Rare and common variants in ROM1 and PRPH2 genes trans-modify Stargardt/ABCA4 disease.

Over 1,500 variants in the ABCA4 locus cause phenotypes ranging from severe, early-onset retinal degeneration to very late-onset maculopathies. The resulting ABCA4/Stargardt disease is the most prevalent Mendelian eye disorder, although its underlying clinical heterogeneity, including penetrance of many alleles, are not well-understood. We hypothesized that a share of this complexity is explained by trans-modifiers, i.e., variants in unlinked loci, which are currently unknown. We sought to identify these by performing exome sequencing in a large cohort for a rare disease of 622 cases and compared variation in seven genes known to clinically phenocopy ABCA4 disease to cohorts of ethnically matched controls. We identified a significant enrichment of variants in 2 out of the 7 genes. Moderately rare, likely functional, variants, at the minor allele frequency (MAF) <0.005 and CADD>25, were enriched in ROM1, where 1.3% of 622 patients harbored a ROM1 variant compared to 0.3% of 10,865 controls (p = 2.41E04; OR 3.81 95% CI [1.77; 8.22]). More importantly, analysis of common variants (MAF>0.1) identified a frequent haplotype in PRPH2, tagged by the p.Asp338 variant with MAF = 0.21 in the matched general population that was significantly increased in the patient cohort, MAF 0.25, p = 0.0014. Significant differences were also observed between ABCA4 disease subgroups. In the late-onset subgroup, defined by the hypomorphic p.Asn1868Ile variant and including c.4253+43G>A, the allele frequency for the PRPH2 p.Asp338 variant was 0.15 vs 0.27 in the remaining cohort, p = 0.00057. Known functional data allowed suggesting a mechanism by which the PRPH2 haplotype influences the ABCA4 disease penetrance. These associations were replicated in an independent cohort of 408 patients. The association was highly statistically significant in the combined cohorts of 1,030 cases, p = 4.00E-05 for all patients and p = 0.00014 for the hypomorph subgroup, suggesting a substantial trans-modifying role in ABCA4 disease for both rare and common variants in two unlinked loci.

Cis-acting modifiers in the ABCA4 locus contribute to the penetrance of the major disease-causing variant in Stargardt disease.

Over 1200 variants in the ABCA4 gene cause a wide variety of retinal disease phenotypes, the best known of which is autosomal recessive Stargardt disease (STGD1). Disease-causing variation encompasses all mutation categories, from large copy number variants to very mild, hypomorphic missense variants. The most prevalent disease-causing ABCA4 variant, present in ~ 20% of cases of European descent, c.5882G > A p.(Gly1961Glu), has been a subject of controversy since its minor allele frequency (MAF) is as high as ~ 0.1 in certain populations, questioning its pathogenicity, especially in homozygous individuals. We sequenced the entire ~140Kb ABCA4 genomic locus in an extensive cohort of 644 bi-allelic, i.e. genetically confirmed, patients with ABCA4 disease and analyzed all variants in 140 compound heterozygous and 10 homozygous cases for the p.(Gly1961Glu) variant. A total of 23 patients in this cohort additionally harbored the deep intronic c.769-784C > T variant on the p.(Gly1961Glu) allele, which appears on a specific haplotype in ~ 15% of p.(Gly1961Glu) alleles. This haplotype was present in 5/7 of homozygous cases, where the p.(Gly1961Glu) was the only known pathogenic variant. Three cases had an exonic variant on the same allele with the p.(Gly1961Glu). Patients with the c.[769-784C > T;5882G > A] complex allele exhibit a more severe clinical phenotype, as seen in compound heterozygotes with some more frequent ABCA4 mutations, e.g. p.(Pro1380Leu). Our findings indicate that the c.769-784C > T variant is major cis-acting modifier of the p.(Gly1961Glu) allele. The absence of such additional allelic variation on most p.(Gly1961Glu) alleles largely explains the observed paucity of affected homozygotes in the population.

Identification and Rescue of Splice Defects Caused by Two Neighboring Deep-Intronic ABCA4 Mutations Underlying Stargardt Disease.

