ABCA4 gene mutations in Japanese patients with Stargardt disease and retinitis pigmentosa

PURPOSE: To evaluate photoreceptor cell-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) gene mutations in Japanese patients with Stargardt disease (STGD) and the correlation of these mutations to clinical phenotypes. METHODS: Serum was obtained from 10 unrelated Japanese patients with STGD and 96 unrelated Japanese patients with autosomal recessive retinitis pigmentosa (arRP). All 50 ABCA4 gene exons of the patients with STGD were screened for mutations by a combination of single-strand conformation polymorphism analysis and polymerase chain reaction (PCR) direct-sequencing techniques. By restriction enzyme digestion, primer extension analysis, and PCR direct sequencing techniques, the patients with arRP were screened for three segregated, presumably null ABCA4 gene mutations observed in Japanese patients with STGD. RESULTS: Three novel, presumably null mutations of the ABCA4 gene, IVS7-45_952delinsTCTGACC, IVS12+2T-->G, and 1894delA, were identified. The Arg2149stop mutation that had been found in a white patient with STGD in a prior study was also found in a Japanese patient. Two arRP-affected siblings and two unrelated patients with STGD were found to be homozygous for the same IVS12+2T-->G mutation, and three other arRP-affected siblings were carriers of the IVS12+2T-->G mutation and/or the IVS7-45_952delinsTCTGACC mutation. These three siblings with arRP showed only atrophic degeneration in the macula early after the onset of the disease, and STGD had been diagnosed. CONCLUSIONS: Three novel ABCA4 gene mutations were identified in Japanese patients with STGD and arRP. Mutations in the ABCA4 gene can cause panretinal degeneration that changes its clinical appearance from STGD to arRP over time.     

Mutation spectrum and founder chromosomes for the ABCA4 gene in South African patients with Stargardt disease

PURPOSE: To assess the mutation spectrum of ABCA4 underlying Stargardt disease (STGD) in South Africa (SA) and to determine whether there is a single or a few founder chromosomes in SA STGD families. METHODS: Sixty-four probands exhibiting the STGD phenotype were screened for mutations in the 50 exons of ABCA4 by single-strand conformational polymorphism-heteroduplex analysis sequencing and restriction fragment length polymorphism analysis. Microsatellite marker haplotyping was used to determine the ancestry in 10 families. RESULTS: Fifty-seven ABCA4 disease-associated alleles were identified that comprised 16 different sequence variants, of which two were novel, in 40 individuals of the cohort of 64 subjects. The most common variants identified included the C1490Y, L2027F, R602W, V256splice, R152X, and 2588G-->C mutations. The C1490Y variant was the most common disease-associated variant identified (19/64 subjects) and was absent in 392 control chromosomes. At least 10 ABCA4 disease-associated haplotypes were identified. Two of these haplotypes, which carried the C1490Y mutation, were identified in three unrelated families. CONCLUSIONS: Results suggest that ABCA4 is the major gene underlying STGD in the cohort investigated. Five of the six common sequence variants identified were at a higher frequency in the SA cohort than reported in published data on individuals of similar ancestry. The mutation and haplotype data suggests that there are several ancestral haplotypes underlying STGD in SA. There seems to be at least two different origins for the common C1490Y mutation, as well as two for the R602W mutation, thereby suggesting several founder effects for STGD in SA.     

Dark and light adaptation of pineal photoreceptors

Dark and light adaptation of pineal photoreceptors was studied in the isolated pineal organ of the rainbow trout, Salmo gairdneri. After intracellular recording, the photoreceptors were iontophoretically injected with Lucifer yellow CH or with horseradish peroxidase for morphological characterization. Pineal photoreceptor cells responded to light with a hyperpolarization whose amplitude was graded with intensity. Following a 30-60 s bleach, receptor responsiveness was greatly reduced with a gradual recovery in the dark. Recovery of membrane potential was complete within 2-4 min in the dark. In response to flashes the hyperpolarizing response increased in darkness in amplitude and duration over a period of more than 30 min and the voltage-intensity curves continuously shifted to lower intensities. After exposure to strong light the time-course of dark adaptation, determined with a threshold criterion, was monophasic and receptor sensitivity increased by at least 5-6 log units. The results show that pineal photoreceptors exhibited the full characteristics of dark adaptation processes previously ascribed to cells proximal to the receptors, i.e. to ganglion cells. Exposure to steady illumination of different intensities induced graded and sustained hyperpolarizations of the receptor membrane potential. The incremental voltage range of responses to test flashes superimposed on the backgrounds was reduced. Voltage-intensity curves were shifted to higher intensities with increasing background illumination indicating that adaptation occurred over a range of about 2.5 log units before the receptors saturated.     

