Determinants of disease penetrance in PRPF31-associated retinopathy

Retinitis pigmentosa 11 (RP11) is caused by dominant mutations in PRPF31, however a significant proportion of mutation carriers do not develop retinopathy. Here, we investigated the relationship between CNOT3 polymorphism, MSR1 repeat copy number and disease penetrance in RP11 patients and non-penetrant carriers (NPCs). We further characterized PRPF31 and CNOT3 expression in fibroblasts from eight RP11 patients and one NPC from a family carrying the c.1205C>T variant. Retinal organoids (ROs) and retinal pigment epithelium (RPE) were differentiated from induced pluripotent stem cells derived from RP11 patients, an NPC and a control subject. All RP11 patients were homozygous for the 3-copy MSR1 repeat in the PRPF31 promoter, while 3/5 NPCs carried a 4-copy MSR1 repeat. The CNOT3 rs4806718 genotype did not correlate with disease penetrance. PRFP31 expression declined with age in adult cadaveric retina. PRPF31 and CNOT3 expression was reduced in RP11 fibroblasts, RO and RPE compared with controls. Both RP11 and NPC RPE displayed shortened primary cilia compared with controls, however a subpopulation of cells with normal cilia lengths was present in NPC RPE monolayers. Our results indicate that RP11 non-penetrance is associated with the inheritance of a 4-copy MSR1 repeat, but not with CNOT3 polymorphisms

Stargardt disease and progress in therapeutic strategies

Background: Stargardt disease (STGD1) is an autosomal recessive retinal dystrophy due to mutations in ABCA4, characterized by subretinal deposition of lipofuscin-like substances and bilateral centrifugal vision loss. Despite the tremendous progress made in the understanding of STGD1, there are no approved treatments to date. This review examines the challenges in the development of an effective STGD1 therapy. Materials and Methods: A literature review was performed through to June 2021 summarizing the spectrum of retinal phenotypes in STGD1, the molecular biology of ABCA4 protein, the in vivo and in vitro models used to investigate the mechanisms of ABCA4 mutations and current clinical trials.Results: STGD1 phenotypic variability remains an challenge for clinical trial design and patient selection. Pre-clinical development of therapeutic options has been limited by the lack of animal models reflecting the diverse phenotypic spectrum of STDG1. Patient-derived cell lines have facilitated the characterization of splice mutations but the clinical presentation is not always predicted by the effect of specific mutations on retinoid metabolism in cellular models. Current therapies primarily aim to delay vision loss whilst strategies to restore vision are less well developed. Conclusions: STGD1 therapy development can be accelerated by a deeper understanding of genotype-phenotype correlations

Using induced pluripotent stem cell-derived retinal pigment epithelial cells to model splicing defects of ABCA4 c.5461-10T > C detected in an Australian Stargardt disease cohort

Purpose : Stargardt disease (STGD1, OMIM: 248200) is mainly caused by missense, frameshifting or nonsense mutations in the ATP-binding cassette transporter gene, ABCA4. However, sequence variants that alter splicing are also pathogenic. Herein, we describe an in vitro investigation of aberrant splicing in ABCA4 variants detected in a STGD1 cohort using patient-derived fibroblast-based assay. In addition, retinal pigment epithelium (RPE) cells differentiated from patient-derived induced pluripotent stem cells (iPSC) were used to further validate such splicing errors. Methods : A cohort of 68 patients clinically diagnosed with STGD1 were recruited in this study. Genomic DNA obtained from recruited STGD1 patients was analysed by a commercial Stargardt/Macular dystrophy screening panel, targeting all exons of ABCA4 and flanking intronic regions, as well as already-known deep-intronic variants of ABCA4. Fibroblasts were propagated from 68 patients, total RNA was extracted and ABCA4 transcript structure was analysed by RT-PCR. The iPSCs reprogrammed from 2 patients carrying heterozygous c.[5461-10T>C;5603A>T] alleles were differentiated into RPE cells and the ABCA4 transcripts re-examined by RT-PCR. Results : A total of 73 unique ABCA4 alleles were identified. Biallelic ABCA4 variants were detected in 66 patients (66/68, 97.06%) and 2 patients (2/68, 2.94%) had a single ABCA4 variant detected. Only exons 13-50 of ABCA4 could be readily amplified from fibroblast RNA. In this region, 9 out of 55 (16.36%) variants, carried by 19 patients (28%), resulted in aberrant splicing. The most prevalent splice variant, c.5461-10T>C, is complexed with c.5603A>T and carried heterozygously by 7 patients (10%). This variant results in mature ABCA4 mRNA transcripts missing exon 39, or exons 39 and 40. The splicing defect was also evident in patient-derived iPSC-RPE cells. Conclusions : Patient-derived fibroblasts are useful for identifying ABCA4 splicing variants affecting exons 13-50. The iPSC-RPE cells provide a feasible platform for further validating retina-specific splice variants of ABCA4 that may be amendable to splice intervention therapies

Genotypic and phenotypic spectrum of foveal hypoplasia : a multicenter study

Purpose To characterize the genotypic and phenotypic spectrum of foveal hypoplasia (FH). Design Multicenter, observational study. Participants A total of 907 patients with a confirmed molecular diagnosis of albinism, PAX6, SLC38A8, FRMD7, AHR, or achromatopsia from 12 centers in 9 countries (n = 523) or extracted from publicly available datasets from previously reported literature (n = 384). Methods Individuals with a confirmed molecular diagnosis and availability of foveal OCT scans were identified from 12 centers or from the literature between January 2011 and March 2021. A genetic diagnosis was confirmed by sequence analysis. Grading of FH was derived from OCT scans. Main Outcome Measures Grade of FH, presence or absence of photoreceptor specialization (PRS+ vs. PRS–), molecular diagnosis, and visual acuity (VA). Results The most common genetic etiology for typical FH in our cohort was albinism (67.5%), followed by PAX6 (21.8%), SLC38A8 (6.8%), and FRMD7 (3.5%) variants. AHR variants were rare (0.4%). Atypical FH was seen in 67.4% of achromatopsia cases. Atypical FH in achromatopsia had significantly worse VA than typical FH (P < 0.0001). There was a significant difference in the spectrum of FH grades based on the molecular diagnosis (chi-square = 60.4, P < 0.0001). All SLC38A8 cases were PRS– (P = 0.003), whereas all FRMD7 cases were PRS+ (P < 0.0001). Analysis of albinism subtypes revealed a significant difference in the grade of FH (chi-square = 31.4, P < 0.0001) and VA (P = 0.0003) between oculocutaneous albinism (OCA) compared with ocular albinism (OA) and Hermansky–Pudlak syndrome (HPS). Ocular albinism and HPS demonstrated higher grades of FH and worse VA than OCA. There was a significant difference (P < 0.0001) in VA between FRMD7 variants compared with other diagnoses associated with FH. Conclusions We characterized the phenotypic and genotypic spectrum of FH. Atypical FH is associated with a worse prognosis than all other forms of FH. In typical FH, our data suggest that arrested retinal development occurs earlier in SLC38A8, OA, HPS, and AHR variants and later in FRMD7 variants. The defined time period of foveal developmental arrest for OCA and PAX6 variants seems to demonstrate more variability. Our findings provide mechanistic insight into disorders associated with FH and have significant prognostic and diagnostic value