The splicing factor Prpf31 is required for hematopoietic stem and progenitor cell expansion during zebrafish embryogenesis.

Pre-mRNA splicing is a precise regulated process and is crucial for system development and homeostasis maintenance. Mutations in spliceosomal components have been found in various hematopoietic malignancies (HMs) and have been considered as oncogenic derivers of HMs. However, the role of spliceosomal components in normal and malignant hematopoiesis remains largely unknown. Pre-mRNA processing factor 31 (PRPF31) is a constitutive spliceosomal component, which mutations are associated with autosomal dominant retinitis pigmentosa. PRPF31 was found to be mutated in several HMs, but the function of PRPF31 in normal hematopoiesis has not been explored. In our previous study, we generated a prpf31 knockout (KO) zebrafish line and reported that Prpf31 regulates the survival and differentiation of retinal progenitor cells by modulating the alternative splicing of genes involved in mitosis and DNA repair. In this study, by using the prpf31 KO zebrafish line, we discovered that prpf31 KO zebrafish exhibited severe defects in hematopoietic stem and progenitor cell (HSPC) expansion and its sequentially differentiated lineages. Immunofluorescence results showed that Prpf31-deficient HSPCs underwent malformed mitosis and M phase arrest during HSPC expansion. Transcriptome analysis and experimental validations revealed that Prpf31 deficiency extensively perturbed the alternative splicing of mitosis-related genes. Collectively, our findings elucidate a previously undescribed role for Prpf31 in HSPC expansion, through regulating the alternative splicing of mitosis-related genes.

The Retinal Pigment Epithelium: Cells That Know the Beat!

The retinal pigment epithelium (RPE) ensures different functions crucial for photoreceptor survival, and thus for vision, such as photoreceptor outer segments (POS) phagocytosis and retinal adhesion. Both follow a circadian rhythm with an activity peak occurring respectively 1.5-2 and 3.5 h after light onset. Interestingly, we showed that two rodent models, ß5-/- and Prpf31+/- mice, display distinct alterations in both functions leading to different phenotypes. Indeed, the phagocytic peak totally disappears in ß5 knockout mice but is attenuated and shifted in Prpf31+/- mice. Conversely, the retinal adhesion peak only attenuated in ß5-/- mice is lost in Prpf31+/- mice. These distinct alterations have different consequences on retinal homeostasis proportional to the observed defects: ß5-/- mice progressively lose vision and accumulate RPE lipofuscin deposits, while Prpf31+/- mice develop RPE metabolic dysfunctions and gradual structural modifications indicative of cellular stress. Hence, animal models are useful to understand the importance of the proper regulation of these functions.

Clinical characterization of patients with PRPF31-related retinitis pigmentosa and asymptomatic carriers: a cross-sectional study.

BACKGROUND/AIM: To describe the clinical phenotype of retinitis pigmentosa (RP) caused by PRPF31-variants and clinical characterization of asymptomatic PRPF31 carriers. MATERIALS AND METHODS: We conducted a descriptive cross-sectional deep phenotyping study. We included subjects with PRPF31 variants predicted to be disease-causing, both individuals with RP and asymptomatic carriers. Participants underwent a comprehensive clinical examination of standard visual function parameters (visual acuity, contrast sensitivity, Goldmann visual field), full-field stimulus threshold (FST), full-field electroretinogram (ff-ERG), and a structural investigation with slit lamp and multimodal imaging. We used Spearman correlation analyses to evaluate associations between quantitative outcomes. RESULTS: We included 21 individuals with disease-causing PRPF31-variants: 16 symptomatic and 5 asymptomatic subjects. The symptomatic subjects demonstrated a typical RP phenotype with constricted visual fields, extinguished ff-ERG, and disrupted outer retinal anatomy. FST was impaired and correlated significantly with other outcome measures in RP subjects. Structure-function correlations with Spearman correlation analysis showed moderate correlation coefficients due to a few outliers in each analysis. The asymptomatic individuals had normal best-corrected visual acuity and visual fields, but showed reduced ff-ERG amplitudes, borderline FST sensitivity, and structural abnormalities on OCT and fundoscopy. CONCLUSIONS: RP11 has a typical RP phenotype but varies in terms of severity. FST measurements correlated well with other functional and structural metrics and may be a reliable outcome measure in future trials as it is sensitive to a broad range of disease severities. Asymptomatic carriers showed sub-clinical disease manifestations, and our findings underline that reported non-penetrance in PRPF31-related RP is not an all-or-none phenomenon.

