Form Of Autosomal Dominant Retinitis Pigmentosa Research Articles

Retinitis Pigmentosa Alleles, in PRPF31 and PRPF6, Effects on Pre‐Messenger RNA Splicing

Retinitis pigmentosa (RP) is a genetically heterogeneous group of retinal disorders that affect 1 in 4,000 individuals in the United States. Patients with RP suffer from progressive degeneration of the light‐sensitive photoreceptors in the retina, which eventually leads to severe vision loss. The autosomal dominant form of RP (adRP) has been linked to eight splicing‐related proteins that affect the accuracy and efficiency of pre‐mRNA splicing. Among these splicing proteins, PRPF31 and PRPF6 are essential to the formation of the pre‐spliceosome. The purpose of this study is to examine the mechanisms by which adRP mutations in PRPF31 and PRPF6 affect pre‐mRNA splicing and to distinguish if these effects are widespread or locally more severe to the retina. This was done by constructing adRP allele orthologs, found first in human adRP patients, into two model organisms, Saccharomyces cerevisiae, and Mus musculus cell lines. First, we utilized S.cerevisiae to examine these mutations' effects on cell growth indicating a possible splicing defect and then examined splicing through in vitro splicing assays. Second, a mouse retinal ganglion precursor‐like cell line was utilized to identify various mRNAs effects of adRP mutants on splicing function and its effects on the phenotypic expression for PRPF6. In order to understand the role of pre‐mRNA splicing in photoreceptor gene expression and function, we have identified photoreceptor genes of interest whose pre‐mRNA splicing can potentially be affected by mutations in PRPF6. This is done by analyzing these genes of interest through splicing assays and immunofluorescent assays. Overall, this project adds a unique approach to studying how adRP alleles affect pre‐messenger RNA splicing.

Intravitreal Administration of Stanniocalcin-1 Rescues Photoreceptor Degeneration with Reduced Oxidative Stress and Inflammation in a Porcine Model of Retinitis Pigmentosa

To investigate the effect of stanniocalcin-1 (STC-1), a secreted polypeptide exhibiting multiple functions in cell survival and death, on photoreceptor degeneration in a porcine model of retinitis pigmentosa (RP). P23H transgenic pigs (TG P23H) and wild-type hybrid littermates were obtained from the National Swine Resource and Research Center. Human recombinant STC-1 was injected intravitreally every 2 weeks from postnatal day 15 (P15) to P75. The contralateral eye was injected with balanced salt solution as a control. Electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed to evaluate retinal function and morphology in vivo at P90. Retinal tissue was collected for histologic analysis and molecular assays to evaluate the antioxidative and anti-inflammatory mechanisms by which STC-1 may rescue photoreceptor degeneration. Intravitreal injection of STC-1 improved retinal function in TG P23H pigs with increased photopic and flicker ERG a- and b-wave amplitudes. Greater integrity of the ellipsoid zone (EZ) band on SD-OCT and morphologic rescue with preservation of cone photoreceptors were observed in STC-1-treated TG P23H pigs. STC-1 altered gene expression in TG P23H pig retina on microarray analysis and increased photoreceptor specific gene expression by reverse transcription-polymerase chain reaction analysis. STC-1 significantly decreased oxidative stress and the expressions of NLRP3 inflammasome, cleaved caspase-1, and IL-1β in TG P23H pig retina. Intravitreal administration of STC-1 enhances cone photoreceptor function, improves EZ integrity, and reduces retinal degeneration through antioxidative and anti-inflammatory effects in a large animal (pig) model of the most common form of autosomal dominant RP in the United States.

A new rhodopsin R135W mutation induces endoplasmic reticulum stress and apoptosis in retinal pigment epithelial cells

Rhodopsin mutations are associated with the autosomal-dominant form of retinitis pigmentosa (RP). Here we report simultaneous occurrence of RP associated with bilateral nanophthalmos and acute angle-closure glaucoma in patient with a new mutation in rhodopsin (R135W). ARPE-19 cells were transfected with myc-tagged wild-type (WT) and R135W rhodopsin constructs. The half-life of WT and R135W rhodopsin was analyzed via cycloheximide chase analysis. We found that R135W rhodopsin was accumulated in the endoplasmic reticulum (ER) and induced unfolded protein response (UPR) and apoptosis. Moreover, chaperone HSP70 alleviated ER stress and prevented apoptosis induced by R135W rhodopsin by attenuating UPR signaling. These findings reveal the novel pathogenic mechanism of RP and suggest that chaperone HSP70 has potential therapeutic significance for RP.