Sequence analysis of the coding regions and splice site sequences in inherited retinal diseases is not able to uncover ∼40% of the causal variants. Whole-genome sequencing can identify most of the non-coding variants, but their interpretation is still very challenging, in particular when the relevant gene is expressed in a tissue-specific manner. Deep-intronic variants in ABCA4 have been associated with autosomal-recessive Stargardt disease (STGD1), but the exact pathogenic mechanism is unknown. By generating photoreceptor precursor cells (PPCs) from fibroblasts obtained from individuals with STGD1, we demonstrated that two neighboring deep-intronic ABCA4 variants (c.4539+2001G>A and c.4539+2028C>T) result in a retina-specific 345-nt pseudoexon insertion (predicted protein change: p.Arg1514Leufs∗36), likely due to the creation of exonic enhancers. Administration of antisense oligonucleotides (AONs) targeting the 345-nt pseudoexon can significantly rescue the splicing defect observed in PPCs of two individuals with these mutations. Intriguingly, an AON that is complementary to c.4539+2001G>A rescued the splicing defect only in PPCs derived from an individual with STGD1 with this but not the other mutation, demonstrating the high specificity of AONs. In addition, a single AON molecule rescued splicing defects associated with different neighboring mutations, thereby providing new strategies for the treatment of persons with STGD1. As many genes associated with human genetic conditions are expressed in specific tissues and pre-mRNA splicing may also rely on organ-specific factors, our approach to investigate and treat splicing variants using differentiated cells derived from individuals with STGD1 can be applied to any tissue of interest.

CLINICAL CHARACTERIZATION OF STARGARDT DISEASE PATIENTS WITH THE p.N1868I ABCA4 MUTATION.

PURPOSE: To investigate the Stargardt disease phenotype associated with an unusually common and "extremely hypomorphic" ABCA4 variant, p.N1868I. METHODS: The charts of 27 patients with p.N1868I on one allele and a severe/deleterious mutation on the other allele were reviewed. Subjective age of onset, best-corrected visual acuity, and stage of disease were recorded for all 27 patients, 18 of whom had multiple visits. When available, fundus photography, spectral domain optical coherence tomography, fundus autofluorescence, full-field electroretinograms, Goldmann visual fields, and fluorescein angiography were included. Five families with multiple affected members were analyzed. RESULTS: The median age at symptom onset was 41.5 years, and 3 p.N1868I patients had not developed visual symptoms as of the most recent eye examination. Median best-corrected visual acuity in the better-seeing eye at baseline was 20/25, and the median duration from symptom onset to legal blindness was 25 years. The five families described in this study demonstrated clinically significant intrafamilial variability, and affected family members who did not share the p.N1868I variant had relatively more severe phenotypes. CONCLUSION: This study demonstrates the consistency of foveal sparing, the variation in age at onset, the intrafamilial variability, and the prognosis with regard to visual acuity in p.N1868I-associated Stargardt disease.

The Rapid-Onset Chorioretinopathy Phenotype of ABCA4 Disease.

PURPOSE: To characterize patients affected by a uniquely severe, rapid-onset chorioretinopathy (ROC) phenotype of ABCA4 disease. DESIGN: Comparative cohort study. PARTICIPANTS: Sixteen patients were selected from a large clinically diagnosed and genetically confirmed cohort (n = 300) of patients diagnosed with ABCA4 disease. MAIN OUTCOME MEASURES: Phenotypic characteristics were assessed on color fundus photographs, short-wavelength autofluorescence (488-nm), and near-infrared autofluorescence (NIR-AF, 787-nm) images. Subfoveal thickness measurements were obtained from enhanced-depth imaging OCT. Generalized retinal function was determined with full-field electroretinogram (ffERG) testing, and lipofuscin accumulation was assessed by quantitative autofluorescence (qAF). RESULTS: All patients exhibited advanced disease features, including pigment migration in the macula and retinal vessel attenuation at an early age, and reported a symptomatic onset, on average, at 7.4 years (average for ABCA4 disease is 21.9 years, P < 0.0001). Deterioration of the macula was observed to begin with an intense, homogeneous signal on short-wavelength autofluorescence, which corresponds to an attenuated NIR-AF signal and progresses to a patchy, coalescing pattern of chorioretinal atrophy within the subsequent decade. Measurement of choroidal thickness revealed a more rapid thinning of choriocapillaris with age of Sattler's layer compared with the rate in most other patients with ABCA4 disease (P < 0.001). Levels of qAF in the macula before atrophy were above both the 95% confidence intervals for healthy individuals and patients with Stargardt disease (STGD1) (>1000 qAF units). Severe attenuation of cone responses and notable decreases in rod responses were detected by ffERG. Sequencing of the ABCA4 gene revealed exclusively deleterious, null mutations, including stop codons; frameshift deletions; variants in canonical splice sites, which completely abolish splicing; and known deleterious missense alleles. CONCLUSIONS: The ROC phenotype is a unique classification of ABCA4 disease, which is caused by deleterious null biallelic ABCA4 mutations and is characterized by the rapid deterioration of retinal pigment epithelium and photoreceptor layers in the macula and significant choroidal thinning within the first 2 decades of life.

