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.

Targeted sequencing and in vitro splice assays shed light on ABCA4-associated retinopathies missing heritability.

The ABCA4 gene is the most frequently mutated Mendelian retinopathy-associated gene. Biallelic variants lead to a variety of phenotypes, however, for thousands of cases the underlying variants remain unknown. Here, we aim to shed further light on the missing heritability of ABCA4-associated retinopathy by analyzing a large cohort of macular dystrophy probands. A total of 858 probands were collected from 26 centers, of whom 722 carried no or one pathogenic ABCA4 variant, while 136 cases carried two ABCA4 alleles, one of which was a frequent mild variant, suggesting that deep-intronic variants (DIVs) or other cis-modifiers might have been missed. After single molecule molecular inversion probes (smMIPs)-based sequencing of the complete 128-kb ABCA4 locus, the effect of putative splice variants was assessed in vitro by midigene splice assays in HEK293T cells. The breakpoints of copy number variants (CNVs) were determined by junction PCR and Sanger sequencing. ABCA4 sequence analysis solved 207 of 520 (39.8%) naive or unsolved cases and 70 of 202 (34.7%) monoallelic cases, while additional causal variants were identified in 54 of 136 (39.7%) probands carrying two variants. Seven novel DIVs and six novel non-canonical splice site variants were detected in a total of 35 alleles and characterized, including the c.6283-321C>G variant leading to a complex splicing defect. Additionally, four novel CNVs were identified and characterized in five alleles. These results confirm that smMIPs-based sequencing of the complete ABCA4 gene provides a cost-effective method to genetically solve retinopathy cases and that several rare structural and splice altering defects remain undiscovered in Stargardt disease cases.

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.

Establishment of the iPSC line CUIMCi005-A from a patient with Stargardt disease for retinal organoid culture.

Pathogenic variation in the ABCA4 gene is the underlying cause of Stargardt disease, the most common inherited retinal degeneration. We established an induced pluripotent stem cell line for retinal organoid research from a patient with mild disease features who is compound heterozygous for the frequent c.5882G>A (p.Gly1961Glu) missense variant and a c.4947delC (p.Glu1650Argfs*12) frameshift variant. Peripheral blood mononuclear cells were reprogrammed using a non-integrating Sendai virus approach. G-banded karyotyping was normal (46, XY) and mycoplasma testing was negative. Immunohistochemistry and RT-qPCR were performed to verify the expression of pluripotency and stemness markers (LIN28, NANOG, OCT4 and SOX2) and trilineage differentiation.

A pathogenic in-frame deletion-insertion variant in BEST1 phenocopies Stargardt disease.

Here, we describe affected members of a 2-generation family with a Stargardt disease-like phenotype caused by a 2-base pair deletion insertion, c.1014_1015delGAinsCT;p.(Trp338_Asn339delinsCysTyr), in BEST1. The variant was identified by whole-exome sequencing, and its pathogenicity was verified through chloride channel recording using WT and transfected mutant HEK293 cells. Clinical examination of both patients revealed similar phenotypes at 2 different disease stages that were attributable to differences in their age at presentation. Hyperautofluorescent flecks along the arcades were observed in the proband, while the affected mother exhibited more advanced retinal pigment epithelium (RPE) loss in the central macula. Full-field electroretinogram testing was unremarkable in the daughter; however, moderate attenuation of generalized cone function was detected in the mother. Results from electrooculogram testing in the daughter were consistent with widespread dysfunction of the RPE characteristic of Best disease. Whole-cell patch-clamp recordings revealed a statistically significant decrease in chloride conductance of the mutant compared with WT cells. This report on a mother and daughter with a BEST1 genotype that phenocopies Stargardt disease broadens the clinical spectrum of BEST1-associated retinopathy.

Longitudinal Analysis of a Resolving Foveomacular Vitelliform Lesion in ABCA4 Disease.

