Quantitative Fundus Autofluorescence and Optical Coherence Tomography in ABCA4 Carriers.

The adenosine triphosphate-binding cassette, subfamily A, member 4 (ABCA4) gene, which is located on the short arm of chromosome 1, encodes for a membrane-associated protein located in outer segment (OS) disc membranes of rod and cone photoreceptors.1,2 ABCA4, originally identified as rim protein,3 participates in the transfer of retinaldehyde from the interior of the OS disc to the cytosol, after photobleaching of rhodopsin. Failure of this process leads to an increased formation of bisretinoid fluorophores due to condensation reactions of retinaldehyde; these fluorophores subsequently accumulate in the retinal pigment epithelium (RPE) as lipofuscin.4–6 Numerous toxic effects of bisretinoid have been demonstrated in in vitro studies.7 Additionally, in Abca4−/− mice that accumulate bisretinoids such as A2E in abundance, photoreceptor cells degenerate.8 Although accumulation of RPE lipofuscin, albeit at lower levels, is also part of the normal aging process, it is widely accepted that excessive accumulation of RPE lipofuscin is the damaging agent in recessive Stargardt disease (STGD1).9,10 The role of lipofuscin accumulation in age-related macular degeneration (AMD), a complex multifactorial disease, remains to be determined. Homozygous and compound heterozygous ABCA4 mutations are associated with multiple retinal dystrophy phenotypes including autosomal recessive STGD1, cone-rod dystrophy-3, and retinitis pigmentosa-19.11–14 Although establishing genotype–phenotype correlations has met with difficulty, given the extraordinary allelic heterogeneity in ABCA4 with currently more than 800 known disease-associated genetic variants, ABCA4-related disease is thought to be inversely correlated with the residual ABCA4 activity. For instance, patients with a severe reduction in ABCA4 activity manifest early and severe phenotypic changes. Approximately 5% of individuals of European descent carry a disease-associated ABCA4 allele.15,16 This high carrier frequency has implications for the extent to which ABCA4 variants contribute to the burden of retinal disease. Carriers of ABCA4 mutations may be at an increased risk of developing AMD,17–20 may reveal subtle visual dysfunction in psychophysical and electrophysiological tests,21 and may demonstrate moderate to severe fundus changes.17,22 The increased accumulation of lipofuscin in the RPE of patients with biallelic mutations in ABCA4 has been documented by histology,9 by spectrofluorometry,23 and more recently by quantitative autofluorescence (qAF).24 It is still unknown, however, whether individuals heterozygous for ABCA4 mutations also have elevated lipofuscin levels due to reduced ABCA4 activity. Data from the Abca4 mouse model support this hypothesis. Thus we and others25,26 have found that in mice heterozygous for a null mutation in Abca4, A2E levels are elevated relative to those in wild-type. In this study we used qAF to measure the effect of monoallelic ABCA4 mutations on RPE lipofuscin levels. Essential to the qAF methodology are an internal fluorescent reference installed in the confocal scanning laser ophthalmoscope (cSLO) and a rigorous protocol for image acquisition. Measurement of fundus AF levels is enabled by an internal reference, which is imaged simultaneously with the fundus to account for changes in laser power over time and to adjust for differences in the sensitivity setting of the cSLO.27 In addition, fundus AF gray levels are corrected for magnification and optic media density. We also analyzed spectral-domain optical coherence tomography (SD-OCT) scans acquired from carriers of ABCA4 mutations to assess whether structural alterations could be observed.