Clinical exome analysis and targeted gene repair of the c.1354dupT variant in iPSC lines from patients with PROM1-related retinopathies exhibiting diverse phenotypes.

BACKGROUND: Inherited retinal dystrophies (IRD) are one of the main causes of incurable blindness worldwide. IRD are caused by mutations in genes that encode essential proteins for the retina, leading to photoreceptor degeneration and loss of visual function. IRD generates an enormous global financial burden due to the lack of understanding of a significant part of its pathophysiology, molecular diagnosis, and the near absence of non-palliative treatment options. Patient-derived induced pluripotent stem cells (iPSC) for IRD seem to be an excellent option for addressing these questions, serving as exceptional tools for in-depth studies of IRD pathophysiology and testing new therapeutic approaches. METHODS: From a cohort of 8 patients with PROM1-related IRD, we identified 3 patients carrying the same variant (c.1354dupT) but expressing three different IRD phenotypes: Cone and rod dystrophy (CORD), Retinitis pigmentosa (RP), and Stargardt disease type 4 (STGD4). These three target patients, along with one healthy relative from each, underwent comprehensive ophthalmic examinations and their genetic panel study was expanded through clinical exome sequencing (CES). Subsequently, non-integrative patient-derived iPSC were generated and fully characterized. Correction of the c.1354dupT mutation was performed using CRISPR/Cas9, and the genetic restoration of the PROM1 gene was confirmed through flow cytometry and western blotting in the patient-derived iPSC lines. RESULTS: CES revealed that 2 target patients with the c.1354dupT mutation presented monoallelic variants in genes associated with the complement system or photoreceptor differentiation and peroxisome biogenesis disorders, respectively. The pluripotency and functionality of the patient-derived iPSC lines were confirmed, and the correction of the target mutation fully restored the capability of encoding Prominin-1 (CD133) in the genetically repaired patient-derived iPSC lines. CONCLUSIONS: The c.1354dupT mutation in the PROM1 gene is associated to three distinct AR phenotypes of IRD. This pleotropic effect might be related to the influence of monoallelic variants in other genes associated with retinal dystrophies. However, further evidence needs to be provided. Future experiments should include gene-edited patient-derived iPSC due to its potential as disease modelling tools to elucidate this matter in question.

Genetic Characterization of 191 Probands with Inherited Retinal Dystrophy by Targeted NGS Analysis.

Inherited retinal diseases (IRDs) represent a frequent cause of blindness in children and adults. As a consequence of the phenotype and genotype heterogeneity of the disease, it is difficult to have a specific diagnosis without molecular testing. To date, over 340 genes and loci have been associated with IRDs. We present the molecular finding of 191 individuals with IRD, analyzed by targeted next-generation sequencing (NGS). For 67 of them, we performed a family segregation study, considering a total of 126 relatives. A total of 359 variants were identified, 44 of which were novel. Genetic diagnostic yield was 41%. However, after stratifying the patients according to their clinical suspicion, diagnostic yield was higher for well-characterized diseases such as Stargardt disease (STGD), at 65%, and for congenital stationary night blindness 2 (CSNB2), at 64%. Diagnostic yield was higher in the patient group where family segregation analysis was possible (68%) and it was higher in younger (55%) than in older patients (33%). The results of this analysis demonstrated that targeted NGS is an effective method for establishing a molecular genetic diagnosis of IRDs. Furthermore, this study underlines the importance of segregation studies to understand the role of genetic variants with unknow pathogenic role.

Clinical and analytical validation of an 82-gene comprehensive genome-profiling panel for identifying and interpreting variants responsible for inherited retinal dystrophies.

