Quantitative Fundus Autofluorescence Distinguishes ABCA4-Associated and Non–ABCA4-Associated Bull's-Eye Maculopathy

doi:10.1016/j.ophtha.2014.08.017

Ophthalmology

Volume 122, Issue 2, February 2015, Pages 345-355

https://doi.org/10.1016/j.ophtha.2014.08.017 Get rights and content

Purpose

Quantitative fundus autofluorescence (qAF) and spectral-domain optical coherence tomography (SD OCT) were performed in patients with bull's-eye maculopathy (BEM) to identify phenotypic markers that can aid in the differentiation of ABCA4-associated and non–ABCA4-associated disease.

Design

Prospective cross-sectional study at an academic referral center.

Subjects

Thirty-seven BEM patients (age range, 8–60 years) were studied. All patients exhibited a localized macular lesion exhibiting a smooth contour and qualitatively normal-appearing surrounding retina without flecks. Control values consisted of previously published data from 277 healthy subjects (374 eyes; age range, 5–60 years) without a family history of retinal dystrophy.

Methods

Autofluorescence (AF) images (30°, 488-nm excitation) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference to account for variable laser power and detector sensitivity. The grey levels (GLs) from 8 circularly arranged segments positioned at an eccentricity of approximately 7° to 9° in each image were calibrated to the reference (0 GL), magnification, and normative optical media density to yield qAF. In addition, horizontal SD OCT images through the fovea were obtained. All patients were screened for ABCA4 mutations using the ABCR600 microarray, next-generation sequencing, or both.

Main Outcome Measures

Quantitative AF, correlations between AF and SD OCT, and genotyping for ABCA4 variants.

Results

ABCA4 mutations were identified in 22 patients, who tended to be younger (mean age, 21.9±8.3 years) than patients without ABCA4 mutations (mean age, 42.1±14.9 years). Whereas phenotypic differences were not obvious on the basis of qualitative fundus AF and SD OCT imaging, with qAF, the 2 groups of patients were clearly distinguishable. In the ABCA4-positive group, 37 of 41 eyes (19 of 22 patients) had qAF₈ of more than the 95% confidence interval for age. Conversely, in the ABCA4-negative group, 22 of 26 eyes (13 of 15 patients) had qAF₈ within the normal range.

Conclusions

The qAF method can differentiate between ABCA4-associated and non–ABCA4-associated BEM and may guide clinical diagnosis and genetic testing.

Section snippets

Patients and Genetic Testing

Thirty-seven BEM patients (age range, 8–60 years) from 35 families were recruited prospectively at the Department of Ophthalmology, Columbia University. All subjects were examined by a retinal specialist (S.H.T.) and had clear media except for some floaters. Patients exhibiting the BEM phenotype were selected on the basis of fundus AF images. All patients exhibited a localized macular lesion exhibiting a smooth contour; outside the macula, the retina was qualitatively normal and without flecks.

Results

ABCA4 mutations were identified in 22 patients, including 21 patients (95%) with both disease-causing ABCA4 variants (Table 1). One patient was homozygous and 13 patients were compound heterozygous for the p.G1961E variant. ABCA4 was excluded as the causal gene in 15 patients because no mutations were detected after complete sequencing of the ABCA4 exons and adjacent intronic sequences. ABCA4-positive patients tended to be younger (mean age, 21.9±8.3 years) than ABCA4-negative patients (mean

Discussion

In this study, we assessed whether qAF, an indirect measure of RPE lipofuscin, could aid in differentiating ABCA4-positve from ABCA4-negative cases of BEM. Quantitative AF clearly distinguished the 2 groups. Specifically, qAF analysis indicated that ABCA4-positive BEM patients have increased lipofuscin levels throughout the posterior pole, whereas patients with BEM resulting from mutations in other genes have qAF levels within normal limits for age. These data reinforce the clinical usefulness

