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PMCID: PMC3293109.-- et. al., Mutations in the CRB1 gene are associated with variable phenotypes of severe retinal dystrophies, ranging from leber congenital amaurosis (LCA) to rod–cone dystrophy, also called retinitis pigmentosa (RP). Moreover, retinal dystrophies resulting from CRB1 mutations may be accompanied by specific fundus features: preservation of the para-arteriolar retinal pigment epithelium (PPRPE) and retinal telangiectasia with exudation (also referred to as Coats-like vasculopathy). In this publication, we report seven novel mutations and classify over 150 reported CRB1 sequence variants that were found in more that 240 patients. The data from previous reports were used to analyze a potential correlation between CRB1 variants and the clinical features of respective patients. This meta-analysis suggests that the differential phenotype of patients with CRB1 mutations is due to additional modifying factors rather than particular mutant allele combination. Hum Mutat 33:306–315, 2012. © 2011 Wiley Periodicals, Inc., The project was financially supported by the Foundation Fighting Blindness (I.A. FFB Grant No: CD-CL-0808-0466-CHNO and the CIC503 recognized as an FFB center, FFB Grant No: C-CMM-0907-0428-INSERM04), Agence Nationale de la Recherche (SSB), Fondation Voir et Entendre (CZ), GIS-maladies rares (CZ), Ville de Paris and Région Ile de France, National Institutes of Health (USA) (KB NIH, Grant No: 1R01EY020902 - 01A1). European Reintegration Grant PERG04-GA-2008-231125 (to K.B.).

Rod-cone dystrophy (RCD), also called retinitis pigmentosa, is characterized by rod followed by cone photoreceptor degeneration, leading to gradual visual loss. Mutations in over 65 genes have been associated with non-syndromic RCD explaining 60% to 70% of cases, with novel gene defects possibly accounting for the unsolved cases. Homozygosity mapping and whole-exome sequencing applied to a case of autosomal recessive non-syndromic RCD from a consanguineous union identified a homozygous variant in WDR34. Mutations in WDR34 have been previously associated with severe ciliopathy syndromes possibly associated with a retinal dystrophy. This is the first report of a homozygous mutation in WDR34 associated with non-syndromic RCD., Doctoral funding from the Ministère de l'Enseignement Supérieur et de la Recherche; Europe exchange 2018 Erasmus; European Reintegration Grant, Grant/Award Number: PERG04-GA-2008-231125; Fondation de France-Berthe Fouassier; Foundation Fighting Blindness, Grant/Award Number: Grant # CD-CL-0808-0466-CHNO CIC503 recogn; Foundation Voir et Entendre; French Agence Nationale de la Recherche, Grant/Award Numbers: IHU FOReSIGHT: ANR-18-IAHU-0001, LIFESENSES: ANR-10-LABX-65; National Eye Institute [R01EY012910 (EAP), R01EY026904 (KMB/EAP) and P30EY014104 (MEEI core support)], the Foundation Fighting

The purpose of this work was to identify the gene defect underlying a relatively mild rod-cone dystrophy (RCD), lacking disease-causing variants in known genes implicated in inherited retinal disorders (IRD), and provide transcriptomic and immunolocalization data to highlight the best candidate. The DNA of the female patient originating from a consanguineous family revealed no large duplication or deletion, but several large homozygous regions. In one of these, a homozygous frameshift variant, c.244_246delins17 p.(Trp82Valfs*4), predicted to lead to a nonfunctional protein, was identified in CCDC51. CCDC51 encodes the mitochondrial coiled-coil domain containing 51 protein, also called MITOK. MITOK ablation causes mitochondrial dysfunction. Here we show for the first time that CCDC51/MITOK localizes in the retina and more specifically in the inner segments of the photoreceptors, well known to contain mitochondria. Mitochondrial proteins have previously been implicated in IRD, although usually in association with syndromic disease, unlike our present case. Together, our findings add another ultra-rare mutation implicated in non-syndromic IRD, whose pathogenic mechanism in the retina needs to be further elucidated.

