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PURPOSE. To investigate the role of two deep-intronic ABCA4 variants, that showed a mild splice defect in vitro and can occur on the same allele as the low penetrant c.5603A>T, in Stargardt disease (STGD1).METHODS. Ophthalmic data were assessed of 18 STGD1 patients who harbored c.769-784C>T or c.4253+43G>A in combination with a severe ABCA4 variant. Subjects carrying c.[769784C>T; 5603A>T] were clinically compared with a STGD1 cohort previously published carrying c.5603A>T noncomplex. We calculated the penetrances of the intronic variants using ABCA4 allele frequency data of the general population and investigated the effect of c.769-784C>T on splicing in photoreceptor progenitor cells (PPCs).RESULTS. Mostly, late-onset, foveal-sparing STGD1 was observed among subjects harboring c.769-784C>T or c.4253+43G>A (median age of onset, 54.5 and 52.0 years, respectively). However, ages of onset, phenotypes in fundo, and visual acuity courses varied widely. No significant clinical differences were observed between the c.[769-784C>T; 5603A>T] cohort and the c.4253+43G>A or the c.5603A>T cohort. The penetrances of c.769-784C>T (20.5%-39.6%) and c.4253+43G>A (35.8%-43.1%) were reduced, when not considering the effect of yet unidentified or known factors in cis, such as c.5603A>T (identified in 7/7 probands with c.769-784C>T; 1/8 probands with c.4253+43G>A). Variant c.769-784C>T resulted in a pseudo-exon insertion in 15% of the total mRNA (i.e., similar to 30% of the c.769-784C>T allele alone).CONCLUSIONS. Two mild intronic ABCA4 variants could further explain missing heritability in late-onset STGD1, distinguishing it from AMD. The observed clinical variability and calculated reduced penetrance urge research into modifiers within and outside of the ABCA4 gene.

To raise awareness of Stargardt disease (STGD1) patients without fundus abnormalities. Medical records were evaluated for age at onset, initial symptoms and diagnosis, reason for delay of diagnosis, age at STGD1 diagnosis, best-corrected visual acuity (BCVA), ophthalmoscopy, fundus photography, fundus autofluorescence (FAF), fluorescein angiography (FA), spectral-domain optical coherence tomography (SD-OCT), full-field electroretinography (ffERG), color vision test, and the presence of ABCA4 variants. In 11.1% of our STGD1 cohort of 280 patients, no fundus abnormalities were observed at first ophthalmic consultation. The median age at onset was 8 years (range, 1–18). There was a median delay in diagnosis of 3 years (range, 0–19) in 27 out of 31 patients, which resulted in a median age at diagnosis of 12 years (range, 7–26). Patients were misdiagnosed with amblyopia, myopia, optic disk pathology, mental health problems, tension headache, neuritis bulbaris, and uveitis. Subtle abnormalities, such as lipofuscin accumulation, were seen on FAF at an earlier disease stage than in ophthalmoscopy. On SD-OCT, this included a thickened external limiting membrane. Color vision tests showed red-green insufficiency in 79% of patients. Reduced ERG amplitudes were only present in 26% (N = 8) and a dark choroid sign in 65% of the patients. Visual acuity considerably fluctuated in the first 5 years after onset. The majority of the patients (65%) carried a least one variant with a severe effect on ABCA4 function. Childhood-onset STGD1 patients were diagnosed with a delay of median 3 years. The presence of accurate competence, equipment, and the possibility for genetic screening is required; therefore, we recommend to refer children with visual complaints without initial fundus abnormalities to a specialized ophthalmologic center. In particular, to diagnose patients at an early stage of disease is of increased importance with the advent of new therapeutic possibilities.

Purpose: Using exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify the missing heritability.Methods: Sequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects.Results: In 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correction of all splicing defects.Conclusion: Our data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants.

