Effects of deficiency in the RLBP1-encoded visual cycle protein CRALBP on visual dysfunction in humans and mice.

Mutations in retinaldehyde-binding protein 1 (RLBP1), encoding the visual cycle protein cellular retinaldehyde-binding protein (CRALBP), cause an autosomal recessive form of retinal degeneration. By binding to 11-cis-retinoid, CRALBP augments the isomerase activity of retinoid isomerohydrolase RPE65 (RPE65) and facilitates 11-cis-retinol oxidation to 11-cis-retinal. CRALBP also maintains the 11-cis configuration and protects against unwanted retinaldehyde activity. Studying a sibling pair that is compound heterozygous for mutations in RLBP1/CRALBP, here we expand the phenotype of affected individuals, elucidate a previously unreported phenotype in RLBP1/CRALBP carriers, and demonstrate consistencies between the affected individuals and Rlbp1/Cralbp-/- mice. In the RLBP1/CRALBP-affected individuals, nonrecordable rod-specific electroretinogram traces were recovered after prolonged dark adaptation. In ultrawide-field fundus images, we observed radially arranged puncta typical of RLBP1/CRALBP-associated disease. Spectral domain-optical coherence tomography (SD-OCT) revealed hyperreflective aberrations within photoreceptor-associated bands. In short-wavelength fundus autofluorescence (SW-AF) images, speckled hyperautofluorescence and mottling indicated macular involvement. In both the affected individuals and their asymptomatic carrier parents, reduced SW-AF intensities, measured as quantitative fundus autofluorescence (qAF), indicated chronic impairment in 11-cis-retinal availability and provided information on mutation severity. Hypertransmission of the SD-OCT signal into the choroid together with decreased near-infrared autofluorescence (NIR-AF) provided evidence for retinal pigment epithelial cell (RPE) involvement. In Rlbp1/Cralbp-/- mice, reduced 11-cis-retinal levels, qAF and NIR-AF intensities, and photoreceptor loss were consistent with the clinical presentation of the affected siblings. These findings indicate that RLBP1 mutations are associated with progressive disease involving RPE atrophy and photoreceptor cell degeneration. In asymptomatic carriers, qAF disclosed previously undetected visual cycle deficiency.

Impairments of Photoreceptor Outer Segments Renewal and Phototransduction Due to a Peripherin Rare Haplotype Variant: Insights from Molecular Modeling.

BACKGROUND: Retinitis pigmentosa punctata albescens (RPA) is a particular form of retinitis pigmentosa characterized by childhood onset night blindness and areas of peripheral retinal atrophy. We investigated the genetic cause of RPA in a family consisting of two affected Egyptian brothers with healthy consanguineous parents. METHODS: Mutational analysis of four RPA causative genes was realized by Sanger sequencing on both probands, and detected variants were subsequently genotyped in their parents. Afterwards, found variants were deeply, statistically, and in silico characterized to determine their possible effects and association with RPA. RESULTS: Both brothers carry three missense PRPH2 variants in a homozygous condition (c.910C > A, c.929G > A, and c.1013A > C) and two promoter variants in RHO (c.-26A > G) and RLBP1 (c.-70G > A) genes, respectively. Haplotype analyses highlighted a PRPH2 rare haplotype variant (GAG), determining a possible alteration of PRPH2 binding with melanoregulin and other outer segment proteins, followed by photoreceptor outer segment instability. Furthermore, an altered balance of transcription factor binding sites, due to the presence of RHO and RLBP1 promoter variants, might determine a comprehensive downregulation of both genes, possibly altering the PRPH2 shared visual-related pathway. CONCLUSIONS: Despite several limitations, the study might be a relevant step towards detection of novel scenarios in RPA etiopathogenesis.

A novel RLBP1 gene geographical area-related mutation present in a young patient with retinitis punctata albescens.