Inherited retinal dystrophies comprise a clinically complex and heterogenous group of diseases characterized by visual impairment due to pathogenic variants of over 300 different genes. Accurately identifying the causative gene and associated variant is crucial for the definitive diagnosis and subsequent selection of precise treatments. Consequently, well-validated genetic tests are required in the clinical practice. Here, we report the analytical and clinical validation of a next-generation sequencing targeted gene panel, the PrismGuide IRD Panel System. This system enables comprehensive genome profiling of 82 genes related to inherited retinal dystrophies. The PrismGuide IRD Panel System demonstrated 100% (n = 43) concordance with Sanger sequencing in detecting single-nucleotide variants, small insertions, and small deletions in the target genes and also in assessing their zygosity. It also identified copy-number loss in four out of five cases. When assessing precision, we evaluated the reproducibility of variant detection with 2,160 variants in 144 replicates and found 100% agreement in terms of single-nucleotide variants (n = 1,584) and small insertions and deletions (n = 576). Furthermore, the PrismGuide IRD Panel System generated sufficient read depth for variant calls across the purine-rich and highly repetitive open-reading frame 15 region of RPGR and detected all five variants tested. These results show that the PrismGuide IRD Panel System can accurately and consistently detect single-nucleotide variants and small insertions and deletions. Thus, the PrismGuide IRD Panel System could serve as useful tool that is applicable in clinical practice for identifying the causative genes based on the detection and interpretation of variants in patients with inherited retinal dystrophies and can contribute to a precise molecular diagnosis and targeted treatments.

Clinical and genetic characterization of patients with eye diseases included in the Spanish Rare Diseases Patient Registry.

BACKGROUND: The low prevalence of rare diseases poses a significant challenge in advancing their understanding. This study aims to delineate the clinical and genetic characteristics of patients with rare eye diseases (RED) enrolled in the Spanish Rare Diseases Patient Registry. METHODS: A total of 864 patients from the registry database were included. Diseases were categorized into inherited retinal dystrophies (n=688); anterior segment diseases (n=48); congenital malformations (n=27); and syndromic diseases with ocular involvement including muscular (n=46), neurological (n=34), or metabolic (n=13); inflammatory diseases (n=4); and tumors (n=4). Data on visual acuity (VA) and/or visual field (VF), symptoms and signs, concurrent diseases in syndromic cases, age of onset and at diagnosis, affected genes, disability rating, inability to work and dependency grade recognition were collected. RESULTS: A mean diagnostic delay of 7 years from symptom onset was observed. Commonly reported symptoms included photophobia, night blindness, and progressive vision loss (≥57% of patients). Cataract was the most prevalent secondary disease (46%), with pseudophakia being the most common ocular surgery (26%). Hearing loss and cardiovascular diseases were the most prevalent concurrent systemic diseases (≥13%). Certificates of disability, incapacity for work, and dependency were held by 87%, 42%, and 19% of patients, respectively. Among the 719 patients with available VA data, 193 (27%) were blind, and 188 (26%) had moderate to severe visual impairment. Over half of the patients (54%) exhibited VF defects, and 216 (25%) had concentric contraction ≤5° or abolished VF. Most had genetic diseases with autosomal recessive (55%), autosomal dominant (30%), X-linked (9%), and mitochondrial (6%) patterns. One patient had mutations in both recessive USH2A and dominant RHO genes simultaneously. Of the 656 patients (75.7%) who underwent genetic testing, only 461 (70.3%) received a positive result (pathogenic or likely pathogenic mutations explaining the phenotype). We found 62 new gene variants related to RED not previously reported in databases of genetic variants related to specific phenotypes. CONCLUSIONS: This study delineates the clinical and genotypic profiles of RED in Spain. Genetic diseases, particularly retinal disorders, predominate, but a significant proportion of affected patients remain genetically undiagnosed, hindering potential gene therapy endeavors. Despite notable improvements in reducing diagnosis delays, it is still remarkable. RED frequently lead to disability and blindness among young populations.

High prevalence of exon-13 variants in USH2A-related retinal dystrophies in Taiwanese population.