PURPOSE: To describe the longitudinal progression and phenotypic association of bilateral foveomacular vitelliform lesions in the setting of ABCA4 disease. DESIGN: Case report and cross-sectional cohort study. PARTICIPANTS: Nineteen patients with confirmed ABCA4 disease exhibiting an optical gap phenotype. METHODS: Multimodal retinal imaging across multiple visits included autofluorescence imaging, spectral-domain OCT (SD-OCT), and OCT angiography. Electro-oculogram (EOG) and full-field electroretinogram testing results were analyzed. Exome sequencing was performed for diagnostic confirmation and the verification of other variations. MAIN OUTCOME MEASURES: Light-peak-to-dark-trough ratio (Arden ratio) on EOG; thickness and en face maps of various retinal layers on SD-OCT; area measurements on 488- and 787-nm autofluorescence images; and the presence of variation in vitelliform-associated genes identified using exome sequencing. RESULTS: A 25-year-old White man presented with bilateral central vision loss due to foveal lesions consisting of vitelliform fluid. The result of EOG testing was inconsistent with bestrophinopathy (Arden ratio = 1.62), and no generalized rod or cone dysfunction was detected on full-field electroretinogram. Exome sequencing identified the pathogenic variants c.5882G>A (p.(Gly1961Glu)) and c.4139C>T (p.(Pro1380Leu)) in ABCA4 and no other vitelliform-associated genes. Significant thinning and abnormal reflectivity of photoreceptor-attributable layers as well as near-infrared autofluorescence abnormalities were found in lesion-adjacent areas. Complete resorption of the vitelliform fluid occurred after 30 months, after which the optical gap lesions exhibited an enlarged and "cavitated" appearance. Phenotypic screening for additional cases from a large ABCA4 disease database (n = 602) identified 18 additional patients at various stages of optical gap lesion formation, most of whom harbored the c.5882G>A (p.(Gly1961Glu)) variant (P < 0.001), although none had apparent vitelliform fluid. At least 5 of the 18 (31.6%) patients exhibited optical gap lesions with the distinct "cavitated" appearance, whereas the lesions remained unperturbed in the other patients over the course of examination. CONCLUSIONS: Foveomacular vitelliform deposition is a mechanistically congruent but rare manifestation of ABCA4 disease. Specifically, this disease phenotype may be clinically associated with the c.5882G>A (p.(Gly1961Glu)) allele and optical gap lesions.

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.

Comparisons Among Optical Coherence Tomography and Fundus Autofluorescence Modalities as Measurements of Atrophy in ABCA4-Associated Disease.

PURPOSE: In ABCA4-associated retinopathy, central atrophy was assessed by spectral domain optical coherence tomography (SD-OCT) and by short-wavelength (SW-AF) and near-infrared (NIR-AF) autofluorescence. METHODS: Patients exhibited a central atrophic lesion characterized by hypoautofluorescence (hypoAF) surrounded either by hyperautofluorescent (hyperAF) rings in both AF images (group 1, 4 patients); or a hyperAF ring in SW-AF but not in NIR-AF images (group 2, 11 patients); or hyperAF rings in neither AF images (group 3, 11 patients). Choroidal hypertransmission and widths of ellipsoid zone (EZ) loss were measured in foveal SD-OCT scans, and in AF images hypoAF and total hypo+hyperAF widths were measured along the same axis. Bland-Altman and repeated measures analysis of variance with Tukey post hoc were applied. RESULTS: For all groups, hypertransmission widths were significantly smaller than EZ loss widths. In Groups 1 and 2, hypertransmission width was not significantly different than SW-hypoAF width, but hypertransmission was narrower than the width of SW-hypo+hyperAF (groups 1, 2) and NIR-hypo+hyperAF (group 1). In group 3, the hypertransmission width was also significantly less than the width of SW-hypoAF and NIR-hypoAF. The EZ loss widths were not significantly different than measurements of total lesion size, the latter being the widths of SW-hypo+hyperAF and NIR-hypo+hyperAF (group 1); widths of NIR-hypoAF and SW-hypo+hyperAF (group 2); and widths of NIR-hypoAF and SW-hypoAF (group 3). CONCLUSIONS: Hypertransmission and SW-hypoAF (except when reflecting total lesion width) underestimate lesion size detected by EZ loss, SW-hypoAF+hyperAF, and NIR-hypo+hyperAF. TRANSLATIONAL RELEVANCE: The findings are significant to the selection of outcome measures in clinical studies.

A genotype-phenotype correlation matrix for ABCA4 disease based on long-term prognostic outcomes.

