Stargardt Disease/Fundus Flavimaculatus
Stargardt disease (STGD) is the most common childhood recessively inherited macular dystrophy. The condition has a genetic basis due to mutations in the ABCA4 gene, on chromosome 1, that encodes a retinal transported protein; it results from the accumulation of visual cycle kinetics-derived byproducts in the retinal pigmented epithelium (RPE) with secondary photoreceptor dysfunction and death.
By German ophthalmologist Karl Stargardt, in 1909.
Incidence and epidemiology
STGD accounts for about 7% of all retinal degenerations, grossly affecting 1 per 10.000 individuals. The disease usually manifests itself in early childhood or adolescence, but later onset has also been reported. As with all autosomal conditions, males and females are equally affected. No race predilection has been noted. The disease progression is slow, but ultimately all experience severe visual disability between the 4th and 7th decade. 
Stargardt is a genetic disorder. In its typical form (STGD1, OMIM #248200) it is caused by mutations involving the ABCA4 gene, through autosomal recessive homozygous or compound heterozygous transmission. Additionally, autosomal dominant transmission (STGD4, OMIM #603786) is possible, through heterozygous mutations in the PROM1 gene (4p).
Stargardt-like macular dystrophies (STGD3, OMIM #600110) involving dominant mutations in the ELOVL4 gene (6q14.1) present with overlapping clinical features but are not currently classified as typical Stargardt disease. The STGD2 was discovered to be the same gene as STGD3, and the term STGD2 was discontinued in 2005.
The ABCA4 gene maps to the short arm of chromosome 1 (1p22.1) and presents with extraordinary allelic heterogeneity, with over 490 disease-associated variants discovered thus far, most of which are missense mutations. As a result, it is not surprising to note that the most frequent ABCA4 disease-associated alleles (i.e. G1961E, G863A/delG863 and A1038V) account for only about 10% of patients, making the gene a rather difficult diagnostic target. Also, the presence of “ethnic group-specific” ABCA4 alleles is responsible for founder phenomena in different areas of the globe. Examples include the T1428M allele which, although extremely rare in populations of European descent, is rather frequent in the Japanese with an estimated frequency of approximately 8%, and the G863A/delG863 allele, which is currently considered a founder mutation in Northern Europe.
ABCA4 spans 150 kb and comprises 50 exons which vary in size from 33 bp to 266 bp. It is a large gene that codes for a 2.273 amino-acid protein.
Spectrum of disease
Given the significant carrier frequency for ABCA4 alleles, reaching as much as 5-10% in the general population, the association between ABCA4 variants and retinal pathology is now broader than previously thought. Indeed, different combinations of ABCA4 alleles are predicted to result in distinct phenotypes, in a continuum of retinal disease manifestations and it appears the severity of disease is deemed inversely proportional to the residual ABCA4 activity. Likewise, there is now up-to-date evidence indicating that ABCA4 is also implicated in the pathogenesis of various other retinal diseases beyond Stargardt, and we now consider age-related macular degeneration (AMD), some cone-rod dystrophies and forms of retinitis pigmentosa, ABCA4-related disorders as well. Accordingly, Stargardt disease would result from partial but not complete inactivation of both alleles, allowing for residual low level ABCA4 expression, whereas retinitis pigmentosa, the most severe of ABCA4-related conditions, would derive from the presence of two null ABCA4 alleles, fully compromising its pattern of genetic expression. These assumptions are further supported by the fact that ABCA4 mutations are thought to be involved in 30 to 60% of autosomal recessive cone-rod dystrophies. Even within the same family, different ABCA4 allelic combinations can be responsible for distinct phenotypes.
It has further been demonstrated that late-onset Stargardt disease is associated with missense mutations that map outside known functional domains of ABCA4, thereby resulting in milder mutant alleles and suggesting that some ABCA4 variants and combinations lead to less severe and later-onset subsets of the disease, like fundus flavimaculatus. Nonetheless, it is important to note that the observed clinical phenotype of Stargardt patients is also significantly influenced by age at time of diagnosis and, accordingly, progression of the disease. Moreover, other genes and/or environmental factors may contribute to ABCA4 expression, influencing the resulting phenotype. Likewise, although there is evidence that ABCA4 is implicated in development of autosomal recessive retinitis pigmentosa and cone-rod dystrophy, it is important to state that these conditions are genetically heterogeneous and their clinical phenotypes may originate from a number of mutations in distinct genes.