AimsTo describe the prevalence of the Charles Bonnet syndrome (CBS) and search for potential CBS risk factors in a Dutch Stargardt disease (STGD1) cohort.MethodsEighty-three patients with STGD1 were screened for CBS. They underwent a full eye examination. All patients completed the social functioning domain of the 36-Item Short Form Health Survey questionnaire. Participants suspected of CBS were interviewed to further evaluate their visual hallucinations.ResultsCBS prevalence was 8.4%. Six out of seven patients with CBS were women. CBS was not associated with age (p=0.279, Mann-Whitney). Patients with CBS had a significant lower social functioning score (p2) as compared with controls (range 0–180 mm2). There was no relation between the position of the scotoma and the location of the visual hallucinations.ConclusionThe relative high CBS prevalence in STGD1 suggests that CBS may be more prevalent in younger ophthalmic patients than currently presumed. In this specific group of patients, we established social isolation and acquired vision impairment as risk factors for CBS. There was a female preponderance among patients with CBS. Age and retinal pigment epithelium atrophy were not identified as significant risk factors. We should actively diagnose CBS in patients of any age who fulfil the criteria for the category vision impairment, especially in cases where social isolation is suspected.

Purpose The purpose of this study was to evaluate the functional relevance of longitudinal changes in hyperautofluorescent areas and flecks in Stargardt disease (STGD1) using short-wavelength autofluorescence (SW-AF) imaging. Methods In this prospective, longitudinal study, 31 patients with STGD1 (56 eyes) underwent microperimetry (MP) and SW-AF imaging twice in 3 to 5 years. A total of 760 MP test points were included in the statistical analysis based on stable fixation and accurate alignment of SW-AF and MP. Autofluorescence intensity was qualitatively assessed in all MP test points. Small circumscriptive hyperautofluorescent lesions were defined as flecks. Longitudinal imaging characteristics observed on SW-AF were classified into the following categories: appearing, disappearing, and stable flecks, stable hyperautofluorescent, and stable background autofluorescence. The relationship between SW-AF intensity changes and MP changes was analyzed using a linear mixed model corrected for baseline sensitivity. Results Retinal sensitivity declined most in locations without change in SW-AF intensity. Functional decline per year was significantly larger in flecks that disappeared (−0.72 ± 1.30 dB) compared to flecks that appeared (−0.34 ± 0.65 dB), if baseline sensitivity was high (≥10 dB; P < 0.01). The correlation between the change observed on SW-AF and the sensitivity change significantly depended on the sensitivity at baseline (P = 0.000). Conclusions Qualitative longitudinal assessment of SW-AF poorly reflected the retinal sensitivity loss observed over the course of 3 to 5 years. Translational Relevance When aiming to assess treatment effect on lesion level, a multimodal end point including MP focused on hyperautofluorescent lesions appears essential but needs further studies on optimizing MP grids, eye-tracking systems, and alignment software.

Stargardt disease is a progressive retinal disorder caused by bi-allelic mutations in the ABCA4 gene that encodes the ATP-binding cassette, subfamily A, member 4 transporter protein. Over the past few years, we and others have identified several pathogenic variants that reside within the introns of ABCA4, including a recurrent variant in intron 36 (c.5196+1137G>A) of which the pathogenicity so far remained controversial. Detailed clinical characterization of this variant confirmed its pathogenic nature, and classified it as an allele of intermediate severity. Moreover, we discovered several additional ABCA4 variants clustering in intron 36. Several of these variants resulted in aberrant splicing of ABCA4, i.e., the inclusion of pseudoexons, while the splicing defects caused by the recurrent c.5196+1137G>A variant strongly increased upon differentiation of patient-derived induced pluripotent stem cells into retina-like cells. Finally, all splicing defects could be rescued by the administration of antisense oligonucleotides that were designed to specifically block the pseudoexon insertion, including rescue in 3D retinal organoids harboring the c.5196+1137G>A variant. Our data illustrate the importance of intronic variants in ABCA4 and expand the therapeutic possibilities for overcoming splicing defects in Stargardt disease., Graphical Abstract, Khan et al. report on the clinical characterization of patients harboring a recurrent deep-intronic variant in ABCA4 underlying retinal disease and demonstrate that this, and other variants close by, lead to splicing defects that can be rescued by antisense oligonucleotides.