Targeted nanopore sequencing enables complete characterisation of structural deletions initially identified using exon-based short-read sequencing strategies.

BACKGROUND: The widespread adoption of exome sequencing has greatly increased the rate of genetic diagnosis for inherited conditions. However, the detection and validation of large deletions remains challenging. While numerous bioinformatics approaches have been developed to detect deletions from whole - exome sequencing and targeted panels, further work is typically required to define the physical breakpoints or integration sites. Accurate characterisation requires either expensive follow - up whole - genome sequencing or the time - consuming, laborious process of PCR walking, both of which are challenging when dealing with the repeat sequences which frequently intersect deletion breakpoints. The aim of this study was to develop a cost-effective, long-range sequencing method to characterise deletions. METHODS: Genomic DNA was amplified with primers spanning the deletion using long-range PCR and the products purified. Sequencing was performed on MinION flongle flowcells. The resulting fast5 files were basecalled using Guppy, trimmed using Porechop and aligned using Minimap2. Filtering was performed using NanoFilt. Nanopore sequencing results were verified by Sanger sequencing. RESULTS: Four cases with deletions detected following comparative read-depth analysis of targeted short-read sequencing were analysed. Nanopore sequencing defined breakpoints at the molecular level in all cases including homozygous breakpoints in EYS, CNGA1 and CNGB1 and a heterozygous deletion in PRPF31. All breakpoints were verified by Sanger sequencing. CONCLUSIONS: In this study, a quick, accurate and cost - effective method is described to characterise deletions identified from exome, and similar data, using nanopore sequencing.

Genetic Modifiers of Non-Penetrance and RNA Expression Levels in PRPF31-Associated Retinitis Pigmentosa in a Danish Cohort.

(1) Background/aims: To examine potential genetic modifiers of disease penetrance in PRPF31-associated retinitis pigmentosa 11 (RP11). (2) Methods: Blood samples from individuals (n = 37) with PRPF31 variants believed to be disease-causing were used for molecular genetic testing and, in some cases (n = 23), also for mRNA expression analyses. Medical charts were used to establish if individuals were symptomatic (RP) or asymptomatic non-penetrant carriers (NPC). RNA expression levels of PRPF31 and CNOT3 were measured on peripheral whole blood using quantitative real-time PCR normalized to GAPDH. Copy number variation of minisatellite repeat element 1 (MSR1) was performed with DNA fragment analysis. (3) Results: mRNA expression analyses on 22 individuals (17 with RP and 5 non-penetrant carriers) revealed no statistically significant differences in PRPF31 or CNOT3 mRNA expression levels between individuals with RP and non-penetrant carriers. Among 37 individuals, we found that all three carriers of a 4-copy MSR1 sequence on their wild-type (WT) allele were non-penetrant carriers. However, copy number variation of MSR1 is not the sole determinant factor of non-penetrance, as not all non-penetrant carriers carried a 4-copy WT allele. A 4-copy MSR1 mutant allele was not associated with non-penetrance. (4) Conclusions: In this Danish cohort, a 4-copy MSR1 WT allele was associated with non-penetrance of retinitis pigmentosa caused by PRPF31 variants. The level of PRPF31 mRNA expression in peripheral whole blood was not a useful indicator of disease status.

Disease progression of retinitis pigmentosa caused by PRPF31 variants in a Nordic population: a retrospective study with up to 36 years follow-up.