B vitamins may protect against age-related macular degeneration (AMD). We evaluated the associations of dietary intake and serum vitamins with the incidence of advanced AMD in the Alienor study. The Alienor study is a prospective population-based cohort of 963 residents of Bordeaux, France, who were 73 years or older at baseline (2006–2008). Examinations were performed every two years over an eight-year period. The incidence of AMD is based on retinal fundus photographs and spectral-domain optical coherence tomography examinations. Among the 861 included participants, 93 developed incident AMD during a median follow-up time of 9.8 years. Participants with normal serum folate (≥10 nmol/L) significantly had a 51% reduced risk for AMD in the fully adjusted Cox model (HR, 0.49 [95% CI, 0.25–0.95], p = 0.036). Participants with a higher dietary intake of B5 and B6 vitamins had a lower risk for developing AMD of up to 28% (HR, 0.72 for 1-SD increase [0.53–0.99], p = 0.049; HR, 0.90 [0.81–0.99], p = 0.049, respectively). This cohort study of older adults suggests a strong association between a normal serum folate status, a high dietary intake of B5 and B6 and a lower risk for developing advanced AMD. Adopting a healthy diet rich in B vitamins may help to reduce vision loss due to AMD.

Contains fulltext : 245098.pdf (Publisher’s version ) (Closed access) PURPOSE: Current prediction models for advanced age-related macular degeneration (AMD) are based on a restrictive set of risk factors. The objective of this study was to develop a comprehensive prediction model applying a machine learning algorithm allowing selection of the most predictive risk factors automatically. DESIGN: Two population-based cohort studies. PARTICIPANTS: The Rotterdam Study I (RS-I; training set) included 3838 participants 55 years of age or older, with a median follow-up period of 10.8 years, and 108 incident cases of advanced AMD. The Antioxydants, Lipids Essentiels, Nutrition et Maladies Oculaires (ALIENOR) study (test set) included 362 participants 73 years of age or older, with a median follow-up period of 6.5 years, and 33 incident cases of advanced AMD. METHODS: The prediction model used the bootstrap least absolute shrinkage and selection operator (LASSO) method for survival analysis to select the best predictors of incident advanced AMD in the training set. Predictive performance of the model was assessed using the area under the receiver operating characteristic curve (AUC). MAIN OUTCOME MEASURES: Incident advanced AMD (atrophic, neovascular, or both), based on standardized interpretation of retinal photographs. RESULTS: The prediction model retained (1) age, (2) a combination of phenotypic predictors (based on the presence of intermediate drusen, hyperpigmentation in one or both eyes, and Age-Related Eye Disease Study simplified score), (3) a summary genetic risk score based on 49 single nucleotide polymorphisms, (4) smoking, (5) diet quality, (6) education, and (7) pulse pressure. The cross-validated AUC estimation in RS-I was 0.92 (95% confidence interval [CI], 0.88-0.97) at 5 years, 0.92 (95% CI, 0.90-0.95) at 10 years, and 0.91 (95% CI, 0.88-0.94) at 15 years. In ALIENOR, the AUC reached 0.92 at 5 years (95% CI, 0.87-0.98). In terms of calibration, the model tended to underestimate the cumulative incidence of advanced AMD for the high-risk groups, especially in ALIENOR. CONCLUSIONS: This prediction model reached high discrimination abilities, paving the way toward making precision medicine for AMD patients a reality in the near future.