Bi-allelic Mutations in KLHL7 Cause a Crisponi/CISS1-like Phenotype Associated with Early-Onset Retinitis Pigmentosa

Crisponi syndrome (CS)/cold-induced sweating syndrome type 1 (CISS1) is a very rare autosomal-recessive disorder characterized by a complex phenotype with high neonatal lethality, associated with the following main clinical features: hyperthermia and feeding difficulties in the neonatal period, scoliosis, and paradoxical sweating induced by cold since early childhood. CS/CISS1 can be caused by mutations in cytokine receptor-like factor 1 (CRLF1). However, the physiopathological role of CRLF1 is still poorly understood. A subset of CS/CISS1 cases remain yet genetically unexplained after CRLF1 sequencing. In five of them, exome sequencing and targeted Sanger sequencing identified four homozygous disease-causing mutations in kelch-like family member 7 (KLHL7), affecting the Kelch domains of the protein. KLHL7 encodes a BTB-Kelch-related protein involved in the ubiquitination of target proteins for proteasome-mediated degradation. Mono-allelic substitutions in other domains of KLHL7 have been reported in three families affected by a late-onset form of autosomal-dominant retinitis pigmentosa. Retinitis pigmentosa was also present in two surviving children reported here carrying bi-allelic KLHL7 mutations. KLHL7 mutations are thus associated with a more severe phenotype in recessive than in dominant cases. Although these data further support the pathogenic role of KLHL7 mutations in a CS/CISS1-like phenotype, they do not explain all their clinical manifestations and highlight the high phenotypic heterogeneity associated with mutations in KLHL7.

Retinitis pigmentosa-associated rhodopsin mutant T17M induces endoplasmic reticulum (ER) stress and sensitizes cells to ER stress-induced cell death

Retinitis pigmentosa (RP) is a group of inherited diseases that primarily affect light‑sensitive rods and cones in the retina. Rhodopsin mutations, including the T17M mutation, are associated with the autosomal dominant form of retinitis pigmentosa (ADRP) and have been linked to abnormal protein folding. However, the molecular mechanisms underlying T17M rhodopsin‑induced retinal degeneration are yet to be elucidated. In the present study, Human embryonic kidney (HEK) 293 and ARPE‑19 cells were transfected with myc‑tagged wild‑type (WT) and T17M rhodopsin constructs. Cells were fixed and stained with anti‑myc antibodies and the localization of WT and T17M rhodopsin was visualized using immunofluorescence microscopy. Turnover rates of WT and T17M rhodopsin were measured using western blot analysis. In addition, endoplasmic reticulum (ER) stress‑induced cell death was analyzed in WT and T17M rhodopsin‑transfected cells using nuclear staining. Misfolded T17M rhodopsin was observed to be abnormally localized in the ER, while WT rhodopsin was predominantly found at the plasma membrane. Protein turnover analysis revealed that T17M rhodopsin was more rapidly degraded by proteasomes than WT rhodopsin. Furthermore, overexpression of T17M rhodopsin was observed to induce cell death and increase cytotoxicity; predisposing cells to ER stress‑induced cell death. These findings show novel insight into the properties of T17M rhodopsin and highlight the role of ER stress in T17M‑associated RP.