PURPOSE. To assess the occurrence and the disease expression of the common p.Asn1868Ile variant in patients with Stargardt disease (STGD1) harboring known, monoallelic causal ABCA4 variants.METHODS. The coding and noncoding regions of ABCA4 were sequenced in 67 and 63 STGD1 probands respectively, harboring monoallelic ABCA4 variants. In case p.Asn1868Ile was detected, segregation analysis was performed whenever possible. Probands and affected siblings harboring p.Asn1868Ile without additional variants in cis were clinically evaluated retrospectively. Two asymptomatic siblings carrying the same ABCA4 variants as their probands were clinically examined. The penetrance of p.Asn1868Ile was calculated using allele frequency data of ABCA4 variants in non-Finnish European individuals.RESULTS. The p.Asn1868Ile variant was found in cis with known variants in 14/67 probands. In 27/67 probands, we identified p.Asn1868Ile without additional variants in cis, in combination with known, mainly severe ABCA4 variants. In 23/27 probands, the trans configuration was established. Among 27 probands and 6/7 STGD1 siblings carrying p.Asn1868Ile, 42% manifested late-onset disease (> 44 years). We additionally identified four asymptomatic relatives carrying a combination of a severe variant and p.Asn1868Ile; ophthalmologic examination in two persons did not reveal STGD1. Based on ABCA4 allele frequency data, we conservatively estimated the penetrance of p.Asn1868Ile, when present in trans with a severe variant, to be below 5%.CONCLUSIONS. A significant fraction of genetically unexplained STGD1 cases carries p.Asn1868Ile as a second variant. Our findings suggest exceptional differences in disease expression or even nonpenetrance of this ABCA4 variant, pointing toward an important role for genetic or environmental modifiers in STGD1.

PURPOSE: It is unknown which retinal cells are involved in the retina-to-sclera signaling cascade causing myopia. As inherited retinal dystrophies (IRD) are characterized by dysfunction of a single retinal cell type and have a high risk of refractive errors, a study investigating the affected cell type, causal gene, and refractive error in IRDs may provide insight herein. DESIGN: Case-control study. METHODS: STUDY POPULATION: Total of 302 patients with IRD. from 2 ophthalmogenetic centers in the Netherlands. REFERENCE POPULATION: Population-based Rotterdam Study-III and Erasmus Rucphen Family Study (N = 5550). Distributions and mean spherical equivalent (SE) were calculated for main affected cell type and causal gene; and risks of myopia and hyperopia were evaluated using logistic regression. RESULTS: Bipolar cell-related dystrophies were associated with the highest risk of SE high myopia 239.7; odds ratio (OR) mild hyperopia 263.2, both P

Purpose: To describe foveal sparing (FS) in central retinal dystrophies (RD). Methods: Participants for this retrospective study were identified from the retinal dystrophy database of the Department of Ophthalmology at Radboud University Medical Center. FS was defined as an intact foveal structure surrounded by at least 180° of chorioretinal atrophy, and a best-corrected visual acuity (BCVA) of 20/200 Snellen). Eligible eyes were identified using fundus autofluorescence (FAF) images, and FS was confirmed using near-infrared reflectance (NIR) imaging and spectral-domain optical coherence tomography when available. Clinical and demographic data were extracted from medical records. We performed quantification of FS and chorioretinal atrophic areas using semiautomated software on fundus autofluorescence and NIR images. We calculated the chronologic change using eye-wise linear regression. Results: We identified 36 patients (56 eyes) with FS. RDs included: Stargardt disease (STGD1;20 patients), central areolar choroidal dystrophy (CACD; 7 patients), mitochondrial retinal dystrophy (MRD; 6 patients), pseudo-Stargardt pattern dystrophy (PSPD; 3 patients). Median age at first presentation was 60 (interquartile range [IQR] 54-63) years. Median BCVA at first presentation ranged from 20/25 Snellen in STGD1, to 20/38 Snellen in MRD. Progression of the chorioretinal atrophic area ranged from 0.26 (0.25-0.28) mm/year in PSPD, to 0.14 (0.11-0.22) in CACD. Change in FS area over time was similar between the different dystrophies. Conclusions: The presence of FS in different RDs suggests a disease-independent mechanism that prolongs the survival of the fovea. The associated preservation of BCVA is important for the individual prognosis and has implications for the design of therapeutic trials for RDs.

