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Background/Objectives: Stargardt disease (STGD1) is an autosomal recessive disorder caused by pathogenic variants in ABCA4 that affects the retina and is characterised by progressive central vision loss. The onset of disease manifestations varies from childhood to early adulthood. Methods: Whole exome (WES), whole gene, and whole genome sequencing (WGS) were performed for a patient with STGD1. Results: WES revealed a heterozygous pathogenic missense variant in ABCA4, but no second pathogenic variant was found. ABCA4 whole-gene sequencing, subsequent WGS, and segregation analysis identified a complex deep-intronic allele (NM_000350.2(ABCA4):c.[1555-5882C>A;1555-5784C>G]) in trans to the missense variant. Minigene assays combined with nanopore sequencing were performed to characterise this deep-intronic complex allele in more detail. Surprisingly, the reference minigene revealed the existence of two pseudoexons in intron 11 of the ABCA4 gene that are included in low-abundance (<1%) transcripts. Both pseudoexons could be confirmed in cDNA derived from wildtype retinal organoids. Despite mild splicing predictions, the variant minigene revealed that the complex deep-intronic allele substantially increased the abundance of transcripts that included the pseudoexon overlapping with the variants. Two antisense oligonucleotides (AONs) were designed to rescue the aberrant splicing events. Both AONs increased the proportion of correctly spliced transcripts, and one of them rescued correct splicing to reference levels. Conclusions: Minigene assays combined with nanopore sequencing proved instrumental in identifying low-abundance transcripts including pseudoexons from wildtype ABCA4 intron 11, one of which was substantially increased by the complex allele. [ABSTRACT FROM AUTHOR]

Background/Objectives: Neural retina leucine zipper (NRL) is a transcription factor involved in the differentiation of rod photoreceptors. Pathogenic variants in the gene encoding NRL have been associated with autosomal dominant retinitis pigmentosa and autosomal recessive clumped pigmentary retinal degeneration. Only a dozen unrelated families affected by recessive NRL-related retinal dystrophy have been described. The purpose of this study was to expand the genotypic spectrum of this disease by reporting clinical and genetic findings of two unrelated families. Methods: Index patients affected by retinal dystrophy were genetically tested by whole-exome sequencing (WES) and whole-genome sequencing (WGS). Segregation analysis within the families was performed for candidate variants. A minigene assay was performed to functionally characterize a variant suspected to affect splicing. Results: Variant filtering revealed homozygous NRL variants in both families. The variant in patient A was a small deletion encompassing the donor splice site of exon 1 of transcript NM_006177.3. The minigene assay revealed that this variant led to two aberrant transcripts that used alternative cryptic donor splice sites located in intron 1. In patient B, a stop-gain variant was identified in the last exon of NRL in a homozygous state due to maternal uniparental disomy of chromosome 14. Conclusions: Our study expands the genotypic spectrum of autosomal recessive NRL-related retinal dystrophy. Moreover, it underscores the importance of actively maintaining bioinformatic pipelines for variant detection and the utility of minigene assays in functionally characterizing candidate splicing variants. [ABSTRACT FROM AUTHOR]

The contribution of splicing variants to molecular diagnostics of inherited diseases is reported to be less than 10%. This figure is likely an underestimation due to several factors including difficulty in predicting the effect of such variants, the need for functional assays, and the inability to detect them (depending on their locations and the sequencing technology used). The aim of this study was to assess the utility of Nanopore sequencing in characterizing and quantifying aberrant splicing events. For this purpose, we selected 19 candidate splicing variants that were identified in patients affected by inherited retinal dystrophies. Several in silico tools were deployed to predict the nature and estimate the magnitude of variant-induced aberrant splicing events. Minigene assay or whole blood-derived cDNA was used to functionally characterize the variants. PCR amplification of minigene-specific cDNA or the target gene in blood cDNA, combined with Nanopore sequencing, was used to identify the resulting transcripts. Thirteen out of nineteen variants caused aberrant splicing events, including cryptic splice site activation, exon skipping, pseudoexon inclusion, or a combination of these. Nanopore sequencing allowed for the identification of full-length transcripts and their precise quantification, which were often in accord with in silico predictions. The method detected reliably low-abundant transcripts, which would not be detected by conventional strategies, such as RT-PCR followed by Sanger sequencing. [ABSTRACT FROM AUTHOR]