BACKGROUND: Autosomal recessive forms of retinitis punctata albescens (RPA) have been described. RPA is characterized by progressive retinal degeneration due to alteration in visual cycle and consequent deposit of photopigments in retinal pigment epithelium. Five loci have been linked to RPA onset. Among these, the retinaldehyde-binding protein 1 gene, RLBP1, is the most frequently involved and several founder mutations were reported. We report results of a genetic molecular investigation performed on a large Sicilian family in which appears a young woman with RPA. RESULTS: The proband is in homozygous condition for a novel RLBP1 single-pair deletion, and her healthy parents, both heterozygous, are not consanguineous. Thenovelc.398delC (p.P133Qfs*258) involves the exon 6 and leads to a premature stop codon, resulting in a truncated protein entirely missing of CRAL-TRIO lipid-binding domain. Pedigree analysis showed other non-consanguineous relatives heterozygous for the same mutation in the family. Extension of mutation research in the native town of the proband revealed its presence also in healthy subjects, in a heterozygous condition. CONCLUSIONS: A novel RLBP1 truncating mutation was detected in a young girl affected by RPA. Although her parents are not consanguineous, the mutation was observed in a homozygous condition. Being them native of the same small Sicilian town of Fiumedinisi, the hypothesis of a geographical area-related mutation was assessed and confirmed.

The rhodopsin-retinochrome system for retinal re-isomerization predates the origin of cephalopod eyes.

BACKGROUND: The process of photoreception in most animals depends on the light induced isomerization of the chromophore retinal, bound to rhodopsin. To re-use retinal, the all-trans-retinal form needs to be re-isomerized to 11-cis-retinal, which can be achieved in different ways. In vertebrates, this mostly includes a stepwise enzymatic process called the visual cycle. The best studied re-isomerization system in protostomes is the rhodopsin-retinochrome system of cephalopods, which consists of rhodopsin, the photoisomerase retinochrome and the protein RALBP functioning as shuttle for retinal. In this study we investigate the expression of the rhodopsin-retinochrome system and functional components of the vertebrate visual cycle in a polyplacophoran mollusk, Leptochiton asellus, and examine the phylogenetic distribution of the individual components in other protostome animals. RESULTS: Tree-based orthology assignments revealed that orthologs of the cephalopod retinochrome and RALBP are present in mollusks outside of cephalopods. By mining our dataset for vertebrate visual cycle components, we also found orthologs of the retinoid binding protein RLBP1, in polyplacophoran mollusks, cephalopods and a phoronid. In situ hybridization and antibody staining revealed that L. asellus retinochrome is co-expressed in the larval chiton photoreceptor cells (PRCs) with the visual rhodopsin, RALBP and RLBP1. In addition, multiple retinal dehydrogenases are expressed in the PRCs, which might also contribute to the rhodopsin-retinochrome system. CONCLUSIONS: We conclude that the rhodopsin-retinochrome system is a common feature of mollusk PRCs and predates the origin of cephalopod eyes. Our results show that this system has to be extended by adding further components, which surprisingly, are shared with vertebrates.

A novel homozygous frameshift variant in the cellular retinaldehyde-binding protein 1 (RLBP1) gene causes retinitis punctata albescens.

BACKGROUND: Retinitis punctata albescens is a form of retinitis pigmentosa characterized by white fleck-like deposits in the fundus, in most cases caused by pathogenic variants in RLBP1 gene. The purpose of this work is to report the phenotypic and genotypic data of a patient with retinitis punctata albescens carrying a deletion in the RLBP1 gene. RESULTS: An 8-year-old Caucasian female has been complaining of nyctalopia for the last 2 years. No other ocular symptoms were present. No relevant past medical or familiar history was described. At clinical examination, the patient's best-corrected visual acuity was 20/20 in both eyes. Anterior segment evaluation and intraocular pressure were normal in both eyes. At fundoscopy, multiple punctate whitish-yellow fleck-like lesions were observed in the proximity of temporal superior and inferior vascular arcades. Scotopic electroretinogram demonstrated severely reduced rod response, without improvement or recovery of rod system function after prolonged dark adaptation. Blood DNA samples of this patient and from her parents were screened for causal variants in RLBP1, RDH5, and PRPH2. CONCLUSION: A probable pathogenic frameshift variant was identified in homozygosity in the RLBP1 gene with an autosomal recessive transmission as another cause of retinitis punctata albescens. This DNA variant will aid ongoing functional studies and add to our understanding of the molecular pathology about RLBP1-associated retinopathies.