Purpose To investigate intersibling phenotypic concordance in Stargardt disease (STGD1). Design Retrospective cohort study. Participants Siblings with genetically confirmed STGD1 and at least 1 available fundus autofluorescence (FAF) image of both eyes. Methods We compared age at onset within families. Disease duration was matched to investigate differences in best-corrected visual acuity (BCVA) and compared the survival time for reaching severe visual impairment ( 1.0 logarithm of the minimum angle of resolution [logMAR]). Central retinal atrophy area was quantified independently by 2 experienced graders using semiautomated software and compared between siblings. Both graders performed qualitative assessment of FAF and spectral-domain (SD) OCT images to identify phenotypic differences. Main Outcome Measures Differences in age at onset, disease duration-matched BCVA, time to severe visual impairment development, FAF atrophy area, FAF patterns, and genotypes. Results Substantial differences in age at onset were present in 5 of 17 families, ranging from 13 to 39 years. Median BCVA at baseline was 0.60 logMAR (range, –0.20 to 2.30 logMAR; Snellen equivalent, 20/80 [range, 20/12–hand movements]) in the right eye and 0.50 logMAR (range, –0.20 to 2.30 logMAR; Snellen equivalent, 20/63 [range, 20/12–hand movements]) in the left eye. Disease duration-matched BCVA was investigated in 12 of 17 families, and the median difference was 0.41 logMAR (range, 0.00–1.10 logMAR) for the right eye and 0.41 logMAR (range, 0.00–1.08 logMAR) for the left eye. We observed notable differences in time to severe visual impairment development in 7 families, ranging from 1 to 29 years. Median central retinal atrophy area was 11.38 mm2 in the right eye (range, 1.98–44.78 mm2) and 10.59 mm2 in the left eye (range, 1.61–40.59 mm2) and highly comparable between siblings. Similarly, qualitative FAF and SD OCT phenotypes were highly comparable between siblings. Conclusions Phenotypic discordance between siblings with STGD1 carrying the same ABCA4 variants is a prevalent phenomenon. Although the FAF phenotypes are highly comparable between siblings, functional outcomes differ substantially. This complicates both sibling-based prognosis and genotype-phenotype correlations and has important implications for patient care and management.

Item does not contain fulltext PURPOSE: To elucidate the functional effect of the ABCA4 variant c.5461-10T-->C, one of the most frequent variants associated with Stargardt disease (STGD1). DESIGN: Case series. PARTICIPANTS: Seventeen persons with STGD1 carrying ABCA4 variants and 1 control participant. METHODS: Haplotype analysis of 4 homozygotes and 11 heterozygotes for c.5461-10T-->C and sequence analysis of the ABCA4 gene for a homozygous proband. Fibroblasts were reprogrammed from 3 persons with STGD1 into induced pluripotent stem cells, which were differentiated into photoreceptor progenitor cells (PPCs). The effect of the c.5461-10T-->C variant on RNA splicing by reverse-transcription polymerase chain reaction was analyzed using PPC mRNA. In vitro assays were performed with minigene constructs containing ABCA4 exon 39. We analyzed the natural history and ophthalmologic characteristics of 4 persons homozygous for c.5461-10T-->C. MAIN OUTCOME MEASURES: Haplotype and rare variant data for ABCA4, RNA splice defects, age at diagnosis, visual acuity, fundus appearance, visual field, electroretinography (ERG) results, fluorescein angiography results, and fundus autofluorescence findings. RESULTS: The frequent ABCA4 variant c.5461-10T-->C has a subtle effect on splicing based on prediction programs. A founder haplotype containing c.5461-10T-->C was found to span approximately 96 kb of ABCA4 and did not contain other rare sequence variants. Patient-derived PPCs showed skipping of exon 39 or exons 39 and 40 in the mRNA. HEK293T cell transduction with minigenes carrying exon 39 showed that the splice defects were the result of the c.5461-10T-->C variant. All 4 subjects carrying the c.5461-10T-->C variant in a homozygous state showed a young age of STGD1 onset, with low visual acuity at presentation and abnormal cone ERG results. All 4 demonstrated severe cone-rod dystrophy before 20 years of age and were legally blind by 25 years of age. CONCLUSIONS: The ABCA4 variant c.5461-10T-->C is located on a founder haplotype lacking other disease-causing rare sequence variants. In vitro studies revealed that it leads to mRNA exon skipping and ABCA4 protein truncation. Given the severe phenotype in persons homozygous for this variant, we conclude that this variant results in the absence of ABCA4 activity.