The proneural transcription factor atonal basic helix–loop–helix transcription factor 7 (ATOH7) is expressed in early progenitors in the developing neuroretina. In vertebrates, this is crucial for the development of retinal ganglion cells (RGCs), as mutant animals show an almost complete absence of RGCs, underdeveloped optic nerves, and aberrations in retinal vessel development. Human mutations are rare and result in autosomal recessive optic nerve hypoplasia (ONH) or severe vascular changes, diagnosed as autosomal recessive persistent hyperplasia of the primary vitreous (PHPVAR). To better understand the role of ATOH7 in neuroretinal development, we created ATOH7 knockout and eGFP-expressing ATOH7 reporter human induced pluripotent stem cells (hiPSCs), which were differentiated into early-stage retinal organoids. Target loci regulated by ATOH7 were identified by Cleavage Under Targets and Release Using Nuclease with sequencing (CUT&RUN-seq) and differential expression by RNA sequencing (RNA-seq) of wildtype and mutant organoid-derived reporter cells. Additionally, single-cell RNA sequencing (scRNA-seq) was performed on whole organoids to identify cell type-specific genes. Mutant organoids displayed substantial deficiency in axon sprouting, reduction in RGCs, and an increase in other cell types. We identified 469 differentially expressed target genes, with an overrepresentation of genes belonging to axon development/guidance and Notch signaling. Taken together, we consolidate the function of human ATOH7 in guiding progenitor competence by inducing RGC-specific genes while inhibiting other cell fates. Furthermore, we highlight candidate genes responsible for ATOH7-associated optic nerve and retinovascular anomalies, which sheds light to potential future therapy targets for related disorders. [ABSTRACT FROM AUTHOR]

The purpose of this study was to assess the added diagnostic value of whole genome sequencing (WGS) for patients with inherited retinal diseases (IRDs) who remained undiagnosed after whole exome sequencing (WES). WGS was performed for index patients in 66 families. The datasets were analyzed according to GATK's guidelines. Additionally, DeepVariant was complemented by GATK's workflow, and a novel structural variant pipeline was developed. Overall, a molecular diagnosis was established in 19/66 (28.8%) index patients. Pathogenic deletions and one deep-intronic variant contributed to the diagnostic yield in 4/19 and 1/19 index patients, respectively. The remaining diagnoses (14/19) were attributed to exonic variants that were missed during WES analysis due to bioinformatic limitations, newly described loci, or unclear pathogenicity. The added diagnostic value of WGS equals 5/66 (9.6%) for our cohort, which is comparable to previous studies. This figure would decrease further to 1/66 (1.5%) with a standardized and reliable copy number variant workflow during WES analysis. Given the higher costs and limited added value, the implementation of WGS as a first-tier assay for inherited eye disorders in a diagnostic laboratory remains untimely. Instead, progress in bioinformatic tools and communication between diagnostic and clinical teams have the potential to ameliorate diagnostic yields. [ABSTRACT FROM AUTHOR]

Mutation analyses are performed in 34 patients who received an initial diagnosis of night blindness, and, in two affected siblings, a homozygous nucleotide substitution in CACNA2D4 is detected. A premature stop codon that truncates one-third of the corresponding open reading frame is introduced by mutation, which defines a novel gene defect that causes autosomal recessive cone dystrophy.