BACKGROUND/AIMS: To investigate the natural history of PRPF31-related retinitis pigmentosa (RP11). MATERIALS AND METHODS: We identified individuals with RP11 and collected retrospective data from disease onset to present date including genetics, demographic data, Goldmann visual field areas, and visual acuity measurements. Visual fields were evaluated as summed squared degrees and best-corrected visual acuity was converted to logMAR. We performed linear mixed model regression analysis to evaluate annual disease progression, and survival analysis to evaluate the age of legal blindness. RESULTS: We included 46 subjects with RP11. Median age of disease onset was 10 years (range 5-65). Follow-up spanned from 0 to 36 years with a median of 8 years. Median Goldmann visual field areas decreased by 10.0% per year (95% CI 7.5%-12.4%) with target IV4e, 7.9% (95% CI 4.5% - 11.2%) with target III4e, and 9.3% (95% CI: 7.0% -11.5%) when combining target sizes. Individuals with RP11 maintained good visual acuity until late stage of disease. Legal blindness was reached at a median age of 57 years (95% CI 50-75 years). CONCLUSIONS: PRPF31 variants cause autosomal dominant retinitis pigmentosa that most commonly manifests in childhood with a variable disease progression. Visual field area deteriorates faster than visual acuity and was the major cause of legal blindness in our study population. This study characterizes disease progression in retinitis pigmentosa caused by PRPF31-variants and demonstrates the importance of differentiation between specific genotypes when counselling patients and conducting natural history studies of RP.

A 69 kb Deletion in chr19q13.42 including PRPF31 Gene in a Chinese Family Affected with Autosomal Dominant Retinitis Pigmentosa.

We aimed to identify the genetic cause of autosomal dominant retinitis pigmentosa (adRP) and characterize the underlying molecular mechanisms of incomplete penetrance in a Chinese family affected with adRP. All enrolled family members underwent ophthalmic examinations. Whole-genome sequencing (WGS), multiplex ligation-dependent probe amplification (MLPA), linkage analysis and haplotype construction were performed in all participants. RNA-seq was performed to analyze the regulating mechanism of incomplete penetrance among affected patients, mutation carriers and healthy controls. In the studied family, 14 individuals carried a novel heterozygous large deletion of 69 kilobase (kb) in 19q13.42 encompassing exon 1 of the PRPF31 gene and five upstream genes: TFPT, OSCAR, NDUFA3, TARM1, and VSTM1. Three family members were sequenced and diagnosed as non-penetrant carriers (NPCs). RNA-seq showed significant differential expression of genes in deletion between mutation carriers and healthy control. The RP11 pedigree in this study was the largest pedigree compared to other reported RP11 pedigrees with large deletions. Early onset in all affected members in this pedigree was considered to be a special phenotype and was firstly reported in a RP11 family for the first time. Differential expression of PRPF31 between affected and unaffected subjects indicates a haploinsufficiency to cause the disease in the family. The other genes with significant differential expression might play a cooperative effect on the penetrance of RP11.

Comprehensive analysis of the PRPF31 gene in retinitis pigmentosa patients: Four novel Alu-mediated copy number variations at the PRPF31 locus.

Retinitis pigmentosa (RP) is a monogenic disease characterized by irreversible degeneration of the retina. PRPF31, the second most common causative gene of autosomal dominant RP, frequently harbors copy number variations (CNVs), but the underlying mechanism is unclear. In this study, we summarized the phenotypic and genotypic characteristics of 18 RP families (F01-F18) with variants in PRPF31. The prevalence of PRPF31 variants in our cohort of Chinese RP families was 1.7% (18/1024). Seventeen different variants in PRPF31 were detected, including eight novel variants. Notably, four novel CNVs encompassing PRPF31, with a proportion of 22.2% (4/18), were validated to harbor gross deletions involving Alu/Alu-mediated rearrangements (AAMRs) in the same orientation. Among a total of 12 CNVs of PRPF31 with breakpoints mapped on nucleotide-resolution, 10 variants (83.3%) were presumably mediated by Alu elements. Furthermore, we described the correlation between the genotypes and phenotypes in PRPF31-related RP. Our findings expand the mutational spectrum of the PRPF31 gene and provide strong evidence that Alu elements of PRPF31 probably contribute to the susceptibility to genomic rearrangement in this locus.