Contains fulltext : 190679.pdf (Publisher’s version ) (Open Access) Leber congenital amaurosis (LCA) is a rare inherited retinal disorder affecting approximately 1:50,000 people worldwide. So far, mutations in 25 genes have been associated with LCA, with CEP290 (encoding the Centrosomal protein of 290 kDa) being the most frequently mutated gene. The most recurrent LCA-causing CEP290 mutation, c.2991+1655A>G, causes the insertion of a pseudoexon into a variable proportion of CEP290 transcripts. We previously demonstrated that antisense oligonucleotides (AONs) have a high therapeutic potential for patients homozygously harbouring this mutation, although to date, it is unclear whether rescuing one single allele is enough to restore CEP290 function. Here, we assessed the AON efficacy at RNA, protein and cellular levels in samples that are compound heterozygous for this mutation, together with a protein-truncating mutation in CEP290. We demonstrate that AONs can efficiently restore splicing and increase protein levels. However, due to a high variability in ciliation among the patient-derived cell lines, the efficacy of the AONs was more difficult to assess at the cellular level. This observation points towards the importance of the severity of the second allele and possibly other genetic variants present in each individual. Overall, AONs seem to be a promising tool to treat CEP290-associated LCA, not only in homozygous but also in compound heterozygous carriers of the c.2991+1655A>G variant.

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.

Objective To describe the phenotype and genotype of patients with early-onset Stargardt disease. Design Retrospective cohort study. Participants Fifty-one Stargardt patients with age at onset ≤10 years. Methods We reviewed patient medical records for age at onset, medical history, initial symptoms, best-corrected visual acuity (BCVA), ophthalmoscopy, fundus photography, fundus autofluorescence (FAF), fluorescein angiography (FA), spectral-domain optical coherence tomography (SD-OCT), and full-field electroretinography (ffERG). The ABCA4 gene was screened for mutations. Main Outcome Measures Age at onset, BCVA, fundus appearance, FAF, FA, SD-OCT, ffERG, and presence of ABCA4 mutations. Results The mean age at onset was 7.2 years (range, 1–10). The median times to develop BCVA of 20/32, 20/80, 20/200, and 20/500 were 3, 5, 12, and 23 years, respectively. Initial ophthalmoscopy in 41 patients revealed either no abnormalities or foveal retinal pigment epithelium (RPE) changes in 10 and 9 patients, respectively; the other 22 patients had foveal atrophy, atrophic RPE lesions, and/or irregular yellow-white fundus flecks. On FA, there was a "dark choroid" in 21 out of 29 patients. In 14 out of 50 patients, foveal atrophy occurred before flecks developed. On FAF, there was centrifugal expansion of disseminated atrophic spots, which progressed to the eventual profound chorioretinal atrophy. Spectral-domain OCT revealed early photoreceptor damage followed by atrophy of the outer retina, RPE, and choroid. On ffERG in 26 patients, 15 had normal amplitudes, and 11 had reduced photopic and/or scotopic amplitudes at their first visit. We found no correlation between ffERG abnormalities and the rate of vision loss. Thirteen out of 25 patients had progressive ffERG abnormalities. Finally, genetic screening of 44 patients revealed ≥2 ABCA4 mutations in 37 patients and single heterozygous mutations in 7. Conclusions In early-onset Stargardt, initial ophthalmoscopy can reveal no abnormalities or minor retinal abnormalities. Yellow-white flecks can be preceded by foveal atrophy and may be visible only on FAF. Although ffERG is insufficient for predicting the rate of vision loss, abnormalities can develop. Over time, visual acuity declines rapidly in parallel with progressive retinal degeneration, resulting in profound chorioretinal atrophy. Thus, early-onset Stargardt lies at the severe end of the spectrum of ABCA4 -associated retinal phenotypes.