Purpose: To (i) describe a series of patients with isolated or syndromic nanophthalmos with the underlying genetic causes, including novel pathogenic variants and their functional characterization and (ii) to study the association of retinal dystrophy in patients with MFRP variants, based on a detailed literature review of genotype–phenotype correlations. Methods: Patients with nanophthalmos and available family members received a comprehensive ophthalmological examination. Genetic analysis was based on whole‐exome sequencing and variant calling in core genes including MFRP, BEST1, TMEM98, PRSS56, CRB1, GJA1, C1QTNF5, MYRF and FAM111A. A minigene assay was performed for functional characterization of a splice site variant. Results: Seven patients, aged between three and 65 years, from five unrelated families were included. Novel pathogenic variants in MFRP (c.497C>T, c.899‐3C>A, c.1180G>A), and PRSS56 (c.1202C>A), and a recurrent de novo variant in FAM111A (c.1706G>A) in a patient with Kenny–Caffey syndrome type 2, were identified. In addition, we report co‐inheritance of MFRP‐related nanophthalmos and ADAR‐related Aicardi–Goutières syndrome. Conclusion: Nanophthalmos is a genetically heterogeneous condition, and the severity of ocular manifestations appears not to correlate with variants in a specific gene. However, retinal dystrophy is only observed in patients harbouring pathogenic MFRP variants. Furthermore, heterozygous carriers of MFRP and PRSS56 should be screened for the presence of high hyperopia. Identifying nanophthalmos as an isolated condition or as part of a syndrome has implications for counselling and can accelerate the interdisciplinary care of patients. [ABSTRACT FROM AUTHOR]

PURPOSE: To identify possible correlations betweenthe putative mutations and the clinical characteristics in X-linked retinitispigmentosa, RP2. DESIGN: A retrospective, descriptive clinical study. MATERIAL: The ophthalmological files on affected persons from three Danishfamilies with identified pathogenic mutations in the RP2 gene. RESULTS: Mutation analysis in 14 Danish families with X-linked retinitispigmentosa revealed disease-associated sequence alterations in eight of them.Five mutations were detected in the RP3 gene ( RPGR ) and three inthe RP2 gene. Genotype-phenotype comparison in the three RP2 familiesrevealed striking interfamilial phenotypic differences. Severe phenotypeswere associated with a null mutation Gln26stop and a missense mutation Arg118His.These families differed mutually with respect to retinal appearance. Affectedcarriers had a delayed onset by three decades. Tapetal reflexes were not observedin the carriers. An in-frame deletion ΔSer6 was associated with a milderphenotype. CONCLUSIONS: Interfamilial differences in RP2 phenotype might be relatedto the type and location of the mutational event. Due to a considerable overlapbetween RP2 and RP3 phenotypes, the genotype cannot safely be deduced fromconventional clinical examination methods. [ABSTRACT FROM AUTHOR]

X-linked retinitis pigmentosa (XLRP) is a genetically heterogeneous group of progressive retinal degenerations. The disease process is initiated by premature apoptosis of rod photoreceptor cells in the retina, which leads to reduced visual acuity and, eventually, complete blindness. Mutations in the retinitis pigmentosa GTPase regulator (RPGR), a ubiquitously expressed gene at the RP3 locus in Xp21.1, account for ~20% of all X-linked cases. We have analysed the expression of this gene by northern blot hybridization, cDNA library screening and RT-PCR in various organs from mouse and man. These studies revealed at least 12 alternatively spliced isoforms. Some of the transcripts are tissue specific and contain novel exons, which elongate or truncate the previously reported open reading frame of the mouse and human RPGR gene. One of the newly identified exons is expressed exclusively in the human retina and mouse eye and contains a premature stop codon. The deduced polypeptide lacks 169 amino acids from the C-terminus of the ubiquitously expressed variant, including an isoprenylation site. Moreover, this exon was found to be deleted in a family with XLRP. Our results indicate tissue-dependent regulation of alternative splicing of RPGR in mouse and man. The discovery of a retina-specific transcript may explain why phenotypic abberations in RP3 are confined to the eye. [ABSTRACT FROM AUTHOR]