BackgroundMore than 1500 variants in the ATP-binding cassette, sub-family A, member 4 (ABCA4), locus underlie a heterogeneous spectrum of retinal disorders ranging from aggressive childhood-onset chorioretinopathy to milder late-onset macular disease. Genotype-phenotype correlation studies have been limited in clinical applicability as patient cohorts are typically small and seldom capture the full natural history of individual genotypes. To overcome these limitations, we constructed a genotype-phenotype correlation matrix that provides quantifiable probabilities of long-term disease outcomes associated with specific ABCA4 genotypes from a large, age-restricted patient cohort.MethodsThe study included 112 unrelated patients at least 50 years of age in whom 2 pathogenic variants were identified after sequencing of the ABCA4 locus. Clinical characterization was performed using the results of best corrected visual acuity, retinal imaging, and full-field electroretinogram testing.ResultsFour distinct prognostic groups were defined according to the spatial severity of disease features across the fundus. Recurring genotypes were observed in milder prognoses, including a newly defined class of rare hypomorphic alleles. PVS1 (predicted null) variants were enriched in the most severe prognoses; however, missense variants were present in a larger-than-expected fraction of these patients. Analysis of allele combinations and their respective prognostic severity showed that certain variants, such as p.(Gly1961Glu), and both rare and frequent hypomorphic alleles, were "clinically dominant" with respect to patient phenotypes irrespective of the allele in trans.ConclusionThese results provide much-needed structure to the complex genetic and clinical landscape of ABCA4 disease and add a tool to the clinical repertoire to quantitatively assess individual genotype-specific prognoses in patients.FUNDINGNational Eye Institute, NIH, grants R01 EY028203, R01 EY028954, R01 EY029315, P30 19007 (Core Grant for Vision Research); the Foundation Fighting Blindness USA, grant no. PPA-1218-0751-COLU; and Research to Prevent Blindness.

Reevaluating the Association of Sex With ABCA4 Alleles in Patients With Stargardt Disease.

IMPORTANCE: Probing differences in disease prevalence between sexes is challenging, especially in mendelian diseases. Independent replication of any association study is warranted. OBJECTIVE: To evaluate whether the recently reported association between sex and mild ABCA4 alleles among patients with autosomal recessive Stargardt disease (STGD1) is reproducible. DESIGN, SETTING, AND PARTICIPANTS: Sequencing and clinical data from 644 unrelated patients with genetically confirmed STGD1 were analyzed in a cross-sectional study at the Department of Ophthalmology, Columbia University, New York, New York. Data were collected from June 1999 to October 2020. MAIN OUTCOMES AND MEASURES: Sex, best-corrected visual acuity, and age at onset among patients with STGD1 with and without mild ABCA4 alleles. RESULTS: A total of 644 patients with STGD1 with at least 2 pathogenic variants were included in the study. The mean (SD) age was 38.6 (17.2) years, and 352 participants (54.7%) were female. The proportion of women was slightly higher in the entire cohort and in most allele categories, although none of the differences were statistically significant. The proportion of women carrying the c.5603A>T p.(Asn1868Ile) allele was 7% (95% CI, -9 to 23) higher than in the subgroup not carrying any mild alleles (P = .32). The proportion of women carrying the c.5882G>A p.(Gly1961Glu) allele was 2% (95% CI, -12 to 15) higher than in the subgroup not carrying any mild alleles (P = .77). The difference between the total mild allele subcohort and the no mild allele subcohort was 3% (95% CI, -8 to 14; P = .48). Compared with patients in the no mild allele category, patients with mild alleles exhibited significantly delayed disease onset (mean [SD] age, 23.1 [11.6] for those with the c.5882G>A allele and 31.7 [13.5] years for those with the c.5603A>T allele vs 18.6 [11.8] years for those with no mild alleles; P < .001) and preserved visual acuity (5882G>A subgroup: mean [SD] logMAR, 0.65 [0.66]; 95% CI, 0.63-0.68; c.5603A>T subgroup: 0.64 [0.39]; 95% CI, 0.58-0.70; those with no mild alleles: 1.00 [0.57]; 95% CI, 0.96-1.03; P < .001). CONCLUSIONS AND RELEVANCE: This independent analysis of a larger cohort of individuals with Stargardt disease did not support the association between sex and certain mild ABCA4 alleles. While sex is undoubtedly an important variable in medicine, its putative association with clinical outcomes should be rigorously scrutinized.

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.

Retinal Pigment Epithelium Atrophy in Recessive Stargardt Disease as Measured by Short-Wavelength and Near-Infrared Autofluorescence.