Retinitis Punctata Albescens and RLBP1-Allied Phenotypes: Phenotype-Genotype Correlation and Natural History in the Aim of Gene Therapy.

Purpose: To identify relevant criteria for gene therapy based on clinical and genetic characteristics of rod-cone dystrophy associated with RLBP1 pathogenic variants in a large cohort comprising children and adults. Design: Retrospective cohort study. Participants: Patients with pathogenic variants in RLBP1 registered in a single French reference center specialized in inherited retinal dystrophies. Methods: Clinical, multimodal imaging, and genetic findings were reviewed. Main Outcome Measures: Age of onset; visual acuity; ellipsoid line length; nasal, temporal, and foveal retinal thickness; and pathogenic variants and related phenotypes, including Newfoundland rod-cone and Bothnia dystrophies (NFRCDs), were reappraised. Results: Twenty-one patients (15 families) were included. The most frequent form was NFRCD with 12 patients (8 families) homozygous for the recurrent deletion of exons 7 through 9 in RLBP1 and 5 patients (4 families) with biallelic protein-truncating variants (2 novel: p.Gln16∗ and p.Tyr251∗). A novel combination of the p.Arg234Trp Bothnia variant with a nonsense variant in trans led to Bothnia dystrophy in 2 sisters. One proband carrying the p.Met266Lys Bothnia variant and in trans p.Arg121Trp and a second, with the p.Arg9Cys and p.Tyr111∗ combination, both demonstrated mild retinitis punctata albescens. Independently of genotype, all patients showed a visual acuity of worse than 20/200, an ellipsoid line width of less than 1000 µm, and a mean foveal thickness of less than 130 to 150 µm, with loss of both the interdigitation and ellipsoid lines. Conclusions: The eligibility for RLBP1 gene therapy first should be determined according to the biallelic variant combination using a robust classification as proposed herein. An ellipsoid line width of more than 1200 µm and a central thickness of more than 130 to 150 µm with detectable ellipsoid and interdigitation lines should be 2 prerequisite imaging indicators for gene therapy.

Qualitative Interviews to Better Understand the Patient Experience and Evaluate Patient-Reported Outcomes (PRO) in RLBP1 Retinitis Pigmentosa (RLBP1 RP).

INTRODUCTION: RLBP1 RP is an autosomal recessive form of retinitis pigmentosa (RP), characterized by night blindness, prolonged dark adaptation, constricted visual fields and impaired macular function. This study aimed to better understand the patient experience of RLBP1 RP and evaluate the content validity of existing patient reported outcome (PRO) instruments in this condition. METHODS: Semi-structured concept elicitation and cognitive debriefing interviews were conducted with RLBP1 RP patients in Canada and Sweden. Interviews started with open-ended concept elicitation questioning, and then patients were cognitively debriefed on The National Eye Institute Visual Functioning Questionnaire (NEI VFQ-25), the Low Luminance Questionnaire (LLQ) and four light/dark adaptation items of the Visual Activities Questionnaire (VAQ). Qualitative interviews were also conducted with three expert clinicians. Anonymized, verbatim transcripts were analyzed using thematic analysis. RESULTS: Twenty-one patients were interviewed (Canada n = 10; Sweden n = 11). Symptoms reported included night blindness (n = 21), difficulty adapting to changes in lighting (n = 21) and difficulties seeing in bright lighting (n = 18). Patients experienced substantial impacts on daily activities (n = 21) and physical functioning (n = 17). Patients had difficulty interpreting and selecting a response for some items in the NEI VFQ-25 and LLQ. Some items were not relevant to patients' disease experience. There were both gaps and overlaps in the conceptual coverage of the instruments. CONCLUSIONS: Visual impairment due to RLBP1 RP has a substantial impact on physical functioning and daily activities. To adequately assess all important symptoms and associated functional impacts in RLBP1 RP, it is recommended to either modify one or more existing instruments or to develop a new non-syndromic RP specific instrument.

Progress in treating inherited retinal diseases: Early subretinal gene therapy clinical trials and candidates for future initiatives.

