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Human Molecular Genetics, 2004, Vol. 13, No. 5 525-534
DOI: 10.1093/hmg/ddh048

Mutations in ABCA4 result in accumulation of lipofuscin before slowing of the retinoid cycle: a reappraisal of the human disease sequence

Artur V. Cideciyan1,*, Tomas S. Aleman1, Malgorzata Swider1, Sharon B. Schwartz1, Janet D. Steinberg1, Alexander J. Brucker1, Albert M. Maguire1, Jean Bennett1, Edwin M. Stone2 and Samuel G. Jacobson1

1Scheie Eye Institute, Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA, USA and 2Howard Hughes Medical Institute and Department of Ophthalmology, University of Iowa Hospitals and Clinics, Iowa City, IA, USA

Received October 21, 2003; Accepted December 17, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Mutations in ABCA4, which encodes a photoreceptor specific ATP-binding cassette transporter (ABCR), cause autosomal recessive forms of human blindness due to retinal degeneration (RD) including Stargardt disease. The exact disease sequence leading to photoreceptor and vision loss in ABCA4-RD is not known. Extrapolation from murine and in vitro studies predicts that two of the earliest pathophysiological features resulting from disturbed ABCR function in man would be slowed kinetics of the retinoid cycle and accelerated deposition of lipofuscin in the retinal pigment epithelium (RPE). To determine the human pathogenetic sequence, we studied surrogate measures of retinoid cycle kinetics, lipofuscin accumulation, and rod and cone photoreceptor and RPE loss in ABCA4-RD patients with a wide spectrum of disease severities. There were different extents of photoreceptor/RPE loss and lipofuscin accumulation in different regions of the retina. Slowing of retinoid cycle kinetics was not present in all patients; when present, it was not homogeneous across the retina; and the extent of slowing correlated well with the degree of degeneration. The orderly relationship between these phenotypic features permitted the development of a model of disease sequence in ABCA4-RD. The model predicted lipofuscin accumulation as a key and early component of the disease expression in man, as in mice. In man, however, abnormal slowing of the rod and cone retinoid cycle occurs at later stages of the disease sequence. Knowledge of the human ABCA4 disease sequence will be critical for defining rates of progression, selecting appropriate patients and retinal locations for future therapy, and choosing appropriate treatment outcomes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The ATP-binding cassette (ABC) transporter gene superfamily encodes membrane proteins involved in translocation of substrates across membranes (1). ABCA4 is a member of this superfamily and encodes ABCR (also known as Rim Protein, RmP) which is exclusively localized in the outer segments (OS) of rod and cone photoreceptors (2–5). ABCR is postulated to be involved in the active transport of all-trans-retinal aldehyde (atRAL) across photoreceptors disk membranes (3,6–9). Mutations in ABCA4 cause blinding autosomal recessive human retinal degenerations (3,10–18). Mouse models with partial or complete loss of abcr function show elevated phosphatidylethanolamine (PE) in rod outer segments and retinal pigment epithelium (RPE), accumulation of an adduct of atRAL and PE and slowing of dark adaptation after bright light exposure, an accelerated light-dependent deposition of N-retinylidene-N-retinylethanolamine (A2E) in the RPE as a major fluorophore of lipofuscin, and a very slow photoreceptor degeneration (19–21). Abnormally increased A2E levels may predispose RPE to apoptosis through several mechanisms likely involving photooxidative damage (22,23), and photoreceptors and vision are lost secondary to the dysfunction and/or death of RPE. Additionally, atRAL mediated photooxidative damage of ABCR may exaggerate this pathophysiologic mechanism by further reducing ABCR activity (24).

