Functional analysis of disease-causing NR2E3 mutations

Purpose:We analyzed the transcriptional activity of disease-causing NR2E3 mutant proteins in a heterologous system. NR2E3 belongs to the nuclear receptor superfamily of transcription factors, characterized by evolutionary-conserved DNA-binding (DBD) and ligand-binding (LBD) domains. NR2E3 acts in concert with the transcription factors CRX and NRL to repress cone-specific genes and activate rod-specific genes in rod photoreceptors. During development, NR2E3 is also required to suppress cone cell generation from retinal progenitor cells. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS), the Goldman-Favre syndrome, and, more recently, with autosomal dominant retinitis pigmentosa (adRP). Methods:The different NR2E3 mutants were generated by QuickChangeR mutagenesis and analyzed by transfection in heterologous HEK293T cells. Results:In transactivation assays in HEK293T cells, the adRP-linked p.G56R mutant protein exhibited a more severe effect both in activation of a rhodopsin promoter reporter construct and in repression of M-opsin promoter reporter construct, than the ESCS-linked R76Q, R76W, G88V, R97H, R104Q, R104W mutants of the DBD. In contrast, the ESCS-linked p.R311Q mutant of the LBD behaved like the NR2E3 wild-type protein in these assays. By co-expressing the corepressors atrophin-1 and -2, a differential repression of the M-opsin promoter was observed in presence of the p.R311Q, p.R385P and p.M407K. Interestingly, corepressor expression also affected the activity of CRX, but not NRL, in both rhodopsin and M-opsin transactivation assays. Conclusions:Taken together, these in vitro results suggest a distinct disease mechanism for the adRP-linked mutation, but open the possibility of different mechanisms for the development of ESCS that is clinically characterized by important phenotypic variations.

Risk assessment of recurrence in sporadic retinoblastoma using a molecular-based algorithm.

PURPOSE: Most RB1 mutations are unique and distributed throughout the RB1 gene. Their detection can be time-consuming and the yield especially low in cases of conservatively-treated sporadic unilateral retinoblastoma (Rb) patients. In order to identify patients with true risk of developing Rb, and to reduce the number of unnecessary examinations under anesthesia in all other cases, we developed a universal sensitive, efficient and cost-effective strategy based on intragenic haplotype analysis. METHODS: This algorithm allows the calculation of the a posteriori risk of developing Rb and takes into account (a) RB1 loss of heterozygosity in tumors, (b) preferential paternal origin of new germline mutations, (c) a priori risk derived from empirical data by Vogel, and (d) disease penetrance of 90% in most cases. We report the occurrence of Rb in first degree relatives of patients with sporadic Rb who visited the Jules Gonin Eye Hospital, Lausanne, Switzerland, from January 1994 to December 2006 compared to expected new cases of Rb using our algorithm. RESULTS: A total of 134 families with sporadic Rb were enrolled; testing was performed in 570 individuals and 99 patients younger than 4 years old were identified. We observed one new case of Rb. Using our algorithm, the cumulated total a posteriori risk of recurrence was 1.77. CONCLUSIONS: This is the first time that linkage analysis has been validated to monitor the risk of recurrence in sporadic Rb. This should be a useful tool in genetic counseling, especially when direct RB1 screening for mutations leaves a negative result or is unavailable.

Vitreoretinal surgery for complications of choroidal tumor biopsy.

