Dominant Form Of Retinitis Pigmentosa Research Articles

Microglia Preserve Visual Function in a Mouse Model of Retinitis Pigmentosa with Rhodopsin-P23H Mutant.

Most forms of outer retinal degenerative diseases involve the ectopic accumulation of microglia/macrophages in the subretinal space, including retinitis pigmentosa. However, their role in the loss of photoreceptor function during retinal degeneration remains unknown. Here, we examined the effect of conditional microglial depletion on photoreceptor numbers and visual function in mice with the rhodopsin P23H mutation, a dominant form of retinitis pigmentosa in humans. We found that microglial depletion led to an elevated level of rhodopsin and increased photoreceptor layer thickness. However, overall electrophysiological functions of the retina were reduced with microglial depletion. Therefore, these results identify an essential role of microglia specially in preserving visual function in outer retinal degeneration.

Modeling PRPF31 retinitis pigmentosa using retinal pigment epithelium and organoids combined with gene augmentation rescue

Mutations in the ubiquitously expressed pre-mRNA processing factor (PRPF) 31 gene, one of the most common causes of dominant form of Retinitis Pigmentosa (RP), lead to a retina-specific phenotype. It is uncertain which retinal cell types are affected and animal models do not clearly present the RP phenotype observed in PRPF31 patients. Retinal organoids and retinal pigment epithelial (RPE) cells derived from human-induced pluripotent stem cells (iPSCs) provide potential opportunities for studying human PRPF31-related RP. We demonstrate here that RPE cells carrying PRPF31 mutations present important morphological and functional changes and that PRPF31-mutated retinal organoids recapitulate the human RP phenotype, with a rod photoreceptor cell death followed by a loss of cones. The low level of PRPF31 expression may explain the defective phenotypes of PRPF31-mutated RPE and photoreceptor cells, which were not observed in cells derived from asymptomatic patients or after correction of the pathogenic mutation by CRISPR/Cas9. Transcriptome profiles revealed differentially expressed and mis-spliced genes belonging to pathways in line with the observed defective phenotypes. The rescue of RPE and photoreceptor defective phenotypes by PRPF31 gene augmentation provide the proof of concept for future therapeutic strategies.

Induction of Autophagy Promotes Clearance of RHOP23H Aggregates and Protects From Retinal Degeneration.

Autophagy is a critical metabolic process that acts as a major self-digestion and recycling pathway contributing to maintain cellular homeostasis. An emerging field of research supports the therapeutic modulation of autophagy for treating human neurodegenerative disorders, in which toxic aggregates are accumulated in neurons. Our previous study identified Ezrin protein as an inhibitor of autophagy and lysosomal functions in the retina; thus, in turn, identifying it as a potential pharmacological target for increasing retinal cell clearance to treat inherited retinal dystrophies in which misfolded proteins have accumulated. This study aimed to verify the therapeutic inhibition of Ezrin to induce clearance of toxic aggregates in a mouse model for a dominant form of retinitis pigmentosa (i.e., RHOP23H/+). We found that daily inhibition of Ezrin significantly decreased the accumulation of misfolded RHOP23H aggregates. Remarkably, induction of autophagy, by a drug-mediated pulsatile inhibition of Ezrin, promoted the lysosomal clearance of disease-linked RHOP23H aggregates. This was accompanied with a reduction of endoplasmic reticulum (ER)-stress, robust decrease of photoreceptors' cell death, amelioration in both retinal morphology and function culminating in a better preservation of vision. Our study opens new perspectives for a pulsatile pharmacological induction of autophagy as a mutation-independent therapy paving the way toward a more effective therapeutic strategy to treat these devastating retinal disorders due to an accumulation of intracellular toxic aggregates.

