mean HRF were correlated only with CVI (r = 0.49; P = 0.001), however, in SGD, it correlated with only choroidal area (r = 0.27; P = 0.04). Conclusion: Choroidal HRF were present in both RP and SGD subjects with more HRFs in those with RP. These HRFs were associated with alteration in choroidal vascularity, which further adds into the pathogenesis of these diseases.

Multimodal evaluation of central and peripheral alterations in Stargardt disease: a pilot study.

BACKGROUND: The clinical phenotype of Stargardt disease (STGD) can be extremely heterogeneous, with variable macular and peripheral retinal involvement. The study aim was to correlate peripheral ultrawide field (UWF) involvement with macular alterations, as assessed by structural optical coherence tomography (OCT) and OCT angiography (OCTA), in order to identify potentially different phenotypes. METHODS: The study involved patients with STGD and healthy controls. We performed a complete ophthalmologic assessment and multimodal imaging, including OCT, OCTA, fundus autofluorescence and UWF imaging. Patients with STGD were subdivided according to the peripheral involvement. OCT and OCTA quantitative parameters were analysed. The main outcome of the study was the classification of UWF subtypes and the correlation between UWF subtypes and macular involvement. RESULTS: Seventy STGD eyes (19 male; mean age 41.3±13.2 years) and 70 healthy eyes (35 male; 50%; mean age 41.2±9.8 years) were included in the analyses. Mean best-corrected visual acuity was 0.60±0.45 LogMAR for the STGD group and 0.0±0.0 LogMAR for controls (p<0.01). All clinical and imaging findings proved to be statistically worse in patients with STGD than in the control subjects (p<0.01). UWF types were distributed as follows: type I (49%), type II (34%), type III (17%). Type III patients proved to be significantly worse in terms of visual function and OCT and OCTA imaging parameters. CONCLUSIONS: The UWF autofluorescence performed in the present study suggests that there exist three different STGD phenotypes. Each phenotype is associated with variable OCT and OCTA impairment. Further studies providing a better assessment of the peripheral retinal involvement in STGD are warranted.

Contextual influences in the peripheral retina of patients with macular degeneration.

Macular degeneration (MD) is the leading cause of low vision in the elderly population worldwide. In case of complete bilateral loss of central vision, MD patients start to show a preferred retinal region for fixation (PRL). Previous literature has reported functional changes that are connected with the emergence of the PRL. In this paper, we question whether the PRL undergoes a use-dependent cortical reorganization that alters the range of spatial lateral interactions between low-level filters. We asked whether there is a modulation of the excitatory/inhibitory lateral interactions or whether contextual influences are well accounted for by the same law that describes the integration response in normal viewers. In a group of 13 MD patients and 7 age-matched controls, we probed contextual influences by measuring the contrast threshold for a vertical target Gabor, flanked by two collinear high-contrast Gabors. Contextual influences of the collinear flankers were indicated by the changes in contrast threshold obtained at different target-to-flanker distances (λs) relative to the baseline orthogonal condition. Results showed that MDs had higher thresholds in the baseline condition and functional impairment in the identification tasks. Moreover, at the shortest λ, we found facilitatory rather than inhibitory contextual influence. No difference was found between the PRL and a symmetrical retinal position (non-PRL). By pulling together data from MD and controls we showed that in the periphery this inversion occurs when the target threshold approach the flankers' contrast (about 1:3 ratio) and that for patients it does occur in both the PRL and a symmetrical retinal position (non-PRL). We conclude that contrary to previous interpretations, this modulation doesn't seem to reflect use-dependent cortical reorganization but rather, it might result from a reduction of contrast gain for the target that promotes target-flankers grouping.

Multi-platform imaging in ABCA4-Associated Disease.

Fundus autofluorescence (FAF) imaging is crucial to the diagnosis and monitoring of recessive Stargardt disease (STGD1). In a retrospective cohort study of 34 patients, we compared FAF imaging platforms varying in field size (30° and 55°: blue/SW-AF and NIR-AF; 200°: ultrawide-field, UWF-AF), excitation wavelength (488 nm, blue/SW-AF; 532 nm, UWF-AF and 787 nm, NIR-AF) and image processing. Due to reduced absorption of 532 nm and 787 nm light by macular pigment, foveal sparing was more readily demonstrable by green/UWF-AF and NIR-AF imaging. Prominent in green/UWF-AF images is a central zone of relatively elevated AF that is continuous inferonasal with a demarcation line bordering lower AF nasally and higher AF temporally. This zone and border are more visible in STGD1 than in healthy eyes and more visible with green/UWF-AF. With the development of AF flecks, inferonasal retina is initially spared. Central atrophic areas were larger in NIR-AF images than in blue/SW-AF and green/UWF-AF images and the presence of a contiguous hyperAF ring varied with imaging modality. Flecks visible as hyperAF foci in blue/SW-AF images were also visible in green/UWF-AF but were often hypoAF in NIR-AF. Since disease in STGD1 often extends beyond the 30° and 55° fields, green/UWF-AF has advantages including for pediatric patients. The imaging platforms examined provided complementary information.

Photoreceptor cells as a source of fundus autofluorescence in recessive Stargardt disease.

Bisretinoid fluorophores form in photoreceptor outer segments from nonenzymatic reactions of vitamin A aldehyde. The short-wavelength autofluorescence (SW-AF) of fundus flecks in recessive Stargardt disease (STGD1) suggests a connection to these fluorophores. Through multimodal imaging, we sought to elucidate this link. Flecks observed in SW-AF images often colocalized with foci exhibiting reduced or absent near-infrared autofluorescence signal, the source of which is melanin in retinal pigment epithelial (RPE) cells. With serial imaging, changes in near-infrared autofluorescence (NIR-AF) preceded the onset of fleck hyperautofluorescence in SW-AF images and fleck profiles in NIR-AF images tended to be larger. Flecks in SW-AF and NIR-AF images also corresponded to hyperreflective lesions traversing photoreceptor-attributable bands in horizontal SD-OCT scans. The hyperreflective lesions interrupted adjacent OCT reflectivity bands and were associated with thinning of the outer nuclear layer. These SD-OCT findings are attributable to photoreceptor cell degeneration. Progressive increases and decreases in the SW-AF intensity of flecks were evident in color-coded quantitative fundus autofluorescence maps. In some cases, flecks appeared to spread radially from the fovea to approximately 8° of eccentricity, beyond which a circumferential spread characterized the distribution. Since the NIR-AF signal is derived from melanin and loss of this autofluorescence is indicative of RPE atrophy, the SW-AF of flecks cannot be accounted for by bisretinoid lipofuscin in RPE. Instead, we suggest that the bisretinoid serving as the source of the SW-AF signal, resides in photoreceptors, the cell that is also the site of bisretinoid synthesis.