OBJECTIVE: To determine the outcomes of vitreoretinal surgery after choroidal tumor biopsy. DESIGN: Retrospective, single-center, consecutive case series. PARTICIPANTS: A total of 739 consecutive patients undergoing choroidal tumor biopsy. METHODS: All subjects who underwent transretinal or transscleral choroidal tumor biopsy for diagnostic or prognostic purposes between May 1993 and May 2013 were identified in our database. We then reviewed patients who subsequently required secondary vitreoretinal surgery for complications arising from such biopsies. MAIN OUTCOME MEASURES: Reason for vitreoretinal surgery, association with biopsy procedure, best-corrected visual acuity (BCVA; logarithm of the minimum angle of resolution [logMAR]), intraocular or extrascleral tumor dissemination, resolution of vitreous hemorrhage, reattachment of the retina with a single vitreoretinal procedure, number of additional vitrectomies undertaken, and number of enucleations. RESULTS: A total of 20 of 739 eyes (2.7%) underwent vitreoretinal surgery for complications arising from choroidal tumor biopsy. The tumors consisted of choroidal melanoma in all 20 eyes. The reasons for the secondary surgery included persistent vitreous hemorrhage in 1.9% (14/739), rhegmatogenous retinal detachment in 0.7% (5/739), and endophthalmitis in 0.14% (1/739). Median BCVA improved from 2.0 logMAR (mean, 1.92 logMAR; range, 0.8-2.7 logMAR) before vitrectomy to 0.72 logMAR (mean, 0.88 logMAR; range, -0.14 to 2.7 logMAR) after vitrectomy and 0.76 logMAR (mean, 1.14 logMAR; range, 0.1-3.0 logMAR) at the final visit (P < 0.0001, t test). Permanent resolution of vitreous hemorrhage was achieved in 6 of 14 patients, and reattachment of the retina was achieved in 2 of 5 patients after the first vitrectomy. A median of 1 (mean, 1.5; range, 1-3) additional vitrectomy was performed. Enucleation was necessary in 3 of 20 eyes (15%). There were no cases of intraocular invasion or extrascleral extension after vitrectomy. CONCLUSIONS: Vitrectomy for complications of choroidal tumor biopsy is rare. Such corrective surgery is complex and is best undertaken by specialized ocular oncologists or vitreoretinal surgeons with experience in managing this problem.

Nouvelles stratégies thérapeutiques en oncologie ophtalmo-pédiatrique [New strategies in pediatric ophthalmic oncology]

Le rétinoblastome représente 11% de tous les cancers apparaissant pendant la première année de vie. Pour éviter les effets secondaires de la chimiothérapie systémique ou de la radiothérapie externe, de nouveaux médicaments et de nouvelles techniques de traitement focalisé ont été développés. Des voies d'administration telles la voie périoculaire (topotécan) ou la voie artérielle ophtalmique directe (carboplatine) sont utilisées aujourd'hui dans certains cas résistants. La radiothérapie actuelle, appliquée exclusivement en deuxième intention, fait appel également à des techniques ciblées, permettant d'éviter les tissus sains et de réduire le risque de cancers radio-induits non oculaires. La radiothérapie stéréotaxique conformationnelle ou conformale et la proton-thérapie font ainsi partie du nouvel arsenal thérapeutique du rétinoblastome. Retinoblastoma represents 11% of all cancers during the first year of life. New drugs and focal treatments have been developed in order to avoid the side effects of systemic chemotherapy and external radiotherapy. New targeted and local administration strategies such as periocular chemotherapy (topotecan) or direct ophthalmic artery delivery (carboplatin), are already used today in selected resistant cases. Radiotherapy, presently indicated only as a second-line treatment, is also subject to new techniques, targeting tumors more closely to avoid involving healthy tissue and reduce the risk of radio-induced nonocular tumors. Stereotactic conformal radiotherapy and proton therapy may thus be included in the new range of treatment methods in retinoblastoma

Double Concentric Autofluorescence Ring in NR2E3-p.G56R-linked Autosomal Dominant Retinitis Pigmentosa.

Purpose: We report an unusual appearance of fundus autofluorescence (FAF) associated with NR2E3-p.G56R-linked autosomal dominant retinitis pigmentosa (ADRP).Methods: Patients were enrolled among three generations in a Swiss family. Molecular diagnosis identified a c.166G>A (p.G56R) mutation. Ophthalmic examination included fundus photography, FAF, near-infrared autofluorescence (NIA), optical coherence tomography (OCT) and visual fields (VF).Results: Fundus examination revealed a wide range of features from unremarkable to attenuated arterial caliber, clumped and spicular pigment deposits in the mid-periphery and optic nerve pallor. FAF showed a double concentric hyperautofluorescent ring: an inner perimacular ring which tended to be smaller in older patients, and an outer ring located along the vascular arcades, which appeared to extend over time towards the periphery and eventually became hypoautofluorescent. The inner and outer hyperautofluorescent rings were seen both on NIA and FAF at a similar localization. There was also a spatial correspondence between the loss of photoreceptor inner segment and outer segment junction on OCT and the area delimited by both double FAF and NIA rings. VF showed either midperipheral annular scotoma or constricted visual field loss in advanced cases, correlating with dystrophic non-functional retinal regions demarcated by the hyperautofluorescent annuli. A double ring of hyperautofluorescence was observed in all but one patient of two additional families, but not in patients harboring mutations in other ADRP genes, including PRPF3, RHO, RP1, PRPH2, PROM1 and CTRP5.Conclusions: The presence of a double concentric hyperautofluorescent ring of FAF may represent a highly penetrant early phenotypic marker of NR2E3-p.G56R-linked ADRP.