Frequent hypomorphic alleles account for a significant fraction of ABCA4 disease and distinguish it from age-related macular degeneration.

BACKGROUND: Variation in the ABCA4 gene is causal for, or associated with, a wide range of phenotypes from early onset Mendelian retinal dystrophies to late-onset complex disorders such as age-related macular degeneration (AMD). Despite substantial progress in determining the causal genetic variation, even complete sequencing of the entire open reading frame and splice sites of ABCA4 identifies biallelic mutations in only 60%-70% of cases; 20%-25% remain with one mutation and no mutations are found in 10%-15% of cases with clinically confirmed ABCA4 disease. This study was designed to identify missing causal variants specifically in monoallelic cases of ABCA4 disease. METHODS: Direct sequencing and analysis were performed in a large familial ABCA4 disease cohort of predominately European descent (n=643). Patient phenotypes were assessed from clinical and retinal imaging data. RESULTS: We determined that a hypomorphic ABCA4 variant c.5603A>T (p.Asn1868Ile), previously considered benign due to high minor allele frequency (MAF) (~7%) in the general population, accounts for 10% of the disease, >50% of the missing causal alleles in monoallelic cases, ~80% of late-onset cases and distinguishes ABCA4 disease from AMD. It results in a distinct clinical phenotype characterised by late-onset of symptoms (4th decade) and foveal sparing (85%). Intragenic modifying effects involving this variant and another, c.2588G>C (p.Gly863Ala) allele, were also identified. CONCLUSIONS: These findings substantiate the causality of frequent missense variants and their phenotypic outcomes as a significant contribution to ABCA4 disease, particularly the late-onset phenotype, and its clinical variation. They also suggest a significant revision of diagnostic screening and assessment of ABCA4 variation in aetiology of retinal diseases.

HYPERREFLECTIVE DEPOSITION IN THE BACKGROUND OF ADVANCED STARGARDT DISEASE.

PURPOSE: To describe an unusual manifestation of hyperreflective deposits in the subretinal space in a group of patients with clinically and genetically confirmed Stargardt disease. METHODS: Retrospective review of color fundus, autofluorescence, infrared reflectance, red-free images, and spectral domain optical coherence tomography in 296 clinically diagnosed and genetically confirmed (2 expected disease-causing mutations in ABCA4) patients with Stargardt disease. Full-field electroretinogram (ffERG), medical history, and genotype data (in silico predictions) were further analyzed from the selected cohort. RESULTS: Eight of 296 patients (2.7%) were found to exhibit small crystalline deposits that were detectable on certain imaging modalities, such as color, infrared reflectance and red-free images, but not autofluorescence. The deposits were most prevalent in the superior region of the macula, and spectral domain optical coherence tomography revealed their presence in the subretinal space. All patients presented with these findings at a notably advanced disease stage with abnormal ffERG and a high proportion of highly deleterious ABCA4 alleles. CONCLUSION: Hyperreflective subretinal deposits may be a manifestation of advanced ABCA4 disease, particularly in regions susceptible to disease-related changes, such as lipofuscin accumulation.