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A total of 259 participants aged ≥6 years with disease-causing variants in the ABCA4 gene were enrolled from 9 centers and followed over a 24-month period. FAF images were obtained every 6 months, and areas of definitely decreased autofluorescence (DDAF) and decreased autofluorescence (DAF) were quantified. Progression rates were estimated from linear mixed models with time as the independent variable.
A total of 488 study eyes of 259 participants (88.8% with both eyes) were enrolled and images from 432 eyes were followed for 24 months. The overall estimated progression of DDAF was 0.74 mm²/y (95% CI 0.64-0.85, P < .0001) and that of DAF was 0.64 mm²/y (95% CI 0.57-0.71) over a 24-month period in univariate analysis. Growth rates were strongly dependent on baseline lesion area. After square root transformation, the DDAF growth rate was not dependent on baseline lesion radius (P = .11), whereas the DAF growth rate was dependent (P < .0001). Genotype was not found to significantly impact the growth rate of DDAF or DAF lesions.
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Genetic diseases affecting the retina are highly heterogeneous. Although fundoscopy remains a diagnostic mainstay, retinal imaging provides additional important information and has become an essential part of the management of these conditions. These in vivo imaging techniques have rapidly evolved over the past decades due to advances in electronics, optics and computer technology. Key strengths include their high resolution and the potential to document retinal pathologies that are barely visible or cannot be detected on fundus examination alone, as well as to accurately document findings/progression over time. Fundus imaging can also be used to consolidate the diagnosis of a specific retinal disorder; multimodal imaging allows in-depth analysis of phenotypic heterogeneity and differential diagnoses (e.g. distinguishing localised from panretinal diseases) and may even indicate mutations in particular genes. Imaging can also offer novel insights into the pathogenesis of retinal conditions and provides surrogate outcome measures to assess the efficacy of therapeutic interventions. This chapter focuses on imaging modalities that are key for phenotyping genetic disorders affecting the retina. This includes established imaging techniques like optical coherence tomography (OCT) and short-wavelength fundus autofluorescence (SW-AF), but also more recently developed imaging techniques such as near-infrared fundus autofluorescence (NIR-AF), quantitative fundus autofluorescence (qAF) and adaptive optics (AO).
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A normal FAF signal, therefore, does not necessarily reflect an intact photoreceptor–RPE complex, but may rather correspond to a structurally intact-appearing RPE cell monolayer with or without the presence of intact photoreceptors. QAF imaging has been shown to be useful for differential diagnosis in retinal dystrophies, e.g., qAF allows for differentiation between ABCA4-associated and non-ABCA4-associated retinal disease (Duncker et al., 2015c). Moreover, PRPH2/RDS- and ABCA4-associated disease exhibiting phenotypic overlap may be partially discriminated when qAF values are corrected for age and race.
Fundus autofluorescence (FAF) imaging is an in vivo imaging method that allows for topographic mapping of naturally or pathologically occurring intrinsic fluorophores of the ocular fundus. The dominant sources are fluorophores accumulating as lipofuscin in lysosomal storage bodies in postmitotic retinal pigment epithelium cells as well as other fluorophores that may occur with disease in the outer retina and subretinal space. Photopigments of the photoreceptor outer segments as well as macular pigment and melanin at the fovea and parafovea may act as filters of the excitation light. FAF imaging has been shown to be useful with regard to understanding of pathophysiological mechanisms, diagnostics, phenotype-genotype correlation, identification of prognostic markers for disease progression, and novel outcome parameters to assess efficacy of interventional strategies in chorio-retinal diseases. More recently, the spectrum of FAF imaging has been expanded with increasing use of green in addition to blue FAF, introduction of spectrally-resolved FAF, near-infrared FAF, quantitative FAF imaging and fluorescence life time imaging (FLIO). This article gives an overview of basic principles, FAF findings in various retinal diseases and an update on recent developments.
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To investigate lipofuscin-related quantitative autofluorescence measures and their association with demographic characteristics, retinal structure, retinal function and genotype in ABCA4-related retinopathy (Stargardt disease 1).
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A total of 77 patients with ABCA4-related retinopathy and 110 control subjects underwent quantitative fundus autofluorescence (qAF) imaging using a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference to measure qAF as surrogate for lipofuscin accumulation. Measures of qAF were correlated with demographic characteristics, structural alterations on optical coherence tomography and fundus autofluorescence imaging, retinal function assessed by full-field electroretinography (ERG) and fundus-controlled perimetry, and genotype.
Most patients (76.6%) had qAF levels >95% prediction interval of the age-related control group, with best discrimination between cases and control subjects in younger patients. Reduced discrimination based on qAF measures was associated with mild disease, more advanced disease with dark flecks, or older age because of the physiological age-related increase in qAF and a ceiling effect in patients. Nullizygous patients presented with high qAF levels earlier in life compared with those with at least 1 milder ABCA4 variant. Within the sectors of qAF measurements, at approximately 7-9° eccentricity, increased qAF without flecks or with only bright flecks was associated with topographically related preserved retinal thickness and fundus-controlled perimetry results, and with normal full-field ERG recordings. All 3 parameters were increasingly abnormal with the development of dark flecks and decreasing qAF.
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: Supplemental material is available at www.aaojournal.org.

: Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

: Supported in part by the National Eye Institute, National Institutes of Health, Bethesda, Maryland (grant nos. EY024091, EY021163, EY019861, and P30EY019007); Foundation Fighting Blindness (Owings Mills, MD; grant no. C-CL-0710); and a grant from Research to Prevent Blindness, New York, New York, to the Department of Ophthalmology, Columbia University. During initial stages of the work, Dr. Delori was supported partially by the National Eye Institute (grant no. EY015520).

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Original articleQuantitative Fundus Autofluorescence Distinguishes ABCA4-Associated and Non–ABCA4-Associated Bull's-Eye Maculopathy

Purpose

Design

Subjects

Methods

Main Outcome Measures

Results

Conclusions

Section snippets

Patients and Genetic Testing

Results

Discussion

Exp Eye Res

Ophthalmology

Prog Retin Eye Res

Ophthalmology

Ophthalmology

Ophthalmology

Cell

Fundus autofluorescence and mfERG for early detection of retinal alterations in patients using chloroquine/hydroxychloroquine

Invest Ophthalmol Vis Sci

Clinical variations in assessment of bull's-eye maculopathy

Arch Ophthalmol

ABCA4 mutations and discordant ABCA4 alleles in patients and siblings with bull's-eye maculopathy

Br J Ophthalmol

Chloroquine retinopathy. Evaluation by fluorescein fundus angiography

Arch Ophthalmol

The PROM1 mutation p.R373C causes an autosomal dominant bull's eye maculopathy associated with rod, rod-cone, and macular dystrophy

Invest Ophthalmol Vis Sci

A detailed study of the phenotype of an autosomal dominant cone-rod dystrophy (CORD7) associated with mutation in the gene for RIM1

Br J Ophthalmol

An autosomal dominant bull's-eye macular dystrophy (MCDR2) that maps to the short arm of chromosome 4

Invest Ophthalmol Vis Sci

Functional characteristics of patients with retinal dystrophy that manifest abnormal parafoveal annuli of high density fundus autofluorescence; a review and update

Doc Ophthalmol

Functional correlates of fundus autofluorescence abnormalities in patients with RPGR or RIMS1 mutations causing cone or cone rod dystrophy

Br J Ophthalmol

In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics

Invest Ophthalmol Vis Sci

Quantitative fundus autofluorescence in healthy eyes

Invest Ophthalmol Vis Sci

Age-related accumulation and spatial distribution of lipofuscin in RPE of normal subjects

Invest Ophthalmol Vis Sci

Original article
Quantitative Fundus Autofluorescence Distinguishes ABCA4-Associated and Non–ABCA4-Associated Bull's-Eye Maculopathy