Substantial evidence supports the fact that some ABCA4 heterozygote carriers may have an increased risk of developing AMD. In fact, it was shown in a multicenter international study that heterozygotes for the G1961E ABCA4 allele had a fivefold increased risk of developing AMD, and that carriers of the D2177N variant had a threefold increased risk. However, although some ABCA4 mutations may have an influence in the development of AMD, they represent only a minor cause. Indeed, several smaller-sized mutation screening studies, and most of the co-segregation studies in AMD families, failed to establish a direct correlation between the disease and ABCA4. Thus, this relationship between AMD and ABCA4 remains speculative.
Interestingly, a Portuguese group identified that heterozygote carriers of some ABCA4 mutations could have insufficient ABCA4 expression not affecting visual acuity, but leading to subnormal visual function, as revealed by psychophysical and electrophysiological approaches. In that way, normal carrier Stargardt relatives were found to have intermediate visual performance, between age-matched control subjects and their diseased relatives. Thus, relatives of Stargardt patients should be periodically followed-up, because their visual function as a group seems to be subnormal.
The ATP-binding cassette (ABC) superfamily comprises a broad and heterogeneous group of proteins specialized in the active transport of various substrates across cellular membranes, against a concentration gradient. These substrates include amino acids, small peptides, ions, metals, lipids and fatty acid derivatives, steroids, organic anions, vitamins and drugs, among others. ABC proteins exist in virtually every living organism and are involved in various human diseases. Grossly, their molecular structure consists of two transmembrane domains that provide a pathway for substrate translocation and two ATP-binding domains that bind and hydrolyze ATP, thereby supplying the energy required for substrate transport. At least 48 genes are known to encode ABC transporters across the genome. Moreover, the ABC superfamily is further organized into seven subfamilies, ranging from ABCA to ABCG. In particular, the ABCA subfamily has recently been implicated in severe inherited diseases involving defects in lipid transport. That is the case of ABCA4, the gene linked to Stargardt disease.
Although ABC transporters are present across the entire human organism, it is interesting to observe that ABCA4 localizes specifically to the retina. The molecule localizes precisely to the disc membranes in cone and rod outer segments, where it participates in the retinoid cycle (involving a number of enzymatic reactions), through which the retina is able to recycle 11-cis-retinal, the visual chromophore, thus returning the photoreceptor to its dark adapted state, enabling further phototransduction. These reactions take place in a sequential manner on the two outermost cellular layers of the human retina, both the photoreceptor cell layer, where ABCA4 is expressed, and the RPE. It is theorized that ABCA4 works as a “flippase” actively transporting N-retinylidene-phosphatidylethanolamine (N-retinylidene-PE), a retinal byproduct, across the disc membranes but, although this is widely accepted, it remains to be experimentally confirmed.
Much of our understanding about ABCA4 function derives from genetic knockout experiments in the genetically engineered abca4 mouse. It is curious to observe that mice, either homozygous or heterozygous for abca4 maintain normal retinal appearance and photoreceptor structure, including normally organized outer segments, in contrast to what is often observed in other degenerative retinal conditions. Also, their electrophysiological studies tend to remain normal but a delayed rod dark adaptation is a constant and characteristic feature. From a biochemical standpoint, abca4 mice fail to transport N-retinylidene-PE across disc membranes, leading to its progressive entrapment inside the discs. N-retinylidene-PE reacts with available all-trans-retinal to form an intermediate byproduct, Di-retinoid-pyridinium-phosphatidylethanolamine (A2PE). Upon photoreceptor renewal, byproduct-loaded outer segments are taken up by the adjacent RPE, where further metabolism converts A2PE to Di-retinoid-pyridinium-ethanolamine (A2E), a vitamin A dimer, which becomes permanently trapped in the RPE due to the impossibility of further hydrolization. A2E is a major component of lipofuscin, a hallmark of cellular degeneration. Already, these experiments indicate that ABCA4 is not required for normal photoreceptor structure or morphogenesis. In fact, it seems to play a metabolic role, being responsible for the removal of retinoid byproducts from disc membranes after photobleaching of rhodopsin, preventing retinoid accumulation in the subcellular space.
Studies in the abca4 knockout mouse support the pathophysiological model of human disease. Thus, mutant ABCA4 would become unable to transport N-retinylidene-PE across disc membranes leading to its progressive accumulation inside disc lumina. All-trans-retinal would also become excessively accumulated. When in excess, all-trans-retinal can re-associate with opsin to form a complex that activates the visual cascade, although less efficiently than photoactivated rhodopsin. This low level of activity could explain the prolonged dark adaptation usually found in Stargardt patients and in abca4 knockout mice, and explains the residual vision often observed in Stargardt patients.
