Mutations In RPGRORF15 Research Articles

Retinal degeneration in rpgra mutant zebrafish.

Introduction: Pathogenic mutations in RPGR ORF15, one of two major human RPGR isoforms, were responsible for most X-linked retinitis pigmentosa cases. Previous studies have shown that RPGR plays a critical role in ciliary protein transport. However, the precise mechanisms of disease triggered by RPGR ORF15 mutations have yet to be clearly defined. There are two homologous genes in zebrafish, rpgra and rpgrb. Zebrafish rpgra has a single transcript homologous to human RPGR ORF15; rpgrb has two major transcripts: rpgrb ex1-17 and rpgrb ORF15, similar to human RPGR ex1-19 and RPGR ORF15, respectively. rpgrb knockdown in zebrafish resulted in both abnormal development and increased cell death in the dysplastic retina. However, the impact of knocking down rpgra in zebrafish remains undetermined. Here, we constructed a rpgra mutant zebrafish model to investigate the retina defect and related molecular mechanism. Methods: we utilized transcription activator-like effector nuclease (TALEN) to generate a rpgra mutant zebrafish. Western blot was used to determine protein expression. RT-PCR was used to quantify gene transcription levels. The visual function of embryonic zebrafish was detected by electroretinography. Immunohistochemistry was used to observe the pathological changes in the retina of mutant zebrafish and transmission electron microscope was employed to view subcellular structure of photoreceptor cells. Results: A homozygous rpgra mutant zebrafish with c.1675_1678delins21 mutation was successfully constructed. Despite the normal morphological development of the retina at 5 days post-fertilization, visual dysfunction was observed in the mutant zebrafish. Further histological and immunofluorescence assays indicated that rpgra mutant zebrafish retina photoreceptors progressively began to degenerate at 3-6 months. Additionally, the mislocalization of cone outer segment proteins (Opn1lw and Gnb3) and the accumulation of vacuole-like structures around the connecting cilium below the OSs were observed in mutant zebrafish. Furthermore, Rab8a, a key regulator of opsin-carrier vesicle trafficking, exhibited decreased expression and evident mislocalization in mutant zebrafish. Discussion: This study generated a novel rpgra mutant zebrafish model, which showed retinal degeneration. our data suggested Rpgra is necessary for the ciliary transport of cone-associated proteins, and further investigation is required to determine its function in rods. The rpgra mutant zebrafish constructed in this study may help us gain a better understanding of the molecular mechanism of retinal degeneration caused by RPGR ORF15 mutation and find some useful treatment in the future.

Retrospective Natural History Study of RPGR-Related Cone- and Cone-Rod Dystrophies While Expanding the Mutation Spectrum of the Disease.

Variants in the X-linked retinitis pigmentosa GTPase regulator gene (RPGR) and, specifically, in its retinal opening reading frame-15 isoform (RPGRORF15) may cause rod-cone (RCD), cone, and cone-rod dystrophies (CDs and CRDs). While RPGR-related RCDs have been frequently evaluated, the characteristics and progression of RPGR-related CD/CRDs are largely unknown. Therefore, the goal of our work was to perform genotype–phenotype correlations specifically in RPGRORF15-related CD/CRDs. This retrospective longitudinal study included 34 index patients and two affected relatives with a molecular diagnosis of RPGR-related CD/CRDs. Patients were recruited at the “Quinze-Vingts” Hospital, Paris, France and screened for mutations in RPGRORF15 at the Institut de la Vision, Paris, France. We identified 29 distinct variants, of which 27 were truncating. All were located in the 3′ half of the RPGRORF15 transcript. Twenty of them were novel. Fifteen subjects were affected by CD, the remaining had CRD. When analyzing the longitudinal data, a progressive decline in visual acuity (VA) was noted, with more than 60% of the patients reaching VA ≥ 1 LogMar in the best eye after the fifth decade of life. To our knowledge, this is the largest described study of a cohort of CD/CRD patients affected by RPGRORF15 variants. Longitudinal data showed a rapidly progressive disease, possibly locating an optimal window of intervention for future therapies in younger ages.

Homozygous females for a X-linked RPGR-ORF15 mutation in an Iranian family with retinitis pigmentosa

Mutations in Retinitis pigmentosa GTPase regulator gene (RPGR) are the most common cause of X-linked retinitis pigmentosa (RP). Almost 60% of disease-causing RPGR mutations are located in ORF-15 region which cannot be detected by Next Generation Sequencing (NGS) due to the existence of highly repetitive regions. An Iranian family with a priori diagnosis of autosomal dominant RP was studied by Sanger sequencing of ORF15 of RPGR gene after an inconclusive NGS result. A frameshift two-base-pair deletion (c.2323_2324del, p.Arg775Glufs*59) in this region was segregating in both affected hemizygous males and affected homozygous females. To our knowledge, this is the first example of homozygous females for RPGR-ORF15 mutations.