BACKGROUND: Biallelic pathogenic variants in USH2A lead to Usher syndrome or non-syndromic retinitis pigmentosa, and shown to have geographical and ethnical distribution in previous studies. This study provided a deeper understanding of the detailed clinical features using multimodal imaging, genetic spectrum, and genotype-phenotype correlations of USH2A-related retinal dystrophies in Taiwan. RESULTS: In our cohort, the mean age at first visit was 47.66 ± 13.54 years, and the mean age at symptom onset, which was referred to the onset of nyctalopia and/or visual field constriction, was 31.21 ± 15.24 years. Among the variants identified, 23 (50%) were missense, 10 (22%) were splicing variants, 8 (17%) were nonsense, and 5 (11%) were frameshift mutations. The most predominant variant was c.2802T>G, which accounted for 21% of patients, and was located in exon 13. Patients with truncated alleles had significantly earlier symptom onset and seemly poorer disease progression regarding visual acuity, ellipsoid zone line length, and hypofluorescent lesions in the macula than those who had the complete gene. However, the clinical presentation revealed similar progression between patients with and without the c.2802T>G variant. During long-term follow-up, the patients had different ellipsoid zone line progression rates and were almost evenly distributed in the fast, moderate, and slow progression subgroups. Although a younger onset age and a smaller baseline intact macular area was observed in the fast progression subgroup, the results showed no significant difference. CONCLUSIONS: This is the first cohort study to provide detailed genetic and longitudinal clinical analyses of patients with USH2A-related retinal dystrophies in Taiwan. The mutated allele frequency in exon 13 was high in Taiwan due to the predominant c.2802T>G variant. Moreover, truncated variants greatly impacted disease progression and determined the length of therapeutic windows. These findings provide insight into the characteristics of candidates for future gene therapies.

PCYT1A deficiency disturbs fatty acid metabolism and induces ferroptosis in the mouse retina.

BACKGROUND: Inherited retinal dystrophies (IRDs) are a group of debilitating visual disorders characterized by the progressive degeneration of photoreceptors, which ultimately lead to blindness. Among the causes of this condition, mutations in the PCYT1A gene, which encodes the rate-limiting enzyme responsible for phosphatidylcholine (PC) de novo synthesis via the Kennedy pathway, have been identified. However, the precise mechanisms underlying the association between PCYT1A mutations and IRDs remain unclear. To address this knowledge gap, we focused on elucidating the functions of PCYT1A in the retina. RESULTS: We found that PCYT1A is highly expressed in Müller glial (MG) cells in the inner nuclear layer (INL) of the retina. Subsequently, we generated a retina-specific knockout mouse model in which the Pcyt1a gene was targeted (Pcyt1a-RKO or RKO mice) to investigate the molecular mechanisms underlying IRDs caused by PCYT1A mutations. Our findings revealed that the deletion of Pcyt1a resulted in retinal degenerative phenotypes, including reduced scotopic electroretinogram (ERG) responses and progressive degeneration of photoreceptor cells, accompanied by loss of cells in the INL. Furthermore, through proteomic and bioinformatic analyses, we identified dysregulated retinal fatty acid metabolism and activation of the ferroptosis signalling pathway in RKO mice. Importantly, we found that PCYT1A deficiency did not lead to an overall reduction in PC synthesis within the retina. Instead, this deficiency appeared to disrupt free fatty acid metabolism and ultimately trigger ferroptosis. CONCLUSIONS: This study reveals a novel mechanism by which mutations in PCYT1A contribute to the development of IRDs, shedding light on the interplay between fatty acid metabolism and retinal degenerative diseases, and provides new insights into the treatment of IRDs.

Establishment of an induced pluripotent stem cell line LEIi019-A from an early-onset retinal dystrophy patient with the autosomal dominant OTX2 c.259G>A variant.

The human induced pluripotent stem cell (iPSC) line LEIi019-A was generated from a patient with early-onset pattern dystrophy caused by a heterozygous mutation NM_001270525.1:c.259G>A (p.Glu87Lys) in OTX2. Patient-derived dermal fibroblasts were reprogrammed using episomal plasmids containing reprogramming factors OCT4, SOX2, KLF4, MYCL, LIN28, TP53 shRNA and miR-302/367. The iPSC line expressed pluripotency markers, displayed a normal 46,XY karyotype and demonstrated the ability to differentiate into the three primary germ layers, retinal organoids and retinal pigment epithelial cells.