Purpose: To compare the detection of retinal pigment epithelium (RPE) atrophy in short-wavelength (SW-AF) and near-infrared autofluorescence (NIR-AF) images in Stargardt disease (STGD1) patients. Methods: SW-AF and NIR-AF images (115 eyes from 115 patients) were analyzed by two independent graders. Hypoautofluorescent (hypoAF) areas, indicative of RPE atrophy, were measured, and the two modalities were compared. Results: Patients were segregated into four groups: nascent (6 [5%]), widespread (21 [18%]), discrete (55 [48%]), and chorioretinal atrophy (33 [29%]). The areas of hypoAF were larger in NIR-AF compared to SW-AF images in discrete (3.9 vs. 2.2 mm2, P < 0.001) and chorioretinal atrophy (12.7 vs. 11.4 mm2, P = 0.015). Similar findings were observed qualitatively in nascent and widespread atrophy patients. Using the area linear model (ALM), lesion area increased at similar rates in SW-AF and NIR-AF images of discrete atrophy (0.20 vs. 0.32 mm2/y, P = 0.275) and chorioretinal atrophy (1.30 vs. 1.74 mm2/y, P = 0.671). Using the radius linear model (RLM), the lesion effective radius also increased similarly in SW-AF and NIR-AF images in discrete (0.03 vs. 0.05 mm2/y, P = 0.221) and chorioretinal atrophy (0.08 vs. 0.10 mm2/y, P = 0.754) patients. Conclusions: NIR-AF reveals a larger area of RPE atrophy in STGD1 patients compared to SW-AF images, but rates of lesion enlargement in the two modalities are similar. Translational Relevance: Measurements of RPE atrophy by AF imaging are crucial for monitoring STGD1 disease progression and given our findings we advocate greater use of NIR-AF for patients.

Progressive Choriocapillaris Impairment in ABCA4 Maculopathy Is Secondary to Retinal Pigment Epithelium Atrophy.

Purpose: To analyze the progression of choriocapillaris (CC) impairment in recessive Stargardt disease (STGD) and compare it to the progression of retinal pigment epithelium (RPE) atrophy. Methods: Fifty-five patients with a clinical diagnosis of STGD and genetic confirmation of pathogenic biallelic variants in ABCA4 were imaged with short-wavelength fundus autofluorescence (SW-AF) and optical coherence tomography angiography (OCTA) at a single clinic visit, whereas a subset of 12 patients were imaged with the same modalities at two different clinic visits. Results: We observed three stages of CC impairment: an area of bright yet intact macular CC (11 patients), regions of vascular rarefaction and incomplete CC atrophy within an area of bright CC (10 patients), and areas of extensive CC atrophy (26 patients). These changes correlated to the degree of RPE atrophy observed in SW-AF imaging. Furthermore, 8 patients presented with early changes on SW-AF, but healthy CC. Quantitative analyses of the atrophic changes revealed that the area of RPE atrophy is larger (9.6 ± 1.7 mm2 vs. 6.9 ± 1.3 mm2, P < 0.001) and that it progresses at a faster rate (1.1 ± 0.1 mm2/year vs. 0.8 ± 0.2 mm2/year, P = 0.004) than the corresponding area of CC atrophy. Conclusions: CC impairment is progressive and OCTA imaging can be used to demonstrate the stages, which culminate in extensive CC atrophy. Furthermore, CC impairment is secondary to RPE atrophy in STGD. We further advocate the use of SW-AF and OCTA imaging in monitoring the progression of STGD.

Modification of the PROM1 disease phenotype by a mutation in ABCA4.

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.

Spectrum of Disease Severity and Phenotype in Choroideremia Carriers.

PURPOSE: To characterize and bring awareness to the disease spectrum of female choroideremia patients, as severity can vary from mild to severe disease, comparable to that observed in male patients. DESIGN: Retrospective cohort study. METHODS: Twelve female carriers of disease-causing variants in the CHM gene confirmed by molecular genetic sequencing were characterized clinically and imaged with short-wave fundus autofluorescence (SW-FAF), spectral-domain optical coherence tomography (OCT), and color fundus imaging. RESULTS: Twelve unrelated female patients with a clinical and genetic diagnosis of choroideremia carriers were included in this study. Disease severity among these phenotypes ranged from mild to severe, resembling the typical presentation of choroideremia in male patients. Mild disease presented with retinal pigment epithelium mottling, a patchy pattern of hypoautofluorescent speckles on SW-FAF, and intact retinal layers on spectral-domain OCT. Severe disease presented with widespread chorioretinal atrophy as shown by SW-FAF and spectral-domain OCT. Each of the identified genetic variants in CHM was predicted to be disease-causing according to in silico prediction software. Disease progression analysis of 4 patients with follow-up showed a decline in visual acuity for 2 patients, with progression observed on spectral-domain OCT in 1 of the patients. No significant disease progression on SW-FAF was observed for any of the patients. CONCLUSIONS: Female carriers of choroideremia can present with a wide range of clinical phenotypes and disease severity, from mild to severe disease, similar to male subjects. Symptomatic female subjects should be considered for current and upcoming gene replacement therapy clinical trials.

Characteristic Ocular Features in Cases of Autosomal Recessive PROM1 Cone-Rod Dystrophy.