Like in abca4 knockout mice, progressive accumulation of A2E in the RPE as lipofuscin deposits is the histological hallmark of Stargardt disease. In Stargardt patients, lipofuscin can be accumulated up to five times above normal values. Excess A2E in the RPE exerts a negative effect on the epithelium’s function and survival. A2E can act as a biological detergent, compromising normal cellular membrane architecture and inhibiting normal RPE metabolic functions. Additionally, in the presence of oxygen and blue-wavelength light it forms free epoxide radicals which induce RPE cell death. Loss of RPE compromises the photoreceptor layer; beyond giving structural support and promoting photoreceptor renewal, RPE also provides nutritional support to photoreceptors and participates in the regeneration of rhodopsin. Death of RPE cells inevitably leads to irreversible secondary photoreceptor degeneration and, consequently, loss of vision. However, it is also been postulated that photoreceptor loss might actually precede RPE cell death.
Stargardt patients may be asymptomatic but most commonly present with bilateral central visual loss, photophobia, color vision abnormalities, central scotomas and slow dark adaptation.
Vision deterioration is rapidly progressive; its age of onset highly variable, but it most often occurs between childhood and adolescence or early adulthood. At presentation visual acuity may range between 20/20 and 20/400, with prior visual acuity being frequently normal, and very few patients further deteriorate their vision to counting finger or hand motion level. Interestingly, it seems the visual prognosis is highly dependent of age of disease onset, with patients presenting with significantly compromised vision at an earlier age having poorer outcomes.
Patients frequently deny a positive family history. When one is identified, an autosomal recessive pattern of inheritance is most common.
Color vision in Stargardt patients is typically compromised. Using Hardy-Rand-Rittler or Ishihara color plates it is possible to detect a mild red-green dyschromatopsia in patients with Stargardt disease. Moreover, when these patients are submitted to a Farnsworth-Munsell Hue Test, a tritan axis (or short wavelength) deviation may be noted.
Diagnostic evaluation of Stargardt disease is based on family history, visual acuity, fundus examination, visual field testing, fluorescein angiography, fundus autofluorescence (FAF), electroretinography (ERG) and optical-coherence tomography (OCT). Genetic testing is currently not performed on a routine basis.
Stargardt disease affects the macula with variable centrifugal expansion. Fundus examination is frequently normal early on the course of disease, even when patients already complain of vision loss. At this stage, the clinical diagnosis of Stargardt may be missed and patients’ complaints can be easily interpreted as functional visual loss. Later on, typical fundus manifestations arise, including pigment mottling, frank macular atrophy, a bull’s eye maculopathy, sparing of the peripapillary retina and fundus flecks. However, it should be underscored that Stargardt disease presents with highly variable phenotypes, influenced by a combination of several factors.
Fundus flecks are pisiform, round or dot-like yellow-white lesions typically found in Stargardt patients and should be seen at a given point in order to consider the clinical diagnosis. Flecks translate the accumulation of lipofuscin in the RPE but may also represent areas of regional depigmentation and atrophy. Fleck distribution may change over time, does not correlate well with the visual loss, and there seems to be no intra-familiar concordance. Flecks may form individual or confluent patterns and have a typical central distribution in Stargardt patients, with variable mid-periphery involvement.
Visual field testing in Stargardt patients is often normal in early disease stages. Over time, relative central scotomas develop, further progressing to absolute central scotomas, in a variable fashion. Typical Stargardt patients usually preserve their peripheral visual fields. However, in severe cases, with widespread retinal atrophy, visual constriction can occur. Another particularly important finding is the change in preferred retinal locus of fixation. Early in Stargardt natural history, most patients maintain foveal fixation, but as disease progresses absolute central scotomas develop and the preferred retinal fixation becomes eccentric, as demonstrated by microperimetry. In most cases, the new eccentric fixation point localizes above the fovea, where contrast sensitivity to low spatial and high temporal frequency stimuli seems best. This is also consistent with the fact that the superior retina has higher densities of ganglion cells. Furthermore, ABCA4-related disorders (including Stargardt disease) tend to spare the structure and function of the parapapillary retina as demonstrated by fundus autofluorescence and OCT. Indeed, a parapapillary ring of normal-appearing fundus autofluorescence can be identified in all disease stages. Additional histological examination shows that the structural abnormalities increase as a function of distance from the optic disc. Remarkably, this area can serve as preferred retinal locus of fixation in up to 30% of patients.