Four Unique Genetic Variants in Three Genes Account for 62.7% of Early-Onset Severe Retinal Dystrophy in Chile: Diagnostic and Therapeutic Consequences.

Leber congenital amaurosis (LCA)/early-onset severe retinal dystrophy (EOSRD) stand as primary causes of incurable childhood blindness. This study investigates the clinical and molecular architecture of syndromic and non-syndromic LCA/EOSRD within a Chilean cohort (67 patients/60 families). Leveraging panel sequencing, 95.5% detection was achieved, revealing 17 genes and 126 variants (32 unique). CRB1, LCA5, and RDH12 dominated (71.9%), with CRB1 being the most prevalent (43.8%). Notably, four unique variants (LCA5 p.Glu415*, CRB1 p.Ser1049Aspfs*40 and p.Cys948Tyr, RDH12 p.Leu99Ile) constituted 62.7% of all disease alleles, indicating their importance for targeted analysis in Chilean patients. This study underscores a high degree of inbreeding in Chilean families affected by pediatric retinal blindness, resulting in a limited mutation repertoire. Furthermore, it complements and reinforces earlier reports, indicating the involvement of ADAM9 and RP1 as uncommon causes of LCA/EOSRD. These data hold significant value for patient and family counseling, pharmaceutical industry endeavors in personalized medicine, and future enrolment in gene therapy-based treatments, particularly with ongoing trials (LCA5) or advancing preclinical developments (CRB1 and RDH12).

Retinal Dystrophies Associated With Peripherin-2: Genetic Spectrum and Novel Clinical Observations in 241 Patients.

Purpose: To describe the clinical, electrophysiological and genetic spectrum of inherited retinal diseases associated with variants in the PRPH2 gene. Methods: A total of 241 patients from 168 families across 15 sites in 9 countries with pathogenic or likely pathogenic variants in PRPH2 were included. Records were reviewed for age at symptom onset, visual acuity, full-field ERG, fundus colour photography, fundus autofluorescence (FAF), and SD-OCT. Images were graded into six phenotypes. Statistical analyses were performed to determine genotype-phenotype correlations. Results: The median age at symptom onset was 40 years (range, 4-78 years). FAF phenotypes included normal (5%), butterfly pattern dystrophy, or vitelliform macular dystrophy (11%), central areolar choroidal dystrophy (28%), pseudo-Stargardt pattern dystrophy (41%), and retinitis pigmentosa (25%). Symptom onset was earlier in retinitis pigmentosa as compared with pseudo-Stargardt pattern dystrophy (34 vs 44 years; P = 0.004). The median visual acuity was 0.18 logMAR (interquartile range, 0-0.54 logMAR) and 0.18 logMAR (interquartile range 0-0.42 logMAR) in the right and left eyes, respectively. ERG showed a significantly reduced amplitude across all components (P < 0.001) and a peak time delay in the light-adapted 30-Hz flicker and single-flash b-wave (P < 0.001). Twenty-two variants were novel. The central areolar choroidal dystrophy phenotype was associated with 13 missense variants. The remaining variants showed marked phenotypic variability. Conclusions: We described six distinct FAF phenotypes associated with variants in the PRPH2 gene. One FAF phenotype may have multiple ERG phenotypes, demonstrating a discordance between structure and function. Given the vast spectrum of PRPH2 disease our findings are useful for future clinical trials.

Identifying an appropriate gene testing tool for inherited retinal dystrophy in Indonesia, a developing country.

