Retinal organoids with X-linked retinoschisis RS1 (E72K) mutation exhibit a photoreceptor developmental delay and are rescued by gene augmentation therapy.

BACKGROUND: X-linked juvenile retinoschisis (XLRS) is an inherited disease caused by RS1 gene mutation, which leads to retinal splitting and visual impairment. The mechanism of RS1-associated retinal degeneration is not fully understood. Besides, animal models of XLRS have limitations in the study of XLRS. Here, we used human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) to investigate the disease mechanisms and potential treatments for XLRS. METHODS: hiPSCs reprogrammed from peripheral blood mononuclear cells of two RS1 mutant (E72K) XLRS patients were differentiated into ROs. Subsequently, we explored whether RS1 mutation could affect RO development and explore the effectiveness of RS1 gene augmentation therapy. RESULTS: ROs derived from RS1 (E72K) mutation hiPSCs exhibited a developmental delay in the photoreceptor, retinoschisin (RS1) deficiency, and altered spontaneous activity compared with control ROs. Furthermore, the delays in development were associated with decreased expression of rod-specific precursor markers (NRL) and photoreceptor-specific markers (RCVRN). Adeno-associated virus (AAV)-mediated gene augmentation with RS1 at the photoreceptor immature stage rescued the rod photoreceptor developmental delay in ROs with the RS1 (E72K) mutation. CONCLUSIONS: The RS1 (E72K) mutation results in the photoreceptor development delay in ROs and can be partially rescued by the RS1 gene augmentation therapy.

Intravitreal Delivery of rAAV2-hSyn-hRS1 Results in Retinal Ganglion Cell-Specific Gene Expression and Retinal Improvement in the Rs1-KO Mouse.

X-linked retinoschisis (XLRS) is a monogenic recessive inherited retinal disease caused by defects in retinoschisin (RS1). It manifests clinically as retinal schisis cavities and a disproportionate reduction of b-wave amplitude compared with the a-wave amplitude. Currently there is no approved treatment. In the last decade, there has been major progress in the development of gene therapy for XLRS. Previous preclinical studies have demonstrated the treatment benefits of hRS1 gene augmentation therapy in mouse models. However, outcomes in clinical trials have been disappointing, and this might be attributed to dysfunctional assembly of RS1 complexes and/or the impaired targeted cells. In this study, the human synapsin 1 gene promoter (hSyn) was used to control the expression of hRS1 to specifically target retinal ganglion cells and our results confirmed the specific expression and functional assembly of the protein. Moreover, our results demonstrated that a single intravitreal injection of rAAV2-hSyn-hRS1 results in architectural restoration of retinal schisis cavities and improvement in vision in a mouse model of XLRS. In brief, this study not only supports the clinical development of the rAAV2-hSyn-hRS1 vector in XLRS patients but also confirms the therapeutic potential of rAAV-based gene therapy in inherited retinal diseases.

Photoreceptor deficits appear at eye opening in Rs1 mutant mouse models of X-linked retinoschisis.

X-linked retinoschisis (XLRS) is an early onset degenerative retinal disease characterized by cystic lesions in the middle layers of the retina. These structural changes are accompanied by a loss of visual acuity and decreased contrast sensitivity. XLRS is caused by mutations in the gene Rs1 which encodes the secreted protein Retinoschisin 1. Young Rs1-mutant mouse models develop key hallmarks of XLRS including intraretinal schisis and abnormal electroretinograms. The electroretinogram (ERG) comprises activity of multiple cellular generators, and it is not known how and when each of these is impacted in Rs1 mutant mice. Here we use an ex vivo ERG system and pharmacological blockade to determine how ERG components generated by photoreceptors, ON-bipolar, and Müller glial cells are impacted in Rs1 mutants and to determine the time course of these changes. We report that ERG abnormalities begin near eye-opening and that all ERG components are involved.

Genotypic and phenotypic diversity in X-linked retinoschisis: Findings from a South Indian patient cohort.