Item does not contain fulltext Variants in ABCA4 are responsible for autosomal-recessive Stargardt disease and cone-rod dystrophy. Sequence analysis of ABCA4 exons previously revealed one causative variant in each of 45 probands. To identify the "missing" variants in these cases, we performed multiplex ligation-dependent probe amplification-based deletion scanning of ABCA4. In addition, we sequenced the promoter region, fragments containing five deep-intronic splice variants, and 15 deep-intronic regions containing weak splice sites. Heterozygous deletions spanning ABCA4 exon 5 or exons 20-22 were found in two probands, heterozygous deep-intronic variants were identified in six probands, and a deep-intronic variant was found together with an exon 20-22 deletion in one proband. Based on ophthalmologic findings and characteristics of the identified exonic variants present in trans, the deep-intronic variants V1 and V4 were predicted to be relatively mild and severe, respectively. These findings are important for proper genetic counseling and for the development of variant-specific therapies.

Contains fulltext : 138884.pdf (Publisher’s version ) (Open Access) PURPOSE: To provide a clinical and genetic description of a patient cohort with Stargardt disease (STGD1) with identifiable foveal sparing. METHODS: Patients with retinal atrophy (defined as an absence of autofluorescence) that surrounded the fovea by at least 180 degrees and did not include the fovea were defined as having foveal sparing; eyes with visual acuity (VA) worse than 20/200 were excluded. We reviewed the medical files and extracted data regarding medical history, VA, ophthalmoscopy, static perimetry, fundus photography, spectral-domain optical coherence tomography (SD-OCT), fluorescein angiography (FA), fundus autofluorescence (FAF), and electroretinography (ERG). We screened each patient's ABCA4 gene for mutations. RESULTS: Seventeen eyes with foveal sparing were identified in 13 unrelated patients. In 4 eyes, the fovea gradually became atrophic after the initial foveal sparing. The mean age at onset was 51 years (range, 32-67 years). Visual acuity was 20/40 or better in all foveal sparing eyes and was 20/25 or better in 41%. Fundus autofluorescence imaging revealed hyperautofluorescent flecks and parafoveal retinal atrophy; SD-OCT revealed sharply delineated atrophy; and perimetry revealed parafoveal scotomas with intact foveal sensitivity. Finally, genetic screening identified mutations in 19 of the 26 ABCA4 gene alleles. CONCLUSIONS: Foveal sparing occurs mainly in patients with late-onset STGD1 and represents the milder end of the clinical spectrum in STGD1. The anatomy, metabolism, and biochemistry of the retina, as well as genetic variations in genes other than ABCA4, can influence the etiology of foveal sparing. Identifying these fovea-protecting factors will facilitate the future development of strategies designed to treat STGD1.

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+5GA, 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.

Hereditary retinal degenerations encompass a group of genetic diseases characterized by extreme clinical variability. Following next-generation sequencing and autozygome-based screening of patients presenting with a peculiar, recessive form of cone-dominated retinopathy, we identified five homozygous variants [p.(Asp594fs), p.(Gln117*), p.(Met712fs), p.(Ile756Phe), and p.(Glu543Lys)] in the polyglutamylase-encoding gene TTLL5, in eight patients from six families. The two male patients carrying truncating TTLL5 variants also suffered from a substantial reduction in sperm motility and infertility, whereas those carrying missense changes were fertile. Defects in this polyglutamylase in humans have recently been associated with cone photoreceptor dystrophy, while mouse models carrying truncating mutations in the same gene also display reduced fertility in male animals. We examined the expression levels of TTLL5 in various human tissues and determined that this gene has multiple viable isoforms, being highly expressed in testis and retina. In addition, antibodies against TTLL5 stained the basal body of photoreceptor cells in rat and the centrosome of the spermatozoon flagellum in humans, suggesting a common mechanism of action in these two cell types. Taken together, our data indicate that mutations in TTLL5 delineate a novel, allele-specific syndrome causing defects in two as yet pathogenically unrelated functions, reproduction and vision.