INTRODUCTION: Inherited retinal dystrophy (IRD) is a group of untreated genetic ocular diseases that mostly affect young people. The number of patients with IRD worldwide, including in developing countries, is growing each year. This literature review aimed to investigate the current utilised genetic screening of IRD worldwide and to propose the most feasible genetics test and diagnostic method for IRD in developing countries, especially Indonesia. MATERIALS AND METHODS: A literature search was performed in PubMed and Google Scholar databases. Papers conducting wide genome sequencing, including panel sequencing (panel-seq), microarray, whole exome sequencing (WES), whole genome sequencing (WGS) and Sanger sequencing on patients with IRD, were included. Papers were sorted into several groups to visualise the sequencing technology's detection rate. Detection rate comparison analysis was done using the meta-regress protocol in the R program. Whereas the number of novel mutations in each testing tool each year was pooled and compared in the graph. RESULTS: After conducting the literature study, 37 papers were sorted from 451 results. Most studies conducted a panel-seq with 16 records followed by WES with seven records. The detection rate of the WES meta-analysis was 0.66, which was slightly better than the panel-seq with 0.55. The number of novel mutation discoveries fluctuated each year with panel-seq as the most prominent finder. Cost factors and the limitation of sequencing devices make panel-seq a more appropriate tool in Indonesia. CONCLUSION: The most effective selection for evaluated genetic testing was WES. Therefore, panel-seq is more suitable for first-tier genetic testing in Indonesia.

Next-generation sequencing to genetically diagnose a diverse range of inherited eye disorders in 15 consanguineous families from Pakistan.

Inherited retinal dystrophies (IRDs) are characterized by photoreceptor dysfunction or degeneration. Clinical and phenotypic overlap between IRDs makes the genetic diagnosis very challenging and comprehensive genomic approaches for accurate diagnosis are frequently required. While there are previous studies on IRDs in Pakistan, causative genes and variants are still unknown for a significant portion of patients. Therefore, there is a need to expand the knowledge of the genetic spectrum of IRDs in Pakistan. Here, we recruited 52 affected and 53 normal individuals from 15 consanguineous Pakistani families presenting non-syndromic and syndromic forms of IRDs. We employed single molecule Molecular Inversion Probes (smMIPs) based panel sequencing and whole genome sequencing to identify the probable disease-causing variants in these families. Using this approach, we obtained a 93% genetic solve rate and identified 16 (likely) causative variants in 14 families, of which seven novel variants were identified in ATOH7, COL18A1, MERTK, NDP, PROM1, PRPF8 and USH2A while nine recurrent variants were identified in CNGA3, CNGB1, HGSNAT, NMNAT1, SIX6 and TULP1. The novel MERTK variant and one recurrent TULP1 variant explained the intra-familial locus heterogeneity in one of the screened families while two recurrent CNGA3 variants explained compound heterozygosity in another family. The identification of variants in known disease-associated genes emphasizes the utilization of time and cost-effective screening approaches for rapid diagnosis. The timely genetic diagnosis will not only identify any associated systemic issues in case of syndromic IRDs, but will also aid in the acceleration of personalized medicine for patients affected with IRDs.

Phototoxicity avoidance is a potential therapeutic approach for retinal dystrophy caused by EYS dysfunction.

Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of IRD. However, the mechanism of photoreceptor cell degeneration by mutant EYS has not been fully elucidated. Here, we generated retinal organoids from induced pluripotent stem cells (iPSCs) derived from patients with EYS-associated retinal dystrophy (EYS-RD). In photoreceptor cells of RD organoids, both EYS and G protein-coupled receptor kinase 7 (GRK7), one of the proteins handling phototoxicity, were not in the outer segment, where they are physiologically present. Furthermore, photoreceptor cells in RD organoids were vulnerable to light stimuli, and especially to blue light. Mislocalization of GRK7, which was also observed in eys-knockout zebrafish, was reversed by delivering control EYS into photoreceptor cells of RD organoids. These findings suggest that avoiding phototoxicity would be a potential therapeutic approach for EYS-RD.

Two case reports of a novel missense mutation in the PNPLA6 gene in two siblings with chorioretinal dystrophy, hypogonadotropic hypogonadism, and cerebellar ataxia.