PURPOSE: Retinoschisis is a distinctive condition characterized by intraretinal layer clefts, primarily associated with X-linked recessive inheritance due to RS1 gene mutations. This study aims to uncover the RS1 mutation spectrum in a cohort of 22 X-linked retinoschisis cases from South India and emphasizes the genotypic and phenotypic associations within patients harboring only RS1 mutations. METHODS: A total of 22 probands were suspected of having X-linked retinoschisis. All study subjects underwent ophthalmic investigations, including assessments of visual acuity, fundus examination, optical coherence tomography (OCT), and electroretinogram (ERG). RS1 gene screening was conducted using Sanger sequencing, and the pathogenicity of the variants was assessed through Sorting Intolerant from Tolerant (SIFT) and PolyPhen-2 in silico tools. RESULTS: The study found that the probands had an average visual acuity of 0.79 ± 0.39 log of minimum angle of resolution (logMAR), ranging from 0.17 to 1.77. During fundus examination, the probands exhibited a characteristic spoke wheel-like pattern in the macular region. Furthermore, OCT analysis revealed distinct alterations in the inner retinal microstructure, and ERG results consistently showed a reduction in b-wave amplitude. Eventually, Sanger sequencing results showed hemizygous mutations in the RS1 gene in only 12 probands, including a novel missense mutation in the RS1 gene's signal sequence. CONCLUSION: This study provides valuable insights into the spectrum of RS1 mutations in X-linked retinoschisis probands from South India. It reveals distinct genotypic-phenotypic associations and highlights the clinical manifestations associated with the disease pathogenesis.

The Road towards Gene Therapy for X-Linked Juvenile Retinoschisis: A Systematic Review of Preclinical Gene Therapy in Cell-Based and Rodent Models of XLRS.

X-linked juvenile retinoschisis (XLRS) is an early-onset progressive inherited retinopathy affecting males. It is characterized by abnormalities in the macula, with formation of cystoid retinal cavities, frequently accompanied by splitting of the retinal layers, impaired synaptic transmission of visual signals, and associated loss of visual acuity. XLRS is caused by loss-of-function mutations in the retinoschisin gene located on the X chromosome (RS1, MIM 30083). While proof-of-concept studies for gene augmentation therapy have been promising in in vitro and rodent models, clinical trials in XLRS patients have not been successful thus far. We performed a systematic literature investigation using search strings related to XLRS and gene therapy in in vivo and in vitro models. Three rounds of screening (title/abstract, full text and qualitative) were performed by two independent reviewers until consensus was reached. Characteristics related to study design and intervention were extracted from all studies. Results were divided into studies using (1) viral and (2) non-viral therapies. All in vivo rodent studies that used viral vectors were assessed for quality and risk of bias using the SYRCLE's risk-of-bias tool. Studies using alternative and non-viral delivery techniques, either in vivo or in vitro, were extracted and reviewed qualitatively, given the diverse and dispersed nature of the information. For in-depth analysis of in vivo studies using viral vectors, outcome data for optical coherence tomography (OCT), immunohistopathology and electroretinography (ERG) were extracted. Meta-analyses were performed on the effect of recombinant adeno-associated viral vector (AAV)-mediated gene augmentation therapies on a- and b-wave amplitude as well as the ratio between b- and a-wave amplitudes (b/a-ratio) extracted from ERG data. Subgroup analyses and meta-regression were performed for model, dose, age at injection, follow-up time point and delivery method. Between-study heterogeneity was assessed with a Chi-square test of homogeneity (I2). We identified 25 studies that target RS1 and met our search string. A total of 19 of these studies reported rodent viral methods in vivo. Six of the 25 studies used non-viral or alternative delivery methods, either in vitro or in vivo. Of these, five studies described non-viral methods and one study described an alternative delivery method. The 19 aforementioned in vivo studies were assessed for risk of bias and quality assessments and showed inconsistency in reporting. This resulted in an unclear risk of bias in most included studies. All 19 studies used AAVs to deliver intact human or murine RS1 in rodent models for XLRS. Meta-analyses of a-wave amplitude, b-wave amplitude, and b/a-ratio showed that, overall, AAV-mediated gene augmentation therapy significantly ameliorated the disease phenotype on these parameters. Subgroup analyses and meta-regression showed significant correlations between b-wave amplitude effect size and dose, although between-study heterogeneity was high. This systematic review reiterates the high potential for gene therapy in XLRS, while highlighting the importance of careful preclinical study design and reporting. The establishment of a systematic approach in these studies is essential to effectively translate this knowledge into novel and improved treatment alternatives.