PRPF31 interacts with PRPH2 confirmed by co-immunoprecipitation and co-localization.

Both PRPF31 and PRPH2 are the causative genes for retinitis pigmentosa. And both of them are associated with the balance of rhodopsin. In this study, we aim to investigate the co-expression and interaction of PRPF31 and PRPH2. We used PRPF31-eGFP, PRPF31-3xFlag and PRPH2-mCherry vectors were transfected into HEK293T and APRE-19 cells. Immunoblotting and co-immunoprecipitation (Co-IP) were used for gene expression validation and protein interaction. Immunofluorescence staining assay was used to test the co-localization analysis of PRPF31 and PRPH2. Co-IP experiments showed that PRPF31 could be pulled down with an anti-PRPH2 antibody. There was co-localization between PRPF31 and PRPH2 in HEK293T, APRE-19 and mouse retina. The Co-IP and co-localization experiments suggest that PRPF31 interacted with PRPH2.

Mesoporous Silica-Based Nanoparticles as Non-Viral Gene Delivery Platform for Treating Retinitis Pigmentosa.

BACKGROUND: Gene therapy is a therapeutic possibility for retinitis pigmentosa (RP), in which therapeutic transgenes are currently delivered to the retina by adeno-associated viral vectors (AAVs). Although their safety and efficacy have been demonstrated in both clinical and preclinical settings, AAVs present some technical handicaps, such as limited cargo capacity and possible immunogenicity in repetitive doses. The development of alternative, non-viral delivery platforms like nanoparticles is of great interest to extend the application of gene therapy for RP. METHODS: Amino-functionalized mesoporous silica-based nanoparticles (N-MSiNPs) were synthesized, physico-chemically characterized, and evaluated as gene delivery systems for human cells in vitro and for retinal cells in vivo. Transgene expression was evaluated by WB and immunofluorescence. The safety evaluation of mice subjected to subretinal injection was assessed by ophthalmological tests (electroretinogram, funduscopy, tomography, and optokinetic test). RESULTS: N-MSiNPs delivered transgenes to human cells in vitro and to retinal cells in vivo. No adverse effects were detected for the integrity of the retinal tissue or the visual function of treated eyes. N-MSiNPs were able to deliver a therapeutic transgene candidate for RP, PRPF31, both in vitro and in vivo. CONCLUSIONS: N-MSiNPs are safe for retinal delivery and thus a potential alternative to viral vectors.

Activation of autophagy reverses progressive and deleterious protein aggregation in PRPF31 patient-induced pluripotent stem cell-derived retinal pigment epithelium cells.

INTRODUCTION: Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. METHODS: We used the induced pluripotent stem cell (iPSC) technology to generate retinal organoids and RPE models from four patients with severe and very severe PRPF31-adRP, unaffected individuals and a CRISPR/Cas9 isogenic control. RESULTS: To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells were carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle and other RP-linked tri-snRNP proteins, which accumulated progressively with time. The mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wild-type levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells, altered morphology of nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin reduced cytoplasmic aggregates, leading to improved cell survival. CONCLUSIONS: Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients.

Landscape of pathogenic variants in six pre-mRNA processing factor genes for retinitis pigmentosa based on large in-house data sets and database comparisons.