BACKGROUND: Boucher Neuhäuser Syndrome (BNS) is a rare disease with autosomal recessive inheritance defined by the classical triad; early-onset ataxia, hypogonadism and chorioretinal dystrophy. CASE PRESENTATION: We present two siblings diagnosed with BNS at midlife, identified with homozygous state of a novel PNPLA6 missense mutation. One healthy sibling and the mother were heterozygous carriers of the mutation. The proband presented with the classical triad and the other sibling presented with visual problems at first. The proband was referred to our department by a private Neurologist, in early adulthood, because of hypogonadism, cerebellar ataxia, axonal neuropathy, and chorioretinal dystrophy for further evaluation. The sibling was referred to our department for evaluation, at childhood, due to visual problems. Later, the patient displayed the triad of ataxia, hypogonadotropic hypogonadism, and chorioretinal dystrophy. The unusual medical history of the two siblings led to further examinations and eventually the diagnosis of the first BNS cases in Cyprus. WES-based ataxia in silico gene panel analysis revealed 15 genetic variants and further filtering analysis revealed the PNPLA6 c.3323G > A variant. Segregation analysis in the family with Sanger sequencing confirmed the PNPLA6 homozygous variant c.3323G > A, p.Arg1108Gln in exon 29. CONCLUSIONS: This highlights the importance of considering rare inherited causes of visual loss, spinocerebellar ataxia, or/and HH in a neurology clinic and the significant role of genetic sequencing in the diagnostic process.

Characteristics of Eyes With CRB1-Associated EOSRD/LCA: Age-Related Changes.

PURPOSE: To evaluate ocular and retinal features of CRB1-associated early onset severe retinal dystrophy/Leber congenital amaurosis (EOSRD/LCA) for age-related changes. DESIGN: Retrospective cohort study. METHODS: Sixteen pediatric patients with biallelic CRB1 EOSRD/LCA who had been followed for up to 18 years were reviewed. Results of comprehensive ophthalmic examinations-including visual acuity, refractive error, dark-adapted visual threshold, Goldmann perimetry, and macular optical coherence tomography (OCT)-were analyzed for significant age-related changes using mixed-effects models. RESULTS: Visual acuity dark-adapted visual sensitivity, and area of seeing visual field (all subnormal from the earliest ages recorded) declined with increasing age. Hyperopia was stable through childhood and adolescence. In CRB1 EOSRD/LCA, OCT extrafoveal inner and outer laminar thicknesses exceeded those in controls but varied little with age, and foveal metrics (depth, breadth, thickness at rim) differed significantly from those in controls, but variations in foveal metrics were not associated with declines in acuity. CONCLUSIONS: From the youngest ages, retinal and visual function is significantly subnormal and becomes progressively compromized. A goal of future therapies should be intervention at young ages, when there is more function to be rescued.

Adaptive Optics Retinal Imaging in RDH12-Associated Early Onset Severe Retinal Dystrophy.

Purpose: RDH12 is among the most common genes found in individuals with early-onset severe retinal (EOSRD). Adaptive optics scanning light ophthalmoscopy (AOSLO) enables resolution of individual rod and cone photoreceptors in the retina. This study presents the first AOSLO imaging of individuals with RDH12-associated EOSRD. Methods: Case series of patients who attended Moorfields Eye Hospital (London, UK). Spectral-domain optical coherence tomography, near-infrared reflectance (NIR), and blue autofluorescence imaging were analyzed. En face image sequences of photoreceptors were recorded using either of two AOSLO modalities. Cross-sectional analysis was undertaken for seven patients and longitudinal analysis for one patient. Results: Nine eyes from eight patients are presented in this case series. The mean age at the time of the assessment was 11.2 ± 6.5 years of age (range 7-29). A subfoveal continuous ellipsoid zone (EZ) line was present in eight eyes. Posterior pole AOSLO revealed patches of cone mosaics. Average cone densities at regions of interest 0.5° to the fovea ranged from 12,620 to 23,660 cells/mm2, whereas intercell spacing ranged from 7.0 to 9.7 µm. Conclusions: This study demonstrates that AOSLO can provide useful high-quality images in patients with EOSRD, even during childhood, with nystagmus, and early macular atrophy. Cones at the posterior pole can appear as scattered islands or, possibly later in life, as a single subfoveal conglomerate. Detailed image analysis suggests that retinal pigment epithelial stress and dysfunction may be the initial step toward degeneration, with NIR being a useful tool to assess retinal well-being in RDH12-associated EOSRD.