Genetic, morphological and electrophysiological findings in a patient with a rare pathogenic variant in the RS1 gene.

PURPOSE: In this study, we report a case of a young adult with X-linked juvenile retinoschisis (XLRS) with a rare pathogenic variant in the RS1 gene (c.522 + 2 T > A). METHODS: Ophthalmological evaluation, optical coherence tomography, full-field and multifocal electroretinograms and extensive genetic screening of genes related to visual loss were carried out in the participant. RESULTS: Clinical ophthalmological exams revealed a mild to moderate impairment of visual acuity. Retinal imaging showed bilateral foveal schisis, as well as normal a-wave, reduction in the b-wave amplitudes in dark- and light- adapted full-field electroretinograms, and abnormal oscillatory potentials. We found also diffuse amplitude reduction in multifocal electroretinogram arrays. A canonical splice variant was identified in the RS1 gene (c.522 + 2 T > A). CONCLUSION: A rare pathogenic variant of the RS1 gene was associated with diffuse retinal involvement (central and peripheral retina), probably in inner retina, and mild to moderate visual acuity impairment. The phenotypical characterization of rare mutations is relevant to provide information about the disease.

Optical Coherence Tomography Feature of Retinoschisis in CRB1-Associated Maculopathy.

This case report describes 2 individuals with hyperreflective columns in the outer nuclear layer observed on optical coherence tomography and possible implications for CRB1-associated maculopathy.

Generation of a gene-corrected isogenic iPSC cell line from an X-linked retinoschisis patient with a hemizygous mutation c.304C > T (p.R102W) in RS1 gene.

X-linked retinoschisis (XLRS) is one of the most common retinal genetic diseases with progressive visual impairment in childhood affecting males. It is manifested with macular and/or peripheral schisis in neural retinas with no effective treatment. Previously, we successfully generated a human-induced pluripotent stem cell (iPSC) line from an XLRS patient carrying the hemizygous RS1 c. 304C > T (p.R102W) mutation. Here, we corrected the c.304C > T mutation in the RS1 gene using CRISPR/Cas9 technology to generate an isogenic control. This cell line is valuable for the study of XLRS.

Do the retinal abnormalities in X-linked juvenile retinoschisis include impaired phototransduction?

X-linked juvenile retinoschisis (XLRS), a hereditary retinal disorder primarily affecting males, is characterized by the formation of cystic spaces between the outer plexiform layer and outer nuclear layer of the retina. Mutations in the RS1 gene, which encodes the extracellular binding protein retinoschisin, are responsible for XLRS pathogenesis. While the role of retinoschisin in maintaining retinal integrity is well established, there is growing evidence suggesting compromised photoreceptor function in XLRS. To investigate the molecular pathways affected by RS1 deficiency, particularly in phototransduction, we performed electroretinographic (ERG) and proteomic analyses on retinae from Rs1 knockout mice, a model of human XLRS. The Rs1 knockout mice had reduced ERG a-wave amplitudes. Correspondingly, differential expression analysis revealed downregulation of proteins crucial for phototransduction, with Ingenuity Pathway Analysis (IPA) highlighting "phototransduction" as the most significantly downregulated biological theme. Compensatory mechanisms were also observed in the IPA, including upregulation of synaptic remodeling, inflammation, cell adhesion, and G-protein signaling. These findings strongly implicate an underrecognized role of photoreceptor dysfunction in XLRS pathology. We speculate that entrapment of mutant retinoschisin protein within photoreceptor inner segments as well as disrupted reciprocal regulation between L-type voltage-gated calcium channels and retinoschisin contribute to the dysfunction in photoreceptors.