Purpose: To define characteristic ocular features in a group of patients with autosomal recessive (AR) PROM1 cone-rod dystrophy (CRD). Methods: Three males and one female from three unrelated families were first seen at the ages of 15 to 22 years and diagnosed with CRD. Clinical testing available for review included full-field electroretinogram (ERG) in three patients, as well as near-infrared autofluorescence (NIR-AF), spectral-domain optical coherence tomography (SD-OCT), and color fundus photography in all four patients. Whole exome sequencing (WES) was performed on all cases, and whole genome sequencing (WGS) was performed in two families. Results: WES found compound heterozygous PROM1 variants in one isolated male, plus heterozygous variants in the remaining patients. WGS uncovered deleterious PROM1 variants in these two families. ERG showed markedly reduced cone-isolated amplitudes and variably reduced rod-isolated amplitudes. The dark-adapted combined rod and cone responses demonstrated notably reduced a-wave amplitudes and moderately reduced b-waves, and the resultant waveform resembled the normal rod-isolated response. On fundus examination, oval-shaped macular lesions were observed, as were several small, circular hypoautofluorescent lesions within the posterior pole on NIR-AF. Three patients showed extramacular circular atrophic lesions. Conclusions: The autofluorescence changes, peripheral retinal abnormalities, and ERG findings have not been emphasized in previous reports of AR PROM1, but they became a recognizable phenotype in this cohort of patients. A similar constellation of findings may be observed in CRD due to CDHR1, a functionally related gene. The pattern of abnormalities reported herein may help to focus genetic screening in patients with these findings.

Late-onset pattern macular dystrophy mimicking ABCA4 and PRPH2 disease is caused by a homozygous frameshift mutation in ROM1.

ROM1 (retinal outer segment membrane protein 1) is a 351-amino acid integral membrane protein on Chromosome 11q, with high structural similarity to PRPH2/RDS. Localized at the rims of photoreceptor outer segments (OSs), it is required for the maintenance of OS structure. Here, we describe a case with a phenotypic manifestation of a homozygous single-base pair deletion, c.712delC (p.Leu238Cysfs*78) in the ROM1 gene, resulting in early termination at exon 2. The variant was detected by whole-exome sequencing (WES) in a 63-yr-old Caucasian woman with late-onset pattern macular dystrophy. Notably, although the phenotype resembles those caused by pathogenic variants in ABCA4 or RDS/PRPH2, no pathogenic variants in these, or any other plausible candidate genes, were identified by WES. Clinical features include the presence of hyperautofluorescent flecks, relative sparing of the central macula, and preserved visual acuity. Reduced visual sensitivity was detected among flecked regions in the retina; however, full-field electroretinogram testing revealed no generalized cone dysfunction. The described first case of the complete loss of ROM1 protein function in the retina suggests its sufficiency for late-onset macular dystrophy. ROM1 and PRPH2 pattern macular dystrophies exhibit phenotype overlap, which may be attributable to their shared role in maintenance of the photoreceptor outer segment structure.

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.

Deep Scleral Exposure: A Degenerative Outcome of End-Stage Stargardt Disease.

PURPOSE: To describe a distinct phenotypic outcome of outer retinal degeneration in a cohort of genetically confirmed patients with recessive Stargardt disease (STGD1). DESIGN: Retrospective case series. METHODS: Twelve patients, who were clinically diagnosed with STGD1 and exhibited a unique degenerative phenotype, were included in the study. Two disease-causing mutations were found in all patients by direct sequencing of the ABCA4 gene. Clinical characterization of patients were defined on fundus photographs, autofluorescence images (488-nm and 532-nm excitation), spectral-domain optical coherence tomography (SD-OCT), and full-field electroretinogram (ffERG) testing. RESULTS: Mean age at initial presentation was 67.8 years and reported age of symptomatic onset was 14.1 years (mean disease duration = 53.8 years). Best-corrected visual acuity ranged from 20/400 to hand motion. All patients exhibited advanced degeneration across the posterior pole resulting in a reflectively pale, blonde fundus owing to unobstructed exposure of the underlying sclera. SD-OCT revealed complete loss of the outer retinal bands (external limiting membrane, ellipsoid zone, interdigitation zone, and retinal pigment epithelium) and choroidal layers. Scotopic and photopic waveforms on ffERG were nonrecordable or severely attenuated in 8 patients who were tested. CONCLUSIONS: Widespread scleral exposure is a clinical outcome in a subset of STGD1 following a long duration of disease progression (∼50 years). The blonde fundus in such cases may exhibit phenotypic overlap and shared therapeutic implications with other aggressive chorioretinal dystrophies such as end-stage choroideremia, gyrate atrophy, or RPE65-Leber congenital amaurosis.