Contains fulltext : 153514.pdf (Publisher’s version ) (Open Access) Retinitis pigmentosa (RP), the most common form of inherited retinal degeneration, is clinically and genetically heterogeneous and can appear as syndromic or non-syndromic. Mucopolysaccharidosis type IIIC (MPS IIIC) is a lethal disorder, caused by mutations in the heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) gene and characterized by progressive neurological deterioration, with retinal degeneration as a prominent feature. We identified HGSNAT mutations in six patients with non-syndromic RP. Whole exome sequencing (WES) in an Ashkenazi Jewish Israeli RP patient revealed a novel homozygous HGSNAT variant, c.370A>T, which leads to partial skipping of exon 3. Screening of 66 Ashkenazi RP index cases revealed an additional family with two siblings homozygous for c.370A>T. WES in three Dutch siblings with RP revealed a complex HGSNAT variant, c.[398G>C; 1843G>A] on one allele, and c.1843G>A on the other allele. HGSNAT activity levels in blood leukocytes of patients were reduced compared with healthy controls, but usually higher than those in MPS IIIC patients. All patients were diagnosed with non-syndromic RP and did not exhibit neurological deterioration, or any phenotypic features consistent with MPS IIIC. Furthermore, four of the patients were over 60 years old, exceeding by far the life expectancy of MPS IIIC patients. HGSNAT is highly expressed in the mouse retina, and we hypothesize that the retina requires higher HGSNAT activity to maintain proper function, compared with other tissues associated with MPS IIIC, such as the brain. This report broadens the spectrum of phenotypes associated with HGSNAT mutations and highlights the critical function of HGSNAT in the human retina.

Author(s): Lambertus, Stanley; Bax, Nathalie M; Fakin, Ana; Groenewoud, Joannes MM; Klevering, B Jeroen; Moore, Anthony T; Michaelides, Michel; Webster, Andrew R; van der Wilt, Gert Jan; Hoyng, Carel B | Abstract: BackgroundEach inherited retinal disorder is rare, but together, they affect millions of people worldwide. No treatment is currently available for these blinding diseases, but promising new options-including gene therapy-are emerging. Arguably, the most prevalent retinal dystrophy is Stargardt disease. In each case, the specific combination of ABCA4 variants (g 900 identified to date) and modifying factors is virtually unique. It accounts for the vast phenotypic heterogeneity including variable rates of functional and structural progression, thereby potentially limiting the ability of phase I/II clinical trials to assess efficacy of novel therapies with few patients. To accommodate this problem, we developed and validated a sensitive and reliable composite clinical trial endpoint for disease progression based on structural measurements of retinal degeneration.Methods and findingsWe used longitudinal data from early-onset Stargardt patients from the Netherlands (development cohort, n = 14) and the United Kingdom (external validation cohort, n = 18). The composite endpoint was derived from best-corrected visual acuity, fundus autofluorescence, and spectral-domain optical coherence tomography. Weighting optimization techniques excluded visual acuity from the composite endpoint. After optimization, the endpoint outperformed each univariable outcome, and showed an average progression of 0.41° retinal eccentricity per year (95% confidence interval, 0.30-0.52). Comparing with actual longitudinal values, the model accurately predicted progression (R2, 0.904). These properties were largely preserved in the validation cohort (0.43°/year [0.33-0.53]; prediction: R2, 0.872). We subsequently ran a two-year trial simulation with the composite endpoint, which detected a 25% decrease in disease progression with 80% statistical power using only 14 patients.ConclusionsThese results suggest that a multimodal endpoint, reflecting structural macular changes, provides a sensitive measurement of disease progression in Stargardt disease. It can be very useful in the evaluation of novel therapeutic modalities in rare disorders.