PRPF31 alternative splicing and expression in human retina

PURPOSE: To provide a mechanistic link between mutations in PRPF31, and essential and ubiquitously expressed gene, and retinitis pigmentosa, a disorder restricted to the eye. METHODS: We investigated the existence of retina-specific PRPF31 isoforms and the expression of this gene in human retina and other tissues, as well as in cultured human cell lines. PRPF31 transcripts were examined by RT-PCR, quantitative PCR, cloning and sequencing. RESULTS: Database searching revealed the presence of a retina-specific PRPF31 isoform in mouse. However, this isoform could not be experimentally identified in transcripts from human retina or from a human whole eye. Nevertheless, four different PRPF31 isoforms, that were common to all analyzed tissues and cell lines, were isolated. Three of these harbored the full-length PRPF31 coding sequence, whereas the fourth was very short and probably non-coding. The amount of PRPF31 mRNA was previously found to be lower in patients with mutations in this gene than in healthy individuals, making it likely that retinal cells are more sensitive to variation in PRPF31 expression. However, quantitative PCR experiments revealed that PRPF31 mRNA levels in human retina were comparable to those detected in other tissues. CONCLUSIONS: Our results show that the retina-restricted phenotype caused by PRPF31 mutations cannot be explained by the presence of tissue-specific isoforms, or by differential expression of PRPF31 in the retina. As a consequence, the etiology of PRPF31-associated retinitis pigmentosa likely relies on other, probably more subtle molecular mechanisms.

Emotional attention as a modulatory system of perception

Report for the scientific sojourn carried out at the University Medical Center, Swiss, from 2010 to 2012. Abundant evidence suggests that negative emotional stimuli are prioritized in the perceptual systems, eliciting enhanced neural responses in early sensory regions as compared with neutral information. This facilitated detection is generally paralleled by larger neural responses in early sensory areas, relative to the processing of neutral information. In this sense, the amygdala and other limbic regions, such as the orbitofrontal cortex, may play a critical role by sending modulatory projections onto the sensory cortices via direct or indirect feedback.The present project aimed at investigating two important issues regarding these mechanisms of emotional attention, by means of functional magnetic resonance imaging. In Study I, we examined the modulatory effects of visual emotion signals on the processing of task-irrelevant visual, auditory, and somatosensory input, that is, the intramodal and crossmodal effects of emotional attention. We observed that brain responses to auditory and tactile stimulation were enhanced during the processing of visual emotional stimuli, as compared to neutral, in bilateral primary auditory and somatosensory cortices, respectively. However, brain responses to visual task-irrelevant stimulation were diminished in left primary and secondary visual cortices in the same conditions. The results also suggested the existence of a multimodal network associated with emotional attention, presumably involving mediofrontal, temporal and orbitofrontal regions Finally, Study II examined the different brain responses along the low-level visual pathways and limbic regions, as a function of the number of retinal spikes during visual emotional processing. The experiment used stimuli resulting from an algorithm that simulates how the visual system perceives a visual input after a given number of retinal spikes. The results validated the visual model in human subjects and suggested differential emotional responses in the amygdala and visual regions as a function of spike-levels. A list of publications resulting from work in the host laboratory is included in the report.