In Silico Functional Meta-Analysis of 5,962 ABCA4 Variants in 3,928 Retinal Dystrophy Cases.

Variants in the ABCA4 gene are associated with a spectrum of inherited retinal diseases (IRDs), most prominently with autosomal recessive (ar) Stargardt disease (STGD1) and ar cone-rod dystrophy. The clinical outcome to a large degree depends on the severity of the variants. To provide an accurate prognosis and to select patients for novel treatments, functional significance assessment of nontruncating ABCA4 variants is important. We collected all published ABCA4 variants from 3,928 retinal dystrophy cases in a Leiden Open Variation Database, and compared their frequency in 3,270 Caucasian IRD cases with 33,370 non-Finnish European control individuals. Next to the presence of 270 protein-truncating variants, 191 nontruncating variants were significantly enriched in the patient cohort. Furthermore, 30 variants were deemed benign. Assessing the homozygous occurrence of frequent variants in IRD cases based on the allele frequencies in control individuals confirmed the mild nature of the p.[Gly863Ala, Gly863del] variant and identified three additional mild variants (p.(Ala1038Val), c.5714+5G>A, and p.(Arg2030Gln)). The p.(Gly1961Glu) variant was predicted to act as a mild variant in most cases. Based on these data, in silico analyses, and American College of Medical Genetics and Genomics guidelines, we provide pathogenicity classifications on a five-tier scale from benign to pathogenic for all variants in the ABCA4-LOVD database.

Analysis of the ABCA4 genomic locus in Stargardt disease.

Autosomal recessive Stargardt disease (STGD1, MIM 248200) is caused by mutations in the ABCA4 gene. Complete sequencing of ABCA4 in STGD patients identifies compound heterozygous or homozygous disease-associated alleles in 65-70% of patients and only one mutation in 15-20% of patients. This study was designed to find the missing disease-causing ABCA4 variation by a combination of next-generation sequencing (NGS), array-Comparative Genome Hybridization (aCGH) screening, familial segregation and in silico analyses. The entire 140 kb ABCA4 genomic locus was sequenced in 114 STGD patients with one known ABCA4 exonic mutation revealing, on average, 200 intronic variants per sample. Filtering of these data resulted in 141 candidates for new mutations. Two variants were detected in four samples, two in three samples, and 20 variants in two samples, the remaining 117 new variants were detected only once. Multimodal analysis suggested 12 new likely pathogenic intronic ABCA4 variants, some of which were specific to (isolated) ethnic groups. No copy number variation (large deletions and insertions) was detected in any patient suggesting that it is a very rare event in the ABCA4 locus. Many variants were excluded since they were not conserved in non-human primates, were frequent in African populations and, therefore, represented ancestral, and not disease-associated, variants. The sequence variability in the ABCA4 locus is extensive and the non-coding sequences do not harbor frequent mutations in STGD patients of European-American descent. Defining disease-associated alleles in the ABCA4 locus requires exceptionally well characterized large cohorts and extensive analyses by a combination of various approaches.

Complex inheritance of ABCA4 disease: four mutations in a family with multiple macular phenotypes.

Over 800 mutations in the ABCA4 gene cause autosomal recessive Stargardt disease. Due to extensive genetic heterogeneity, observed variant-associated phenotypes can manifest tremendous variability of expression. Furthermore, the high carrier frequency of pathogenic ABCA4 alleles in the general population (~1:20) often results in pseudo-dominant inheritance patterns further complicating the diagnosis and characterization of affected individuals. This study describes a genotype/phenotype analysis of an unusual family with multiple macular disease phenotypes spanning across two generations and segregating four distinct ABCA4 mutant alleles. Complete sequencing of ABCA4 discovered two known missense mutations, p.C54Y and p.G1961E. Array comparative genomic hybridization revealed a large novel deletion combined with a small insertion, c.6148-698_c.6670del/insTGTGCACCTCCCTAG, and complete sequencing of the entire ABCA4 genomic locus uncovered a new deep intronic variant, c.302+68C>T. Patients with the p.G1961E mutation had the mildest, confined maculopathy phenotype with peripheral flecks while those with all other mutant allele combinations exhibited a more advanced stage of generalized retinal and choriocapillaris atrophy. This family epitomizes the clinical and genetic complexity of ABCA4-associated diseases. It contained variants from all classes of mutations, in the coding region, deep intronic, both single nucleotide variants and copy number variants that accounted for varying phenotypes segregating in an apparent dominant fashion. Unequivocally defining disease-associated alleles in the ABCA4 locus requires a multifaceted approach that includes advanced mutation detection methods and a thorough analysis of clinical phenotypes.