PURPOSE: Variants in six genes encoding pre-mRNA processing factors (PRPFs) are a common cause of autosomal dominant retinitis pigmentosa (ADRP). This study aims to determine the characteristics of potential pathogenic variants (PPVs) in the six genes. METHODS: Variants in six PRPF genes were identified from in-house exome sequencing data. PPVs were identified based on comparative bioinformatics analysis, clinical phenotypes and the ACMG/AMP guidelines. The features of PPVs were revealed by comparative analysis of in-house data set, gnomAD and previously published literature. RESULTS: Totally, 36 heterozygous PPVs, including 19 novels, were detected from 45 families, which contributed to 4.4% (45/1019) of RP cases. These PPVs were distributed in PRPF31 (17/45, 37.8%), SNRNP200 (12/45, 26.7%), PRPF8 (10/45, 22.2%) and PRPF3 (6/45, 13.3%) but not in PRPF6 or PRPF4. Different types of PPVs were predominant in different PRPF genes, such as loss-of-function variants in PRPF31 and missense variants in the five remaining genes. The clustering of PPVs in specific regions was observed in SNRNP200, PRPF8 and PRPF3. The pathogenicity for certain classes of variants in these genes, such as loss-of-function variants in PRPF6 and missense variants in PRPF31 and PRPF4, requires careful consideration and further validation. The predominant fundus changes were early macular involvement, widespread RPE atrophy and pigmentation in the mid- and far-peripheral retina. CONCLUSION: Systemic comparative analysis may shed light on the characterization of PPVs in these genes. Our findings provide a brief landscape of PPVs in PRPF genes and the associated phenotypes and emphasize the careful classification of pathogenicity for certain types of variants that warrant further characterization.

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.

Predicting lncRNA-Protein Interaction With Weighted Graph-Regularized Matrix Factorization.

Long non-coding RNAs (lncRNAs) are widely concerned because of their close associations with many key biological activities. Though precise functions of most lncRNAs are unknown, research works show that lncRNAs usually exert biological function by interacting with the corresponding proteins. The experimental validation of interactions between lncRNAs and proteins is costly and time-consuming. In this study, we developed a weighted graph-regularized matrix factorization (LPI-WGRMF) method to find unobserved lncRNA-protein interactions (LPIs) based on lncRNA similarity matrix, protein similarity matrix, and known LPIs. We compared our proposed LPI-WGRMF method with five classical LPI prediction methods, that is, LPBNI, LPI-IBNRA, LPIHN, RWR, and collaborative filtering (CF). The results demonstrate that the LPI-WGRMF method can produce high-accuracy performance, obtaining an AUC score of 0.9012 and AUPR of 0.7324. The case study showed that SFPQ, SNHG3, and PRPF31 may associate with Q9NUL5, Q9NUL5, and Q9UKV8 with the highest linking probabilities and need to further experimental validation.

Clinical Evidence for the Importance of the Wild-Type PRPF31 Allele in the Phenotypic Expression of RP11.

PRPF31-associated retinopathy (RP11) is a common form of autosomal dominant retinitis pigmentosa (adRP) that exhibits wide variation in phenotype ranging from non-penetrance to early-onset RP. Herein, we report inter-familial and intra-familial variation in the natural history of RP11 using multimodal imaging and microperimetry. Patients were recruited prospectively. The age of symptom onset, best-corrected visual acuity, microperimetry mean sensitivity (MS), residual ellipsoid zone span and hyperautofluorescent ring area were recorded. Genotyping was performed using targeted next-generation and Sanger sequencing and copy number variant analysis. PRPF31 mutations were found in 14 individuals from seven unrelated families. Four disease patterns were observed: (A) childhood onset with rapid progression (N = 4), (B) adult-onset with rapid progression (N = 4), (C) adult-onset with slow progression (N = 4) and (D) non-penetrance (N = 2). Four different patterns were observed in a family harbouring c.267del; patterns B, C and D were observed in a family with c.772_773delins16 and patterns A, B and C were observed in 3 unrelated individuals with large deletions. Our findings suggest that the RP11 phenotype may be related to the wild-type PRPF31 allele rather than the type of mutation. Further studies that correlate in vitro wild-type PRPF31 allele expression level with the disease patterns are required to investigate this association.