International audience; Melanoma-associated retinopathy (MAR) is a rare paraneoplastic retinal disorder usually occurring in the context of metastatic melanoma. Patients present with night blindness, photopsias and a constriction of the visual field. MAR is an auto-immune disorder characterized by the production of autoantibodies targeting retinal proteins, especially autoantibodies reacting to the cation channel TRPM1 produced in melanocytes and ON-bipolar cells. TRPM1 has at least three different isoforms which vary in the N-terminal region of the protein. In this study, we report the case of three new MAR patients presenting different anti-TRPM1 autoantibodies reacting to the three isoforms of TRPM1 with variable binding affinity. Two sera recognized all isoforms of TRPM1, while one recognized only the two longest isoforms upon immunolocalization studies on overexpressing cells. Similarly, the former two sera reacted with all TRPM1 isoforms on western blot, but an immunoprecipitation enrichment step was necessary to detect all isoforms with the latter serum. In contrast, all sera labelled ON-bipolar cells on Tprm1+/+ but not on Trpm1-/- mouse retina as shown by co-immunolocalization. This confirms that the MAR sera specifically detect TRPM1. Most likely, the anti-TRPM1 autoantibodies of different patients vary in affinity and concentration. In addition, the binding of autoantibodies to TRPM1 may be conformation-dependent, with epitopes being inaccessible in some constructs (truncated polypeptides versus full-length TRPM1) or applications (western blotting versus immunohistochemistry). Therefore, we propose that a combination of different methods should be used to test for the presence of anti-TRPM1 autoantibodies in the sera of MAR patients.

Purpose: To investigate associations of adherence to the Mediterranean diet (MeDi) with incidence of advanced age-related macular degeneration (AMD; the symptomatic form of AMD) in 2 European population-based prospective cohorts. Design: Prospective cohort study of the Rotterdam Study I (RS-I) and the Antioxydants, Lipides Essentiels, Nutrition et Maladies Oculaires (Alienor) Study populations. Participants: Four thousand four hundred forty-six participants 55 years of age or older from the RS-I (The Netherlands) and 550 French adults 73 years of age or older from the Alienor Study with complete ophthalmologic and dietary data were included in the present study. Methods: Examinations were performed approximately every 5 years over a 21-year period (1990–2011) in RS-I and every 2 years over a 4-year period (2006–2012) in the Alienor Study. Adherence to the MeDi was evaluated using a 9-component score based on intake of vegetables, fruits, legumes, cereals, fish, meat, dairy products, alcohol, and the monounsaturated-to-saturated fatty acids ratio. Associations of incidence of AMD with MeDi were estimated using multivariate Cox proportional hazard models. Main Outcomes Measures: Incidence of advanced AMD based on retinal fundus photographs. Results: Among the 4996 included participants, 155 demonstrated advanced incident AMD (117 from the RS-I and 38 from the Alienor Study). The mean follow-up time was 9.9 years (range, 0.6–21.7 years) in the RS-I and 4.1 years (range, 2.5–5.0 years) in the Alienor Study. Pooling data for both the RS-I and Alienor Study, participants with a high (range, 6–9) MeDi score showed a significantly reduced risk for incident advanced AMD compared with participants with a low (range, 0–3) MeDi score in the fully adjusted Cox model (hazard ratio, 0.59; 95% confidence interval, 0.37–0.95; P = 0.04 for trend). Conclusions: Pooling data from the RS-I and Alienor Study, higher adherence to the MeDi was associated with a 41% reduced risk of incident advanced AMD. These findings support the role of a diet rich in healthful nutrient-rich foods such as fruits, vegetables, legumes, and fish in the prevention of AMD., The author(s) have made the following disclosure(s): B.M.J.M.: Consultant e Bausch & Lomb (Rochester, New York); Financial support e Laboratoires Théa (Clermont-Ferrand, France). A.C.-G.: Financial support e Laboratoires Théa (Clermont-Ferrand, France). M.-N.D.: Consultant e Bayer (Leverkusen, Germany), Allergan (Irvine, California), Novartis (Basel, Switzerland); Board membership e Bayer (Leverkusen, Germany), Allergan (Irvine, California), Novartis (Basel, Switzerland). O.H.F.: Financial support e Nestle (Vevey, Switzerland). J.-F.K.: Consultant e Alcon (Hünenberg, Switzerland), Alimera (Alpharetta, Georgia), Novartis (Basel, Switzerland), Roche (Basel, Switzerland), Thea (Clermont-Ferrand, France), Zeiss (Oberkochen, Germany), Bayer (Leverkusen, Germany). C.C.W.K.: Consultant e Bayer (Leverkusen, Germany); Lecturer e Novartis (Basel, Switzerland), Thea Pharma (Clermont-Ferrand, France). C.D.: Consultant e Allergan (Irvine, California), Bausch & Lomb (Rochester, New York), Laboratoires Théa (Clermont-Ferrand, France), Novartis (Basel, Switzerland), Roche (Basel, Switzerland); Financial support e Laboratoires Théa (Clermont-Ferrand, France), Essilor (Charenton le Pont, France). M.B.: Travel fees e Bayer (Leverkusen, Germany); Consultant e Roche (Basel, Switzerland). R.I., H.L., C.M., and E.N.: Employees e F. Hoffmann-La Roche Ltd (Basel, Switzerland). J.M.: Financial support e Bayer (Leverkusen, Germany), Alcon (Hünenberg, Switzerland), Ophthotech (New-York, NY), Notal Vision (Manassas, VA), Novartis (Basel, Switzerland), Roche (Basel, Switzerland). I.L.: Unrestricted research support e OPTOS Plc (Dunfermline, UK). Competing financial interest of members of the EYE-RISK consortium not otherwise disclosed: Verena Arndt, Sebastian Bühren, Tanja Endermann, and Markus Zumbansen are employees of AYOXXA. The EYE-RISK project is supported by the European Union’s Horizon 2020 Research and Innovation Programme (grant no.: 634479). The Rotterdam Study is funded by Erasmus Medical Center and Erasmus University, Rotterdam, The Netherlands; the Organization for the Health Research and Development (ZonMw); the Research Institute for Diseases in the Elderly (RIDE); the Ministry of Education, Culture and Science; the Ministry for Health, Welfare and Sports; the European Commission (DG XII), and the Municipality of Rotterdam, Rotterdam, The Netherlands. Additionally, the ophthalmic research within the Rotterdam Study was supported by the following foundations: Oogfonds; Bartiméus Sonneheerdt Vereniging; Landelijke Stichting voor Blinden en Slechtzienden; Algemene Nederlandse Vereniging Ter Voorkoming Van Blindheid; Novartis Foundation; and MaculaFonds, which contributed through UitZicht (grant nos.: 2015-36 and 2016-19). The funding organizations had no role in the design or conduct of this research and provided unrestricted grants. The Antioxydants, Lipides Essentiels, Nutrition et Maladies Oculaires Study is funded by Laboratoires Théa; Fondation Voir et Entendre; Retina France; Agence Nationale de la Recherche (ANR 2010-PRSP-011 VISA); and Caisse Nationale pour la Solidarité et l’Autonomie.