Drug screening with zebrafish visual behavior identifies carvedilol as a potential treatment for an autosomal dominant form of retinitis pigmentosa

Retinitis Pigmentosa (RP) is a mostly incurable inherited retinal degeneration affecting approximately 1 in 4000 individuals globally. The goal of this work was to identify drugs that can help patients suffering from the disease. To accomplish this, we screened drugs on a zebrafish autosomal dominant RP model. This model expresses a truncated human rhodopsin transgene (Q344X) causing significant rod degeneration by 7 days post-fertilization (dpf). Consequently, the larvae displayed a deficit in visual motor response (VMR) under scotopic condition. The diminished VMR was leveraged to screen an ENZO SCREEN-WELL REDOX library since oxidative stress is postulated to play a role in RP progression. Our screening identified a beta-blocker, carvedilol, that ameliorated the deficient VMR of the RP larvae and increased their rod number. Carvedilol may directly on rods as it affected the adrenergic pathway in the photoreceptor-like human Y79 cell line. Since carvedilol is an FDA-approved drug, our findings suggest that carvedilol can potentially be repurposed to treat autosomal dominant RP patients.

Structure network-based landscape of rhodopsin misfolding by mutations and algorithmic prediction of small chaperone action

Failure of a protein to achieve its functional structural state and normal cellular location contributes to the etiology and pathology of heritable human conformational diseases. The autosomal dominant form of retinitis pigmentosa (adRP) is an incurable blindness largely linked to mutations of the membrane protein rod opsin. While the mechanisms underlying the noxious effects of the mutated protein are not completely understood, a common feature is the functional protein conformational loss. Here, the wild type and 39 adRP rod opsin mutants were subjected to mechanical unfolding simulations coupled to the graph theory-based protein structure network analysis.A robust computational model was inferred and in vitro validated in its ability to predict endoplasmic reticulum retention of adRP mutants, a feature linked to the mutation-caused misfolding. The structure-based approach could also infer the structural determinants of small chaperone action on misfolded protein mutants with therapeutic implications.The approach is exportable to conformational diseases linked to missense mutations in any membrane protein.

Photoreceptor Degeneration in Pro23His Transgenic Rats (Line 3) Involves Autophagic and Necroptotic Mechanisms.

Photoreceptor death contributes to 50% of irreversible vision loss in the western world. Pro23His (P23H) transgenic albino rat strains are widely used models for the most common rhodopsin gene mutation associated with the autosomal dominant form of retinitis pigmentosa. However, the mechanism(s) by which photoreceptor death occurs are not well understood and were the principal aim of this study. We first used electroretinogram recording and optical coherence tomography to confirm the time course of functional and structural loss. Electroretinogram analyses revealed significantly decreased rod photoreceptor (a-wave), bipolar cell (b-wave) and amacrine cell responses (oscillatory potentials) from P30 onward. The cone-mediated b-wave was also decreased from P30. TUNEL analysis showed extensive cell death at P18, with continued labeling detected until P30. Focused gene expression arrays indicated activation of, apoptosis, autophagy and necroptosis in whole retina from P14-18. However, analysis of mitochondrial permeability changes (ΔΨm) using JC-1 dye, combined with immunofluorescence markers for caspase-dependent (cleaved caspase-3) and caspase-independent (AIF) cell death pathways, indicated mitochondrial-mediated cell death was not a major contributor to photoreceptor death. By contrast, reverse-phase protein array data combined with RIPK3 and phospho-MLKL immunofluorescence indicated widespread necroptosis as the predominant mechanism of photoreceptor death. These findings highlight the complexity of mechanisms involved in photoreceptor death in the Pro23His rat model of degeneration and suggest therapies that target necroptosis should be considered for their potential to reduce photoreceptor death.