Exploring microperimetry and autofluorescence endpoints for monitoring disease progression in PRPF31-associated retinopathy.

BACKGROUND: Mutations in the splicing factor pre-messenger RNA processing factor 31 (PRPF31) gene cause autosomal dominant retinitis pigmentosa 11 (RP11) through a haplo-insufficiency mechanism. We describe the phenotype and progression of microperimetry and autofluorescence endpoints in an Indigenous Australian RP11 family. PATIENTS AND METHODS: Ophthalmic examination, optical coherence tomography, fundus autofluorescence and microperimetry were performed at baseline and every 6-12 months. Baseline and annual change in best-corrected visual acuity (BCVA), microperimetry mean sensitivity (MS) and number of scotoma loci, residual ellipsoid zone (EZ) span and hyperautofluorescent ring (HAR) area were reported. Next-generation and Sanger sequencing were performed in available members. RESULTS: 12 affected members from three generations were examined. Mean (SD, range) age at onset of symptoms was 11 (4.5, 4-19) years. MS declined steadily from the third decade and EZ span and HAR area declined rapidly during the second decade. Serial microperimetry showed negligible change in MS over 2-3 years. However, mean EZ span, near-infrared and short-wavelength HAR area reduction was 203 (6.4%) µm/year, 1.8 (8.7%) mm2/year and 1.1 (8.6%) mm2/year, respectively. Genetic testing was performed on 11 affected and 10 asymptomatic members and PRPF31 c.1205 C > A (p.Ser402Ter) mutation was detected in all affected and two asymptomatic members (non-penetrant carriers). CONCLUSIONS: Our findings suggest that in the studied cohort, the optimal window for therapeutic intervention is the second decade of life and residual EZ span and HAR area can be considered as efficacy outcome measures. Further studies on larger samples with different PRPF31 mutations and longer follow-up duration are recommended.

Identification of two novel PRPF31 mutations in Chinese families with non-syndromic autosomal dominant retinitis pigmentosa.

BACKGROUND: Retinitis pigmentosa is a heterogeneous group of inherited retinal diseases leading to progressive vision loss. It has been estimated that the etiology is still unclear in 22%-40% of cases, indicating that many novel pathogenic variations related to RP remain unidentified in many patients. In this study, our aim was to investigate the disease-causing variants and function of the variants in two Chinese families with non-syndromic autosomal dominant retinitis pigmentosa (adRP). METHODS: Clinical data and peripheral blood DNA samples were collected. Whole exome sequencing (WES) was conducted to screen for variations. Then, the expression of green fluorescent protein (GFP)-fused wild-type PRPF31 protein and its variants was evaluated via western blotting and GFP fluorescence detection in vitro. RESULTS: Two novel heterozygous variants of PRPF31 (NM_015629.4): c.855+5G>A and c.849_855del (p.Pro284Ilefs*35) were identified respectively in two families. The variant c.855+5G>A is co-segregated with the disease in adRP-01 family. The pedigree analysis result for c.849_855del (p. Pro284Ilefs*35) shows an inheritance pattern with incomplete penetrance for adRP-02 family. The RT-PCR analysis shows the PRPF31 gene c.855+5G>A leading to the missing from the 997th to the 1405th positions of the PRPF31 gene (NM_015629.4) cDNA. The expressions of the mutant GFP-fused PRPF31 protein were not detected in HEK293 cells or Cos7 cells via western blotting and immunofluorescence. CONCLUSIONS: Our findings identified two novel variants in PRPF31 in two Chinese families with adRP, expanding the mutational spectrum of this gene. Functional analysis reveals that these variants lead to the truncation of the PRPF31 protein.

A c.544_618del75bp mutation in the splicing factor gene PRPF31 is involved in non-syndromic retinitis pigmentosa by reducing the level of mRNA expression.