Emotional attention as a modulatory system of perception

Report for the scientific sojourn carried out at the University Medical Center, Swiss, from 2010 to 2012. Abundant evidence suggests that negative emotional stimuli are prioritized in the perceptual systems, eliciting enhanced neural responses in early sensory regions as compared with neutral information. This facilitated detection is generally paralleled by larger neural responses in early sensory areas, relative to the processing of neutral information. In this sense, the amygdala and other limbic regions, such as the orbitofrontal cortex, may play a critical role by sending modulatory projections onto the sensory cortices via direct or indirect feedback.The present project aimed at investigating two important issues regarding these mechanisms of emotional attention, by means of functional magnetic resonance imaging. In Study I, we examined the modulatory effects of visual emotion signals on the processing of task-irrelevant visual, auditory, and somatosensory input, that is, the intramodal and crossmodal effects of emotional attention. We observed that brain responses to auditory and tactile stimulation were enhanced during the processing of visual emotional stimuli, as compared to neutral, in bilateral primary auditory and somatosensory cortices, respectively. However, brain responses to visual task-irrelevant stimulation were diminished in left primary and secondary visual cortices in the same conditions. The results also suggested the existence of a multimodal network associated with emotional attention, presumably involving mediofrontal, temporal and orbitofrontal regions Finally, Study II examined the different brain responses along the low-level visual pathways and limbic regions, as a function of the number of retinal spikes during visual emotional processing. The experiment used stimuli resulting from an algorithm that simulates how the visual system perceives a visual input after a given number of retinal spikes. The results validated the visual model in human subjects and suggested differential emotional responses in the amygdala and visual regions as a function of spike-levels. A list of publications resulting from work in the host laboratory is included in the report.

The outer limiting membrane (OLM) revisited: clinical implications.

PURPOSE: The outer limiting membrane (OLM) is considered to play a role in maintaining the structure of the retina through mechanical strength. However, the observation of junction proteins located at the OLM and its barrier permeability properties may suggest that the OLM may be part of the retinal barrier. MATERIAL AND METHODS: Normal and diabetic rat, monkey, and human retinas were used to analyze junction proteins at the OLM. Proteome analyses were performed using immunohistochemistry on sections and flat-mounted retinas and western blotting on protein extracts obtained from laser microdissection of the photoreceptor layers. Semi-thin and ultrastructure analyses were also reported. RESULTS: In the rat retina, in the subapical region zonula occludens-1 (ZO-1), junction adhesion molecule (JAM), an atypical protein kinase C, is present and the OLM shows dense labeling of occludin, JAM, and ZO-1. The presence of occludin has been confirmed using western blot analysis of the microdissected OLM region. In diabetic rats, occludin expression is decreased and glial cells junctions are dissociated. In the monkey retina, occludin, JAM, and ZO-1 are also found in the OLM. Junction proteins have a specific distribution around cone photoreceptors and Müller glia. Ultrastructural analyses suggest that structures like tight junctions may exist between retinal glial Müller cells and photoreceptors. CONCLUSIONS: In the OLM, heterotypic junctions contain proteins from both adherent and tight junctions. Their structure suggests that tight junctions may exist in the OLM. Occludin is present in the OLM of the rat and monkey retina and it is decreased in diabetes. The OLM should be considered as part of the retinal barrier that can be disrupted in pathological conditions contributing to fluid accumulation in the macula.

Shorter time to diagnosis and improved stage at presentation in Swiss patients with retinoblastoma treated from 1963 to 2004.

