Late-onset Stargardt disease is a subtype of Stargardt disease type 1 (STGD1), defined by an age of onset of 45 years or older. We describe the disease characteristics, underlying genetics, and disease progression of late-onset STGD1 and highlight the differences from geographic atrophy. Retrospective cohort study. Seventy-one patients with late-onset STGD1. Medical files were reviewed for clinical data including age at onset, initial symptoms, and best-corrected visual acuity. A quantitative and qualitative assessment of retinal pigment epithelium (RPE) atrophy was performed on fundus autofluorescence images and OCT scans. Age at onset, genotype, visual acuity, atrophy growth rates, and loss of external limiting membrane, ellipsoid zone, and RPE. Median age at onset was 55.0 years (range, 45-82 years). A combination of a mild and severe variant in ATP-binding cassette subfamily A member 4 (ABCA4) was the most common genotype (n = 49 [69.0%]). The most frequent allele, c.5603A→T (p.Asn1868Ile), was present in 43 of 71 patients (60.6%). No combination of 2 severe variants was found. At first presentation, all patients have flecks. Foveal-sparing atrophy was present in 33.3% of eyes, whereas 21.1% had atrophy with foveal involvement. Extrafoveal atrophy was present in 38.9% of eyes, and no atrophy was evident in 6.7% of eyes. Time-to-event curves showed a median duration of 15.4 years (95% confidence interval, 11.1-19.6 years) from onset to foveal involvement. The median visual acuity decline was -0.03 Snellen decimal per year (interquartile range [IQR], -0.07 to 0.00 Snellen decimal; 0.03 logarithm of the minimum angle of resolution). Median atrophy growth was 0.590 mm²/year (IQR, 0.046-1.641 mm²/year) for definitely decreased autofluorescence and 0.650 mm²/year (IQR, 0.299-1.729 mm²/year) for total decreased autofluorescence. Late-onset STGD1 is a subtype of STGD1 with most commonly 1 severe and 1 mild ABCA4 variant. The general patient presents with typical fundus flecks and retinal atrophy in a foveal-sparing pattern with preserved central vision. Misdiagnosis as age-related macular degeneration should be avoided to prevent futile invasive treatments with potential complications. In addition, correct diagnosis lends patients with late-onset STGD1 the opportunity to participate in potentially beneficial therapeutic trials for STGD1. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

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.

The U4 small nuclear RNA (snRNA) forms a duplex with the U6 snRNA and, together with U5 and ∼30 proteins, is part of the U4/U6.U5 tri-snRNP complex, located at the core of the major spliceosome. Recently, recurrent de novo variants in the U4 RNA, transcribed from the RNU4-2 gene, and in at least two other RNU genes were discovered to cause neurodevelopmental disorder. We detected inherited and de novo heterozygous variants in RNU4-2 (n.18_19insA and n.56T>C) and in four out of the five RNU6 paralogues (n.55_56insG and n.56_57insG) in 135 individuals from 62 families with non-syndromic retinitis pigmentosa (RP), a rare form of hereditary blindness. We show that these variants are recurrent among RP families and invariably cluster in close proximity within the three-way junction (between stem-I, the 5’ stem-loop and stem-II) of the U4/U6 duplex, affecting its natural conformation. Interestingly, this region binds to numerous splicing factors of the tri-snRNP complex including PRPF3, PRPF8 and PRPF31, previously associated with RP as well. The U4 and U6 variants identified seem to affect snRNP biogenesis, namely the U4/U6 di-snRNP, which is an assembly intermediate of the tri-snRNP. Based on the number of positive cases observed, deleterious variants in RNU4-2 and in RNU6 paralogues could be a significant cause of isolated or dominant RP, accounting for up to 1.2% of all undiagnosed RP cases. This study highlights the role of non-coding genes in rare Mendelian disorders and uncovers pleiotropy in RNU4-2 , where different variants underlie neurodevelopmental disorder and RP.

Missense variants in ABCA4 constitute ~50% of causal variants in Stargardt disease (STGD1). Their pathogenicity is attributed to their direct effect on protein function, whilst their potential impact on pre-mRNA splicing disruption remains poorly understood. Interestingly, synonymous ABCA4 variants have previously been classified as ‘severe’ variants based on in silico analyses. Here, we systemically investigated the role of synonymous and missense variants in ABCA4 splicing by combining computational predictions and experimental assays. To identify variants of interest, we used SpliceAI to ascribe defective splice predictions on a dataset of 5579 biallelic STGD1 probands. We selected those variants with predicted delta scores for acceptor/donor gain > 0.20, and no previous reports on their effect on splicing. Fifteen ABCA4 variants were selected, 4 of which were predicted to create a new splice acceptor site and 11 to create a new splice donor site. In addition, three variants of interest with delta scores < 0.20 were included. The variants were introduced in wild-type midigenes that contained 4–12 kb of ABCA4 genomic sequence, which were subsequently expressed in HEK293T cells. By using RT-PCR and Sanger sequencing, we identified splice aberrations for 16 of 18 analyzed variants. SpliceAI correctly predicted the outcomes for 15 out of 18 variants, illustrating its reliability in predicting the impact of coding ABCA4 variants on splicing. Our findings highlight a causal role for coding ABCA4 variants in splicing aberrations, improving the severity assessment of missense and synonymous ABCA4 variants, and guiding to new treatment strategies for STGD1.

Purpose: The effect of noncoding variants is often unknown in the absence of functional assays. Here, we characterized an ABCA4 intron 7 variant, c.859-25A>G, identified in Palestinian probands with Stargardt disease (STGD) or cone-rod dystrophy (CRD). We investigated the effect of this variant on the ABCA4 mRNA and retinal phenotype, and its prevalence in Palestine. Methods: The ABCA4 gene was sequenced completely or partially in 1998 cases with STGD or CRD. The effect of c.859-25A>G on splicing was investigated in silico using SpliceAI and in vitro using splice assays. Homozygosity mapping was performed for 16 affected individuals homozygous for c.859-25A>G. The clinical phenotype was assessed using functional and structural analyses including visual acuity, full-field electroretinography, and multimodal imaging. Results: The smMIPs-based ABCA4 sequencing revealed c.859-25A>G in 10 Palestinian probands from Hebron and Jerusalem. SpliceAI predicted a significant effect of this putative branchpoint-inactivating variant on the nearby intron 7 splice acceptor site. Splice assays revealed exon 8 skipping and two partial inclusions of intron 7, each having a deleterious effect. Additional genotyping revealed another 46 affected homozygous or compound heterozygous individuals carrying variant c.859-25A>G. Homozygotes shared a genomic segment of 59.6 to 87.9 kb and showed severe retinal defects on ophthalmoscopic evaluation. Conclusions: The ABCA4 variant c.859-25A>G disrupts a predicted branchpoint, resulting in protein truncation because of different splice defects, and is associated with early-onset STGD1 when present in homozygosity. This variant was found in 25/525 Palestinian inherited retinal dystrophy probands, representing one of the most frequent inherited retinal disease-causing variants in West-Bank Palestine.

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.