PURPOSE: A previous study reported a novel c.544_618del75bp mutation in exon 7 of the PRPF31 gene in a Chinese family with autosomal dominant retinal pigmentosa (ADRP). However, the selected pedigree was a small part of the whole family and the function of the c.544_618del75bp mutation was not explored deeply. The aim of the present study was to validate the previous results and explore the functional significance of the c.544_618del75bp mutation. METHODS: We extended the size of the ADRP pedigree and sequenced DNA and cDNA of the PRPF31 gene for all members of the family and 100 healthy controls. Real-time quantitative polymerase chain reaction (PCR) analysis was performed on the cDNA of patients in the family and cell culture, plasmids transfection and western blot analysis were done to evaluate the functional effect of the mutation in vitro. RESULTS: Sanger sequencing showed that the mutation was present in all patients and absent in all normal individuals, except for participant III-9. Bioinformatics analysis revealed that the c.544_618del75bp mutation caused a 25 amino acid deletion in the PRPF31 protein. In addition, the mRNA expression assay revealed that the mRNA expression level of the PRPF31 and RP9 genes were significantly lower in RP patients than controls (p < 0.05). Finally, the in vitro transfection assay demonstrated that the mRNA expression level of the mutant transfection group was significantly lower than the wild-type transfection group (p < 0.05). CONCLUSIONS: Our study suggested that the c.544_618del75bp mutation in the PRPF31 gene was a causative mutation in this ADRP family and affected the expression of RP9 gene by influencing the formation of U4/U6-U5 tri-snRNP, eventually leading to the occurrence of RP.

RNA Splicing Factor Mutations That Cause Retinitis Pigmentosa Result in Circadian Dysregulation.

Circadian clocks regulate multiple physiological processes in the eye, but their requirement for retinal health remains unclear. We previously showed that Drosophila homologs of spliceosome proteins implicated in human retinitis pigmentosa (RP), the most common genetically inherited cause of blindness, have a role in the brain circadian clock. In this study, we report circadian phenotypes in murine models of RP. We found that mice carrying a homozygous H2309P mutation in Pre-mRNA splicing factor 8 (Prpf8) display a lengthened period of the circadian wheel-running activity rhythm. We show also that the daily cycling of circadian gene expression is dampened in the retina of Prpf8-H2309P mice. Surprisingly, molecular rhythms are intact in the eye cup, which includes the retinal pigment epithelium (RPE), even though the RPE is thought to be the primary tissue affected in this form of RP. Downregulation of Prp31, another RNA splicing factor implicated in RP, leads to period lengthening in a human cell culture model. The period of circadian bioluminescence in primary fibroblasts of human RP patients is not significantly altered. Together, these studies link a prominent retinal disorder to circadian deficits, which could contribute to disease pathology.

AAV-Mediated Gene Augmentation Therapy Restores Critical Functions in Mutant PRPF31+/- iPSC-Derived RPE Cells.

Retinitis pigmentosa (RP) is the most common form of inherited vision loss and is characterized by degeneration of retinal photoreceptor cells and the retinal pigment epithelium (RPE). Mutations in pre-mRNA processing factor 31 (PRPF31) cause dominant RP via haploinsufficiency with incomplete penetrance. There is good evidence that the diverse severity of this disease is a result of differing levels of expression of the wild-type allele among patients. Thus, we hypothesize that PRPF31-related RP will be amenable to treatment by adeno-associated virus (AAV)-mediated gene augmentation therapy. To test this hypothesis, we used induced pluripotent stem cells (iPSCs) with mutations in PRPF31 and differentiated them into RPE cells. The mutant PRPF31 iPSC-RPE cells recapitulate the cellular phenotype associated with the PRPF31 pathology, including defective cell structure, diminished phagocytic function, defects in ciliogenesis, and compromised barrier function. Treatment of the mutant PRPF31 iPSC-RPE cells with AAV-PRPF31 restored normal phagocytosis and cilia formation, and it partially restored structure and barrier function. These results suggest that AAV-based gene therapy targeting RPE cells holds therapeutic promise for patients with PRPF31-related RP.