Gene mapping and mutation screeneng in candidate genes in a Chinese family of autosomal dominant retinitis pigmentosa

We wanted to find the gene defect in a Chinese pedigree with autosomal dominant form of retinitis pigmentosa (ADRP). A small Chinese family with retinitis pigmentosa was collected. The genetic analysis of the family suggested an autosomal dominant pattern. Microsatellite (STR) markers tightly linked to candidate genes for ADRP were selected for linkage analysis. We got a maximum LOD score of 0.87 between markers D19S210 and D19S418. Precursor mRNA-processing factor (PRPF) 31, 3, 8, rhodopsin (RHO), peripherin 2 (PRPH2 or RDS), rod outer segment protein 1 (ROM1), neural retina leucine zipper (NRL), cone-rod homeobox-containing (CRX), inosine-5-prime-monophosphate dehydrogenase, type I (IMPDH1) and retinitis pigmentosa 1 (RPI) were amplified by polymerase chain reaction (PCR) and screened by direct sequencing. One new sequence variation was found. It was the missence mutation c.148G > C (D50H) occurred in exon 1 of RDS gene which existed in all the effected individuals and one unaffected family member. The DNA sequence variation didn't cosegregate with the RP disease. We considered this transition was one new polymorphism which we speculate involved in the pathogenesis of ADRP and increased the risk of ADRP. Further study should be conducted to confirm the causative gene of this family.

Structural insights into retinitis pigmentosa from unfolding simulations of rhodopsin mutants

Disease-causing missense mutations in membrane proteins, such as rhodopsin mutations associated with the autosomal dominant form of retinitis pigmentosa (ADRP), are often linked to defects in folding and/or trafficking. The mechanical unfolding of wild-type rhodopsin was compared with that of 20 selected ADRP-linked mutants more or less defective in folding and retinal binding. Rhodopsin fold is characterized by networks of amino acids in the retinal and G-protein binding sites likely to play a role in the stability and function of the protein. The distribution of highly connected nodes in the network reflects the existence of a diffuse intramolecular communication inside and between the 2 poles of the helix bundle, which makes pathogenic mutations share similar phenotypes irrespective of topological and physicochemical differences between them. Because of this communication, the ADRP-linked rhodopsin mutations share a more or less marked ability to impair selected hubs in the protein structure network. The extent of this structural effect relates to the severity of the biochemical defect caused by mutation. The investigative strategy employed in this study is likely to apply to all structurally known membrane proteins particularly susceptible to misassembly-causing mutations.

Progressive renal injury from transgenic expression of human carbonic anhydrase IV folding mutants is enhanced by deficiency of p58 IPK

Mutations in the human carbonic anhydrase IV (hCAIV) have been associated with retinal degeneration in an autosomal-dominant form of retinitis pigmentosa (RP17). Prior in vitro cell culture studies confirmed that all of the RP17-associated hCAIV mutations cause protein misfolding, leading to endoplasmic reticulum (ER) stress-induced apoptosis in cells expressing the mutant proteins. To evaluate the physiological impacts of these folding mutants in other carbonic anhydrase IV-producing tissues, we generated two transgenic mouse lines expressing R219S or R14W hCAIV under control of the endogenous hCAIV promoter. Expression of either of these mutant proteins in kidneys caused progressive renal injury in male transgenic mice as evidenced by an age-dependent increase in the tubule cell apoptosis starting at approximately 20 weeks of age and vacuolization throughout the renal cortex in older mice. Up-regulation of the ER chaperone, BiP, was observed in the cells of the renal cortex of the male transgenic mice, suggesting ER stress as a causal factor for the renal injury. The renal injury inflicted by expression of the folding mutants was markedly enhanced by haploinsufficiency of the ER cochaperone p58(IPK). The transgenic mice expressing the hCAIV folding mutants on a p58(IPK) heterozygous background showed extensive renal tubular apoptosis by approximately 10 weeks of age in both male and female mice. These data indicate that expression of the RP17-associated folding mutants of hCAIV can adversely affect tissues beyond the retina and their in vivo proteotoxicity is sensitive to modulation of the protein folding environment of the ER.