AAV-miR-204 Protects from Retinal Degeneration by Attenuation of Microglia Activation and Photoreceptor Cell Death

Inherited retinal diseases (IRDs) represent a frequent cause of genetic blindness. Their high genetic heterogeneity hinders the application of gene-specific therapies to the vast majority of patients. We recently demonstrated that the microRNA miR-204 is essential for retinal function, although the underlying molecular mechanisms remain poorly understood. Here, we investigated the therapeutic potential of miR-204 in IRDs. We subretinally delivered an adeno-associated viral (AAV) vector carrying the miR-204 precursor to two genetically different IRD mouse models. The administration of AAV-miR-204 preserved retinal function in a mouse model for a dominant form of retinitis pigmentosa (RHO-P347S). This was associated with a reduction of apoptotic photoreceptor cells and with a better preservation of photoreceptor marker expression. Transcriptome analysis showed that miR-204 shifts expression profiles of transgenic retinas toward those of healthy retinas by the downregulation of microglia activation and photoreceptor cell death. Delivery of miR-204 exerted neuroprotective effects also in a mouse model of Leber congenital amaurosis, due to mutations of the Aipl1 gene. Our study highlights the mutation-independent therapeutic potential of AAV-miR204 in slowing down retinal degeneration in IRDs and unveils the previously unreported role of this miRNA in attenuating microglia activation and photoreceptor cell death.

A Small Chaperone Improves Folding and Routing of Rhodopsin Mutants Linked to Inherited Blindness

SummaryThe autosomal dominant form of retinitis pigmentosa (adRP) is a blindness-causing conformational disease largely linked to mutations of rhodopsin. Molecular simulations coupled to the graph-based protein structure network (PSN) analysis and in vitro experiments were conducted to determine the effects of 33 adRP rhodopsin mutations on the structure and routing of the opsin protein. The integration of atomic and subcellular levels of analysis was accomplished by the linear correlation between indices of mutational impairment in structure network and in routing. The graph-based index of structural perturbation served also to divide the mutants in four clusters, consistent with their differences in subcellular localization and responses to 9-cis retinal. The stability core of opsin inferred from PSN analysis was targeted by virtual screening of over 300,000 anionic compounds leading to the discovery of a reversible orthosteric inhibitor of retinal binding more effective than retinal in improving routing of three adRP mutants.

Insights into the pathogenesis of dominant retinitis pigmentosa associated with a D477G mutation in RPE65.

RPE65 is the essential trans-cis isomerase of the classical retinoid (visual) cycle. Mutations in RPE65 give rise to severe retinal dystrophies, most of which are associated with loss of protein function and recessive inheritance. The only known exception is a c.1430G>A (D477G) mutation that gives rise to dominant retinitis pigmentosa with delayed onset and choroidal and macular involvement. Position 477 is distant from functionally critical regions of RPE65. Hence, the mechanism of D477G pathogenicity remains unclear, although protein misfolding and aggregation mechanisms have been suggested. We characterized a D477G knock-in mouse model which exhibited mild age-dependent changes in retinal structure and function. Immunoblot analysis of protein extracts from the eyes of these knock-in mice demonstrated the presence of ubiquitinated RPE65 and reduced RPE65 expression. We observed an accumulation of retinyl esters in the knock-in mice as well as a delay in rhodopsin regeneration kinetics and diminished electroretinography responses, indicative of RPE65 functional impairment induced by the D477G mutation in vivo. However, a cell line expressing D477G RPE65 revealed protein expression levels, cellular localization and retinoid isomerase activity comparable to cells expressing wild-type protein. Structural analysis of an RPE65 chimera suggested that the D477G mutation does not perturb protein folding or tertiary structure. Instead, the mutation generates an aggregation-prone surface that could induce cellular toxicity through abnormal complex formation as suggested by crystal packing analysis. These results indicate that a toxic gain-of-function induced by the D477G RPE65 substitution may play a role in the pathogenesis of this form of dominant retinitis pigmentosa.

Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery.