Fluorescein angiography has currently a limited role in the diagnostic evaluation of Stargardt disease and is not performed on a routine basis; FAF is less invasive and provides similar data. However, it can be useful at initial presentation whenever fundus changes are not obvious. In Stargardt, fluorescein angiography reveals a “dark-choroid” sign in up to 62% of patients. This sign, not exclusive of Stargardt disease, derives from a lack of early choroidal hyperfluorescence, which is blocked by high-grade lipofuscin accumulation in the RPE, thus improving visualization of the small retinal capillaries that become easily evident over the dark, non-fluorescent and high-contrast choroid. Fundus flecks are seen as small irregular hyperfluorescent lesions. Their presence over a “dark-choroid” background further suggests the diagnosis of Stargardt disease.
FAF imaging provides a fast non-invasive way to study the health and viability of the RPE. Abnormally increased FAF represents excessive lipofuscin accumulation in the RPE. Inversely, decreased areas of FAF relate to low level RPE metabolic activity which normally underlies local atrophy with secondary photoreceptor loss. Therefore, FAF is a perfectly adequate exam to stage and diagnose Stargardt especially if combined with ultrastructural data derived from OCT. Indeed, abnormalities of FAF intensity are an early sign of ABCA4-related disease and correlate well with local severity. Abnormally high FAF intensity with all other normal parameters suggests that RPE lipofuscin deposition may be the first pathophysiological event in ABCA4-related disease.
Electrophysiological studies in Stargardt patients reveal that they typically maintain normal or subnormal full-field electroretinographic scotopic (rods) and photopic (cones) responses. However, patients with more widespread disease can present with notably abnormal scotopic and photopic responses on full-field ERG. Given that there is no reliable way to predict the type of functional visual loss based on the fundus examination alone, electrophysiological testing is essential to evaluate patients with Stargardt disease. Specifically, these tests have a prognostic value as patients that present with early peripheral photoreceptor dysfunction have a higher chance of developing greater functional losses. Curiously, there seems to be intra-familial homogeneity in the qualitative pattern of functional loss. Electroretinography can further demonstrate the slow dark adaptation typical of Stargardt patients, correlating with underlying slow rod kinetics. Indeed, it has been found that delay of dark adaptation is strongly correlated with the absolute dark-adapted rod sensitivity at the same retinal locus, suggesting a direct relationship between the extent of local rod photoreceptor degeneration and abnormality of retinoid cycle kinetics. More central retinal locations show slower kinetics and lower sensitivities than more peripheral loci. Cone dark adaptation kinetics has similar results. Likewise, both rod and cone thresholds present comparable loss. Interestingly, retinoid cycle slowing down tends to progress like the underlying retinal degeneration, meaning that younger individuals with less severe disease demonstrate faster photoreceptor responses than older individual with more advanced disease.
Ultrastructural imaging provided by OCT is a fast evolving tool, which has been applied to the real-time, non-invasive, in vivo study of retinal diseases. Newer high-resolution tools using superluminescent diodes and ultrashort pulsed lasers, allow sub-micrometer resolution, further improving image detail. OCT has been increasingly used in the study of Stargardt disease. It allows early detection of lipofuscin accumulation in the RPE and photoreceptor layer disorganization. Combined with FAF data, OCT can provide valuable information regarding disease staging. Recently, it was found that OCT provides a more precise evaluation of local disease severity than FAF, a relationship that is further accentuated as FAF intensity values decrease. Indeed, when areas of absent fluorescence were analyzed using OCT, the degree of photoreceptor loss was greater than expected. These findings lead to the assumption that photoreceptor loss may actually precede RPE cell death, bringing new insights into the yet-to-be-fully-understood pathophysiology of Stargardt disease.
To overcome the genetic screening challenge of the ABCA4 gene, the ABCR400 microarray was developed, and contains all currently known disease-associated genetic variants and many common ABCA4 polymorphisms. Overall detection rates currently range between 65 and 75%. Several laboratories worldwide provide genetic testing of the ABCA4 gene.
Once thought to represent a completely distinct condition, fundus flavimaculatus shares obvious phenotypic similarities with Stargardt disease and it is now the consensus that fundus flavimaculatus and Stargardt disease are genetically linked. The former represents a subset of Stargardt manifestations, with intermediate forms between the two equally considered. It should be pointed that Stargardt and fundus flavimaculatus differ in important aspects. Patients with fundus flavimaculatus often have a later disease onset and slower visual deterioration, making fundus flavimaculatus a milder condition. Surprisingly, comparing fundus photographs of both conditions, one might get the opposite impression because in fundus flavimaculatus there is more widespread retinal involvement. Flecks are more diffusively scattered throughout the posterior pole and extend out to the midperiphery, but the macula is less involved, allowing better visual performance.
