Induced pluripotent stem cells (iPSCs), reprogrammed from human somatic cells, hold the capacity to differentiate into most human body cells. iPSCs can be differentiated into retinal organoids, a ...three-dimensional structured retina containing various retinal cells. Patient-specific retinal organoids provide a powerful disease model to recapitulate the disease to study the pathogenesis of inherited retinal dystrophies, to screen or discover new drugs, and most importantly to supply an unlimited cell source for retinal regeneration.
To report a case of bilateral chorioretinal scarring due to CLN3 heterozygous deletion in an asymptomatic patient.
A 63 year-old patient with a history of well-controlled diabetes presented as a ...referral for diabetic retinopathy. He was asymptomatic with 20/20 visual acuity in both eyes. Exam revealed bilateral multifocal chorioretinal scarring left worse than right, sparing the fovea. He was unable to provide a family history due to adoption, and his remaining medical history and review of systems were noncontributory. Inflammatory and infectious workup was negative; however, genetic testing revealed heterozygous deletion of CLN3 exons 8 and 9. His disease has been nonprogressive at all follow-up appointments.
Mutations of CLN3 can present with retina-specific findings including bull's-eye maculopathy and electroretinogram (ERG) deficits; to our knowledge this patient's presentation is unique among those with CLN3 mutations.
Inherited retinal dystrophies (IRDs) are a group of clinically and genetically heterogeneous diseases involving more than 280 genes and no less than 20 different clinical phenotypes. In this study, ...our aims were to identify the disease-causing gene variants of 319 Chinese patients with IRD, and compare the pros and cons of targeted panel sequencing and whole exome sequencing (WES). Patients were assigned for analysis with a hereditary eye disease enrichment panel (HEDEP) or WES examination based on time of recruitment. This HEDEP was able to capture 441 hereditary eye disease genes, which included 291 genes related to IRD. As
ORF15 was difficult to capture, all samples were subjected to Sanger sequencing for this region. Among the 163 disease-causing variants identified in this study, 73 had been previously reported, and the other 90 were novel. Genes most commonly implicated in different inheritances of IRDs in this cohort were presented. HEDEP and WES achieved diagnostic yield with 41.2% and 33.0%, respectively. In addition, nine patients were found to carry pathogenic mutations in the
ORF15 region with Sanger sequencing. Our study demonstrates that HEDEP can be used as a first-tier test for patients with IRDs.
Stargardt disease is an autosomal recessively inherited retinal disorder commonly caused by pathogenic variants in the ABCA4 gene encoding the ATP-binding cassette subfamily A member 4 (ABCA4) ...protein. Several deep-intronic variants in ABCA4 have been classified as disease causing. By strengthening a cryptic splice site, deep-intronic variant c.5197-557G>T induces the inclusion of a 188-bp intronic sequence in the mature mRNA, resulting in a premature termination codon. Here, we report the design and evaluation of three CRISPR-Cas9 approaches implementing Streptococcus pyogenes Cas9 (single and dual guide RNA) or Streptococcus pyogenes Cas9 nickase (dual guide RNA) for their potential to correct c.5197-557G>T-induced aberrant splicing in minigene splicing assays and patient-derived cone photoreceptor precursor cells. The different strategies were able to rescue correct splicing by up to 83% and increase the overall correctly spliced transcripts by 1.8-fold, demonstrating the successful CRISPR-Cas9-mediated rescue in patient-derived photoreceptor precursor cells of an ABCA4 splicing defect. The results provide initial evidence of possible permanent splicing correction for Stargardt disease, expanding the therapeutic toolbox to counteract deep-intronic pathogenic variants in ABCA4.
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A mutation localized in the intronic part of ABCA4 able to affect the correct reading frame of the gene was targeted by three CRISPR-Cas9-based approaches. Rescue of the correct reading frame was achieved in mutant cone photoreceptor precursor cells, while no detrimental secondary effect was observed in control cells.