What do we know about the pathophysiology of human ABCA4-RD? Clinically-defined disease severity has been postulated to inversely relate to presumed residual ABCR activity (11,25–28). Proposed primary ABCA4 disease features of slowed dark adaptation and accelerated deposition of lipofuscin have been observed in some patients with known or presumed ABCA4-RD (15,16,29–32), but the complex interaction of photoreceptor and RPE degeneration with these features has not been explored. With potential therapies for humans with ABCA4-RD now being considered based on success in the murine model (33), there should be validation of the assumption that there are shared disease mechanisms in human ABCA4-RD and abcr deficient mice. We used surrogate measures of rod and cone photoreceptor loss, retinoid cycle kinetics, lipofuscin accumulation and RPE loss to develop a model of disease sequence in human ABCA4-RD as a first step toward defining in detail the human phenotype resulting from abnormalities in ABCR function.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Spectrum of rod and cone photoreceptor disease expression
A broad disease spectrum was found in our cohort of patients with ABCA4-RD; diseases ranged from delimited central retinopathies to retina-wide degenerations (Supplementary Material Table 1). Central retinal abnormalities typical of milder disease expressions are exemplified using psychophysical measures of sensitivity and crossectional images of microstructure (Fig. 1A). Some patients had relative preservation of foveal function within an area of parafoveal dysfunction (Fig. 1A, upper left, middle panels). In these patients, the preferred locus of fixation was foveal and visual acuities were relatively preserved (range, –0.13 to 0.4 logMAR). The majority (78%) of patients had a central scotoma (Fig. 1A, upper right panel) and used an extrafoveal locus of fixation which tended to be in the superior or peripapillary retina. As expected, these patients had, on average, worse acuities (range, 0.1 to 1.7 logMAR). In vivo microstructure by optical coherence tomography (OCT) showed thinning of the central retina caused by thinning of the photoreceptor nuclear layer and disruption of the signal originating from photoreceptor inner/outer segments (Fig. 1A, lower). The lateral extent of dysfunction by psychophysics corresponded to that of the microstructural abnormalities.



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  		Figure 1. Range of central and peripheral ABCA4-associated disease. (A) Central retinal abnormalities. Psychophysical sensitivity profiles (upper panels) and in vivo microstructure crossections (lower panels) through the anatomical fovea in three patients with increasing severity of central disease. Gray region on profiles represent the normal range; hash marks are the physiological blindspot corresponding to the optic nerve head. White line on the crossectional images represents the mean normal retinal thickness. Brackets delimit the image features corresponding to the photoreceptor layer (PR). F refers to fovea, and T and N refer to temporal and nasal retina, respectively. VA refers to best-corrected visual acuity. (B) Peripheral retinal abnormalities in three representative patients. Maps of rod- and cone-mediated psychophysical sensitivity loss presented on a gray scale; white is within normal limits and black is scotoma. Leading edges of ERG photoresponses (symbols) evoked by 4.6 log scot-td.s blue flash for rods and 4.1 log phot-td.s red flash for cones. Smooth lines show models of rod and cone phototransduction activation for patients (black) and for mean normal (gray). (C) Rod and cone ERG photoresponse maximum amplitudes relate to mean peripheral rod- and cone-mediated sensitivity losses determined psychophysically. Individual patient (P) numbers specified in (A) and (B) or used as symbols in (C) refer to Supplementary Material Table 1.

 
Peripheral retinal abnormalities were quantified with rod- and cone-isolated psychophysics and electroretinogram (ERG) photoresponses (Fig. 1B). Maps of psychophysical sensitivity loss revealed regional retinal variations in dysfunction and a wide range of severity: from central dysfunction only, to a gradient of dysfunction with greatest severity in the central retina and least in the periphery, to severely abnormal rod- and cone-mediated dysfunction across the retina (Fig. 1B). Rod- and cone-isolated ERG photoresponses, the only in vivo measure that can directly quantify photoreceptor function, indicated that the psychophysical defects were photoreceptor-based. A physiologically-based model provided estimates of remaining functional rod and cone photoreceptors across the retina and their average phototransduction activation kinetics (Fig. 1B). The maximum amplitude parameters of the model which are expected to be linearly proportional to the total rod or cone outer segment membrane area, ranged from normal to severely abnormal; sensitivity parameters were either normal or showed minor abnormalities in most patients (Supplementary Material Fig. 1). The maximum amplitude parameters for rods and cones were well correlated (r2=0.69, Supplementary Material Fig. 1) with most of the patients falling near the line of equal loss.

Evidence for a direct photoreceptor contribution to the perceptual dysfunction is shown in the relationship between psychophysical sensitivities and ERG photoresponses (Fig. 1C). Mean peripheral (>30° eccentricity) psychophysical sensitivity losses for rods and cones showed a good correlation (r2, 0.57 and 0.56 for rods and cones, respectively) to respective maximum amplitude measures using electrophysiology (Fig. 1C). These results also validated the use of psychophysical rod and cone sensitivities as local estimates of rod and cone degeneration in our cohort of ABCA4-RD patients.