RPE65-Associated Retinal Dystrophies: Phenotypes and Treatment Effects with Voretigene Neparvovec.

Retinal dystrophies linked to the RPE65 gene are mostly fast-progressing retinal diseases, with childhood onset of night blindness and progressive visual loss up to the middle adult age. Rare phenotypes linked to this gene are known with congenital stationary night blindness or slowly progressing retinitis pigmentosa, as well as an autosomal dominant c.1430A>G (p.Asp477Gly) variant. This review gives an overview of the current knowledge of the clinical phenotypes, as well as experience with the efficacy and safety of the approved gene augmentation therapy voretigene neparvovec.

PRPH2-Related Retinal Dystrophies: Mutational Spectrum in 103 Families from a Spanish Cohort.

PRPH2, one of the most frequently inherited retinal dystrophy (IRD)-causing genes, implies a high phenotypic variability. This study aims to analyze the PRPH2 mutational spectrum in one of the largest cohorts worldwide, and to describe novel pathogenic variants and genotype-phenotype correlations. A study of 220 patients from 103 families recruited from a database of 5000 families. A molecular diagnosis was performed using classical molecular approaches and next-generation sequencing. Common haplotypes were ascertained by analyzing single-nucleotide polymorphisms. We identified 56 variants, including 11 novel variants. Most of them were missense variants (64%) and were located in the D2-loop protein domain (77%). The most frequently occurring variants were p.Gly167Ser, p.Gly208Asp and p.Pro221_Cys222del. Haplotype analysis revealed a shared region in families carrying p.Leu41Pro or p.Pro221_Cys222del. Patients with retinitis pigmentosa presented an earlier disease onset. We describe the largest cohort of IRD families associated with PRPH2 from a single center. Most variants were located in the D2-loop domain, highlighting its importance in interacting with other proteins. Our work suggests a likely founder effect for the variants p.Leu41Pro and p.Pro221_Cys222del in our Spanish cohort. Phenotypes with a primary rod alteration presented more severe affectation. Finally, the high phenotypic variability in PRPH2 hinders the possibility of drawing genotype-phenotype correlations.

Genotype-Phenotype of CRB1-Associated Early-Onset Retinal Dystrophy: Novel Insights on Retinal Architecture and Therapeutic Window for Clinical Trials.

Purpose: The purpose of this study was to investigate the genotypic and phenotypic characteristics of CRB1-associated early onset retinal dystrophy (CRB1-eoRD) and retinal architecture by swept-source optical coherence tomography (SS-OCT). Methods: Eleven probands with CRB1-eoRD were recruited. Clinical information, genetic analysis, and comprehensive ophthalmic examinations including SS-OCT and SS-OCT angiography (SS-OCTA) were conducted. Results: A total of 81.8% (9/11) of CRB1-eoRD presented as Leber congenital amaurosis (LCA). Common clinical manifestations included coin-like yellow-white retinal spots (20/22, 90.9%) and para-arteriolar retinal pigment epithelial retention (12/22, 54.5%). Nineteen different CRB1 variants were detected in our case series, including 12 missense, 3 frameshifts, 3 nonsense, and 1 splicing. Of them, 12 variants had been reported, and 7 were novel. SS-OCT showed thinner central macula (the LCA group, P < 0.0001), thicker total retina (P < 0.0001), thinner outer retina (P < 0.05), and thicker inner retina (P < 0.0001) compared with the healthy control. The inner/outer (I/O) retina thickness ratio of CRB1-eoRD was 3.0, higher than the healthy control of 1.2 and other inherited retinal diseases (IRDs) of 2.2 (P < 0.0001 and P = 0.0027, respectively). SS-OCTA revealed an increased vascular density and perfusion area of the superficial vascular complex and deep vascular complex in CRB1-eoRD. Conclusions: LCA emerges as a frequently occurring phenotype in CRB1-eoRD. The unique features of SS-OCT and SS-OCTA are illustrated, and the novel biomarker, I/O ratio, may facilitate early diagnosis. The insights gained from this study hold significant value in determining the treatment window for potential forthcoming CRB1 gene therapy.