Alterations of slow and fast rod ERG signals in patients with molecularly confirmed Stargardt disease type 1

PURPOSE: To investigate the slow and fast rod signals of the scotopic 15-Hz flicker ERG in patients with molecularly confirmed Stargardt disease type I (STGD1). There is evidence that these slow and the fast rod ERG signals can be attributed to the rod bipolar-AII cell pathway and the rod-cone coupling pathway, respectively. METHODS: Twenty-seven patients with STGD1 with mutations in both alleles of the ABCA4 gene were included. Scotopic ERG response amplitudes and phases to flicker intensities ranging from -3.37 to -0.57 log scotopic troland x sec (log scot td x sec) were measured at a flicker frequency of 15 Hz. In addition, scotopic standard ERGs were obtained. Twenty-two normal subjects served as controls. RESULTS: The amplitudes of both the slow and fast rod ERG signals were significantly reduced in the STGD1 group. The phases of the slow rod signals lagged significantly, whereas those of the fast rod signals did not. The standard scotopic ERG did not reveal significant alterations. CONCLUSIONS: The results provide evidence that a defective ABCA4 transporter can functionally affect both the rod bipolar-AII cell pathway and the rod-cone coupling pathway. In STGD1, the scotopic 15-Hz flicker ERG may reveal subtle abnormalities at different sites within the rod system that remain undetected by standard ERG techniques.     

Retinal Histopathology in Eyes from a Patient with Stargardt disease caused by Compound Heterozygous ABCA4 Mutations*

Background: The goal of this study was to define the histopathology of the retina in donor eyes from a patient with Stargardt disease (STGD1) due to compound mutations in the ABCA4 gene.

Materials and Methods: Eyes were obtained from a 66-year-old female and fixed within 18 hours postmortem. The fundi of the posterior globes were evaluated with macroscopic, SLO and OCT imaging. The perifoveal and peripheral regions were processed for electron microscopy and immunocytochemistry using cell specific antibodies. Two age-similar normal eyes were used as controls. Prior ophthalmic examinations and genetic test results were also reviewed.

Results: All imaging modalities showed scattered bone spicules in the peripheral retina. Atrophy of the RPE was present around the optic nerve as evidenced by the absence of SLO autofluorescence. Histology analysis showed a severely degenerated fovea with little evidence of any retinal layering or remaining RPE. The fovea was severely degenerated, with little evidence of any retinal cell layer, including the RPE. In contrast, retinal nuclear layers were present in the periphery. The perifoveal region contained few cones labeled with cone-specific antibodies; some rhodopsin-labeled cells, reactive glia labeled with GFAP; and decreased autofluorescence of the RPE. The fovea was free of cone-specific labeling, contained a few disorganized rhodopsin-labeled cells and showed substantial GFAP labeling and no autofluorescent material in the retina. The periphery displayed stubby cells labeled with cone-specific antibodies, decreased rhodopsin-labeled cells, increased GFAP staining, and autofluorescent granules in the RPE.

Conclusions: The histopathology of the retina in this patient with Stargardt disease displayed a highly degenerated fovea. In all retinal locations studied, cones were more severely affected than rods.     

Perceptual learning in patients with Stargardt disease

ObjectiveTo evaluate the efficacy of Perceptual Learning in improving the peripheral reading performance of patients with Stargardt disease (STGD).DesignProspective observational randomized study.ParticipantsFourteen consecutive patients (7 females, 7 males; median age of 50.4 ± 12.8 years) with STGD were analyzed and divided into two groups: Group A received “Win-flash” as Perceptual Learning training and Group B was used as control.MethodsSubjects underwent an ophthalmic evaluation at baseline, after perceptual learning training and at 6 months of follow-up. Outcomes measured included reading speed, contrast sensitivity and fixation stability.ResultsReading speed improved of 51,7% after training in group A. Visual acuity, contrast sensitivity and fixation stability enhanced in group A after training from 0.89 (±0.09) LogMAR to 0.75 (±0.2) LogMAR (t(6)= 3.6, p= 0.001), from 0.8 (±0.3) LogC (0.6 ? 0.9) to 1.3 (±0.3) LogC (t(13)=3.17, p= 0.003) and from 59.3 % (± 24.3) to 71.5 % (± 20.4) (t(13)=1.8 p= 0.04), respectively. No changes were found in group B. At 6-monts of follow-up, visual acuity and contrast sensitivity decreased in group A.ConclusionsSTGD patients receiving “Win-flash training”, as PL technique, showed an improvement of reading performance on a real-world task. Early follow-up for perceptual learning re-intervention should be considered.     

