Notch inhibition promotes regeneration and immunosuppression supports cone survival in a zebrafish model of inherited retinal dystrophy

Photoreceptor degeneration leads to irreversible vision loss in humans with retinal dystrophies such as Retinitis Pigmentosa. Whereas photoreceptor loss is permanent in mammals, zebrafish possesses the ability to regenerate retinal neurons and restore visual function. Following acute damage, Muller glia (MG) re-enter the cell cycle and produce multipotent progenitors whose progeny differentiate into mature neurons. Both MG reprogramming and proliferation of retinal progenitor cells require reactive microglia and associated inflammatory signaling. Paradoxically, MG in zebrafish models of photoreceptor degeneration fail to re-enter the cell cycle and regenerate lost cells. Here, we used the zebrafish cep290 mutant to demonstrate that progressive cone degeneration generates an immune response but does not stimulate MG proliferation. Acute light damage triggered photoreceptor regeneration in cep290 mutants but cones were only restored to pre-lesion densities. Using irf8 mutant zebrafish, we found that the chronic absence of microglia reduced inflammation and rescued cone degeneration in cep290 mutants. Finally, single-cell RNA-sequencing revealed sustained expression of notch3 in MG of cep290 mutants and inhibition of Notch signaling induced MG to re-enter the cell cycle. Our findings provide new insights on the requirements for MG to proliferate and the potential for immunosuppression to prolong photoreceptor survival.

Genetic and Phenotypic Landscape of PRPH2-Associated Retinal Dystrophy in Japan

Peripherin-2 (PRPH2) is one of the causative genes of inherited retinal dystrophy. While the gene is relatively common in Caucasians, reports from Asian ethnicities are limited. In the present study, we report 40 Japanese patients from 30 families with PRPH2-associated retinal dystrophy. We identified 17 distinct pathogenic or likely pathogenic variants using next-generation sequencing. Variants p.R142W and p.V200E were relatively common in the cohort. The age of onset was generally in the 40’s; however, some patients had earlier onset (age: 5 years). Visual acuity of the patients ranged from hand motion to 1.5 (Snellen equivalent 20/13). The patients showed variable phenotypes such as retinitis pigmentosa, cone-rod dystrophy, and macular dystrophy. Additionally, intrafamilial phenotypic variability was observed. Choroidal neovascularization was observed in three eyes of two patients with retinitis pigmentosa. The results demonstrate the genotypic and phenotypic variations of the disease in the Asian cohort.

Clinical Features and Natural History in a Cohort of Chinese Patients with RPE65-Associated Inherited Retinal Dystrophy

RPE65-associated inherited retinal dystrophy (RPE65-IRD) is an early-onset retinal degeneration. The aim of this study was to describe the clinical features and natural course of this disease in a Chinese patient cohort with RPE65 biallelic variants. Thirty patients from 29 unrelated families with biallelic disease-causing RPE65 variants underwent full ophthalmic examinations. Thirteen were followed up over time. An additional 57 Chinese cases from 49 families were retrieved from the literature to analyze the relationship between best-corrected visual acuity (BCVA) and age. Our 30 patients presented age-dependent phenotypic characteristics. Multiple white dots were a clinical feature of young patients, while maculopathy, epiretinal membrane, and bone spicules were common in adult patients. Among the 84 patients, BCVA declined with age in a nonlinear, positive-acceleration relationship (p < 0.001). All patients older than 40 years met the WHO standard for low vision. Longitudinal observation revealed a slower visual acuity loss in patients younger than 20 years than those in their third or fourth decade of life. Our study detailed the clinical features and natural course of disease in Chinese patients with RPE65-IRD. Our results indicated that these patients have a relatively stable BCVA in childhood and adolescence, but eyesight deteriorates rapidly in the third decade of life. These findings may facilitate the implementation of gene therapy in China.