Stargardt disease remains an incurable condition. Current therapeutic options include photoprotection and low-vision aids. Pharmacological slow-down of the visual cycle, gene therapy and other treatment options aim to prevent lipofuscin accumulation and represent prospects of long-term visual rescue.
Unbound all-trans-retinal induces photo-oxidative damage to the unusually sensitive ABCA4, further compromising its function. Given that Stargardt patients already have impaired ABCA4 function and increased levels of all-trans-retinal trapped inside their photoreceptors, it is logical to think that these patients would be extremely sensitive to light exposure. Furthermore, it has been demonstrated that di-retinoid-pyridinium-ethanolamine (A2E) does not accumulate in the RPE of abca4 knockout mice kept in total darkness. Thus, Stargardt patients should be advised to avoid direct sunlight exposure. Ultraviolet-blocking sunglasses are a useful option.
Vitamin A supplementation has been regarded as a therapeutic option for certain retinal degenerative conditions such as retinitis pigmentosa. However, recent data suggests that in ABCA4-mediated disease, vitamin A supplementation accelerates the accumulation of lipofuscin pigments in the RPE. Long-term vitamin supplementation increases the formation of vitamin A dimers which favor lipofuscin synthesis and deposition. Therefore, Stargardt patients should avoid vitamin A supplementation. In contrast, long-term administration of deuterium-enriched vitamin A (i.e. C20-D3-vitamin-A), may have a beneficial effect since this compound prevented vitamin A dimerization in the murine model of Stargardt disease.
Isotretinoin has reportedly been capable of dampening A2E deposition in the RPE of abca4 knockout mice. Considerable side effects associated with chronic intake of isotretinoin prevent its chronic use in humans.
Stargardt disease is a preferred target for gene replacement therapy. Like in Leber Congenital Amaurosis (LCA), from which the most astonishing results of human ocular gene therapy have been obtained, the replacement of the mutant ABCA4 gene by its wild-type counterpart may produce positive results, as those observed in RPE65 gene trials.
Animal testing using gene therapy for Stargardt disease remains limited. Kong and co-workers were able to successfully treat the Stargardt phenotype in abca4 knockout mice using lentiviral gene therapy. Each mouse received a single unilateral subretinal injection of ABCA4-carrying equine infectious anemia viral vectors, which resulted in significant rescue of the retinal phenotype. Treated eyes showed marked reduction in retinal A2E accumulation. Moreover, 1 year after gene transfer, A2E accumulation in treated eyes matched the A2E levels of normal wild-type controls.
The relatively large ABCA4 gene (6.8 kb) presents a unique packaging challenge with the available viral vectors. Lentiviral vectors are the most suitable for ABCA4 gene transfer. Other packaging options include AAV2/5 chimeras and new vector systems, such as the Hd-Ad vectors or other non-viral vectors.
Given Stargardt is a retinal degeneration, identification of viable photoreceptors plays a central role in the selection of patients amenable to gene therapy. Thus, retinal imaging is essential to assess photoreceptor viability, when selecting patients for gene therapy. High-definition OCT and Adaptive Optics technology enhances lateral resolution in retinal images up to 3-4μm, allowing the visualization of individual photoreceptors.
Years of gene therapy research for LCA and underlying RPE65 mutations, have produced incomparable breakthroughs, which will invariably serve as a foundation for further research involving other retinal dystrophies. In December 2009, Oxford BiomedicaTM announced that StarGenTM, a gene-based therapy that uses the company’s LentiVector® technology for the treatment of Stargardt disease, has received orphan designation from the Committee for Orphan Medicinal Products of the European Medicines Agency (EMEA). In collaboration with Sanofi-Aventis, both companies advanced StarGenTM into PhaseI/II development in 2010. The US charity, Foundation Fighting Blindness, is also supporting the programme and previously funded preclinical development. With this initiative, Oxford BiomedicaTM is deemed to bring considerable hope for the 600 new cases of Stargardt disease diagnosed every year and for many other Stargardt patients that currently await treatment for their visually-debilitating condition. Further trials to seek the efficacy of complement inhibitors, stem cell transplantation and ABCA4 gene therapy are in order.
To Prof. Eduardo J. G. Duarte Silva for his significant contribution.
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