Audo, Isabelle et al., Inherited retinal diseases are a group of clinically and genetically heterogeneous disorders for which a significant number of cases remain genetically unresolved. Increasing knowledge on underlying pathogenic mechanisms with precise phenotype-genotype correlation is, however, critical for establishing novel therapeutic interventions for these yet incurable neurodegenerative conditions. We report phenotypic and genetic characterization of a large family presenting an unusual autosomal dominant retinal dystrophy. Phenotypic characterization revealed a retinopathy dominated by inner retinal dysfunction and ganglion cell abnormalities. Whole-exome sequencing identified a missense variant (c.782A>C, p.Glu261Ala) in ITM2B coding for Integral Membrane Protein 2B, which co-segregates with the disease in this large family and lies within the 24.6 Mb interval identified by microsatellite haplotyping. The physiological role of ITM2B remains unclear and has never been investigated in the retina. RNA in situ hybridization reveals Itm2b mRNA in inner nuclear and ganglion cell layers within the retina, with immunostaining demonstrating the presence of the corresponding protein in the same layers. Furthermore, ITM2B in the retina co-localizes with its known interacting partner in cerebral tissue, the amyloid ß precursor protein, critical in Alzheimer disease physiopathology. Interestingly, two distinct ITM2B mutations, both resulting in a longer protein product, had already been reported in two large autosomal dominant families with Alzheimer-like dementia but never in subjects with isolated retinal diseases. These findings should better define pathogenic mechanism(s) associated with ITM2B mutations underlying dementia or retinal disease and add a new candidate to the list of genes involved in inherited retinal dystrophies., The project was supported by GIS-maladies rares (C.Z.), Retina France (part of the 100-Exome Project) (I.A., J.-A.S. and C.Z.), Foundation Voir et Entendre (C.Z.), Foundation Fighting Blindness (FFB) grant CD-CL-0808-0466-CHNO (I.A. and the CIC503, recognized as an FFB centre), FFB grant C-CMM-0907-0428-INSERM04, Ville de Paris and Region Ile de France and by the French State programme ‘Investissements d’Avenir’ managed by the Agence Nationale de la Recherche (LIFESENSES: ANR-10-LABX-65)