Consanguinity-based analysis of exome sequencing yields likely genetic causes in patients with inherited retinal dystrophy

Background Consanguineous families have a relatively high prevalence of genetic disorders caused by bi-allelic mutations in recessive genes. This study aims to evaluate the effectiveness and efficiency of a consanguinity-based exome sequencing approach to capturing genetic mutations in inherited retinal dystrophy families with consanguineous marriages. Methods Ten unrelated consanguineous families with a proband affected by inherited retinal dystrophy were recruited in this study. All participants underwent comprehensive ophthalmic examinations. Whole exome sequencing was performed, followed by a homozygote-prior strategy to rapidly filter disease-causing mutations. Bioinformatic prediction of pathogenicity, Sanger sequencing and co-segregation analysis were carried out for further validation. Results In ten consanguineous families, a total of 10 homozygous mutations in 8 IRD genes were identified, including 2 novel mutations, c.1654_1655delAG (p. R552Afs*5) in gene FAM161A in a patient diagnosed with retinitis pigmentosa, and c.830T > C (p.L277P) in gene CEP78 in a patient diagnosed with cone and rod dystrophy. Conclusion The genetic etiology in consanguineous families with IRD were successfully identified using consanguinity-based analysis of exome sequencing data, suggesting that this approach could provide complementary insights into genetic diagnoses in consanguineous families with variant genetic disorders.

Genotype Profile of Global EYS-Associated Inherited Retinal Dystrophy and Clinical Findings in a Large Chinese Cohort

PurposeThe aim of this study was to probe the global profile of the EYS-associated genotype-phenotype trait in the worldwide reported IRD cases and to build a model for predicting disease progression as a reference for clinical consultation.MethodsThis retrospective study of 420 well-documented IRD cases with mutations in the EYS gene included 39 patients from a genotype-phenotype study of inherited retinal dystrophy (IRD) conducted at the Beijing Institute of Ophthalmology and 381 cases retrieved from global reports. All patients underwent ophthalmic evaluation. Mutations were revealed using next-generation sequencing, followed by Sanger DNA sequencing and real-time quantitative PCR analysis. Multiple regression models and statistical analysis were used to assess the genotype and phenotype characteristics and traits in this large cohort.ResultsA total of 420 well-defined patients with 841 identified mutations in the EYS gene were successfully obtained. The most common pathogenic variant was a frameshift c.4957dupA (p.S1653Kfs∗2) in exon 26, with an allele frequency of 12.7% (107/841), followed by c.8805C &gt; A (p.Y2935X) in exon 43, with an allele frequency of 5.9% (50/841). Two new hot spots were identified in the Chinese cohort, c.1750G &gt; T (p.E584X) and c.7492G &gt; C (p.A2498P). Several EYS mutation types were identified, with CNV being relatively common. The mean age of onset was 20.54 ± 11.33 (4–46) years. Clinical examinations revealed a typical progression of RPE atrophy from the peripheral area to the macula.ConclusionThis large global cohort of 420 IRD cases, with 262 distinct variants, identified genotype-phenotype correlations and mutation spectra with hotspots in the EYS gene.

USH2A gene variants cause Keratoconus and Usher syndrome phenotypes in Pakistani families

Background Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy, affecting approximately 1 in 4000 individuals worldwide. The most common form of syndromic RP is Usher syndrome (USH) accounting for approximately 20–30 % of RP cases. Mutations in the USH2A gene cause a significant proportion of recessive non-syndromic RP and USH type II (USH2). This study aimed to determine the causative role of the USH2A gene in autosomal recessive inherited ocular diseases and to establish genotype-phenotype correlation associated with USH2A variants. Methods We performed direct Sanger sequencing and co-segregation analysis of the USH2A gene to identify disease causing variants in a non-syndromic RP family, two USH2 families and two Keratoconus (KC) families. Results Disease causing variants in the USH2A gene were identified in two families displayed KC and USH2 phenotypes. A novel variant c.4029T > G, p.Asn1343Lys in the USH2A gene was detected in a Pakistani family with KC phenotype. In addition, a missense variant (c.7334 C > T, p. Ser2445Phe) in the USH2A gene was found segregating in another Pakistani family with USH2 phenotype. Homozygosity of identified missense USH2A variants was found associated with autosomal recessive inherited KC and USH2 phenotypes in investigated families. These variants were not detected in ethnically matched healthy controls. Moreover, the USH2A variants were predicted to be deleterious or potentially disease causing by PolyPhen-2, PROVEAN and SIFT. Conclusions This study provided first evidence for association of a novel USH2A variant with KC phenotype in a Pakistani family as well as established the phenotype-genotype correlation of a USH2A variant (c.7334 C > T, p. Ser2445Phe) with USH2 phenotype in another Pakistani family. The phenotype-genotype correlations established in present study may improve clinical diagnosis of affected individuals for better management and counseling.