Identification and molecular characterization of two recurrent missense mutations in the RS1 gene in two families with X-linked retinoschisis from North India.

X-linked retinoschisis (XLR) is a rare medical condition that involves in the splitting of neurosensory layers and the impairment of vision in the retina. In majority of the XLR cases, pathogenic variants in Retinoschisin 1 (RS1) gene have been implicated in males with an early age of onset during early childhood. In the present study, we have recruited two North Indian families having multiple affected male members, who were diagnosed with XLR. The entire protein-coding region of RS1 was screened by PCR-Sanger sequencing and two recurrent pathogenic variants (p.I81N and p.R102Q) were unraveled. The in vitro study of these variants demonstrated the aggregation of mutant RS1 within the endoplasmic reticulum. Furthermore, mutant forms of this protein showed significant intracellular retention, which was evident by the absence of retinoschisin protein fractions in the extracellular media. These inferences were also supported by extensive bioinformatics analysis of the mutants, which showed dramatic conformational changes in the local structure of retinoschisin. Thus, our study suggests that the identified pathogenic variants interfere with proper protein folding, leading to anomalous structural changes ultimately resulting in intracellular retention of retinoschisin within the retina.

A Carrier Female Manifesting an Unusual X-Linked Retinoschisis Phenotype Associated with the Pathogenic Variant c.266delA, p.(Tyr89LeufsTer37) in RS1, and Skewed X-Inactivation.

X-linked retinoschisis (XLRS) is the most common juvenile macular degeneration in males. Unlike most other X-linked retinal dystrophies, carrier heterozygous females are very rarely reported to show clinical features of the disease. Herein, we describe unusual retinal features in a 2-year-old female infant with family history and genetic testing consistent with XLRS.

Acute angle-closure in juvenile X-linked retinoschisis.

Juvenile X-linked retinoschisis (JXR), the most common inherited retinal disorder in young males, presents with a wide range of phenotypic variations. Acute angle closure in children with JXR has been reported in the literature only once before. We present a case of acute-angle closure, temporally associated with pharmacologic dilation, in a 12-year-old boy with JXR.

Management of angle-closure glaucoma with X-linked retinoschisis: a case report.

BACKGROUND: X-linked retinoschisis (XLRS), due to mutations in the RS1 gene, is a common genetically determined form of macular degeneration. This report describes an unusual case of angle-closure glaucoma (ACG) with XLRS and discusses the treatment. CASE PRESENTATION: A 39-year-old Chinese man with an X chromosome-recessive inherited c.489G > A variant in the RS1 gene was diagnosed as XLRS and ACG, presenting with cystic macular lesions, shallow anterior chamber depth (ACD), and angle-closure with uncontrolled intraocular pressure (IOP). Malignant glaucoma occurred following trabeculectomy combining phacoemulsification with intraocular lens (IOL) implantation and goniosynechialysis. Subsequent anterior vitrectomy and irido-zonulo-hyaloid-vitrectomy (IZHV) effectively lowered IOP and deepened ACD, but the cystic cavity became larger. CONCLUSIONS: There is a potential risk of malignant glaucoma in ACG patients with XLRS after filtering surgery. Although anterior vitrectomy can effectively resolve aqueous misdirection, the macular retinoschisis may get worse. Awareness of this risk may aid in surgical planning and postoperative management in these patients.

The genetic spectrum and clinical features of X-linked juvenile retinoschisis in Central China.