Restoration of RPGR expression in vivo using CRISPR/Cas9 gene editing

Mutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) cause the X-linked form of inherited retinal degeneration, and the majority are frameshift mutations in a highly repetitive, purine-rich region of RPGR known as the OFR15 exon. Truncation of the reading frame in this terminal exon ablates the functionally important C-terminal domain. We hypothesized that targeted excision in ORF15 by CRISPR/Cas9 and the ensuing repair by non-homologous end joining could restore RPGR reading frame in a portion of mutant photoreceptors thereby correcting gene function in vivo. We tested this hypothesis in the rd9 mouse, a naturally occurring mutant line that carries a frameshift mutation in RPGRORF15, through a combination of germline and somatic gene therapy approaches. In germline gene-edited rd9 mice, probing with RPGR domain-specific antibodies demonstrated expression of full length RPGRORF15 protein. Hallmark features of RPGR mutation-associated early disease phenotypes, such as mislocalization of cone opsins, were no longer present. Subretinal injections of the same guide RNA (sgRNA) carried in AAV sgRNA and SpCas9 expression vectors restored reading frame of RPGRORF15 in a subpopulation of cells with broad distribution throughout the retina, confirming successful correction of the mutation. These data suggest that a simplified form of genome editing mediated by CRISPR, as described here, could be further developed to repair RPGRORF15 mutations in vivo.

RPGR isoform imbalance causes ciliary defects due to exon ORF15 mutations in X-linked retinitis pigmentosa (XLRP).

Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause severe retinal ciliopathy, X-linked retinitis pigmentosa. Although two major alternatively spliced isoforms, RPGRex1-19 and RPGRORF15, are expressed, the relative importance of these isoforms in disease pathogenesis is unclear. Here, we analyzed fibroblast samples from eight patients and found that all of them form longer cilia than normal controls, albeit to different degrees. Although all mutant RPGRORF15 messenger RNAs (mRNAs) are unstable, their steady-state levels were similar or higher than those in the control cells, suggesting there may be increased transcription. Three of the fibroblasts that had higher levels of mutant RPGRORF15 mRNA also exhibited significantly higher levels of RPGRex1-19 mRNA. Four samples with unaltered RPGRex1-19 levels carried mutations in RPGRORF15 that resulted in this isoform being relatively less stable. Thus, in all cases, the RPGRex1-19/RPGRORF15 isoform ratio was increased, and this was highly correlative to the cilia extension defect. Moreover, overexpression of RPGRex1-19 (mimicking the increase in RPGRex1-19 to RPGRORF15 isoform ratio) or RPGRORF15 (mimicking reduction of the ratio) resulted in significantly longer or shorter cilia, respectively. Notably, the cilia length defect appears to be attributable to both the loss of the wild-type RPGRORF15 protein and to the higher levels of the RPGRex1-19 isoform, indicating that the observed defect is due to the altered isoform ratios. These results suggest that maintaining the optimal RPGRex1-9 to RPGRORF15 ratio is critical for cilia growth and that designing strategies that focus on the best ways to restore the RPGRex1-19/RPGRORF15 ratio may lead to better therapeutic outcomes.

Disruption of RPGR protein interaction network is the common feature of RPGR missense variations that cause XLRP

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease with severe vision impairment leading to blindness. About 10-15% of RP cases are caused by mutations in the RPGR gene, with RPGR mutations accounting for 70% of X-linked RP cases. The mechanism by which RPGR mutations cause photoreceptor cell dysfunction is not well understood. In this study, we show that the two isoforms of RPGR (RPGR1-19 and RPGRORF15) interact with endogenous PDE6D, INPP5E, and RPGRIP1L. The RPGR1-19 isoform contains two PDE6D binding sites with the C-terminal prenylation site being the predominant PDE6D binding site. The C terminus of RPGR1-19 that contains the prenylation site regulates its interaction with PDE6D, INPP5E, and RPGRIP1L. Only the RPGR1-19 isoform localizes to cilia in cultured RPE1 cells. Missense variations found in RPGR patients disrupt the interaction between RPGR isoforms and their endogenous interactors INPP5E, PDE6D, and RPGRIP1L. We evaluated a RPGR missense variation (M58K) found in a family with X-linked retinitis pigmentosa (XLRP) and show that this missense variation disrupts the interaction of RPGR isoforms with their endogenous interactors. The M58K variation also disrupts the ciliary localization of the RPGR1-19 isoform. Using this assay, we also show that some of the RPGR missense variants reported in the literature might not actually be disease causing. Our data establishes an in vitro assay that can be used to validate the potential pathogenicity of RPGR missense variants.

