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To analyze the clinical characteristics, natural history, and genetics of CRB1-associated retinal dystrophies.Multicenter international retrospective cohort study.Review of clinical notes, ophthalmic images, and genetic testing results of 104 patients (91 probands) with disease-causing CRB1 variants. Macular optical coherence tomography (OCT) parameters, visual function, fundus characteristics, and associations between variables were the main outcome measures.The mean age of the cohort at the first visit was 19.8 ± 16.1 (median 15) years, with a mean follow-up of 9.6 ± 10 years. Based on history, imaging, and clinical examination, 26 individuals were diagnosed with retinitis pigmentosa (RP; 25%), 54 with early-onset severe retinal dystrophy / Leber congenital amaurosis (EOSRD/LCA; 52%), and 24 with macular dystrophy (MD; 23%). Severe visual impairment was most frequent after 40 years of age for patients with RP and after 20 years of age for EOSRD/LCA. Longitudinal analysis revealed a significant difference between baseline and follow-up best-corrected visual acuity in the 3 subcohorts. Macular thickness decreased in most patients with EOSRD/LCA and MD, whereas the majority of patients with RP had increased perifoveal thickness.A subset of individuals with CRB1 variants present with mild, adult-onset RP. EOSRD/LCA phenotype was significantly associated with null variants, and 167_169 deletion was exclusively present in the MD cohort. The poor OCT lamination may have a degenerative component, as well as being congenital. Disease symmetry and reasonable window for intervention highlight CRB1 retinal dystrophies as a promising target for trials of novel therapeutics.

Contains fulltext : 251966.pdf (Publisher’s version ) (Open Access) 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.

Background Retinitis pigmentosa (RP) is a heterogeneous group of inherited ocular diseases that result in progressive retinal degeneration. This study aims to describe different Swept-source Optical Coherence Tomographic (SS-OCT) changes in Palestinian RP patients and to explore possible correlations with Visual Acuity (VA). Methods A cross-sectional observational study was conducted on Retinitis Pigmentosa patients diagnosed with RP in a tertiary eye hospital. Full history and ocular examination were made. SS-OCT imaging was done for all eyes assessing the presence of cystoid macular edema, epiretinal membrane, macular holes, and external limiting membrane, ellipsoid zone status. Also, central macular thickness and choroidal vascular thickness were measured. Results The study was run on 161 eyes of 81 patients; 53 males and 28 females. The average age at examination was 26.1 (6–78) years. Twenty-six eyes (16.1%) were of syndromic RP patients, mostly Usher syndrome; 20 eyes (12.4%). The mean Logaritmic minimal angle of resolution (LogMAR) of Best Corrected Visual Acuity (BCVA)of the study sample was 0.66 ± 0.7. The most prevalent change was cystoid macular edema [28 eyes, (17.4%)], followed by epiretinal membrane [17eye, (10.6%)]. A macular hole was noted only in one eye (0.6%). Ellipsoid zone and external limiting membrane were absent in 55 eyes (35.0%) and 60 eyes 37.5%. Vitreous hyperreflective foci were found in 35 eyes (43.8%). LogMAR of BCVA was associated significantly with cystoid macular edema (p = 0.001), ellipsoid zone(p = 0.001), and external limiting membrane (p = 0.001). Conclusions Detailed SS-OCT assessment in Palestinian patients diagnosed with RP identified different morphologies from other populations. Cystoid macular edema and vitreous hyperreflective foci may reflect signs of early or intermediate stages of the disease. Disease progression can be monitored by measuring the length/width (area) of ellipsoid zone +/− external limiting membrane and choroidal vascular thickness, which should be evaluated serially using high-resolution OCT.