Was the degree of photoreceptor degeneration related to the genotype? We tested the hypothesis that there was a relationship between presumed residual ABCR activity and disease severity (11,25–28) in 15 patients where plausible disease-causing variants in the ABCA4 gene were detected in both alleles. A relationship between mutation locus and overall severity of degeneration was not obvious (Supplementary Material Fig. 2). Of interest were four compound heterozygotes with G1961E change in one allele (P6, P11, P12, P15): those with frameshift mutations in the second allele (2005delAT or 4531insC) showed less degeneration at older ages (55 and 29, respectively) than those with point mutations (R2149L or E1122K) at younger ages (48 and 19, respectively). Similarly, a compound heterozygote (P4) with V849A and R408X mutations showed less severe disease than a patient (P7) with V849A and R2107H changes. Alleles demonstrated to have major abnormalities in vitro (7) could result in mild disease (P10: E1087K/G1961E) or severe disease (P5: G818E/L541P+A1038V; or P9: N965S/N965S). Therefore, the inverse relationship postulated between residual ABCR activity and clinically-defined disease severity (11,25–28) was not supported in terms of quantitative measures of disease severity as defined here.

Retinoid cycle kinetic abnormalities coincide with, not precede, retinal degeneration
Maps of psychophysical sensitivity loss demonstrated that photoreceptor degeneration could vary across the retina (Fig. 1B). This prompted the use of co-localized estimates of retinal degeneration, retinoid cycle kinetics and lipofuscin accumulation to understand the human ABCA4-RD disease sequence.

Recovery kinetics of psychophysical sensitivity after a bright adapting flash (dark adaptation kinetics) were used to probe retinoid cycle kinetics. Patients were first studied at a single retinal location: 30° eccentric to the anatomical fovea. The rod segment of retinoid cycle kinetics at this locus ranged from normal to dramatically slow. We used the time to reach criterion sensitivity as a quantitative measure of dark adaptation and divided the patients' results into three groups: normal, mild slowing and moderate/severe slowing (Fig. 2A, left, middle and right panels, respectively). Delay of dark adaptation was highly correlated with the absolute dark-adapted rod sensitivity at the same retinal locus (r2=0.8, Fig. 2B) suggesting a strong relationship between the extent of local rod photoreceptor degeneration and abnormality of retinoid cycle kinetics. Cone dark adaptation kinetics showed a similar range of variation among patients (Fig. 2A, insets) which, in turn, was related to dark-adapted cone sensitivity (r2=0.55, Fig. 2C). Not unexpectedly, rod and cone thresholds were highly correlated (r2=0.63, Fig. 2D) and showed mostly equal loss similar to retina-wide results of ERG photoresponses (Supplementary Material Fig. 1).



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  		Figure 2. Retinoid cycle kinetics as estimated with psychophysical dark adaptation. (A) Recovery of rod sensitivity following a flash at time zero isomerizing nearly all visual pigment in representative patients grouped according to kinetics: normal (left), mild slowing (middle) and moderate/severe slowing (right, note change in horizontal axis). Dark-adapted sensitivities shown before time zero. Gray lines represent the normal range. Insets, recovery of cone sensitivity. (B) Delay of rod adaptation is strongly related to loss of dark-adapted rod sensitivity measured before the light exposure. (C) Delay of cone adaptation is strongly related to loss of dark-adapted cone sensitivity measured during the cone plateau phase. (D) Comparison of rod and cone sensitivity losses. Line is the hypothetical equal loss line. (E) Dark adaptation at two retinal locations (4 and 30° eccentric) measured during the same session. Central locations shows greater delay than the more peripheral locations in both patients. (F) Dark adaptation at a location 30° eccentric to fovea showing discordant results in two pairs of siblings.

 
The intersubject variation of dark-adaptation abnormality had two possible explanations: (i) differences in genotype; or (ii) differing extents of retinal degeneration at the tested locus. The latter hypothesis was readily testable by measuring intra-retinal variation of dark adaptation kinetics, recognizing that degeneration varied across the retina in most patients but the molecular defect was expected to be invariant. When we determined dark adaptation kinetics at multiple retinal loci simultaneously in a subset (n=19) of ABCA4-RD patients, we found there could be dramatic intraretinal variation of recovery kinetics (Fig. 2E). More central locations showed slower kinetics and lower sensitivities than more peripheral loci, which showed faster kinetics and higher sensitivities. These data support the notion that an important contributor to retinoid cycle abnormalities was the extent of local photoreceptor degeneration. Further support came from affected siblings sharing the same ABCA4 genotype but displaying different extents of retinoid cycle abnormality at the same midperipheral locus (Fig. 2F). The younger siblings tended to show less recovery slowing than the older siblings suggesting retinoid cycle abnormalities to be progressive like the retinal degeneration. A contribution of the genotype to a primary dark-adaptation abnormality remains a theoretical possibility although contributory evidence was not available in our data.