CLINICAL AND GENETIC CHARACTERISTICS OF MALE PATIENTS WITH RPGR-ASSOCIATED RETINAL DYSTROPHIES: A Long-Term Follow-up Study.

To describe the phenotype and clinical course of patients with RPGR-associated retinal dystrophies, and to identify genotype-phenotype correlations. A multicenter medical records review of 74 male patients with RPGR-associated retinal dystrophies. Patients had retinitis pigmentosa (RP; n = 52; 70%), cone dystrophy (COD; n = 5; 7%), or cone-rod dystrophy (CORD; n = 17; 23%). The median follow-up time was 11.6 years (range 0-57.1). The median age at symptom onset was 5.0 years (range 0-14 years) for patients with RP and 23.0 years (range 0-60 years) for patients with COD/CORD. The probability of being blind (best-corrected visual acuity <0.05) at the age of 40 was 20% and 55% in patients with RP and COD/CORD, respectively. RPGR-ORF15 mutations were associated with high myopia (P = 0.01), which led to a faster best-corrected visual acuity decline in patients with RP (P < 0.001) and COD/CORD (P = 0.03). Patients with RP with RPGR-ORF15 mutations had a faster visual field decline (P = 0.01) and thinner central retina (P = 0.03) than patients with mutations in exon 1 to 14. Based on best-corrected visual acuity survival probabilities, the intervention window for gene therapy for RPGR-associated retinal dystrophies is relatively broad in patients with RP. RPGR-ORF15 mutations were associated with COD/CORD and with a more severe phenotype in RP. High myopia is a risk factor for faster best-corrected visual acuity decline.

Development of High-Throughput Clinical Testing ofRPGRORF15 Using a Large Inherited Retinal Dystrophy Cohort

Mutations in the ORF15 region of RPGR account for approximately half of all X-linked retinitis pigmentosa cases. However, a robust high-throughput method for the detection of ORF15 mutations has yet to be validated. We set out to develop the first clinically validated next-generation sequencing (NGS) method for the detection of mutations in this difficult-to-sequence region, including test accuracy and coverage data. As part of a blind-test, 145 research samples, previously tested by Sanger sequencing, and 81 clinical samples were evaluated using NGS of long-range PCR products fragmented with Illumina's Nextera library preparation kit (method 1), or with Centrillion's OneTube technology, supplemented with duplication analysis using an ORF15-specific in-silico array (method 2). DNA fragments were analyzed using Agilent's DNA 1000 assay, and sequencing was done on Illumina's MiSeq 2×150 or HiSeq2500 2×100. NextGENe by SoftGenetics was used for data analysis and variant calling. The Nextera library preparation method produced 24 cases of discordance due to (in order of decreasing occurrence) false-negatives, incorrectly called variants, and a false-positive. Subsequent use of a new, OneTube NGS library preparation method, supplemented with duplication analyses, resolved discordance between Sanger and NGS data in all cases. This improvement in variant detection accuracy was largely attributed to improvement in random fragmentation offered by the enzymatic OneTube method, resulting in more complete coverage of the highly repetitive ORF15 region. Minimum coverage was roughly 320 reads for Nextera and 6800 reads for OneTube (normalized for total read counts). This paper documents the first clinically validated NGS method for reliable, high-throughput sequencing of RPGR ORF15. Sensitivity and specificity of the new method were 100%, with the caveat of unclear zygosity calling for one large duplication case. These findings demonstrate a reliable and practical implementation for NGS-based diagnosis of RPGR ORF15 mutations. They also provide the foundation for targeted, high-coverage sequencing of any other repetitive regions within the genome.