International audience; GNAT1, encoding the transducin subunit Gα, is an important element of the phototransduc-tion cascade. Mutations in this gene have been associated with autosomal dominant and autosomal recessive congenital stationary night blindness. Recently, a homozygous trun-cating GNAT1 mutation was identified in a patient with late-onset rod-cone dystrophy. After exclusion of mutations in genes underlying progressive inherited retinal disorders, by targeted next generation sequencing, a 32 year-old male sporadic case with severe rod-cone dystrophy and his unaffected parents were investigated by whole exome sequencing. This led to the identification of a homozygous nonsense variant, c.963C>A p.(Cys321*) in GNAT1, which was confirmed by Sanger sequencing. The mother was heterozygous for this variant whereas the variant was absent in the father. c.963C>A p.(Cys321*) is predicted to produce a shorter protein that lacks critical sites for the phototransduction cascade. Our work confirms that the phenotype and the mode of inheritance associated with GNAT1 variants can vary from autosomal dominant, autosomal recessive congenital stationary night blindness to autosomal recessive rod-cone dystrophy.

Audo, Isabelle, et al., Congenital stationary night blindness (CSNB) is a heterogeneous retinal disorder characterized by visual impairment under low light conditions. This disorder is due to a signal transmission defect from rod photoreceptors to adjacent bipolar cells in the retina. Two forms can be distinguished clinically, complete CSNB (cCSNB) or incomplete CSNB; the two forms are distinguished on the basis of the affected signaling pathway. Mutations in NYX, GRM6, and TRPM1, expressed in the outer plexiform layer (OPL) lead to disruption of the ON-bipolar cell response and have been seen in patients with cCSNB. Whole-exome sequencing in cCSNB patients lacking mutations in the known genes led to the identification of a homozygous missense mutation (c.1807C>T [p.His603Tyr]) in one consanguineous autosomal-recessive cCSNB family and a homozygous frameshift mutation in GPR179 (c.278delC [p.Pro93Glnfs57]) in a simplex male cCSNB patient. Additional screening with Sanger sequencing of 40 patients identified three other cCSNB patients harboring additional allelic mutations in GPR179. Although, immunhistological studies revealed Gpr179 in the OPL in wild-type mouse retina, Gpr179 did not colocalize with specific ON-bipolar markers. Interestingly, Gpr179 was highly concentrated in horizontal cells and Müller cell endfeet. The involvement of these cells in cCSNB and the specific function of GPR179 remain to be elucidated., The project was supported by GIS-maladies rares (C.Z.), Retina France ([part of the 100-Exome Project] I.A., C.P.H., J.-A.S., H.D. and C.Z.), Foundation Voir et Entendre (C.Z.), Agence National de la Recherche (S.S.B), Foundation Fighting Blindness (FFB) grant CD-CL-0808-0466-CHNO (I.A. and the CIC503, recognized as an FFB center), FFB grant C-CMM-0907-0428-INSERM04, Ville de Paris and Région Ille de France, the French Association against Myopathy (AFM) grant KBM-14390 (O.P.), and National Institutes of Health grant 1R01EY020902-01A1 (K.B.).