Due to improved phenotyping and genetic characterization, the field of 'incurable' and 'blinding' inherited retinal diseases (IRDs) has moved substantially forward. Decades of ascertainment of IRD patient data from Philadelphia and Toronto centers illustrate the progress from Mendelian genetic types to molecular diagnoses. Molecular genetics have been used not only to clarify diagnoses and to direct counseling but also to enable the first clinical trials of gene-based treatment in these diseases. An overview of the recent reports of gene augmentation clinical trials by subretinal injections is used to reflect on the reasons why there has been limited success in this early venture into therapy. These first-in human experiences have taught that there is a need for advancing the techniques of delivery of the gene products - not only for refining further subretinal trials, but also for evaluating intravitreal delivery. Candidate IRDs for intravitreal gene delivery are then suggested to illustrate some of the disorders that may be amenable to improvement of remaining central vision with the least photoreceptor trauma. A more detailed understanding of the human IRDs to be considered for therapy and the calculated potential for efficacy should be among the routine prerequisites for initiating a clinical trial.

Nonclinical Safety Evaluation of scAAV8-RLBP1 for Treatment of RLBP1 Retinitis Pigmentosa.

Retinitis pigmentosa is a form of retinal degeneration usually caused by genetic mutations affecting key functional proteins. We have previously demonstrated efficacy in a mouse model of RLBP1 deficiency with a self-complementary AAV8 vector carrying the gene for human RLBP1 under control of a short RLBP1 promoter (CPK850).1 In this article, we describe the nonclinical safety profile of this construct as well as updated efficacy data in the intended clinical formulation. In Rlbp1-/- mice dosed at a range of CPK850 levels, a minimum efficacious dose of 3 × 107 vg in a volume of 1 µL was observed. For safety assessment in these and Rlbp1+/+ mice, optical coherence tomography (OCT) and histopathological analysis indicated retinal thinning that appeared to be dose-dependent for both Rlbp1 genotypes, with no qualitative difference noted between Rlbp1+/+ and Rlbp1-/- mice. In a non-human primate study, RLBP1 mRNA expression was detected and dose dependent intraocular inflammation and retinal thinning were observed. Inflammation resolved slowly over time and did not appear to be exacerbated in the presence of anti-AAV8 antibodies. Biodistribution was evaluated in rats and satellite animals in the non-human primate study. The vector was largely detected in ocular tissues and low levels in the optic nerve, superior colliculus, and lateral geniculate nucleus, with limited distribution outside of these tissues. These data suggest that an initial subretinal dose of ∼3 × 107 vg/µL CPK850 can safely be used in clinical trials.

Ral Signals through a MAP4 Kinase-p38 MAP Kinase Cascade in C. elegans Cell Fate Patterning.

C. elegans vulval precursor cell (VPC) fates are patterned by an epidermal growth factor (EGF) gradient. High-dose EGF induces 1° VPC fate, and lower dose EGF contributes to 2° fate in support of LIN-12/Notch. We previously showed that the EGF 2°-promoting signal is mediated by LET-60/Ras switching effectors, from the canonical Raf-MEK-ERK mitogen-activated protein (MAP) kinase cascade that promotes 1° fate to the non-canonical RalGEF-Ral that promotes 2° fate. Of oncogenic Ras effectors, RalGEF-Ral is by far the least well understood. We use genetic analysis to identify an effector cascade downstream of C. elegans RAL-1/Ral, starting with an established Ral binding partner, Exo84 of the exocyst complex. Additionally, RAL-1 signals through GCK-2, a citron-N-terminal-homology-domain-containing MAP4 kinase, and PMK-1/p38 MAP kinase cascade to promote 2° fate. Our study delineates a Ral-dependent developmental signaling cascade in vivo, thus providing the mechanism by which lower EGF dose is transduced.

Phenotype variations of retinal dystrophies caused by mutations in the RLBP1 gene.