To describe the phenotype and genotype of patients with early-onset Stargardt disease.Retrospective cohort study.Fifty-one Stargardt patients with age at onset ≤10 years.We reviewed patient medical records for age at onset, medical history, initial symptoms, best-corrected visual acuity (BCVA), ophthalmoscopy, fundus photography, fundus autofluorescence (FAF), fluorescein angiography (FA), spectral-domain optical coherence tomography (SD-OCT), and full-field electroretinography (ffERG). The ABCA4 gene was screened for mutations.Age at onset, BCVA, fundus appearance, FAF, FA, SD-OCT, ffERG, and presence of ABCA4 mutations.The mean age at onset was 7.2 years (range, 1-10). The median times to develop BCVA of 20/32, 20/80, 20/200, and 20/500 were 3, 5, 12, and 23 years, respectively. Initial ophthalmoscopy in 41 patients revealed either no abnormalities or foveal retinal pigment epithelium (RPE) changes in 10 and 9 patients, respectively; the other 22 patients had foveal atrophy, atrophic RPE lesions, and/or irregular yellow-white fundus flecks. On FA, there was a "dark choroid" in 21 out of 29 patients. In 14 out of 50 patients, foveal atrophy occurred before flecks developed. On FAF, there was centrifugal expansion of disseminated atrophic spots, which progressed to the eventual profound chorioretinal atrophy. Spectral-domain OCT revealed early photoreceptor damage followed by atrophy of the outer retina, RPE, and choroid. On ffERG in 26 patients, 15 had normal amplitudes, and 11 had reduced photopic and/or scotopic amplitudes at their first visit. We found no correlation between ffERG abnormalities and the rate of vision loss. Thirteen out of 25 patients had progressive ffERG abnormalities. Finally, genetic screening of 44 patients revealed ≥2 ABCA4 mutations in 37 patients and single heterozygous mutations in 7.In early-onset Stargardt, initial ophthalmoscopy can reveal no abnormalities or minor retinal abnormalities. Yellow-white flecks can be preceded by foveal atrophy and may be visible only on FAF. Although ffERG is insufficient for predicting the rate of vision loss, abnormalities can develop. Over time, visual acuity declines rapidly in parallel with progressive retinal degeneration, resulting in profound chorioretinal atrophy. Thus, early-onset Stargardt lies at the severe end of the spectrum of ABCA4-associated retinal phenotypes.

Contains fulltext : 168272.pdf (Publisher’s version ) (Open Access) Purpose: Asymmetry in disease progression between left and right eyes can occur in Stargardt disease (STGD1), and this needs to be considered in novel therapeutic trials with a fellow-eye paired controlled design. This study investigated the inter-eye discordance of best-corrected visual acuity (BCVA) and progression of RPE atrophy in STGD1. Methods: We performed a retrospective cohort study collecting 68 STGD1 patients (136 eyes) with >/=1 ABCA4 variants and >/=0.5-year follow-up on BCVA and fundus autofluorescence. We compared inter-eye correlations of RPE atrophy progression between early-onset (/=45) STGD1 and ABCA4 variant combinations by chi2 tests. We identified associations of discordant baseline BCVA and RPE atrophy with discordant RPE atrophy progression by odds ratios (OR). We defined discordance by differences >1.5 interquartile ranges +/- first/third interquartiles. Results: Progression of RPE atrophy correlated moderately between eyes (rho = 0.766), which decreased with later onset (P = 9.8 x 10-7) and lower pathogenicity of ABCA4 combinations (P = 0.007). Twelve patients (17.6%) had discordant inter-eye RPE atrophy progression, associated with baseline discordance of RPE atrophy (OR, 6.50 [1.35-31.34]), but not BCVA (OR, 0.33 [0.04-2.85]). Conclusions: Lower inter-eye correlations are more likely found in late-onset STGD1 and patients carrying low pathogenic ABCA4 combinations. To achieve the highest power in a therapeutic trial, early-phase studies should minimize inter-eye discordance by selecting early-onset STGD1 patients carrying severe ABCA4 variants without evidence of asymmetry at baseline.