et al., Rod-cone dystrophies (retinitis pigmentosa [RP]) are a clinically and genetically heterogeneous group of inherited retinal disorders characterized by photoreceptor degeneration. RP1 is a major gene underlying autosomal dominant (ad) RP, though prevalence figures vary depending on the origin of the cases from 0-10% of all adRP. Some mutations in RP1 also lead to autosomal recessive (ar) RP. Herein, we review all previously reported and several novel RP1 mutations in relation to the associated phenotype in RP1 patients from a French adRP cohort. Prevalence studies from this cohort show that 5.3% of the cases have RP1 mutations. This is in accordance with other studies reported from United Kingdom and United States. The majority of mutations represent truncating mutations that are located in a hot spot region of the gene. Similarly, we identified in total four novel deletions and nonsense mutations, of which two may represent recurrent mutations in this population. In addition, a novel missense mutation of uncertain pathogenicity was identified. Including our findings to date, 47 RP1 mutations are known to cause adRP. Variable penetrance of the disease was observed in our and other cohorts. Most patients with RP1 mutations show classical signs of RP with relatively preserved central vision and visual field., Department of Paris, Foundation Fighting Blindness (CD-CL-0808-0466-CHNO to I.A. and the CIC503 recognized as an FFB center C-CMM-0907-0428-INSERM04); ANR (to S.S.B.); NIHR Biomedical Research Centre for Ophthalmology; The Special Trustees of Moorfields Eye Hospital London; Foundation Voir et Entendre (to C.Z.); French Ministry of Health (PHRC# 2008-A01238-47 to C.H.)

International audience; The exquisite sensitivity and frequency tuning of hearing depend on the correct structure and functioning of the auditory sensory hair cells, the neighbouring supporting cells, and the homeostasis of their ionic environment. The increasing number of adhesion proteins identified as causing hearing impairment in humans and mice when defective is consistent with a critical role for cellcell junctions between neighbouring epithelial cells of the cochlea, and of fibrous links within the hair bundle, the sensory hair cell structure responsible for sound reception. Classical cadherins and/or associated adherens-junction proteins, such as p120-catenin or nectin 3, have been shown to be essential for establishment of the regular mosaic cellular pattern of the auditory sensory epithelium. Two cadherinrelated proteins, protocadherin-15 and cadherin-23, are key components of both lateral links and tip-links in hair bundles; they are essential components of the mechanoelectrical transduction machinery. Studies of the role of these adhesion proteins and of the pathogenesis of the forms of deafness caused by defects of these proteins have provided considerable insight into the development and functioning of the auditory sensory epithelium, and of the hair cells in particular.

A detrimental perceptive consequence of damaged auditory sensory hair cells consists in a pronounced masking effect exerted by low-frequency sounds, thought to occur when auditory threshold elevation substantially exceeds 40 dB. Here, we identified the submembrane scaffold protein Nherf1 as a hair-bundle component of the differentiating outer hair cells (OHCs). Nherf1(-/-) mice displayed OHC hair-bundle shape anomalies in the mid and basal cochlea, normally tuned to mid- and high-frequency tones, and mild (22-35 dB) hearing-threshold elevations restricted to midhigh sound frequencies. This mild decrease in hearing sensitivity was, however, discordant with almost nonresponding OHCs at the cochlear base as assessed by distortion-product otoacoustic emissions and cochlear microphonic potentials. Moreover, unlike wild-type mice, responses of Nherf1(-/-) mice to high-frequency (20-40 kHz) test tones were not masked by tones of neighboring frequencies. Instead, efficient maskers were characterized by their frequencies up to two octaves below the probe-tone frequency, unusually low intensities up to 25 dB below probe-tone level, and growth-of-masker slope (2.2 dB/dB) reflecting their compressive amplification. Together, these properties do not fit the current acknowledged features of a hypersensitivity of the basal cochlea to lower frequencies, but rather suggest a previously unidentified mechanism. Low-frequency maskers, we propose, may interact within the unaffected cochlear apical region with midhigh frequency sounds propagated there via a mode possibly using the persistent contact of misshaped OHC hair bundles with the tectorial membrane. Our findings thus reveal a source of misleading interpretations of hearing thresholds and of hypervulnerability to low-frequency sound interference.

