Quantitative Fundus Autofluorescence Distinguishes ABCA4-associated and non-ABCA4-associated Bull's-Eye Maculopathy

INTRODUCTION

Varying definitions of Bull's-Eye maculopathy (BEM) have been reported^1-4 since the term was first introduced in 1966 to describe the fundus appearance of chloroquine retinopathy.⁵ The classical BEM phenotype presents as concentric parafoveal rings of increased and decreased fundus autofluorescence (AF).^6-8 However the rings in BEM can be less obvious due to mottling, bright or reduced foveal AF and/or hyperautofluorescence that is diffuse and the lesion can be round or elliptical.^6,8,9 The BEM phenotype can also progress to extensive macular atrophy.⁶ Some patients who are homozygous for disease-causing mutations in the ABCA4 gene can present with the BEM phenotype.^2,4 However, BEM is not specific to ABCA4 and can also be caused by mutations in phototransduction-related genes (e.g. GUCA1A, RPGR);^10,11 mutations in a gene (PROM1)⁶ that encodes a protein involved in outer segment morphogenesis; and mutations in a gene (RIM1)⁷ that encodes a photoreceptor synaptic protein. Thus pinpointing the underlying cause of BEM in an individual patient can be challenging.

Advances in non-invasive imaging have facilitated the diagnosis and differentiation of retinal dystrophies. Two imaging modalities that have proven especially valuable in this regard are spectral domain optical coherence tomography (SD-OCT) and fundus autofluorescence (AF). By SD-OCT, structural changes of the retina can be studied almost at a cellular level. Fundus autofluorescence (AF) emerges from RPE lipofuscin,¹² a mixture of fluorophores with spectral emission features that reflect the bisretinoid constituents that have been characterized.¹³ RPE lipofuscin accumulates with age in healthy eyes^14,15 and its formation is accentuated in recessive Stargardt disease (STGD1).^16-19 Disease-related processes can also alter the distribution of the AF signal. Through image registration, SD-OCT and AF can be correlated.

Using quantitative fundus AF (qAF), an approach we have developed to measure fundus AF intensities, we recently reported increased qAF levels in STGD1 patients with confirmed ABCA4 mutations.²⁰ In these patients, qAF was elevated even at young ages and in qualitatively unaffected appearing fundus areas. Whether qAF levels are also increased in retinal dystrophies related to mutations other than ABCA4 and to what extent lipofuscin is involved in the pathogenesis of these conditions, remains to be determined.

The objective of this study was to investigate whether qAF, a non-invasive approach to measuring RPE lipofuscin in vivo, could aid in distinguishing ABCA4-associated from non-ABCA4-associated disease in BEM, a phenotype that is common to some retinal dystrophies. In addition, we were interested as to whether based on qualitative assessment of AF and SD-OCT images a distinction of ABCA4-positive and ABCA4-negative patients is feasible.

METHODS

RESULTS

ABCA4 mutations were identified in 22 patients including 21 cases (95%) with both disease-causing ABCA4 variants (Table). One patient (P1) was homozygous and 13 patients were compound heterozygous for the p.G1961E variant. ABCA4 was excluded as the causal gene in 15 patients since 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 age: 42.1 ±14.9 years) (unpaired t-test; p<0.0001). There was no difference in visual acuities between the two groups (unpaired t-test; right eyes: p=0.88, left eyes: p=0.13). International Society for Clinical Electrophysiology of Vision (ISCEV) standardized full-field electroretinography (ERG) was performed in 16/22 ABCA4-positive patients. All of these patients were classified as isolated maculopathy (normal full-field ERG).^3,26 Of the 15 ABCA4-negative patients, 10 manifested generalized cone dysfunction; 3 had normal ERGs and 2 did not participate in ERG testing.