Lipofuscin accumulation is the earliest detectable abnormality
Autofluorescence (AF) imaging was used to estimate the distribution of lipofuscin accumulation within the RPE. In normal subjects, AF intensity peaks on a ring ~10° eccentric to the fovea and falls gradually towards the fovea and towards the periphery. The eccentricity of the peak AF is smaller in the nasal retina where the optic nerve head is located. This smooth topography is modified by steep but orderly changes occurring near the fovea and optic nerve head. The reduction at the fovea is believed to be due to a combination of factors that include absorption by the macular pigment, increased RPE melanin, as well as a reduction in lipofuscin content (31,34). The reduction observed at the peripapillary ring has not been extensively analyzed, and the lack of AF within the optic nerve head is due to the lack of RPE cells in this region.

AF images of some patients (e.g. P29, P44) were indistinguishable from those in normal subjects (Fig. 3A) suggesting the mildest disease expression of ABCA4-RD may not always include lipofuscin accumulation. Most patients, however, showed AF abnormalities near the fovea and distributed throughout the central retina (Fig. 3A and B). Two types of abnormalities were apparent on AF images: intensity and texture. AF intensities could vary dramatically across the retina and range from supernormal to undetectable and include normal values (Fig. 3A and B). Similarly, ‘local’ parameters mean AF intensity and AF texture varied dramatically across the retina as shown in a representative patient (Fig. 3C). Notable were those regions where mean AF intensity was within normal limits but AF texture was abnormally elevated.



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  		Figure 3. Lipofuscin accumulation as estimated with fundus autofluorescence (AF) images. (A) Standardized images of AF intensity mapped to a color scale (shown on the right) in a representative normal subject and five patients. Black represents no image data and purple is the dark-level of the detector. (B) Horizontal and vertical profiles of AF intensity in the five patients shown in (A). Gray lines represent the range of normal results. T, N, I, S, refer to temporal, nasal, inferior and superior retina, respectively. (C) AF intensity image of a representative patient mapped to a color scale as in (A), and derived local parameters of mean AF, AF texture and AF texture index showing regions within (green) or outside (red) normal limits. (D–F) Mean AF, AF texture and AF texture index values as a function of rod adaptation delay. Green curves are fit by eye.

 
Could regional variation in mean AF intensity and texture have a relationship to dark adaptation kinetics (which is related to local extent of retinal degeneration)? At a locus near 30° eccentricity where co-localized and measurable data were available in the largest subset of the patients, some of the highest mean AF intensities (e.g. P42) were associated with near normal kinetics and some of the lowest mean AF intensities with either normal (e.g. P29) or extremely slow (e.g. P32) dark adaptation kinetics (Fig. 3D). There appeared to be a non-monotonic relationship suggesting the existence of another contributing variable. AF texture also showed a non-monotonic relationship with rod dark adaptation delay; the extent of decrease in texture was smaller that the decrease in mean intensity with increasing delay (Fig. 3E). AF texture index, which was derived as a unitless ratio of AF texture to mean AF intensity, showed a monotonic but nonlinear relationship with the rod adaptation delay. In this group of ABCA4-RD patients, a local AF texture index of >8% was associated with an abnormal delay in rod adaptation (Fig. 3F).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Disease model of human ABCA4-associated retinal degeneration
A number of clinically diagnosed retinal diseases have been associated with ABCA4 mutations (3,10–18), suggesting phenotypic diversity despite commonality of molecular causation. We asked whether it was possible to gain understanding of the disease process resulting from ABCA4 mutations using in vivo mechanism-based probes of function and structure within retinal regions. Our observations point to an orderly disease sequence in human ABCA4-RD. Six stages of ABCA4-RD were definable. Stages I and VI, the two extremes, were incontrovertible. At Stage I, retinal regions had normal structure and function of photoreceptors and RPE, as shown by normal parameters for rod and cone sensitivities, dark adaptation kinetics, and AF intensity and texture. At the other extreme, Stage VI, there were regions with complete degeneration of photoreceptors and RPE, as shown by undetectable visual function and AF. Intervening stages were defined as having progressively greater numbers of abnormalities (Fig. 4).