OBJECTIVES: Retinoblastoma is the most frequent intraocular malignancy in children. Early diagnosis is essential for globe salvage and patient survival. The aim of our study was to determine how time to diagnosis of retinoblastoma has evolved over a 40-year period in Switzerland. METHOD AND PATIENTS: A retrospective study of 139 Swiss patients with retinoblastoma was performed comparing 3 periods: (1) 1963-1983; (2) 1984-1993; and (3) 1994-2004. Factors taken into account were gender, laterality of retinoblastoma, age at first symptoms, type and first observer of symptoms, time to diagnosis, age at diagnosis, disease stage, and family history. RESULTS: Thirty-seven patients (26.6%) were treated in period 1, 44 (31.7%) in period 2, and 58 (41.7%) in period 3. Overall, the diagnostic interval decreased in a significant way from 6.97 months in period 1 to 3.58 in period 2 and to 2.25 in period 3. When looking separately at unilateral and bilateral disease, the decrease of the diagnostic interval remained statistically significant in unilateral retinoblastoma; there was also a significant reduction in the number of patients with advanced group E disease (Murphree classification) (61.5% in period 1, 46.7% in period 2, 22.2% in period 3). In bilateral disease, the same observations were made to a lesser extent. However, there were no cases with group E disease in 10 patients with positive family history. Leukocoria (48.2%) and strabismus (20.1%) were the 2 most frequent symptoms throughout the 3 periods. The only factors that statistically influenced the chances of having a diagnosis of group E disease were the diagnostic interval and period of diagnosis. CONCLUSIONS: Progress has been made in the diagnosis of retinoblastoma in Switzerland, notably in unilateral disease. Improvement to a lesser extent has also been observed in bilateral cases but without statistical significance. Greater effort is needed to teach physicians-in-training to recognize the importance of ocular symptoms and refer patients earlier.

Melastatin Expression in Ocular Melanocytic Proliferations

Purpose: Melastatin (MLSN-1) belongs to the transient receptor potential (TRP) superfamilly of calcium-permeable channels, and has been reported to be a melanocyte-specific gene. In human cutaneous melanoma, MLSN-1 mRNA expression displays a pattern of inverse correlation to disease free survival. We describe the patterns of MLSN-1 mRNA expression in conjunctival nevi, conjunctival melanoma, and uveal melanoma. Methods: In situ hybridization using two S35-labelled riboprobes for MLSN-1 was performed on formalin-fixed, paraffin-embedded tissues. A control probe for H4 histone was used to confirm mRNA integrity in these archival tissues. The 21 ocular melanocytic lesions studied included 5 conjunctival nevi, 6 conjunctival melanomas, and 10 enucleated eyes with uveal melanoma. The minimal requirement for interpretation of MLSN-1 mRNA loss was the presence of only background signal in a focus of at least 5 adjacent melanocytic cells. Results: Ubiquitous expression of MLSN-1 mRNA was found in conjunctival melanocytes in the non-lesional epithelium adjacent to the conjunctival melanocytic proliferations and in all 5 conjunctival nevi studied. Four different patterns of MLSN-1 mRNA expression were observed in conjunctival melanomas: one case showed complete preservation of MLSN-1 mRNA, two cases showed diffuse scattered loss of MLSN-1 mRNA, two cases showed focal clonal loss of MLSN-1 mRNA expression, and one case had no detected MLSN-1 mRNA. In uveal melanomas, MLSN-1 mRNA expression was partially preserved in two cases, lost by a clearly delimited subset of tumor cells (focal clonal loss) in four cases, and was not detectable in the entire tumor in four cases. MLSN-1 mRNA expression was also found in the normal iris, ciliary and choroidal melanocytes as well as in the retinal pigmented epithelium and in the inner nuclear layer of the retina. Conclusions: The patterns of MLSN-1 mRNA expression in the ocular melanocytic proliferations are similar to those reported in cutaneous melanocytic proliferations. In the conjunctiva, MLSN-1 mRNA expression appeared to correlate with tumor progression; all the benign conjunctival nevi had preserved expression of MLSN-1 mRNA and most of the conjunctival melanomas partial or complete loss of expression. In uveal melanoma, patterns of melastatin expression ranging from partial preservation to complete loss were found. Additional studies of a large number of ocular melanocytic proliferations may show a correlation with tumor progression and prognosis similar to that observed in cutaneous melanoma.

Ocular and systemic bio-distribution of rhodamine-conjugated liposomes loaded with VIP injected into the vitreous of Lewis rats.