Quantifying fixation in patients with Stargardt disease

Fixational eye movements in 60 eyes of 30 patients with ABCA4-associated Stargardt disease were recorded by a Scanning Laser Ophthalmoscope (SLO). The results were quantified by two new fixation quality measures expressing the eccentricity of the preferred retinal locus (PRL) non-parametrically, and fixation stability by a dynamic index. 46 eyes (77%) fixated eccentrically; in 32 eyes (70% of the eccentrically fixating eyes) the PRL was located above the central retinal lesion. PRL eccentricity correlated positively with logMAR visual acuity (r=.72; p<.0001) and negatively with fixation stability (r=-.58; p<.0001). Multiple PRL were found only in three eyes.     

A novel mutation in the ABCR gene in four patients with autosomal recessive Stargardt disease

PURPOSE: To identify additional mutations in the ABCR gene and describe the clinical features of four affected siblings with autosomal recessive Stargardt disease. METHODS: A cohort of eight siblings was identified for study. Four of these individuals were diagnosed with Stargardt disease based on clinical evaluation and fluorescein angiography. Blood samples were obtained from seven of eight siblings, including all those affected. All 50 exons of the ABCR gene were analyzed by single-stranded confirmation polymorphism analysis, followed by direct sequencing of observed variants, to identify mutations in the ABCR gene. RESULTS: We identified a previously unreported kindred of eight siblings, four of whom had mutations in both of their ABCR alleles. A previously described G-to-C transversion of nucleotide 2588, predicting a Gly863Ala amino acid substitution, and a novel G-to-A transition of nucleotide 161, resulting in a Cys54Tyr substitution, were identified. These mutations co-segregated with the affected members of this family. Three of the siblings demonstrated clinical features characteristic of classic Stargardt disease, with bilateral regions of macular atrophy associated with yellow-white "flavimaculatus" flecks in the posterior pole at the level of the retinal pigment epithelium. The fourth affected sibling showed features of early Stargardt disease, with a beaten-bronze appearance to both maculas, as well as perimacular flecks. In all four affected patients, fluorescein angiography showed a characteristic peripheral dark choroid. CONCLUSIONS: We have identified both a previously described and a novel mutation in the ABCR gene in four patients with autosomal recessive Stargardt disease. In-depth knowledge of the ABCR mutation spectrum in patients with Stargardt disease will provide for more efficient screening and may provide potential therapies for Stargardt disease and other retinal diseases.     

Epiretinal membrane removal in patients with Stargardt disease

Epiretinal membranes (ERMs) in Stargardt disease have been known to undergo spontaneous separation in children. Results of surgical intervention in adult patients with Stargardt disease have rarely been reported. A retrospective review of results of surgical intervention for ERM causing visual impairment in two adult patients of Stargardt disease was carried out. Both patients developed ERM in one eye during their follow-up period with the resultant drop in their preexisting visual acuity. Postsurgery, restoration of foveal contour with some improvement in visual acuity was observed in both patients. No adverse effect of surgery was noted.     

Occurrence of full-thickness macular hole complicating Stargardt disease with ABCR mutation

PURPOSE: To report an unusual episode of full-thickness macular hole complicating Stargardt disease with an ABCR mutation. METHODS: Case report . RESULTS: Fundus examination of a 20-year-old healthy man showed typical fundus manifestation with yellowish-round or fish-like flecks associated with vitreous macular adhesion and a round punched-out area in the right eye. Optical coherence tomography (OCT) illustrated a full-thickness macular hole. Molecular genetic examination of the ABCR gene showed two heterozygous missense mutations: R1108C (CGC-->TGC) in exon 22 and a splicing mutation IVS6--> 1GT - described in the literature in association with Stargardt disease. CONCLUSIONS: Macular hole was once described in other inherited retinal degenerations (Best disease and Bietti crystal line retinopathy). The pathogenesis gives rise to a host of speculations: widespread alteration of the retinal pigment epithelium; inflammatory mechanisms; a minor trauma which might cause subretinal fibrosis. Surgical procedures were not performed on our patient after his ophthalmologic history and findings were considered.     