Recessive Stargardt disease phenocopying hydroxychloroquine retinopathy.

PURPOSE: To describe a series of patients with Stargardt disease (STGD1) exhibiting a phenotype usually associated with hydroxychloroquine (HCQ) retinopathy on spectral domain-optical coherence tomography (SD-OCT). METHODS: Observational case series from Columbia University Medical Center involving eight patients with genetically-confirmed STGD1. Patients selected for the study presented no history of HCQ use. Horizontal macular SD-OCT scans and accompanying 488-nm autofluorescence (AF) images, color fundus photographs, and full-field electroretinograms were analyzed. RESULTS: All study patients exhibited an abrupt thinning of the parafoveal region or disruption of the outer retinal layers on SD-OCT resembling the transient HCQ retinopathy phenotype. Funduscopy and AF imaging revealed variations of bull's eye maculopathy (BEM). Five patients exhibited local fleck-like deposits around the lesion. Genetic screening confirmed two disease-causing ABCA4 mutations in five patients and one mutation in three patients. CONCLUSIONS: A transient SD-OCT phenotype ascribed to patients with HCQ retinopathy is associated with an early subtype of STGD1. This finding may also present with HCQ retinopathy-like BEM lesions on AF imaging and funduscopy. A possible phenotypic overlap is unsurprising, given certain shared mechanistic disease processes between the two conditions. A thorough work-up, including screening of genes that are causal in retinal dystrophies associated with foveal sparing, may prevent misdiagnosis of more ambiguous cases.

Quantitative fundus autofluorescence distinguishes ABCA4-associated and non-ABCA4-associated bull's-eye maculopathy.

PURPOSE: Quantitative fundus autofluorescence (qAF) and spectral-domain optical coherence tomography (SD OCT) were performed in patients with bull's-eye maculopathy (BEM) to identify phenotypic markers that can aid in the differentiation of ABCA4-associated and non-ABCA4-associated disease. DESIGN: Prospective cross-sectional study at an academic referral center. SUBJECTS: Thirty-seven BEM patients (age range, 8-60 years) were studied. All patients exhibited a localized macular lesion exhibiting a smooth contour and qualitatively normal-appearing surrounding retina without flecks. Control values consisted of previously published data from 277 healthy subjects (374 eyes; age range, 5-60 years) without a family history of retinal dystrophy. METHODS: Autofluorescence (AF) images (30°, 488-nm excitation) were acquired with a confocal scanning laser ophthalmoscope equipped with an internal fluorescent reference to account for variable laser power and detector sensitivity. The grey levels (GLs) from 8 circularly arranged segments positioned at an eccentricity of approximately 7° to 9° in each image were calibrated to the reference (0 GL), magnification, and normative optical media density to yield qAF. In addition, horizontal SD OCT images through the fovea were obtained. All patients were screened for ABCA4 mutations using the ABCR600 microarray, next-generation sequencing, or both. MAIN OUTCOME MEASURES: Quantitative AF, correlations between AF and SD OCT, and genotyping for ABCA4 variants. RESULTS: ABCA4 mutations were identified in 22 patients, who tended to be younger (mean age, 21.9±8.3 years) than patients without ABCA4 mutations (mean age, 42.1±14.9 years). Whereas phenotypic differences were not obvious on the basis of qualitative fundus AF and SD OCT imaging, with qAF, the 2 groups of patients were clearly distinguishable. In the ABCA4-positive group, 37 of 41 eyes (19 of 22 patients) had qAF8 of more than the 95% confidence interval for age. Conversely, in the ABCA4-negative group, 22 of 26 eyes (13 of 15 patients) had qAF8 within the normal range. CONCLUSIONS: The qAF method can differentiate between ABCA4-associated and non-ABCA4-associated BEM and may guide clinical diagnosis and genetic testing.