Visual Function in Carriers of X-Linked Retinitis Pigmentosa

To determine the frequency and severity of visual function loss in female carriers of X-linked retinitis pigmentosa (XLRP). Case series. Two hundred seventy-six XLRP carriers with cross-sectional data (n= 242) and longitudinal data (n= 34; median follow-up, 16 years; follow-up range, 3-37 years). Half of the carriers were from RPGR- or RP2-genotyped families. Retrospective medical records review. Visual acuities, visual field areas, final dark adaptation thresholds, and full-field electroretinography (ERG) responses to 0.5-Hz and 30-Hz flashes. In genotyped families, 40% of carriers showed a baseline abnormality on at least 1 of 3 psychophysical tests. There was a wide range of function among carriers. For example, 3 of 121 (2%) genotyped carriers were legally blind because of poor visual acuity, some as young as 35 years. Visual fields were less affected than visual acuity. In all carriers, the average ERG amplitude to 30-Hz flashes was approximately 50% of normal, and the average exponential rate of amplitude loss over time was half that of XLRP males (3.7%/year vs. 7.4%/year, respectively). Among obligate carriers with affected fathers, sons, or both, 53 of 55 (96%) had abnormal baseline ERG results. Some carriers who initially had completely normal fundi in both eyes went on to experience moderately decreased vision, although not legal blindness. Among carriers with RPGR mutations, those with mutations in ORF15, compared with those in exons 1-14, had worse final dark adaptation thresholds and lower 0.5-Hz and 30-Hz ERG amplitudes. Most carriers of XLRP had mildly or moderately reduced visual function but rarely became legally blind. In most cases, obligate carriers could be identified by ERG testing. Carriers of RPGR ORF15 mutations tended to have worse visual function than carriers of RPGR exon 1 through 14 mutations. Because XLRP carrier ERG amplitudes and decay rates over time were on average half of those of affected men, these observations were consistent with the Lyon hypothesis of random X-inactivation.

RPGR-Associated Retinal Degeneration in Human X-Linked RP and a Murine Model

We investigated the retinal disease due to mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene in human patients and in an Rpgr conditional knockout (cko) mouse model. XLRP patients with RPGR-ORF15 mutations (n = 35, ages at first visit 5-72 years) had clinical examinations, and rod and cone perimetry. Rpgr-cko mice, in which the proximal promoter and first exon were deleted ubiquitously, were back-crossed onto a BALB/c background, and studied with optical coherence tomography and electroretinography (ERG). Retinal histopathology was performed on a subset. Different patterns of rod and cone dysfunction were present in patients. Frequently, there were midperipheral losses with residual rod and cone function in central and peripheral retina. Longitudinal data indicated that central rod loss preceded peripheral rod losses. Central cone-only vision with no peripheral function was a late stage. Less commonly, patients had central rod and cone dysfunction, but preserved, albeit abnormal, midperipheral rod and cone vision. Rpgr-cko mice had progressive retinal degeneration detectable in the first months of life. ERGs indicated relatively equal rod and cone disease. At late stages, there was greater inferior versus superior retinal degeneration. RPGR mutations lead to progressive loss of rod and cone vision, but show different patterns of residual photoreceptor disease expression. Knowledge of the patterns should guide treatment strategies. Rpgr-cko mice had onset of degeneration at relatively young ages and progressive photoreceptor disease. The natural history in this model will permit preclinical proof-of-concept studies to be designed and such studies should advance progress toward human therapy.

Rd9 Is a Naturally Occurring Mouse Model of a Common Form of Retinitis Pigmentosa Caused by Mutations in RPGR-ORF15

Animal models of human disease are an invaluable component of studies aimed at understanding disease pathogenesis and therapeutic possibilities. Mutations in the gene encoding retinitis pigmentosa GTPase regulator (RPGR) are the most common cause of X-linked retinitis pigmentosa (XLRP) and are estimated to cause 20% of all retinal dystrophy cases. A majority of RPGR mutations are present in ORF15, the purine-rich terminal exon of the predominant splice-variant expressed in retina. Here we describe the genetic and phenotypic characterization of the retinal degeneration 9 (Rd9) strain of mice, a naturally occurring animal model of XLRP. Rd9 mice were found to carry a 32-base-pair duplication within ORF15 that causes a shift in the reading frame that introduces a premature-stop codon. Rpgr ORF15 transcripts, but not protein, were detected in retinas from Rd9/Y male mice that exhibited retinal pathology, including pigment loss and slowly progressing decrease in outer nuclear layer thickness. The levels of rhodopsin and transducin in rod outer segments were also decreased, and M-cone opsin appeared mislocalized within cone photoreceptors. In addition, electroretinogram (ERG) a- and b-wave amplitudes of both Rd9/Y male and Rd9/Rd9 female mice showed moderate gradual reduction that continued to 24 months of age. The presence of multiple retinal features that correlate with findings in individuals with XLRP identifies Rd9 as a valuable model for use in gaining insight into ORF15-associated disease progression and pathogenesis, as well as accelerating the development and testing of therapeutic strategies for this common form of retinal dystrophy.