PURPOSE: Local delivery of therapeutic molecules encapsulated within liposomes is a promising method to treat ocular inflammation. The purpose of the present study was to define the biodistribution of rhodamine-conjugated liposomes loaded with vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, following their intravitreal (IVT) injection in normal rats. METHODS: Healthy seven- to eight-week-old Lewis male rats were injected into the vitreous with empty rhodamine-conjugated liposomes (Rh-Lip) or with VIP-loaded Rh-Lip (VIP-Rh-Lip; 50 mM of lipids with an encapsulation efficiency of 3.0+/-0.4 mmol VIP/mol lipids). Twenty-four h after IVT injection, the eyes, the cervical, mesenteric, and inguinal lymph nodes (LN), and spleen were collected. The phenotype and distribution of cells internalizing Rh-Lip and VIP-Rh-Lip were studied. Determination of VIP expression in ocular tissues and lymphoid organs and interactions with T cells in cervical LN was performed on whole mounted tissues and frozen tissue sections by immunofluorescence and confocal microscopy. RESULTS: In the eye, 24 h following IVT injection, fluorescent liposomes (Rh-Lip and VIP-Rh-Lip) were detected mainly in the posterior segment of the eye (vitreous, inner layer of the retina) and to a lesser extent at the level of the iris root and ciliary body. Liposomes were internalized by activated retinal Müller glial cells, ocular tissue resident macrophages, and rare infiltrating activated macrophages. In addition, fluorescent liposomes were found in the episclera and conjunctiva where free VIP expression was also detected. In lymphoid organs, Rh-Lip and VIP-Rh-Lip were distributed almost exclusively in the cervical lymph nodes (LN) with only a few Rh-Lip-positive cells detected in the spleen and mesenteric LN and none in the inguinal LN. In the cervical LN, Rh-Lip were internalized by resident ED3-positive macrophages adjacent to CD4 and CD8-positive T lymphocytes. Some of these T lymphocytes in close contact with macrophages containing VIP-Rh-Lip expressed VIP. CONCLUSIONS: Liposomes are specifically internalized by retinal Müller glial cells and resident macrophages in the eye. A limited passage of fluorescent liposomes from the vitreous to the spleen via the conventional outflow pathway and the venous circulation was detected. The majority of fluorescent liposomes deposited in the conjunctiva following IVT injection reached the subcapsular sinus of the cervical LN via conjuntival lymphatics. In the cervical LN, Rh-Lip were internalized by resident subcapsular sinus macrophages adjacent to T lymphocytes. Detection of VIP in both macrophages and T cells in cervical LN suggests that IVT injection of VIP-Rh-Lip may increase ocular immune privilege by modulating the loco-regional immune environment. In conclusion, our observations suggest that IVT injection of VIP-loaded liposomes is a promising therapeutic strategy to dampen ocular inflammation by modulating macrophage and T cell activation mainly in the loco-regional immune system.

CNOT3 is a modifier of PRPF31 mutations in retinitis pigmentosa with incomplete penetrance.

Heterozygous mutations in the PRPF31 gene cause autosomal dominant retinitis pigmentosa (adRP), a hereditary disorder leading to progressive blindness. In some cases, such mutations display incomplete penetrance, implying that certain carriers develop retinal degeneration while others have no symptoms at all. Asymptomatic carriers are protected from the disease by a higher than average expression of the PRPF31 allele that is not mutated, mainly through the action of an unknown modifier gene mapping to chromosome 19q13.4. We investigated a large family with adRP segregating an 11-bp deletion in PRPF31. The analysis of cell lines derived from asymptomatic and affected individuals revealed that the expression of only one gene among a number of candidates within the 19q13.4 interval significantly correlated with that of PRPF31, both at the mRNA and protein levels, and according to an inverse relationship. This gene was CNOT3, encoding a subunit of the Ccr4-not transcription complex. In cultured cells, siRNA-mediated silencing of CNOT3 provoked an increase in PRPF31 expression, confirming a repressive nature of CNOT3 on PRPF31. Furthermore, chromatin immunoprecipitation revealed that CNOT3 directly binds to a specific PRPF31 promoter sequence, while next-generation sequencing of the CNOT3 genomic region indicated that its variable expression is associated with a common intronic SNP. In conclusion, we identify CNOT3 as the main modifier gene determining penetrance of PRPF31 mutations, via a mechanism of transcriptional repression. In asymptomatic carriers CNOT3 is expressed at low levels, allowing higher amounts of wild-type PRPF31 transcripts to be produced and preventing manifestation of retinal degeneration.