P23H is the most common mutation in the RHODOPSIN (RHO) gene leading to a dominant form of retinitis pigmentosa (RP), a rod photoreceptor degeneration that invariably causes vision loss. Specific disruption of the disease P23H RHO mutant while preserving the wild-type (WT) functional allele would be an invaluable therapy for this disease. However, various technologies tested in the past failed to achieve effective changes and consequently therapeutic benefits. We validated a CRISPR/Cas9 strategy to specifically inactivate the P23H RHO mutant, while preserving the WT allele in vitro. We, then, translated this approach in vivo by delivering the CRISPR/Cas9 components in murine Rho+/P23H mutant retinae. Targeted retinae presented a high rate of cleavage in the P23H but not WT Rho allele. This gene manipulation was sufficient to slow photoreceptor degeneration and improve retinal functions. To improve the translational potential of our approach, we tested intravitreal delivery of this system by means of adeno-associated viruses (AAVs). To this purpose, the employment of the AAV9-PHP.B resulted the most effective in disrupting the P23H Rho mutant. Finally, this approach was translated successfully in human cells engineered with the homozygous P23H RHO gene mutation. Overall, this is a significant proof-of-concept that gene allele specific targeting by CRISPR/Cas9 technology is specific and efficient and represents an unprecedented tool for treating RP and more broadly dominant genetic human disorders affecting the eye, as well as other tissues.

Phenotypic characterization of P23H and S334ter rhodopsin transgenic rat models of inherited retinal degeneration

We produced 8 lines of transgenic (Tg) rats expressing one of two different rhodopsin mutations in albino Sprague-Dawley (SD) rats. Three lines were generated with a proline to histidine substitution at codon 23 (P23H), the most common autosomal dominant form of retinitis pigmentosa in the United States. Five lines were generated with a termination codon at position 334 (S334ter), resulting in a C-terminal truncated opsin protein lacking the last 15 amino acid residues and containing all of the phosphorylation sites involved in rhodopsin deactivation, as well as the terminal QVAPA residues important for rhodopsin deactivation and trafficking. The rates of photoreceptor (PR) degeneration in these models vary in proportion to the ratio of mutant to wild-type rhodopsin. The models have been widely studied, but many aspects of their phenotypes have not been described. Here we present a comprehensive study of the 8 Tg lines, including the time course of PR degeneration from the onset to one year of age, retinal structure by light and electron microscopy (EM), hemispheric asymmetry and gradients of rod and cone degeneration, rhodopsin content, gene dosage effect, rapid activation and invasion of the outer retina by presumptive microglia, rod outer segment disc shedding and phagocytosis by the retinal pigmented epithelium (RPE), and retinal function by the electroretinogram (ERG). The biphasic nature of PR cell death was noted, as was the lack of an injury-induced protective response in the rat models. EM analysis revealed the accumulation of submicron vesicular structures in the interphotoreceptor space during the peak period of PR outer segment degeneration in the S334ter lines. This is likely due to the elimination of the trafficking consensus domain as seen before as with other rhodopsin mutants lacking the C-terminal QVAPA. The 8 rhodopsin Tg lines have been, and will continue to be, extremely useful models for the experimental study of inherited retinal degenerations.

Rhodopsin T17M Mutant Inhibits Complement C3 Secretion in Retinal Pigment Epithelium via ROS Induced Downregulation of TWIST1

Rhodopsin mutations cause autosomal dominant form of retinitis pigmentosa (RP). T17M rhodopsin predisposes cells to endoplasmic reticulum stress induced apoptosis. However, the pathogenic role of T17M rhodopsin in RP is not completely understood. Complement C3 has a protective role in RP pathogenesis. This study aimed to investigate whether T17M rhodopsin regulates C3 secretion in retinal pigment epithelium. The human retinal pigment epithelial cell line (ARPE-19) was engineered to overexpress wide-type (WT) and T17M rhodopsin. Gene expression was detected by RT-PCR and Western blot analysis. C3 secretion was detected by ELISA. The overexpression of T17M rhodopsin significantly induced ROS and reduced C3 secretion and transcription in ARPE-19 cells, but ROS scavengers could partially rescue reduced C3 secretion and transcription. Mechanistically, we found that ROS suppressed transcription factor TWIST1 which is responsible for activated transcription of C3. In conclusion, our data provide the first evidence that T17M rhodopsin mutant disrupts C3 secretion via the induction of ROS and the suppression of TWIST1. These findings reveal novel insight into the pathogenic role of mutant rhodopsin in RP. J. Cell. Biochem. 118: 4914-4920, 2017. © 2017 Wiley Periodicals, Inc.