Identification of NovelRPGRORF15 Mutations in X-linked Progressive Cone-Rod Dystrophy (XLCORD) Families

To test the incidence of mutations in RPGR ORF15 in six families with X-linked progressive retinal degeneration (cone-rod dystrophy [XLCORD], macular or cone dystrophy) and to undertake a detailed phenotypic assessment of families in whom ORF15 mutations were identified. To amplify and sequence ORF15 in its entirety, a cloning strategy was developed. Families with mutations in ORF15 underwent electrophysiological testing, color vision assessment, color fundus photography, and fundus autofluorescence (AF) imaging. Novel protein truncation mutations were identified in two families. In family A, a 2-bp mutation was identified in ORF15+A1094C G1095T, predicted to result in a truncated protein (E364D/E365X). In family B, a G-to-T transversion (ORF15+1176G>T) resulted in a nonsense mutation (G392X). Characteristics of phenotype in both families included progressive deterioration of central vision and subsequently night vision, mild photophobia, and moderate to high myopia. Ophthalmoscopic abnormalities were generally confined to the macula. A parafoveal ring of increased AF was observed, and electrophysiological evidence of a greater generalized abnormality in cone than rod responses were consistent with a cone-rod dystrophy phenotype. The cloning strategy for ORF15 facilitated comprehensive sequence analysis in patients. Two families were identified with nonsense mutations, and clinical evaluation revealed them both to have a similar phenotype. The presence of a parafoveal ring of increased AF was an early indicator of affected status in these families. No disease-causing mutations in ORF15 were detected in four other families, suggesting that ORF15 mutations may not be the most common cause of XLCORD.

Clinical studies of X-linked retinitis pigmentosa in three Swedish families with newly identified mutations in the RP2 and RPGR-ORF15 genes

Purpose: To describe new disease-causing RP2 and RPGR-ORF15 mutations and their corresponding clinical phenotypes in Swedish families with X-linked retinitis pigmentosa (XLRP) and to establish genotype-phenotype correlations by studying the clinical spectrum of disease in families with a known molecular defect. Methods: Seventeen unrelated families with RP and an apparent X-linked pattern of disease inheritance were identified from the Swedish RP registry and screened for mutations in the RP2 and RPGR (for the RP3 disease) genes. These families had been previously screened for the RPGR exons 1–19, and disease-causing mutations were identified in four of them. In the remaining 13 families, we sequenced the RP2 gene and the newly discovered RPGR-ORF exon. Detailed clinical evaluations were then obtained from individuals in the three families with identified mutations. Results: Mutations in RP2 and RPGRORF15 were identified in three of the 13 families. Clinical evaluations of affected males and carrier females demonstrated varying degrees of retinal dysfunction and visual handicap, with early onset and severe disease in the families with mutations in the ORF15 exon of the RPGR gene. Conclusions: A total of seven mutations in the RP2 and RPGR genes have been discovered so far in Swedish XLRP families. All affected individuals express a severe form of retinal degeneration with visual handicap early in life, although the degree of retinal dysfunction varies both in hemizygous male patients and in heterozygous carrier females. Retinal disease phenotypes in patients with mutations in the RPGR-ORF15 were more severe than in patients with mutations in RP2 or other regions of the RPGR .

A Comprehensive Mutation Analysis of RP2 and RPGR in a North American Cohort of Families with X-Linked Retinitis Pigmentosa

X-linked retinitis pigmentosa (XLRP) is a clinically and genetically heterogeneous degenerative disease of the retina. At least five loci have been mapped for XLRP; of these, RP2 and RP3 account for 10%-20% and 70%-90% of genetically identifiable disease, respectively. However, mutations in the respective genes, RP2 and RPGR, were detected in only 10% and 20% of families with XLRP. Mutations in an alternatively spliced RPGR exon, ORF15, have recently been shown to account for 60% of XLRP in a European cohort of 47 families. We have performed, in a North American cohort of 234 families with RP, a comprehensive screen of the RP2 and RPGR (including ORF15) genes and their 5' upstream regions. Of these families, 91 (39%) show definitive X-linked inheritance, an additional 88 (38%) reveal a pattern consistent with X-linked disease, and the remaining 55 (23%) are simplex male patients with RP who had an early onset and/or severe disease. In agreement with the previous studies, we show that mutations in the RP2 gene and in the original 19 RPGR exons are detected in <10% and approximately 20% of XLRP probands, respectively. Our studies have revealed RPGR-ORF15 mutations in an additional 30% of 91 well-documented families with X-linked recessive inheritance and in 22% of the total 234 probands analyzed. We suggest that mutations in an as-yet-uncharacterized RPGR exon(s), intronic changes, or another gene in the region might be responsible for the disease in the remainder of this North American cohort. We also discuss the implications of our studies for genetic diagnosis, genotype-phenotype correlations, and gene-based therapy.