Purpose: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. Methods: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. Results: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. Conclusion: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases. Fil: Khan, Mubeen. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Cornelis, Stéphanie S.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Del Pozo Valero, Marta. Hospital Universitario Fundación Jiménez Díaz; España. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Whelan, Laura. Trinity College; Estados Unidos Fil: Runhart, Esmee H.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Mishra, Ketan. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Bults, Femke. Radboud University Nijmegen Medical Centre; Países Bajos Fil: AlSwaiti, Yahya. St John of Jerusalem Eye Hospital Group; Palestina (ANP) Fil: AlTalbishi, Alaa. St John of Jerusalem Eye Hospital Group; Palestina (ANP) Fil: De Baere, Elfride. University of Ghent; Bélgica Fil: Banfi, Sandro. Seconda Universita Degli Studi Di Napoli; Italia Fil: Banin, Eyal. The Hebrew University of Jerusalem; Israel Fil: Bauwens, Miriam. University of Ghent; Bélgica Fil: Ben Yosef, Tamar. The Ruth And Bruce Rappaport Faculty Of Medicine; Israel Fil: Boon, Camiel J. F.. Leiden University. Leiden University Medical Center; Países Bajos Fil: van den Born, L. Ingeborgh. Rotterdam Ophthalmic Institute; Países Bajos Fil: Defoort, Sabine. Universite Lille; Francia Fil: Devos, Aurore. Universite Lille; Francia Fil: Dockery, Adrian. Trinity College; Estados Unidos Fil: Dudakova, Lubica. Charles University and General University Hospital; República Checa Fil: Fakin, Ana. Charles University and General University Hospital; República Checa Fil: Farrar, G. Jane. Trinity College; Estados Unidos Fil: Ferraz Sallum, Juliana Maria. Universidade Federal de Sao Paulo; Brasil Fil: Fujinami, Kaoru. UCL Institute of Ophthalmology; Reino Unido Fil: Gilissen, Christian. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Glavac, Damjan. University of Ljubljana; Eslovenia Fil: Gorin, Michael B.. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: Greenberg, Jacquie. University of Cape Town; Sudáfrica Fil: Hayashi, Takaaki. The Jikei University School of Medicine; Japón Fil: Hettinga, Ymkje M.. Bartiméus Diagnostic Center for Complex Visual Disorders; Países Bajos Fil: Hoischen, Alexander. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Hoyng, Carel B.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Hufendiek, Karsten. University Eye Hospital Hannover Medical School; Alemania Fil: Jägle, Herbert. University Regensburg; Alemania Fil: Kamakari, Smaragda. OMMA Ophthalmological Institute of Athens; Grecia Fil: Karali, Marianthi. Seconda Universita Degli Studi Di Napoli; Italia Fil: Kellner, Ulrich. No especifíca; Fil: Klaver, Caroline C. W.. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Kousal, Bohdan. Charles University and General University Hospital; República Checa Fil: Lamey, Tina M.. University of Western Australia; Australia Fil: MacDonald, Ian M.. University of Alberta; Canadá Fil: Matynia, Anna. University of California at Los Angeles. School of Medicine; Estados Unidos Fil: McLaren, Terri L.. University of Western Australia; Australia Fil: Mena, Marcela D.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Meunier, Isabelle. Université Montpellier II; Francia Fil: Miller, Rianne. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Newman, Hadas. Universitat Tel Aviv; Israel Fil: Ntozini, Buhle. University of Cape Town; Sudáfrica Fil: Oldak, Monika. No especifíca; Fil: Pieterse, Marc. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Podhajcer, Osvaldo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Puech, Bernard. Universite Lille; Francia Fil: Ramesar, Raj. University of Cape Town; Sudáfrica Fil: Rüther, Klaus. No especifíca; Fil: Salameh, Manar. No especifíca; Fil: Salles, Mariana Vallim. Universidade de Sao Paulo; Brasil Fil: Sharon, Dror. The Hebrew University of Jerusalem; Israel Fil: Simonelli, Francesca. Seconda Universita Degli Studi Di Napoli; Italia Fil: Spital, Georg. No especifíca; Fil: Steehouwer, Marloes. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Szaflik, Jacek P.. No especifíca; Fil: Thompson, Jennifer A.. No especifíca; Fil: Thuillier, Caroline. Universite Lille; Francia Fil: Tracewska, Anna M.. No especifíca; Fil: van Zweeden, Martine. Radboud University Nijmegen Medical Centre; Países Bajos Fil: Vincent, Andrea L.. University of Auckland; Nueva Zelanda Fil: Zanlonghi, Xavier. No especifíca; Fil: Liskova, Petra. Charles University and General University Hospital; República Checa Fil: Stöhr, Heidi. Universitat Regensburg; Alemania Fil: De Roach, John N.. University of Western Australia; Australia Fil: Ayuso, Carmen. Hospital Universitario Fundación Jiménez Díaz; España Fil: Roberts, Lisa. University of Cape Town; Sudáfrica Fil: Weber, Bernhard H. F.. Universitat Regensburg; Alemania Fil: Dhaenens, Claire Marie. Universite Lille; Francia Fil: Cremers, Frans P. M.. Radboud University Nijmegen Medical Centre; Países Bajos