488. In Vitro and In Vivo Genome Editing of the RHO Gene to Downregulate Dominant Mutations

Although progresses have been made in the understanding of the genetic basis for Retinitis Pigmentosa (RP), the development of therapeutic intervention is still lagging behind. Gene therapy was successfully applied to retina degeneration but only to recessive mutations. Rhodopsin (RHO) mutations represent a common cause of RP, accounting for 25% of adRP and 8 to 10% of all RP (Hartong et al., 2006) with more that 100 different mutations identified so far. RHO is a G-protein coupled receptor with localization restricted to rod outer segments where the phototransduction cascade initiates. Data on the pathogenic mechanism of mutant RHO are still controversial. Accumulation of mutant RHO in different subcellular compartments, and among those the ER, may trigger unfolded protein response (UPR) devoted to cytoprotective outputs to permit cells to reduce protein synthesis and up-regulate chaperons to cope with stress (Lin et al., 2007). In the autosomal dominant RP the mutation in only one allele is sufficient to the onset of retinal degeneration. To treat this kind of disorder a gene addition therapy is not suitable, as the mutant allele, mRNA, or protein product must be silenced beforehand. We aimed at developing genome editing tools to knock out the RHO defective alleles by introducing a double strand breaks (DSB) into the target gene. Two gRNAs were designed in the first exon on the RHO gene encompassing the P23H mutation. The two gRNAs were tested singularly or together in vitro on HeLa clones stably expressing P23H RHO. We demonstrated insertions or deletions (indels) in the genomic DNA specifically in the RHO gene by Cel I assay, TIDE and sequencing. Indels caused strong reduction of the RHO mRNA and of RHO protein up to 90%. The higher effect was obtained with the two gRNAs together. The two gRNAs were then in vivo expressed with Cas9 in photoreceptors of transgenic mice expressing the human P23H Rho gene by electroporation. Targeted cells were tracked by co-expression of EGFP. EGFP+ cells were FACS-sorted and indels in the human P23H RHO gene were analyzed by sequencing. We were able to detect up to 30% of genome editing in vivo. No editing was scored on murine Rho allele. We also detected reduction of RHO mRNA expression as well as RHO protein. Finally, we developed new tools to downregulate mutant RHO in dominant forms of RP. The CRISPR/Cas9 system reveled a high efficiency and should be tested for knock-down followed by gene replacement approaches.

High-throughput screening assays to identify small molecules preventing photoreceptor degeneration caused by the rhodopsin P23H mutation.

High-throughput screening (HTS) is one of the major techniques for discovering promising molecules for drug development. Rhodopsin mutations cause the most common autosomal dominant form of retinitis pigmentosa, an inherited retinal degenerative disease that currently has no effective treatment. To find an optimal pharmacological treatment for rhodopsin-associated retinitis pigmentosa, we performed two cell-based HTSs with mammalian cells expressing the P23H rod opsin mutant and identified two sets of novel compounds for further validation and characterization. The first HTS screen identified pharmacological chaperones of P23H opsin that increased its translocation from the endoplasmic reticulum to the plasma membrane. The second HTS screen selected small molecules that enhanced the clearance of the mutant opsin while vision could be sustained by the healthy gene allele expressing wild-type rhodopsin. Here we describe the methodology of these two HTS assays in detail.

Chemical chaperone 4-phenylbutyrate prevents endoplasmic reticulum stress induced by T17M rhodopsin.