ABCA4-positive and –negative patients shared similar AF features (Figs. 2 and 3; AF images of all patients available at http://aaojournal.org). For instance we found that in both groups of patients the BEM lesion could be distinctly circular (e.g. ABCA4+ve patients 8, 9; ABCA4-ve patients 26, 30, 31, 33, 37) or elliptical (e.g. ABCA4+ve patients 3, 7, 16; ABCA4-ve patients 27, 32). The parafovea could be mottled (e.g. ABCA4+ve patients 3, 4, 9, 15, 17; ABCA4-ve patients 25, 26, 30, 33) and the hyperautofluorescent ring can be bright with well-defined borders (e.g. ABCA4+ve patients 5, 7; ABCA4-ve patients 25, 26, 28), relatively faint (e.g. ABCA4+ve patients 11,13, 19, 21; ABCA4-ve patients 27, 29, 30, 31, 37) or it could be diffuse (e.g. ABCA4+ve patients 18, 22; ABCA4-ve patients 23, 34). The fovea could also appear bright (e.g. ABCA4+ve patient 2, 6, 9, 16; ABCA4-ve patient 30, 32, 33, 35) or dark (e.g. ABCA4+ve patients 14, 18; ABCA4-ve patients 25, 27, 28). Lesion size also varied amongst the patients while there was a remarkable similarity between fellow eyes with respect to lesion size, shape and AF pattern.

DISCUSSION

In the present 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 two groups. Specifically, qAF analysis indicated that ABCA4-positive BEM patients have increased lipofuscin levels throughout the posterior pole, while patients with BEM due to mutations in other genes have qAF levels within normal limits for age. These data reinforce the clinical utility of qAF. Due to the association between ABCA4-related disease and a dark choroid in fluorescein angiography, the latter imaging modality can also benefit diagnosis. However, qAF has the advantage of being non-invasive. It is also worth noting that In the case of G1961E mutations in ABCA4 neither fluorescein angiography nor qAF may be helpful imaging modalities; a dark choroid is absent in these individuals²⁷ and in the presence of the G1961E mutation, qAF values can be within the normal range for age (Fig. 5).

The ABCA4 disease pathway is known. Functioning ABCA4 protein²⁸ is needed to remove N-retinylidene-phosphatidylethanolamine (NRPE), formed by the binding of retinaldehyde to phosphatidylethanolamine (PE), from the outer segment disc membranes of rods and cones. Failure of this process, such as in STGD1, permits NRPE to accumulate in excess such that a second molecule of retinaldehyde can react with NRPE leading to the formation of A2E and related bisretinoids. These molecules are phagocytosed and stored in RPE cells as lipofuscin and have potential for cellular damage.¹³ The disease pathways in ABCA4-negative BEM patients are less clear and the phenotypic similarities between ABCA4-positive and ABCA4-negative patients are currently difficult to explain. Moreover, while lipofuscin accumulation is likely involved in the primary pathogenesis of ABCA4-related disease, whether or not this is the case for non-ABCA4-disease is not known.

Although retinal changes were most obvious in the central macula, we also observed ONL thinning in a transition zone at the lesion border in all but one patient. In a subgroup of patients, the ONL in the transition zone was altered by the presence of hyperreflective material. The ELM was also pronounced outside the central part of the lesion in some ABCA4-positive patients. A case of ELM thickening in a young STGD1 patient without any obvious RPE changes has been reported before²⁹ and it was suggested that ELM thickening could be an early disease marker. While in our study cohort all patients with an OEL were ABCA4-positive, other studies have shown that OEL can also be associated with mutations in other genes, e.g. in achromatopsia.³⁰ We identified several quantifiable features by SD-OCT, however, none of these clearly distinguished ABCA4-positive and ABCA4-negative cases.

Since all photoreceptors of ABCA4-positive cases have a defective visual cycle, it is perhaps not surprising, that retinal changes also extended into areas that appeared unaffected on AF imaging. It is interesting to consider however, why the central macula appears to be more susceptible to the damaging effects of the ABCA4 mutations than the rest of the retina. One possibility is that certain ABCA4 mutations, for instance, the p.G1961E variant, may elicit a stronger effect on cones than rods. Although p.G1961E is the most frequent ABCA4 variant in STGD1 (allele frequency ~10%),^31,32 it is still remarkable that 14/22 (64%) ABCA4-positive BEM patients carried this variant. It has been noted before, however, that the p.G1961E appears to be associated with the BEM phenotype and a milder disease spectrum.^2,20,31,33 The notion that STGD1 patients with a BEM phenotype have less widespread disease is further supported by Fig. 7, which shows the qAF₈ levels of the white ABCA4-positve BEM patients in comparison to previously published²⁰ white STGD1 patients with other phenotypes than BEM, including patients with extramacular fundus changes. ABCA4-positve BEM patients have higher qAF levels than normal subjects (and ABCA4-negative BEM patients) but can have relatively low qAF levels when compared to other STGD1 patients.