Missing heritability in human diseases represents a major challenge. Although whole-genome sequencing enables the analysis of coding and non-coding sequences, substantial costs and data storage requirements hamper its large-scale use to (re)sequence genes in genetically unsolved cases. The ABCA4 gene implicated in Stargardt disease (STGD1) has been studied extensively for 22 years, but thousands of cases remained unsolved. Therefore, single molecule molecular inversion probes were designed that enabled an automated and cost-effective sequence analysis of the complete 128-kb ABCA4 gene. Analysis of 1,054 unsolved STGD and STGD-like probands resulted in bi-allelic variations in 448 probands. Twenty-seven different causal deep-intronic variants were identified in 117 alleles. Based on in vitro splice assays, the 13 novel causal deep-intronic variants were found to result in pseudo-exon (PE) insertions (n=10) or exon elongations (n=3). Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions that were accompanied by flanking exon deletions. Structural variant analysis revealed 11 distinct deletions, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. Integrated complete gene sequencing combined with transcript analysis, identified pathogenic deep-intronic and structural variants in 26% of bi-allelic cases not solved previously by sequencing of coding regions. This strategy serves as a model study that can be applied to other inherited diseases in which only one or a few genes are involved in the majority of cases.

Purpose Usher syndrome (USH) is the most common cause for deaf-blindness. It is genetically and clinically heterogeneous and prevalent in populations with high consanguinity rate. We aim to characterize the set of genes and mutations that cause USH in the Israeli and Palestinian populations. Methods Seventy-four families with USH were recruited (23 with USH type 1 [USH1], 33 with USH2, seven with USH3, four with atypical USH, and seven families with an undetermined USH type). All affected subjects underwent a full ocular evaluation. A comprehensive genetic analysis, including Sanger sequencing for the detection of founder mutations, homozygosity mapping, and whole exome sequencing in large families was performed. Results In 79% of the families (59 out of 74), an autosomal recessive inheritance pattern could be determined. Mutation detection analysis led to the identification of biallelic causative mutations in 51 (69%) of the families, including 21 families with mutations in USH2A, 17 in MYO7A, and seven in CLRN1. Our analysis revealed 28 mutations, 11 of which are novel (including c.802G>A, c.8558+1G>T, c.10211del, and c.14023A>T in USH2A; c.285+2T>G, c.2187+1G>T, c.3892G>A, c.5069_5070insC, c.5101C>T, and c.6196C>T in MYO7A; and c.15494del in GPR98). Conclusions We report here novel homozygous mutations in various genes causing USH, extending the spectrum of causative mutations. We also prove combined sequencing techniques as useful tools to identify novel disease-causing mutations. To the best of our knowledge, this is the largest report of a genetic analysis of Israeli and Palestinian families (n = 74) with different USH subtypes.