PURPOSE: X-linked juvenile retinoschisis (XLRS) is the most common congenital retinoschisis in rare vitreoretinopathy and causes visual disturbances. The study aimed to explore possible genetic mutations associated with XLRS and assess the clinical characteristics in Chinese families. METHODS: Seventeen cases and thirty-four eyes of probands and thirty-nine cases and seventy-eight eyes of their guardians were recruited. Peripheral blood DNA was extracted and PCR-amplified for retinal disease second-generation panel sequencing to screen for mutated genes. Pathogenicity was referred to the guidelines of the American College of Medical Genetics and Genomics (ACMG). RESULTS: A total of 17 male patients were included, with an average age of 9.73 years (range, 5 ~ 27 years). Clinical data indicate typical macular retinoschisis (97.06%), peripheral retinoschisis (46.67%), retinal holes (32.35%). Fifteen mutations (10 missense mutations, 4 shift mutations, and 3 nonsense mutations) of RS1 gene were identified, including 5 novel mutations. In novel mutations, amino acid conservation analysis shows W33, W50, E62, and G70 were highly conserved, and software predicts mutations to be pathogenic. SWISS-MODEL protein prediction software showed protein structural changes in proband 13. CONCLUSIONS: We have identified and described five novel mutations in the RS1 gene and their corresponding clinical manifestations. These findings not only expand the range of known RS1 mutations and associated clinical phenotypes but also provide a basis for mechanistic studies and diagnosis of XLRS.

A paradigm shift in the treatment of refractory angle closure glaucoma in a patient with X-linked juvenile retinoschisis.

BACKGROUND: X-linked retinoschisis (XLRS) is a rare inherited bilateral retinal degeneration caused by mutations in RS1 gene, occurring exclusively in men. Various ocular complications associated with XLRS are reported, and angle closure glaucoma in these eyes is one such complication that is refractory and needs surgery for intraocular pressure control. Glaucoma surgery in these eyes often results in refractory malignant glaucoma with its serious sequelae. Several surgical modifications to prevent this complication have been tried with no or limited success. METHODOLOGY: In this report, we present a case of XLRS in a young male with a 22-year follow-up. We have described the natural history and progression of retinal disease and glaucoma. RESULTS: Refractory angle closure glaucoma in our patient was treated with core vitrectomy, phacoemulsification with intraocular lens implantation, and irido-zonulo-hyaloido-vitrectomy. This helped in successful deepening of anterior chamber, good IOP control, and preventing malignant glaucoma. CONCLUSION: Our case highlights the role of vitrectomy in managing the secondary angle closure glaucoma in eyes with X-LRS.

X-Linked Retinoschisis.

X-linked retinoschisis (XLRS) is an inherited vitreoretinal dystrophy causing visual impairment in males starting at a young age with an estimated prevalence of 1:5000 to 1:25,000. The condition was first observed in two affected brothers by Josef Haas in 1898 and is clinically diagnosed by characteristic intraretinal cysts arranged in a petaloid "spoke-wheel" pattern centered in the macula. When clinical electroretinogram (ERG) testing began in the 1960s, XLRS was noted to have a characteristic reduction of the dark-adapted b-wave amplitude despite normal or usually nearly normal a-wave amplitudes, which became known as the "electronegative ERG response" of XLRS disease. The causative gene, RS1, was identified on the X-chromosome in 1997 and led to understanding the molecular and cellular basis of the condition, discerning the structure and function of the retinoschisin protein, and generating XLRS murine models. Along with parallel development of gene delivery vectors suitable for targeting retinal diseases, successful gene augmentation therapy was demonstrated by rescuing the XLRS phenotype in mouse. Two human phase I/II therapeutic XLRS gene augmentation studies were initiated; and although these did not yield definitive improvement in visual function, they gave significant new knowledge and experience, which positions the field for further near-term clinical testing with enhanced, next-generation gene therapy for XLRS patients.

SYMPTOMATIC EARLY-ONSET X-LINKED RETINOSCHISIS: Clinical Presentation and Outcomes.