Clinical and molecular characteristics of childhood-onset Stargardt disease.

PURPOSE: To describe the clinical and molecular characteristics of patients with childhood-onset Stargardt disease (STGD). DESIGN: Retrospective case series. PARTICIPANTS: Forty-two patients who were diagnosed with STGD in childhood at a single institution between January 2001 and January 2012. METHODS: A detailed history and a comprehensive ophthalmic examination were undertaken, including color fundus photography, autofluorescence imaging, spectral-domain optical coherence tomography (SD-OCT), and pattern and full-field electroretinograms. The entire coding region and splice sites of ABCA4 were screened using a next-generation, sequencing-based strategy. The molecular genetic findings of childhood-onset STGD patients were compared with those of adult-onset patients. MAIN OUTCOME MEASURES: Clinical, imaging, electrophysiologic, and molecular genetic findings. RESULTS: The median ages of onset and the median age at baseline examination were 8.5 (range, 3-16) and 12.0 years (range, 7-16), respectively. The median baseline logarithm of the minimum angle of resolution visual acuity was 0.74. At baseline, 26 of 39 patients (67%) with available photographs had macular atrophy with macular/peripheral flecks; 11 (28%) had macular atrophy without flecks; 1 (2.5%) had numerous flecks without macular atrophy; and 1 (2.5%) had a normal fundus appearance. Flecks were not identified at baseline in 12 patients (31%). SD-OCT detected foveal outer retinal disruption in all 21 patients with available images. Electrophysiologic assessment demonstrated retinal dysfunction confined to the macula in 9 patients (36%), macular and generalized cone dysfunction in 1 subject (4%), and macular and generalized cone and rod dysfunction in 15 individuals (60%). At least 1 disease-causing ABCA4 variant was identified in 38 patients (90%), including 13 novel variants; ≥2 variants were identified in 34 patients (81%). Patients with childhood-onset STGD more frequently harbored 2 deleterious variants (18% vs 5%) compared with patients with adult-onset STGD. CONCLUSIONS: Childhood-onset STGD is associated with severe visual loss, early morphologic changes, and often generalized retinal dysfunction, despite often having less severe fundus abnormalities on examination. One third of children do not have flecks at presentation. The relatively high proportion of deleterious ABCA4 variants supports the hypothesis that earlier onset disease is often owing to more severe variants in ABCA4 than those found in adult-onset disease.

Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common form of irreversible blindness in developed countries. Variants in the factor H gene (CFH, also known as HF1), which encodes a major inhibitor of the alternative complement pathway, are associated with the risk for developing AMD. Here we test the hypothesis that variation in genes encoding other regulatory proteins of the same pathway is associated with AMD. We screened factor B (BF) and complement component 2 (C2) genes, located in the major histocompatibility complex class III region, for genetic variation in two independent cohorts comprising approximately 900 individuals with AMD and approximately 400 matched controls. Haplotype analyses identify a statistically significant common risk haplotype (H1) and two protective haplotypes. The L9H variant of BF and the E318D variant of C2 (H10), as well as a variant in intron 10 of C2 and the R32Q variant of BF (H7), confer a significantly reduced risk of AMD (odds ratio = 0.45 and 0.36, respectively). Combined analysis of the C2 and BF haplotypes and CFH variants shows that variation in the two loci can predict the clinical outcome in 74% of the affected individuals and 56% of the controls. These data expand and refine our understanding of the genetic risk for AMD.

Genetic and clinical analysis of ABCA4-associated disease in African American patients.