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  		Figure 4. Model of ABCA4 disease sequence. Data from midperipheral retinal region of different patients ordered according to disease sequence in the upper five panels. Lowest panel shows the disease severity in these patients. Stages of disease are distinguished by vertical lines. Stages I–V are shown; Stage VI refers to complete degeneration of photoreceptors and RPE, and is not shown. Up represents greater or faster than normal.

 
Increased AF intensity was the only abnormality that occurred with normal values for all other parameters, suggesting that elevated RPE lipofuscin, defined as Stage II disease (Fig. 4), may be the first pathophysiological process detectable in human ABCA4-RD. Abnormal accumulation of lipofuscin has been demonstrated in Stargardt disease (STGD) by histopathology (35,36), and by non-invasive methods (16,31,32). The current work is the first to provide evidence for an association of this disease effect with otherwise normal photoreceptors and RPE. Retinal regions with Stage II disease may not be differentiable from normal aged retina, suggesting an acceleration of the rate of normal age-related lipofuscin accumulation in ABCA4-RD (31,37). This rate of accumulation shows some macroscopic variation in the normal retina (37) and it is tempting to speculate that the same intraretinal molecular and environmental gradients that contribute to this normal variation also play a role in the retinal distribution of ABCA4 disease. Furthermore, microscopic intraretinal variation in the rate of normal age-related lipofuscin accumulation may also exist although this has not been experimentally explored to date.

Stage III disease would correspond to retinal regions displaying abnormal increase of AF texture, the microscopic spatial variation in intensity, in addition to abnormal increase in mean AF intensity (Fig. 4). The appearance of regions we term Stage III disease on AF imaging were qualitatively similar to published images (16,32,38). AF texture has not been previously quantified or related to co-localized extent of retinal degeneration. Increased AF texture may result initially from microscopic variations in rates of lipofuscin accumulation or from apical condensation of melanin granules observed in RPE cells laden with lipofuscin (35). Increased AF texture associated with more severe stages of disease may include loss of RPE cells, individually or in small patches, after they have reached a threshold insult due to a combination of lipofuscin accumulation, epigenetic and environmental factors, as well as retinal regional properties. As RPE cells are lost, surviving cells may spread to cover the area of exposed Bruch's membrane (36) and thus explain the reductions in mean AF intensity observed in severely affected retinal regions (below).

Stage IV in the ABCA4 disease sequence would involve partial degeneration of photoreceptors and slowing of the retinoid cycling in addition to the RPE abnormalities of Stages II and III; abnormalities would be present in all measured parameters. Coincidence of rod and cone degeneration and slowing of rod and cone retinoid cycles suggests significant contributions to this stage from RPE loss. This is further supported by Stage V disease where there is a counterintuitive return of mean AF intensity to normal levels as retinoid cycle abnormalities and photoreceptor degeneration become more pronounced (Fig. 4). The reduction of AF intensity may be secondary to the reduction in the number of living RPE cells or reduced shedding of OS membrane as the photoreceptors degenerate.

Factors in addition to the primary molecular defect are likely to be defining the rate of progression in each region, making the overall retina-wide phenotype not a simple sum of parts. Consistent with this postulation, the retina-wide index of disease severity does not correlate with the local disease stage except for stage V (Fig. 4).

Delayed dark adaptation is not a characteristic manifestation in ABCA4-associated retinal degeneration
Kinetics of dark adaptation ranged from normal to dramatically abnormal in our cohort of ABCA4-RD, a result consistent with earlier studies of ABCA4 retinopathies (15) when taken together with studies in ungenotyped patients with STGD, fundus flavimaculatus, or cone-rod dystrophy (CRD) (29,30,39–42). Two possible causes may be proposed for the wide range of dark adaptation kinetics observed in ABCA4-RD: dark adaptation delay is a primary manifestation related to the extent of the loss of ABCR activity, or a secondary phenotype related to the extent of disease. The variation of dark-adaptation kinetics observed intraretinally and between affected siblings, and the existence of an orderly relationship between the amount of local retinal and RPE degeneration and the degree of dark adaptation delay provide support for a dominant role caused by a secondary manifestation. Furthermore, in those patients with ABCA4-RD and demonstrably normal dark adaptation kinetics, it can be stated that ABCA4 mutations causing overt human retinopathy do not necessarily result in a detectable delay in dark-adaptation kinetics. However, our data do not rule out a (minor) component of dark adaptation delay resulting directly from the loss of ABCR activity in those patients with slow kinetics.