Phenotypic Characterization of a Large Family With RP10 Autosomal-Dominant Retinitis Pigmentosa: An Asp226Asn Mutation in the IMPDH1 Gene

To evaluate the clinical features associated with the RP10 form of autosomal-dominant retinitis pigmentosa in 11 affected members of various ages from one family with a defined IMPDH1 mutation (Asp226Asn). Prospective, observational case series. Visual function assessment included visual acuity, color vision, visual field, dark adaptometry, full-field electroretinography (ffERG), and multifocal electroretinography (mfERG). Ophthalmologic examinations, fundus photography, and optical coherence tomographic scans were also performed. Blood samples were obtained to screen for basic immune function. Visual acuity was slightly reduced in the teenage years and substantially reduced in association with cystoid macular edema (CME) at all ages. Color defects were observed in three patients (one teen, two adults). Dark-adapted thresholds were elevated. Visual fields were markedly constricted by age 40 (<or=20 degrees). Rod and cone a-wave and b-wave ffERG responses were small or nondetectable by age 20, with greater rod than cone loss at all ages. The normal to significantly delayed ffERG cone b-wave implicit times in different patients were explained by their mfERG implicit times from the central retina. The amplification factors (log S) and recovery kinetics derived from the full-field rod a-waves were normal. Optical coherence tomography revealed subretinal fluid accumulation in the majority of eyes. Cystoid macular edema was diagnosed in four patients. No unusual immunologic findings were noted. The Asp226Asn mutation is associated with a severe, early-onset form of retinal degeneration in members of this family.

Chemical chaperones protect from effects of apoptosis-inducing mutation in carbonic anhydrase IV identified in retinitis pigmentosa 17.

Carbonic anhydrase (CA) IV is a glycosylphosphotidylinositol-anchored enzyme highly expressed on the plasma face of microcapillaries and especially strongly expressed in the choriocapillaris of the human eye. In collaboration with scientists at the University of Cape Town (Rondebosch, South Africa), we recently showed that the R14W mutation in the signal sequence of CA IV, which they identified in patients with the retinitis pigmentosa (RP) 17 form of autosomal dominant RP, results in accumulation of unfolded protein in the endoplasmic reticulum (ER), leading to ER stress, the unfolded protein response, and apoptosis in a large fraction of transfected COS-7 cells expressing mutant, but not wild-type, CA IV. Here we present experiments showing that several well characterized CA inhibitors largely prevent the adverse effects of expressing R14W CA IV in transfected COS-7 cells. Specifically, CA inhibitors prevent the accelerated turnover of the mutant protein, the up-regulation of Ig-binding protein, double-stranded RNA-regulated protein kinase-like ER kinase, and CCAAT/enhancer-binding protein homologous protein (markers of the unfolded protein response and ER stress), the inhibition of production of other secretory proteins expressed from COS-7-transfecting plasmids, and the induction of apoptosis, all characteristics of transfected cells expressing R14W CA IV. Furthermore, treatment with 4-phenylbutyric acid, a nonspecific chemical chaperone used in other protein-folding disorders, also dramatically reduces the apoptosis-inducing effect of expressing R14W CA IV cDNA in transfected COS-7 cells. These experiments suggest a promising approach to treatment of RP17 that might delay the onset or possibly prevent this autosomal dominant form of RP.

Structural and functional impairment of endocytic pathways by retinitis pigmentosa mutant rhodopsin-arrestin complexes.

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous degenerative eye disease. Mutations at Arg135 of rhodopsin are associated with a severe form of autosomal dominant RP. This report presents evidence that Arg135 mutant rhodopsins (e.g., R135L, R135G, and R135W) are hyperphosphorylated and bind with high affinity to visual arrestin. Mutant rhodopsin recruits the cytosolic arrestin to the plasma membrane, and the rhodopsin-arrestin complex is internalized into the endocytic pathway. Furthermore, the rhodopsin-arrestin complexes alter the morphology of endosomal compartments and severely damage receptor-mediated endocytic functions. The biochemical and cellular defects of Arg135 mutant rhodopsins are distinct from those previously described for class I and class II RP mutations, and, hence, we propose that they be named class III. Impaired endocytic activity may underlie the pathogenesis of RP caused by class III rhodopsin mutations.

Mutations in a novel retina-specific gene cause autosomal dominant retinitis pigmentosa.