PURPOSE: Mutations in the RLBP1 gene encoding the cellular retinaldehyde-binding protein (CRALBP) cause autosomal recessive progressive retinopathy, such as retinitis punctata albescens (RPA), Bothnia-type dystrophy (BD), Newfoundland rod-cone dystrophy (NFRCD), retinitis pigmentosa (RP) and fundus albipunctatus (FA). We present the clinical heterogeneity and genetic findings of seven patients from five families with RLBP1 mutations, including three novel mutations. METHODS: Seven patients underwent complete ophthalmological examination including psychophysical tests (visual acuity, colour vision, visual field, dark adaptation) and electrophysiology (Ganzfeld and multifocal ERG). Additionally, fundus photography, autofluorescence (FAF) and spectral domain optical coherence tomography (OCT) recordings were performed. Genomic DNA was analysed by high-throughput sequencing for all RP-related genes in a diagnostic set-up. RESULTS: The patients presented with variable phenotypes, including RPA, BD, RP and a mild form of NFRCD. No detectable or severely depressed responses in electrophysiological examinations were seen in all cases. Visual field constriction was variable among individuals. Severely reduced visual acuity was only observed in the patient presenting with BD. The other patients retained mild to moderate reduction of visual function. Despite the morphological differences, central retinal thinning - as a common feature - could be observed. CONCLUSIONS: The fact that different mutations in RLBP1 are correlated with quite different morphological and functional characteristics outlines the complexity of the protein. Identifying new mutations and comparing the different phenotypes may help to better understand the function of the protein and the consequences in pathological changes that involve RPE and choroid.

Genotype-phenotype correlations in Bothnia dystrophy caused by RLBP1 gene sequence variations.

PURPOSE: To evaluate phenotypes caused by different RLBP1 mutations in autosomal recessive retinitis pigmentosa of Bothnia type. METHODS: Compound heterozygotes for mutations in the RLBP1 gene [c.677T>A]+[c.700C>T] (p.M226K+p.R234W), n = 10, aged 7-84 years, and homozygotes c.677T>A (p.M226K), n = 2, aged 63 and 73 years, were studied using visual acuity (VA), low-contrast VA, visual fields (VFs) and optical coherence tomography (OCT). Retrospective VA and VFs, standardized dark adaptation and full-field electroretinograms (ERGs) were analysed and prolonged dark adaptometry and ERG (at 24 hr) were performed. RESULTS: Progressive decline of VA and VF areas was age-dependent. Retinal degenerative maculopathy, peripheral degenerative changes and retinitis punctata albescens (RPA) were present. Early retinal thinning in the central foveal, foveal (Ø 1 mm), and inner ring (Ø 3 mm) in the macular region, with homogenous, high-reflectance RPA changes, was visualized in and adjacent to the retinal pigment epithelium/choriocapillaris using OCT. Reduced dark adaptation and affected ERGs were present in all ages. Prolonged dark adaptation and ERG (at 24 hr), an increase in final threshold, and ERG rod and mixed rod/cone responses were found. CONCLUSIONS: The two RLBP1 genotypes presented a phenotypical and electrophysiological expression of progressive retinal disease similar to that previously described in homozygotes for the c.700C>T (p.R234W) RLBP1 mutation. The uniform phenotypical expression of RLBP1 mutations is relevant information for the disease and of importance in planning future treatment strategies.

Self-reported quality of life in patients with retinitis pigmentosa and maculopathy of Bothnia type.

PURPOSE: To assess vision-related quality-of-life subscales with objective measurements of visual function in patients affected with retinitis pigmentosa of Bothnia type (BD). METHODS: Forty-nine patients answered the NEI-VFQ-25 questionnaire. High- and low-contrast distance acuity (VA), near VA, and visual fields (VF) were measured. Weighted VA (WVA) and low-contrast (10%) VA (WLCVA), binocular VF areas, and central scotoma were calculated. Adjusted mean subscale scores were calculated and associations analyzed. RESULTS: Subscale scores for general, far, and near vision, social functioning, and color vision were lowest while general health, ocular pain, and mental health were highest in the BD phenotype. The correlations were substantial and similar for WVA, WLCVA, and near vision. The degree of measured VF impairment had few associations with the different adjusted subscale scores. CONCLUSION: The NEI VFQ-25 subscales were well associated with clinical vision measures depending on VA. The progression of VF defects typical for the BD phenotype does not seem to affect the self-perceived quality of life, which might indicate adaptability to this type of progressive VF loss. The BD phenotype has a significant impact on multiple domains of daily life, but there are no signs of accelerating depression related to the increasing visual impairment.