Dark adaptation in ABCA4-RD can be viewed in the context of results in other human retinopathies with known molecular cause. Mutations in RHO, GRK1, SAG and RDH5, established as critically involved in the retinoid cycle (43), result in adaptation delays that are invariant across the retina (44–47). On the other hand, mutations in TIMP3 and CTRP5, believed to cause a subRPE deposit with ensuing secondary degeneration of photoreceptors and RPE, result in adaptation delays that show inter-subject and intra-retinal variation and progression on serial measurements (48–50). Most instructive may be the comparison of ABCA4-RD dark adaptation results with those from patients with mutations in the gene encoding Peripherin-RDS, another rod and cone specific rim protein. Peripherin-RDS mutations are associated with dark adaptation abnormalities that show dramatic inter-subject and intra-retinal variation, ranging from normal to severely abnormal (51,52). ABCA4-RD results are more similar to those in patients with Peripherin-RDS mutations than to those of TIMP3 or CTRP5 in terms of showing a distinct relationship between local rod and cone degeneration and delay of dark adaptation. This may not be surprising since the clinical phenotype of Peripherin-RDS and ABCA4 disease show some overlap (53) and the diseases both have abnormal lipofuscin accumulation (54). We propose that regional retinal slowing of the retinoid cycle could be a marker of insult to RPE or photoreceptors or to the milieu between them in ABCA4- and Peripherin-RDS-associated RD.

Abcr knockout mice show elevated PE, accumulation of an adduct of atRAL and PE and slowing of dark adaptation after bright light exposure, an accelerated light-dependent deposition of A2E in the RPE as a major fluorophore of lipofuscin, and a very slow photoreceptor degeneration. An age or stage with normal dark adaptation has not been reported to date in mice with partial or complete loss of abcr function (19–21), as compared to the normal dark adaptation kinetics observed in a subset of patients in the current work. Quantitative differences between humans and mice for this phenotypic feature, however, are not unique to ABCA4-RD. Differences have also been observed in Grk1 deficiency (45,55) and in partial loss of Rpe65 function (56,57). In terms of ABCA4 disease, differences between mice and man may be due to interspecies differences in molecular reactions defining the rate-limiting step during different phases of dark adaptation. Hypothetical contributors include deactivation of activated rhodopsin, diffusion of atRAL out of photoreceptors, isomerization, esterification, oxidation of 11-cis-retinol, and regeneration of rhodopsin (43) but comparative reaction rates of the retinoid cycle for humans and mice are currently not known.

Current and future value of a disease model
The disease model we propose for ABCA4-RD, although determined years after extensive research into basic mechanisms of the abcr-deficient mouse, should stimulate a reappraisal of the relationship between disease pathways in the murine and human retinopathies. For example, slowed dark adaptation in mice with partial or complete loss of abcr function at stages of little or no retinal degeneration is not consistent with the human phenotype, although it has been considered to be so previously (19–21,33). The disease model of ABCA4-RD should also be valuable in choosing retinal regions appropriate for potential treatments and for defining outcome measures. A treatment based on the ability of systemic retinoic acid to slow the retinoid cycle, for example, has shown success in the abcr deficient mouse (33) and this substance (or others acting through similar mechanisms) may be considered for trial in patients. It will be important to differentiate slowing of dark adaptation predicted to occur due to the ‘treatment’ and possibly the same effect predicted to occur with the progression of the ABCA4 disease stage. Among potential outcome measures for future treatment, the finding of lipofuscin accumulation at the earliest stages may make this the disease expression worthy of monitoring for change.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Subjects and molecular genetic analyses
The study population consisted of patients with clinical diagnoses of STGD or CRD [age=39±16 years, n=47; phenotype classification (13,18); Supplementary Material Table 1] and one or more changes in the ABCA4 gene considered to be disease-causing variants. Details of the molecular genetic analysis and interpretation of the allelic variation have been published (14). Research procedures were in accordance with institutional guidelines and the Declaration of Helsinki. All patients signed informed consent.

Visual function and structure studies
A complete eye examination, including best-corrected ETDRS visual acuity, was performed in all subjects. Preferred locus of fixation with respect to the anatomical fovea was documented using fundus imaging and all psychophysical tests were adjusted to each patient's preferred fixation locus.