Autosomal recessive Stargardt disease (STGD1) is caused by hundreds of mutations in the ABCA4 gene, which are often specific to racial and ethnic groups. Here, we investigated the ABCA4 variation and their phenotypic expression in a cohort of 44 patients of African American descent, a previously under-characterized racial group. Patients were screened for mutations in ABCA4 by next-generation sequencing and array-comparative genomic hybridization (aCGH), followed by analyses for pathogenicity by in silico programs. Thorough ophthalmic examination was performed on all patients. At least two (expected) disease-causing alleles in the ABCA4 gene were identified in 27 (61.4%) patients, one allele in 11 (25%) patients, and no ABCA4 mutations were found in six (13.6%) patients. Altogether, 39 different disease-causing ABCA4 variants, including seven new, were identified on 65 (74%) chromosomes, most of which were unique for this racial group. The most frequent ABCA4 mutation in this cohort was c.6320G>A (p.(R2107H)), representing 19.3% of all disease-associated alleles. No large copy number variants were identified in any patient. Most patients reported later onset of symptoms. In summary, the ABCA4 mutation spectrum in patients of West African descent differs significantly from that in patients of European descent, resulting in a later onset and "milder" disease.

The value of retinal imaging with infrared scanning laser ophthalmoscopy in patients with stargardt disease.

PURPOSE: To demonstrate the value of infrared scanning laser ophthalmoscopy (SLO) for determining structural retinal and choroidal changes in patients with Stargardt disease and its comparison to findings on short-wavelength fundus autofluorescence (SW-AF) imaging, spectral-domain optical coherence tomography, and microperimetry measurements. METHODS: Forty-four eyes of 22 patients with Stargardt disease were studied using infrared-SLO, spectral-domain optical coherence tomography, macular microperimetry, SW-AF, electroretinography, and fundus photography. RESULTS: Although SW-AF imaging outlined the regions of retinal pigment epithelial (RPE) atrophy (hypofluorescence) and enhanced the visibility of more funduscopically apparent flecks (hyperfluorescence), infrared-SLO imaging outlined the regions of choroidal, and RPE, atrophic changes. Degenerative changes in photoreceptor and RPE cell layers, evident on spectral-domain optical coherence tomography imaging, were associated with either hyporeflective or hyperreflective images on infrared-SLO imaging, depending on whether both RPE and choroidal atrophy (hyperreflectance) or solely RPE atrophy (hyporeflectance) was present. Threshold elevations on microperimetry testing corresponded to both RPE and choroidal atrophy on infrared-SLO imaging and RPE atrophy on SW-AF. CONCLUSION: Although SW-AF identifies regions of RPE atrophy, infrared-SLO also identifies the involvement of the choroid that has important implications for the potential improvement in visual function from treatment. Thus, infrared-SLO imaging offers an additional advantage beyond that obtained with SW-AF.

Psychophysical measurement of rod and cone thresholds in stargardt disease with full-field stimuli.

PURPOSE: To investigate psychophysical thresholds in Stargardt disease with the full-field stimulus test (FST). METHODS: Visual acuity, spectral domain optical coherence tomography, full-field electroretinogram, and FST measurements were made in 1 eye of 24 patients with Stargardt disease. Dark-adapted rod FST thresholds were measured with short-wavelength stimuli, and cone FST thresholds were obtained from the cone plateau phase of dark adaptation using long-wavelength stimuli. Correlation coefficients were calculated for FST thresholds versus macular thickness, visual acuity, and electroretinogram amplitudes. RESULTS: The Stargardt disease patients' FST cone thresholds correlated significantly with visual acuity, macular thickness, and electroretinogram cone response amplitudes (all P < 0.01). The patients' FST rod thresholds correlated with electroretinogram rod response amplitudes (P < 0.01) but not macular thickness (P = 0.05). All patients with Stargardt disease with flecks confined to the macula, and most of the patients with flecks extending outside of the macula had normal FST thresholds. All patients with extramacular atrophic changes had elevated FST cone thresholds and most had elevated FST rod thresholds. CONCLUSION: Full-field stimulus test rod and cone threshold elevation in patients with Stargardt disease correlated well with measures of structure and function, as well as ophthalmoscopic retinal appearance. The Full-field stimulus test appears to be a useful tool for assessing rod and cone function in Stargardt disease.

Towards Uncovering the Role of Incomplete Penetrance in Maculopathies through Sequencing of 105 Disease-Associated Genes.