PURPOSE: To describe clinical characteristics and outcomes of children with early-onset X-linked retinoschisis. METHODS: In this retrospective consecutive case series, we included children diagnosed with symptomatic X-linked retinoschisis younger than 2 years. Presenting signs, clinical characteristics, treatments, and outcomes were recorded. RESULTS: Seven patients (14 eyes) with a mean age of 17.14 ± 6.28 months were included. Strabismus was the most common presenting symptom (6 of 7 patients, 86%). Clinical signs at the first diagnosis included peripheral retinoschisis in 13 eyes (13/14, 93%), of which 5 (5/13, 38%) were bullous, vitreous hemorrhage in 3 eyes (3/14, 21%), and retinal detachment in 3 eyes (3/14, 21%). The macula was involved in all eyes: It was detached in 2 eyes (2/14, 14%) and involved in the peripheral schisis in 4 eyes (4/14, 29%). In all remaining eyes, optical coherence tomography revealed foveoschisis. Six eyes (6/14, 42%) received surgery. At the last follow-up, visual acuity, when available, ranged from no light perception to 20/40, and no children had persistent retinal detachment. CONCLUSION: Children with early-onset X-linked retinoschisis had severe forms. All children had peripheral retinoschisis which was often bullous and extended to the macula. Diagnosis is often clinical but handheld optical coherence tomography can be helpful in atypical forms. Complications requiring surgical management are frequent.

Clinical and genetic features of retinoschisis in 120 families with RS1 mutations.

BACKGROUND/AIMS: X-linked retinoschisis (XLRS), associated with RS1, is the most common type of X-linked retinopathy in children. This study aimed to identify clinical and genetic features of retinoschisis in 120 families with RS1 variants in China. METHODS: RS1 variants were collected from our in-house exome data and were predicted by multiple-step bioinformatics analysis. Clinical data of 122 patients from 120 families with potential pathogenic RS1 variants were analysed and summarised, respectively. RESULT: Totally, 79 hemizygous variants (53 missense, 25 truncation and 1 indel), were detected. All except one (78/79, 98.7%), including 22 novels, were classified as potential pathogenic and detected exclusively in 120 families with retinoschisis. Clinical data demonstrated an average age of presentation at 5 years (1 month-41 years). Macular changes were classified as macular schisis (87.5%), macular atrophy (10.7%), normal (0.9%) and unclassified (0.9%). Patients with macular atrophy had older age but similar visual acuity compared with macular schisis. Peripheral retinal changes included flat retinoschisis (52.4%), bullous retinoschisis (BRS) (10.7%) and normal-like (36.9%) patients. Spontaneous regression was observed in two patients with BRS on follow-up examination. Visual acuity in the peripheral retinoschisis group was worse than that without peripheral retinoschisis. CONCLUSION: Almost all rare RS1 variants were potential pathogenic. All patients with RS1 pathogenic variants showed detectable characteristics in the macula and/or peripheral retina. Our data on RS1 variants and associated clinical phenotypes may be of value for clinical diagnosis and genetic test of retinoschisis.

X-linked retinoschisis: mutation spectrum and genotype-phenotype relationship in an Italian pediatric cohort.

BACKGROUND: X-linked juvenile retinoschisis (×LRS) is an X-linked vitreoretinal degenerative disease that consists of variable phenotypes ranging from severe early-onset defects to subtle abnormalities diagnosed in elderly patients. XLRS is caused by a loss of function of the protein Retinoschisin (RS1), which is essential to preserve retinal integrity and function of photoreceptor-bipolar synapse. The literature data so far mostly agree on the absence of a clear genotype-phenotype correlation in XLRS. We reviewed clinical and molecular characteristics of a cohort of Italian pediatric XLRS patients to assess the presence of a correlation between genotype and phenotype severity. MATERIALS AND METHODS: We retrospectively examined clinical and genetic features of a cohort of 27 XLRS patients. In this study we included patients with a diagnosis of XLRS confirmed by fundus photography, spectral domain optical coherence tomography, and molecular analysis and with an onset of less than 10 years of age. We sorted RS1 variants according to their effect of RS1 structure and function in three separate groups. RESULTS: According to previous studies, we did not observe a conclusive genotype-phenotype correlation in our cohort; nevertheless, we noticed that patients harboring RS1 variants leading to RS1-secreted mutants show a more homogeneous phenotype, with an overall good visual acuity, compared to the other two groups. CONCLUSIONS: Our data support the hypothesis that secretion profile of RS1 could influence the severity of the phenotype. More extensive and functional studies are needed to acquire notions in view of the opportunity of gene replacement therapy for XLRS patients.