CLCN2
encodes a two-pore homodimeric chloride channel protein (CLC-2) that is widely expressed in human tissues. The association between
Clcn2
and the retina is well-established in mice, as ...loss-of-function of CLC-2 can cause retinopathy in mice; however, the ocular phenotypes caused by
CLCN2
mutations in humans and the underlying mechanisms remain unclear. The present study aimed to define the ocular features and reveal the pathogenic mechanisms of
CLCN2
variants associated with retinal degeneration in humans using an in vitro overexpression system, as well as patient-induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) cells and retinal organoids (ROs). A patient carrying the homozygous c.2257C > T (p.R753X) nonsense
CLCN2
mutation was followed up for > 6 years. Ocular features were comprehensively characterized with multimodality imaging and functional examination. The patient presented with severe bilateral retinal degeneration with loss of photoreceptor and RPE. In vitro
,
mutant CLC-2 maintained the correct subcellular localization, but with reduced channel function compared to wild-type CLC-2 in HEK293T cells. Additionally, patient iPSC-derived RPE cells carrying the
CLCN2
mutation exhibited dysfunctional ClC-2 chloride channels and outer segment phagocytosis. Notably, these functions were rescued following the repair of the
CLCN2
mutation using the CRISPR-Cas9 system. However, this variant did not cause significant photoreceptor degeneration in patient-derived ROs, indicating that dysfunctional RPE is likely the primary cause of biallelic
CLCN2
variant-mediated retinopathy. This study is the first to establish the confirmatory ocular features of human
CLCN2
-related retinal degeneration, and reveal a pathogenic mechanism associated with biallelic CLCN2 variants, providing new insights into the cause of inherited retinal dystrophies.
Pathogenic variants in DNA-damage regulated autophagy modulator 2 gene (DRAM2) cause a rare autosomal recessive retinal dystrophy and its disease course is not well understood. We present two ...Slovenian patients harboring a novel DRAM2 variant and a detailed review of all 23 other patients described to date. Whole exome and whole genome sequencing were performed in the two patients, and both underwent ophthalmological examination with a 2-year follow-up. PubMed was searched for papers with clinical descriptions of DRAM2 retinopathy. Patient 1 was homozygous for a novel variant, p.Met1?, and presented with the acute onset of photopsia and retina-wide retinopathy at the age of 35 years. The patient was first thought to have an autoimmune retinopathy and was treated with mycophenolate mofetil, which provided some symptomatic relief. Patient 2 was compound heterozygous for p.Met1? and p.Leu246Pro and presented with late-onset maculopathy at the age of 59 years. On review, patients with DRAM2 retinopathy usually present in the third decade with central visual loss, outer retinal layer loss on optical coherence tomography and a hyperautofluorescent ring on fundus autofluorescence. Either cone–rod or rod–cone dystrophy phenotype is observed on electroretinography, reflecting the importance of DRAM2 in both photoreceptor types. Non-null variants can result in milder disease.
The retinoid isomerohydrolase RPE65 has received considerable attention worldwide since a successful clinical gene therapy was approved in 2017 as the first treatment for vision loss associated with ...RPE65-mediated inherited retinal disease. Identifying patients with RPE65 mutations is a prerequisite to assessing the patients' eligibility to receive RPE65-targeted gene therapies, and it is necessary to identify individuals who are most likely to benefit from gene therapies. This study aimed to investigate the RPE65 mutations frequency in the Chinese population and to determine the genetic and clinical characteristics of these patients.
Only 20 patients with RPE65 mutations were identified, and RPE65 mutations were determined to be the 14th most common among all patients with genetic diagnoses. Ten novel variants and two hotspots associated with FAP were identified. A literature review revealed that a total of 57 patients of Chinese origin were identified with pathogenic mutations in the RPE65 gene. The mean best Snellen corrected visual acuity was worse (mean 1.3 ± 1.3 LogMAR) in patients older than 20 years old than in those younger than 15 years old (0.68 ± 0.92 LogMAR). Bone spicule-like pigment deposits (BSLPs) were observed in six patients; they were older than those without BSLP and those with white-yellow dots. Genotype-phenotype analysis revealed that truncating variants seem to lead to a more severe clinical presentation, while best corrected visual acuity testing and fundus changes did not correlate with specific RPE65 variants or mutation types.
This study provides a detailed clinical-genetic assessment of patients with RPE65 mutations of Chinese origin. These results may help to elucidate RPE65 mutations in the Chinese population and may facilitate genetic counseling and the implementation of gene therapy in China.