Allele-Specific Knockout by CRISPR/Cas to Treat Autosomal Dominant Retinitis Pigmentosa Caused by the G56R Mutation in NR2E3

Retinitis pigmentosa (RP) is an inherited retinal dystrophy that causes progressive vision loss. The G56R mutation in NR2E3 is the second most common mutation causing autosomal dominant (ad) RP, a transcription factor that is essential for photoreceptor development and maintenance. The G56R variant is exclusively responsible for all cases of NR2E3-associated adRP. Currently, there is no treatment for NR2E3-related or, other, adRP, but genome editing holds promise. A pertinent approach would be to specifically knockout the dominant mutant allele, so that the wild type allele can perform unhindered. In this study, we developed a CRISPR/Cas strategy to specifically knockout the mutant G56R allele of NR2E3 and performed a proof-of-concept study in induced pluripotent stem cells (iPSCs) of an adRP patient. We demonstrate allele-specific knockout of the mutant G56R allele in the absence of off-target events. Furthermore, we validated this knockout strategy in an exogenous overexpression system. Accordingly, the mutant G56R-CRISPR protein was truncated and mis-localized to the cytosol in contrast to the (peri)nuclear localizations of wild type or G56R NR2E3 proteins. Finally, we show, for the first time, that G56R iPSCs, as well as G56R-CRISPR iPSCs, can differentiate into NR2E3-expressing retinal organoids. Overall, we demonstrate that G56R allele-specific knockout by CRISPR/Cas could be a clinically relevant approach to treat NR2E3-associated adRP.

Chronic untreated retinal detachment in a patient with choroideremia provides insight into the disease process and potential therapy

Aim: We present the case of a 72-year-old male with advanced choroideremia and a left chronic rhegmatogenous retinal detachment, which to our knowledge is the first formal report of a retinal detachment in this disease. Background: Choroideremia is a rare X-linked inherited retinal dystrophy, caused by mutations in the CHM gene which encodes Rab escort protein 1 (REP1), and affected males typically experience a progressive centripetal loss of vision. The disease pathology is caused by a primary retinal pigment epithelium degeneration, which leads to secondary loss of photoreceptors and choriocapillaris. This in turn leads to fusion of the degenerate outer retinal layers resulting in a retinopexy that is known to make subretinal gene therapy particularly challenging in these patients. Conclusion: Although retinal gene therapy is commonly targeted to the macular area in choroideremia, the observation of a rhegmatogenous retinal detachment indicates that the peripheral retina may not fuse with the residual choroid as occurs in the equatorial and macular regions. If this hypothesis is correct, targeting gene therapy to the retinal periphery even in advanced cases may be feasible and could potentially be used to preserve navigational vision.

Mutation Screening in the miR-183/96/182 Cluster in Patients With Inherited Retinal Dystrophy

Inherited retinal dystrophy (IRD) is a heterogenous blinding eye disease and affects more than 200,000 Americans and millions worldwide. By far, 270 protein-coding genes have been identified to cause IRD when defective. However, only one microRNA (miRNA), miR-204, has been reported to be responsible for IRD when a point-mutation occurs in its seed sequence. Previously, we identified that a conserved, polycistronic, paralogous miRNA cluster, the miR-183/96/182 cluster, is highly specifically expressed in all photoreceptors and other sensory organs; inactivation of this cluster in mice resulted in syndromic IRD with multi-sensory defects. We hypothesized that mutations in the miR-183/96/182 cluster in human cause IRD. To test this hypothesis, we perform mutation screening in the pre-miR-183, -96, -182 in &gt;1000 peripheral blood DNA samples of patients with various forms of IRD. We identified six sequence variants, three in pre-miR-182 and three in pre-miR-96. These variants are in the pre-miRNA-182 or -96, but not in the mature miRNAs, and are unlikely to be the cause of the IRD in these patients. In spite of this, the nature and location of these sequence variants in the pre-miRNAs suggest that some may have impact on the biogenesis and maturation of miR-182 or miR-96 and potential roles in the susceptibility to diseases. Although reporting on negative results so far, our study established a system for mutation screening in the miR-183/96/182 cluster in human for a continued effort to unravel and provides deeper insight into the potential roles of miR-183/96/182 cluster in human diseases.