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

Outcome of ABCA4 disease-associated alleles in autosomal recessive retinal dystrophies: retrospective analysis in 420 Spanish families.

OBJECTIVE: To provide a comprehensive overview of all detected mutations in the ABCA4 gene in Spanish families with autosomal recessive retinal disorders, including Stargardt's disease (arSTGD), cone-rod dystrophy (arCRD), and retinitis pigmentosa (arRP), and to assess genotype-phenotype correlation and disease progression in 10 years by considering the type of variants and age at onset. DESIGN: Case series. PARTICIPANTS: A total of 420 unrelated Spanish families: 259 arSTGD, 86 arCRD, and 75 arRP. METHODS: Spanish families were analyzed through a combination of ABCR400 genotyping microarray, denaturing high-performance liquid chromatography, and high-resolution melting scanning. Direct sequencing was used as a confirmation technique for the identified variants. Screening by multiple ligation probe analysis was used to detect possible large deletions or insertions in the ABCA4 gene. Selected families were analyzed further by next generation sequencing. MAIN OUTCOME MEASURES: DNA sequence variants, mutation detection rates, haplotypes, age at onset, central or peripheral vision loss, and night blindness. RESULTS: Overall, we detected 70.5% and 36.6% of all expected ABCA4 mutations in arSTGD and arCRD patient cohorts, respectively. In the fraction of the cohort where the ABCA4 gene was sequenced completely, the detection rates reached 73.6% for arSTGD and 66.7% for arCRD. However, the frequency of possibly pathogenic ABCA4 alleles in arRP families was only slightly higher than that in the general population. Moreover, in some families, mutations in other known arRP genes segregated with the disease phenotype. CONCLUSIONS: An increasing understanding of causal ABCA4 alleles in arSTGD and arCRD facilitates disease diagnosis and prognosis and also is paramount in selecting patients for emerging clinical trials of therapeutic interventions. Because ABCA4-associated diseases are evolving retinal dystrophies, assessment of age at onset, accurate clinical diagnosis, and genetic testing are crucial. We suggest that ABCA4 mutations may be associated with a retinitis pigmentosa-like phenotype often as a consequence of severe (null) mutations, in cases of long-term, advanced disease, or both. Patients with classical arRP phenotypes, especially from the onset of the disease, should be screened first for mutations in known arRP genes and not ABCA4.

Insights Into PROM1-Macular Disease Using Multimodal Imaging.

Purpose: To describe the features of genetically confirmed PROM1-macular dystrophy in multimodal images. Methods: Thirty-six (36) eyes of 18 patients (5-66 years; mean age, 42.4 years) were prospectively studied by clinical examination and multimodal imaging. Short-wavelength autofluorescence (SW-AF) and quantitative fundus autofluorescence (qAF) images were acquired with a scanning laser ophthalmoscope (HRA+OCT, Heidelberg Engineering) modified by insertion of an internal autofluorescent reference. Further clinical testing included near-infrared autofluorescence (NIR-AF; HRA2, Heidelberg Engineering) with semiquantitative analysis, spectral domain-optical coherence tomography (HRA+OCT) and full-field electroretinography. All patients were genetically confirmed by exome sequencing. Results: All 18 patients presented with varying degrees of maculopathy. One family with individuals affected across two generations exhibited granular fleck-like deposits across the posterior pole. Areas of granular deposition in SW-AF and NIR-AF corresponded to intermittent loss of the ellipsoid zone, whereas discrete regions of hypoautofluorescence corresponded with a loss of outer retinal layers in spectral-domain optical coherence tomography scans. For 18 of the 20 eyes, qAF levels within the macula were within the 95% confidence intervals of healthy age-matched individuals; nor was the mean NIR-AF signal increased relative to healthy eyes. Conclusions: Although PROM1-macular dystrophy (Stargardt disease 4) can exhibit phenotypic overlap with recessive Stargardt disease, significantly increased SW-AF levels were not detected. As such, elevated bisretinoid lipofuscin may not be a feature of the pathophysiology of PROM1 disease. The qAF approach could serve as a method of early differential diagnosis and may help to identify appropriate disease targets as therapeutics become available to treat inherited retinal disease.