Inherited retinal diseases are a common cause of visual impairment in children and young adults, often resulting in severe loss of vision in later life. The most frequent form of inherited retinopathy is retinitis pigmentosa (RP), with an approximate incidence of 1 in 3,500 individuals worldwide. RP is characterized by night blindness and progressive degeneration of the midperipheral retina, accompanied by bone spicule-like pigmentary deposits and a reduced or absent electroretinogram (ERG). The disease process culminates in severe reduction of visual fields or blindness. RP is genetically heterogeneous, with autosomal dominant, autosomal recessive and X-linked forms. Here we have identified two mutations in a novel retina-specific gene from chromosome 8q that cause the RP1 form of autosomal dominant RP in three unrelated families. The protein encoded by this gene is 2,156 amino acids and its function is currently unknown, although the amino terminus has similarity to that of the doublecortin protein, whose gene (DCX) has been implicated in lissencephaly in humans. Two families have a nonsense mutation in codon 677 of this gene (Arg677stop), whereas the third family has a nonsense mutation in codon 679 (Gln679stop). In one family, two individuals homozygous for the mutant gene have more severe retinal disease compared with heterozygotes.

Disruption of Conserved Rhodopsin Disulfide Bond by Cys 187Tyr Mutation Causes Early and Severe Autosomal Dominant Retinitis Pigmentosa

To determine the molecular basis of an early and severe form of autosomal dominant retinitis pigmentosa and to characterize the associated phenotype. Visual function evaluation included electrophysiologic and psychophysical testing. Molecular genetic analysis included determining the DNA sequence of sections of the rhodopsin gene amplified by polymerase chain reaction and screening for changes single-nucleotide by allele-specific oligonucleotide hybridization. Affected family members are heterozygous for a unique Cys187Tyr rhodopsin mutation which disrupts a highly conserved disulfide bond essential to normal rhodopsin function. The retinitis pigmentosa (RP) phenotype includes early and severe retinal dysfunction. The full-field electroretinogram showed only negligible remaining rod and cone responses by 22 years of age. Visual fields were constricted severely by early middle-age years. Macular dysfunction caused reduced visual acuity in early adult years, and macular atrophy was present in older age. The severity of phenotype generally correlated with age, with the exception of an affected 44-year-old patient who had better visual acuity, fields, electroretinogram, and dark-adapted thresholds than did three younger affected relatives, ranging in age from 22 to 38 years. An early onset, blinding form of autosomal dominant RP results from a rhodopsin Cys187Tyr mutation that eliminates a residue necessary for the formation of a highly conserved disulfide bond essential to normal rhodopsin function. The fact that one family member is significantly less affected than his younger relatives suggests that genetic or environmental factors can modulate the phenotype.

Retinitis pigmentosa families showing apparent X linked inheritance but unlinked to the RP2 or RP3 loci.

Three families with retinitis pigmentosa (RP) are described in which the disorder shows apparent X linked inheritance but does not show linkage to the RP2 and RP3 regions of the short arm of the X chromosome. The families are also inconsistent with a localisation of the disease gene between DXS164 and DXS28. In one case, reassessment of the family in the light of these results suggested that the family may have an autosomal dominant form of RP. The remaining two families are consistent with X linkage and suggest the possibility of a new X linked RP (XLRP) locus. These families highlight the difficulties in determining the mode of inheritance on the basis of pedigree structure and clinical data alone. Molecular genetics plays an important role in confirming the mode of inheritance and in detecting potential misclassifications, particularly in a group of disorders as heterogeneous as RP. They emphasise that caution is required in genetic counselling of RP families, particularly in the absence of any molecular genetic analysis.

Gene of type II autosomal dominant retinitis pigmentosa maps on the long arm of chromosome 3

Linkage analysis has been performed on a large Australian family segregating for the autosomal dominant form of retinitis pigmentosa (ADRP). The majority of patients had no subjective symptoms of night blindness until their second decade and good visual acuity until late in life. The disease in this family has been classified as Type II ADRP according to the subdivisions provided by both Massof and Finkelstein and Fishman and colleagues. Linkage (Omax:0.08 at Zmax:4.78) is here demonstrated between the disease locus and D3S47 (a marker locus on the long arm of chromosome 3), which showed in an earlier study very close linkage without recombination to the disease locus in an Irish pedigree with a clinically more severe and early onset (Type I) ADRP.