It is perhaps not a coincidence that most ABCA4-positive patients in our study clustered at young ages. In STGD1, the BEM phenotype may in some cases be a transient stage and patients will eventually develop more extensive fundus changes. This is illustrated in Fig. 8 wherein disease progression over a period of 2.5 years is shown for the brother of P15, who also carries the complex allele L541P/A1038V. At the initial visit (Fig. 8A), this patient could readily have been classified as presenting with BEM. However, we decided not to include the patient in this study since some peripheral flecks were already visible. At the subsequent visits 1.2 (Fig. 8B) and 2.5 (Fig. 8C) years later, the flecks were more pronounced and numerous.

A question that is not only of interest for the interpretation of the AF signal in BEM but also for other retinal dystrophies, is what may be the basis for the high AF ring that surrounds the low AF part of the central lesion. We found that, similar to retinitis pigmentosa,³⁴ the ISe was discontinued at the outer border of or within the high AF ring. Thus, the high AF ring may reflect an accelerated production of bisretinoids within degenerating photoreceptors.³⁵ As an alternative explanation, Freund et al. recently suggested that the high AF ring may result from a “window defect”.³⁶ They proposed that loss of the outer retina might cause less absorption of the AF signal originating from the RPE. However, photopigment, the major absorber of the fundus AF exciting and emitted light, was bleached before image acquisition in our protocol.

A limitation of this study is that retinal layer thicknesses on SD-OCT were not analyzed quantitatively. By retinal layer segmentation one could confirm whether the ONL was thinned throughout the retina or whether the thinning was limited to the central part of the lesion and the adjacent transition zone.³⁷

While finding the definitive causal genes in the ABCA4-negative patients is beyond the scope of this study, we have investigated the genetic causality in most ABCA4-negative cases by whole-exome sequencing (WES, 7/15 patients) or targeted candidate gene sequencing (4/15 patients). While these studies have not yielded the causal gene in most cases, we found that: 1) the genetic causality varies widely in the ABCA4-negative group; 2) except for one patient with an RPGR mutation, in all cases we excluded known genes that have previously been associated with the BEM phenotype (PROM1, GUCA1A, etc.); 3) in some cases we unexpectedly found disease-causing mutations in known retinal disease genes such as CRX³⁸ and CNGA3, which have never been associated with the BEM phenotype; i.e., suggesting phenotypic expansion (BEM phenotype in genes known to cause a very different disease phenotype). Since almost all of our patients exhibit sporadic disease with no available family members, we were not able to identify the causal gene, even by WES, in most cases since, unless one finds clearly pathogenic variants in known disease genes, it is impossible to distinguish between many candidate genes that result from WES studies of a single patient. In summary, we found a very likely causal gene in 5/15 cases, excluded known BEM-associated genes in most cases, and identified multiple possible new causal genes in 4/15 cases that would need detailed follow-up analyses.

A common challenge in the diagnosis of retinal dystrophies is that mutations in different genes can result in very similar phenotypes. Identifying the causal gene has implications with respect to the patient's prognosis and will be critical when personalized treatment options such as gene therapy become available. Quantitative AF cannot circumvent the need for genetic testing of causal genes such as ABCA4. However, clinical criteria such as qAF that provides some indication of how likely a specific gene is the cause of a patient's phenotype, can be a valuable clinical tool. For instance, in patients with a clinical diagnosis of recessive Stargardt disease (STGD1), complete sequencing of exons and adjacent intronic sequences in the ABCA4 gene, may identify only one disease-causing mutation in 15-20% of cases and no mutations in 10-15% of individuals.³² In these groups of patients, qAF analysis can be particularly valuable. While qAF levels in the normal range do not completely exclude the possibility of ABCA4 mutations, they substantially increase the likelihood of finding causal mutations in genes other than ABCA4.

Quantitative AF is clinically useful to recognize BEM patients with qAF levels above the normal limits for age, as in these patients one might expect to confirm the presence of ABCA4 mutations with genotyping. Thus the qAF method could perhaps guide genetic testing and help to counsel patients at a stage where genetic results are not yet available. Ongoing longitudinal qAF studies involving STGD1 patients hold promise in providing valuable information regarding the natural course of the disease. Quantitative AF may also help to ascertain the role of lipofuscin in different disorders. Whether the qAF approach will be useful as an outcome measure in clinical trials, remains to be determined.

Supplementary Material