PURPOSE: To describe the phenotypic variabilityin a Polish Norrie disease (ND) family associated with the missense mutationA63D. METHODS: A patient with spared vision from a Polish ND family underwentdetailed ophthalmological examinations including slit-lamp biomicroscopy,ultrasound (USG), angiography, Goldmann kinetic visual field, and electroretinography(ERG). Mutation screening was carried out using the single-strand conformationpolymorphism (SSCP) technique and subsequent DNA sequencing of the codingpart of the ND gene. RESULTS: A mutation was detected (exon 3, A63D) in alarge Polish family with 12 affected males, all but one presenting with classicalND symptoms. In one male, partially preserved vision was observed up to 40years of age (distance acuity of the right eye 1/50 and left eye 2/50). Slit-lampexamination revealed remnants of a persistent primary vitreous and hyaloidartery. Upon angiography, the retina was vascularized within the posteriorpole but not in the periphery. The ERG revealed pathological changes characteristicfor chorioretinal degenerations. CONCLUSION: Within one family, individualswith identical sequence alterations in the ND gene can show remarkable phenotypicvariablity of the ocular symptoms. These findings indicate the involvementof additional factors (epigenetic or genetic) in ocular pathogenesis of ND. [ABSTRACT FROM AUTHOR]

Optic nerve hypoplasia (ONH) is a congenital optic nerve abnormality caused by underdevelopment of retinal ganglion cells (RGCs). Despite being a rare disease, ONH is the most common optic disk anomaly in ophthalmological practice. So far, mutations in several genes have been identified as causative; however, many cases of ONH remain without a molecular explanation. The early transcription factor atonal basic-helix-loop-helix (bHLH) transcription factor 7 (ATOH7) is expressed in retinal progenitor cells and has a crucial role in RGC development. Previous studies have identified several mutations in the ATOH7 locus in cases of eye developmental diseases such as non-syndromic congenital retinal non-attachment and persistent hyperplasia of the primary vitreous. Here we present two siblings with a phenotype predominated by bilateral ONH, with additional features of foveal hypoplasia and distinct vascular abnormalities, where whole-exome sequencing identified two compound heterozygous missense mutations affecting a conserved amino acid residue within the bHLH domain of ATOH7 (NM&#95;145178.3:c.175G>A; p.(Ala59Thr) and c.176C>T; p.(Ala59Val)). ATOH7 expression constructs with patient single nucleotide variants were cloned for functional characterization. Protein analyses revealed decreased protein amounts and significantly enhanced degradation in the presence of E47, a putative bHLH dimerization partner. Protein interaction assays revealed decreased heterodimerization and DNA-binding of ATOH7 variants, resulting in total loss of transcriptional activation of luciferase reporter gene expression. These findings strongly support pathogenicity of the two ATOH7 mutations, one of which is novel. Additionally, this report highlights the possible impact of altered ATOH7 dimerization on protein stability and function., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Purpose: To establish mouse models for RPGR-associated diseases by generating and characterizing an Rpgr mutation (in-frame deletion of exon 4) in two different genetic backgrounds (BL/6 and BALB/c)., Methods: Gene targeting in embryonic stem (ES) cells was performed to introduce a in-frame deletion of exon 4 in the Rpgr gene (Rpgr(DeltaEx4)). Subsequently, the mutation was introduced in two different inbred mouse strains by successive breeding. Mutant and wild-type mice of both strains were characterized by electroretinography (ERG) and histology at five time points (1, 3, 6, 9, and 12 months). RPGR transcript amounts were assessed by quantitative RT-PCR. A variety of photoreceptor proteins, including RPGR-ORF15, RPGRIP, PDE6delta/PrBPdelta, rhodopsin, and cone opsin, were localized on retinal sections by immunohistochemistry., Results: Mislocalization of rhodopsin and cone opsin was an early pathologic event in mutant mice of both lines. In contrast, RPGR-ORF15 as well as RPGRIP1 and PDE6delta/PrBPdelta showed similar localizations in mutant and wild-type animals. Functional and histologic studies revealed a mild rod-dominated phenotype in mutant male mice on the BL/6 background, whereas a cone-dominated phenotype was observed for the same mutation in the BALB/c background., Conclusions: Both Rpgr mutant mouse lines developed retinal disease with a striking effect of the genetic background. Cone-specific modifiers might influence the retinal phenotype in the BALB/c strain. The two lines provide models to study RPGR function in rods and cones, respectively.