BackgroundRhodopsin mutations are associated with the autosomal dominant form of retinitis pigmentosa. T17M mutation in rhodopsin predisposes cells to endoplasmic reticulum (ER) stress and induces cell death. This study aimed to examine whether chemical chaperone 4-phenylbutyrate prevents ER stress induced by rhodopsin T17M.ResultsARPE-19 cells were transfected with myc-tagged wild-type (WT) and T17M rhodopsin constructs. Turnover of WT and T17M rhodopsin was measured by cycloheximide chase analysis. The activity of ubiquitin-proteasome system was evaluated by GFPU reporter. We found that T17M rhodopsin was misfolded, ubiqutinated and eliminated by ER-associated degradation pathway (ERAD) in ARPE-19 cells. Accumulated T17M rhodopsin induced unfolded protein response, but had no effect on the activity of ubiquitin proteasome system. Moreover, chemical chaperone 4-phenylbutyrate facilitated the turnover of T17M rhodopsin and prevented apoptosis and ER stress induced by T17M rhodopsin.ConclusionsChemical chaperone could attenuate UPR signaling and ER stress induced by T17M rhodopsin and has potential therapeutic significance for retinitis pigmentosa.

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.

Clinical assessment and molecular genetics of an autosomal dominant retinitis pigmentosa in a Bulgarian Roma family

AbstractPurpose To make a clinical assessment and molecular genetic analysis in patients with autosomal dominant form of retinitis pigmentosa (adRP) in a Bulgarian Roma family.Methods Clinical assessment and genealogical analysis in a Bulgarian Roma family suggested the presence of RP with autosomal dominant inheritance with at least 12 affected in 4 generations. Best corrected visual acuity; kinetic Goldmann perimetry; direct and indirect ophthalmoscopy; ERG; fluorescein angiography. The molecular genetic analysis involved screening of 15 known adRP genes using microarray panel of Asper Biotech in the index patient.Results T in exon 4 of the RP1 gene, leading to an amino acid substitution T373I was found in heterozygous condition.adRP is a severe and genetically heterogeneous retinal degeneration. We present a Bulgarian Roma family with typical clinical symptoms of RP and heterozygous change in the RP1 gene, which has previously been described as a possible disease causing mutation in a Pakistani family with adRP and in homozygous condition leading to a severe arRP in 2 consanguineous families of Pakistani origin.Conclusion The clinical and genetic analysis of additional affected and unaffected family members is ongoing. This will allow better genotype‐phenotype correlations to be made.

Phenotype associated with mutation in the recently identified autosomal dominant retinitis pigmentosa KLHL7 gene.

To characterize the clinical phenotype, with an emphasis on electrophysiologic findings, in a family with autosomal dominant retinitis pigmentosa caused by mutation in the recently identified KLHL7 gene. Eleven patients from a single family were selected from the Swedish retinitis pigmentosa register. Four patients had been examined 13 to 17 years earlier and underwent further ophthalmologic examination, including visual acuity, fundus inspection, Goldmann perimetry, full-field electroretinography (ERG), multifocal ERG, and optical coherence tomography. KLHL7 mutation was identified by sequence analysis. In most examined family members, the fundus showed minor abnormalities. Full-field ERG demonstrated reduced cone and rod function, but rod responses were preserved in some patients late in life. Follow-up (<or=17 years) demonstrated slowly progressive retinal degeneration. In an adolescent family member, cone and rod function was initially normal, but retinitis pigmentosa was confirmed by electrophysiology 17 years later. Optical coherence tomography and multifocal ERG demonstrated macular abnormalities of varying degree among family members. Genetic analysis revealed a heterozygous exon 6 change (c.458C>T) in 7 family members. Observed in 2 Scandinavian families to date, KLHL7 mutation has recently been associated with autosomal dominant retinitis pigmentosa. Clinical examination with long-term follow-up verified a phenotype with a varying degree of retinal photoreceptor dysfunction and, in some family members, with late onset and preserved rod function until late in life. Clinical Relevance Patients with minor retinal abnormalities and normal ERG findings early in life can harbor an autosomal dominant form of retinitis pigmentosa with a varying degree of visual impediment. Some patients with late onset may retain night vision for many years.

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.