Our goal was to design a customized microarray, arrEYE, for high-resolution copy number variant (CNV) analysis of known and candidate genes for inherited retinal dystrophy (iRD) and retina-expressed ...noncoding RNAs (ncRNAs).
arrEYE contains probes for the full genomic region of 106 known iRD genes, including those implicated in retinitis pigmentosa (RP) (the most frequent iRD), cone–rod dystrophies, macular dystrophies, and an additional 60 candidate iRD genes and 196 ncRNAs. Eight CNVs in iRD genes identified by other techniques were used as positive controls. The test cohort consisted of 57 patients with autosomal dominant, X-linked, or simplex RP.
In an RP patient, a novel heterozygous deletion of exons 7 and 8 of the HGSNAT gene was identified: c.634-408_820+338delinsAGAATATG, p.(Glu212Glyfs*2). A known variant was found on the second allele: c.1843G>A, p.(Ala615Thr). Furthermore, we expanded the allelic spectrum of USH2A and RCBTB1 with novel CNVs.
The arrEYE platform revealed subtle single-exon to larger CNVs in iRD genes that could be characterized at the nucleotide level, facilitated by the high resolution of the platform. We report the first CNV in HGSNAT that, combined with another mutation, leads to RP, further supporting its recently identified role in nonsyndromic iRD.
Genet Med19 4, 457–466.
The inherited retinal dystrophies (IRDs) are a clinically and genetically complex group of disorders primarily affecting the rod and cone photoreceptors or other retinal neuronal layers, with ...emerging therapies heralding the need for accurate molecular diagnosis. Targeted capture and panel-based strategies examining the partial or full exome deliver molecular diagnoses in many IRD families tested. However, approximately one in three families remain unsolved and unable to obtain personalised recurrence risk or access to new clinical trials or therapy. In this study, we investigated whole genome sequencing (WGS), focused assays and functional studies to assist with unsolved IRD cases and facilitate integration of these approaches to a broad molecular diagnostic clinical service. The WGS approach identified variants not covered or underinvestigated by targeted capture panel-based clinical testing strategies in six families. This included structural variants, with notable benefit of the WGS approach in repetitive regions demonstrated by a family with a hybrid gene and hemizygous missense variant involving the opsin genes,
and
. There was also benefit in investigation of the repetitive GC-rich ORF15 region of
. Further molecular investigations were facilitated by focused assays in these regions. Deep intronic variants were identified in
and
, with functional RNA based studies of the
variant revealing activation of a cryptic splice acceptor site. While targeted capture panel-based methods are successful in achieving an efficient molecular diagnosis in a proportion of cases, this study highlights the additional benefit and clinical value that may be derived from WGS, focused assays and functional genomics in the highly heterogeneous IRDs.
Background.
New pathogenetic treatment options, such as gene therapy, are now used to treat previously uncurable diseases. However, price of such treatment is high, especially in the case of orphan ...diseases, where costs may many-fold exceed the prices for other types of medication. This raises a question of optimal way of financing gene therapy in Russia.
Objective:
to evaluate economic consequences of centralizing procurement of gene therapy in 2021–2030 in the case of the drug indicated for treatment of biallelic RPE65 mutation-associated retinal dystrophy.
Material and methods.
Voretigene neparvovec is a new gene therapy that is used to treat RPE65 mutation-associated Leber congenital amaurosis and isolated retinitis pigmentosa. We estimated the number of patients (children and adults) that could be treated with voretigene neparvovec in 2021–2030 in Russia using demographic forecasting method, literature and expert data. Budget costs of treatment were estimated for two scenarios: status-quo, where gene therapy is purchased by regions for higher price, and centralized scenario with federal procurements and lower price for the drug.
Results.
Up to 100 children and 56 adults could be treated with voretigene neparvovec in 2021–2030 in Russia. Centralizing procurements at the expense of federal budget may save up to 20.7% or 1.8 billion rub. (1.13 billion rub. for children and 0.67 billion rub. for adults), compared to regional procurements.
Conclusion.
Centralizing procurements of expensive drugs intended for gene therapy of orphan diseases may save budget costs of the Russian Federation, compared to status-quo decentralized purchases.
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