Electroretinographic findings in patients with Stargardt disease and fundus flavimaculatus

PURPOSE: To characterize the clinical and electroretinogram (ERG) features of our cohort of patients with Stargardt disease (STGD) exhibiting coding sequence variations in the ABCA4 gene. METHODS: Review of 76 patients with the clinical diagnosis of Stargardt disease/fundus flavimaculatus (STGD/FF) from the University of Iowa Department of Ophthalmology and Visual Sciences (41 patients) and the Casey Eye Institute (35 patients). Clinical examination, Goldmann perimetry, and electroretinography were performed on all 76 patients. Patients were divided into three groups on the basis of their funduscopic and electroretinographic features: (1) a normal ERG by the standards of the laboratory; (2) minimal rod or cone abnormalities; (3) severe ERG dysfunction. The latter category was further subdivided on the basis of a cone-dominated loss of function (C > R or "cone-rod dystrophy") or diffuse depression of rods and cones (C = R). Mutational analysis of the coding sequence of the ABCA4 gene was performed by single strand conformation polymorphism analysis followed by automated DNA sequencing. Each electroretinographic group was analyzed for the presence of disease causing changes using exact tests of binomial proportions corrected for multiple comparisons by Bonferroni method. Quantitative polymerase chain reaction (QPCR) was performed on patients who were homozygous for disease causing changes in the ABCA4 gene to rule out the possibility of deletions. RESULTS: Overall, 56 of 76 patients (and 77 of 152 alleles) exhibited coding sequence variations that were compatible with high-penetrance disease-causing mutations. The most common of these were His423Arg (9), frameshift mutations (7), Ala1038Val (7), and Pro1380Leu (6). Although no patients with His423Arg presented with normal ERGs, no significant correlation was observed between specific sequence variations and the electroretinographic characteristics or fundus appearance. However, a significantly greater fraction of patients with normal ERG studies failed to exhibit detectable disease-causing coding sequence variations in the ABCA4 gene identified on either allele (P = 0.0006). CONCLUSION: STGD/FF patients in our cohort exhibit a wide range of electroretinographic abnormalities, some of which are more prevalent than previously suspected. No direct correlation between clinical appearance, electrophysiologic characteristics and specific ABCA4 alleles could be identified, although a significantly lower number of our cohort with a normal ERG exhibited detectable coding sequence variations in the ABCA4 gene. However, four patients with ERG dysfunction were homozygous for a His423Arg change proven by QPCR not to be an artifact of a deletion. The presence of electrophysiologic dysfunction is not uncommon in our cohort of patients with STGD. Thus, the ERG provides clinically important information of retinal function for STGD/FF and, as such, is still indicated as part of the evaluation of these patients.     

Modification of the PROM1 disease phenotype by a mutation in ABCA4

ABSTRACT

Background: The extensive phenotypic heterogeneity of monogenic diseases can be largely traced to intragenic variation; however, recent advances in clinical detection and gene sequencing have uncovered the emerging role of non-allelic variation (i.e. genetic trans-modifiers) in shaping disease phenotypes. Identifying these associations are not only of significant diagnostic value, but also provides scientific insight into the expanded molecular etiology of rare diseases. This reports describes the discordant clinical manifestation of a family segregating mutations in ABCA4 and PROM1.

Methods: Three patients across a two generation family underwent multimodal imaging and functional testing of the retina including color photography, fundus autofluorescence (AF), spectral domain-optical coherence tomography (SD-OCT) and full-field electroretinography (ffERG). Genetic characterization was carried out by direct Sanger and whole exome sequencing.

Results: Clinical examination revealed similar retinal degenerative phenotypes in the proband and her mother. Despite being younger, the proband’s phenotype was more advanced and exhibited additional features related to Stargardt disease not found in the mother. Whole exome sequencing identified a pathogenic missense variant in PROM1, c.400C > T, p.(Arg134Cys), as the underlying cause of retinal disease in both the proband and mother. Sequencing of the ABCA4 locus uncovered a single disease-causing variant, c.5714 + 5G > A in the daughter segregating from the father who, surprisingly, also exhibited very subtle disease changes associated with STGD1 despite being a heterozygous carrier.

Conclusions: Harboring an additional heterozygous ABCA4 mutation increases severity and confers STGD1-like features in patients with PROM1 disease which provides supporting evidence for their shared pathophysiology and potential treatment prospects.