Rod- and cone-mediated sensitivities were measured across the visual field on a 12-degree grid using a modified automated perimeter (58). Sensitivity losses were determined at each locus compared to mean normal values and mean peripheral sensitivity losses were calculated for all loci at eccentricities of 30° or greater. Cone-mediated sensitivity profiles horizontally across the anatomical fovea were obtained on a 2° grid in all patients with foveal fixation and a subset with extrafoveal fixation. OCT (OCT1 or OCT3, Zeiss Humphrey Instruments, Dublin, CA) scans were performed and analyzed (59) to estimate in vivo microstructure in central retinal crossections.

Dark (bleaching) adaptation functions were measured psychophysically at a location 30° eccentric to the anatomical fovea; in a subset of patients, dark adaptation kinetics at a more central locus (4–10° eccentric) was measured simultaneously with the more peripheral locus (44–52). The kinetics of the dark adaptation functions were quantified as the time to reach a criterion sensitivity which was set at 1.8 log units below mean normal value (dark-adapted for rods, cone-plateau for cones).

ERG photoresponses were recorded under dark- and light-adapted conditions. The leading edges were analyzed with a model of rod and cone phototransduction activation and parameters of maximum amplitude and sensitivity were derived (45–47); standardized ERGs were also performed.

Retina-wide extent of retinal degeneration was quantified for each patient as a percentile rank within the study population (100% referring to the patient with most severe disease) based on equal contributions of three rankings: peripheral rod function, peripheral cone function and best corrected visual acuity. Multiple measures were used for the peripheral function rankings to assure monotonity. Peripheral rod function ranking was the average of three rankings: rod photoresponse amplitude, mean peripheral rod sensitivity loss and standardized ERG b-wave amplitude. Peripheral cone function ranking was the average of three rankings: cone photoresponse amplitude, mean peripheral cone sensitivity loss and standardized 30 Hz ERG amplitude.

Autofluorescence imaging
Spatial distribution of retinal AF was obtained with a confocal scanning laser ophthalmoscope (HRA, Heidelberg Engineering, Dossenheim, Germany) as described (60). The illumination light was at 488 nm and fluorescence was detected above 500 nm. All images were acquired with a lateral magnification wherein a 30x30° square field was sampled onto 512x512 pixels. The imaging protocol typically consisted of 6–10 overlapping regions. At each region, 20–40 consecutive images were obtained, frames with obvious blinks, or eye movements were discarded and the remaining images were averaged after manual alignment using retinal landmarks. A wide field composite AF image was formed from the resulting noise-reduced images. Images from left eyes were transformed into equivalent right eyes. A rigid body transformation was calculated and applied to register the anatomical fovea and the center of the optic nerve head to predetermined locations based on mean normal results (applied scale factors=0.95±0.07; rotation=–1.2±4.5°). AF intensity of each image was normalized by the mean intensity within a 30x30 pixel region at the center of the optic nerve (applied normalization factors=1.0±0.26).

In addition to the AF intensity at each pixel within the standardized image co-ordinates, three ‘local’ parameters were derived using a moving block size of 25x25 pixels: mean AF intensity, AF texture and AF texture index. Mean AF intensity corresponded to the moving average values and provided a macroscopic topography of the AF change across the retina. AF texture corresponded to the moving standard deviation and provided a measure of microscale topography of AF. AF texture index was defined as AF texture divided by mean AF intensity. Normal statistics for AF intensity and three local parameters were determined from a group of normal subjects (age=43±11 years, n=8) and used to interpret data from patients. Pixels corresponding to major blood vessels were manually specified and not included in the calculation of local parameters.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
The work was supported by grants from the National Institutes of Health (EY-13203, -13385, -13729, -10820, -12156), Macula Vision Research Foundation, Foundation Fighting Blindness, Macular Disease Foundation, F.M. Kirby Foundation, Sam B. Williams Fund, and the Mackall Foundation. A.V.C. and J.B. are Research to Prevent Blindness William and Mary Greve Scholars. We thank Elaine DeCastro, Leigh Gardner, Jessica Emmons, John Chico, Elaine Smilko, Elizabeth Windsor, Alejandro Roman and Jean Andorf for their valuable assistance.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Scheie Eye Institute, University of Pennsylvania, 51 North 39th Street, Philadelphia, PA 19104, USA. Email: cideciya{at}mail.med.upenn.edu


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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