International audience; The Usher syndrome (USH) is the most prevalent cause of inherited deaf-blindness. Three clinical subtypes, USH1–3, have been defined, and ten USH genes identified. The hearing impairment due to USH gene defects has been shown to result from improper organisation of the hair bundle, the sound receptive structure of sensory hair cells. In contrast, the cellular basis of the visual defect is less well understood as this phenotype is absent in almost all the USH mouse models that faithfully mimic the human hearing impairment. Structural and molecular interspecies discrepancies regarding photoreceptor calyceal processes and the association with the distribution of USH1 proteins have recently been unravelled, and have led to the conclusion that a defect in the USH1 protein complex-mediated connection between the photoreceptor outer segment and the surrounding calyceal processes (in both rods and cones), and the inner segment (in rods only), probably causes the USH1 retinal dystrophy in humans.; Le syndrome de Usher (USH) constitue la première cause de cécité-surdité héréditaire. Trois sous-types cliniques, USH1–3, ont été́ définis, et dix gènes USH ont été identifiés. La déficience auditive due aux défauts des gènes USH résulte d’une désorganisation de la touffe ciliaire des cellules sensorielles. À l’inverse, la base cellulaire du défaut visuel est beaucoup moins bien comprise, car il manque dans presque tous les modèles murins de USH, qui reproduisent pourtant fidèlement la déficience auditive. Les différences inter-espèces structurelles concernant les processus caliciels de la cellule photoréceptrice et leur association avec la localisation des protéines USH1 ont récemment été mises en évidence. Elles ont conduit à la conclusion qu’un défaut dans la connexion médiée par les protéines USH1 entre le segment externe du photorécepteur et, d’une part, les processus caliciels (à la fois dans les bâtonnets et les cônes) et, d’autre part, le segment interne (dans les bâtonnets seulement) est probablement à l’origine de la dystrophie rétinienne USH1 chez l’homme.

Background Imerslund-Gräsbeck Syndrome (IGS) is a rare genetic disorder characterised by juvenile megaloblastic anaemia. IGS is caused by mutations in either of the genes encoding the intestinal intrinsic factor-vitamin B12 receptor complex, cubam. The cubam receptor proteins cubilin and amnionless are both expressed in the small intestine as well as the proximal tubules of the kidney and exhibit an interdependent relationship for post-translational processing and trafficking. In the proximal tubules cubilin is involved in the reabsorption of several filtered plasma proteins including vitamin carriers and lipoproteins. Consistent with this, low-molecular-weight proteinuria has been observed in most patients with IGS. The aim of this study was to characterise novel disease-causing mutations and correlate novel and previously reported mutations with the presence of low-molecular-weight proteinuria. Methods Genetic screening was performed by direct sequencing of the CUBN and AMN genes and novel identified mutations were characterised by in silico and/or in vitro investigations. Urinary protein excretion was analysed by immunoblotting and high-resolution gel electrophoresis of collected urines from patients and healthy controls to determine renal phenotype. Results Genetic characterisation of nine IGS patients identified two novel AMN frameshift mutations alongside a frequently reported AMN splice site mutation and two CUBN missense mutations; one novel and one previously reported in Finnish patients. The novel AMN mutations were predicted to result in functionally null AMN alleles with no cell-surface expression of cubilin. Also, the novel CUBN missense mutation was predicted to affect structural integrity of the IF-B12 binding site of cubilin and hereby most likely cubilin cell-surface expression. Analysis of urinary protein excretion in the patients and 20 healthy controls revealed increased urinary excretion of cubilin ligands including apolipoprotein A-I, transferrin, vitamin D-binding protein, and albumin. This was, however, only observed in patients where plasma membrane expression of cubilin was predicted to be perturbed. Conclusions In the present study, mutational characterisation of nine IGS patients coupled with analyses of urinary protein excretion provide additional evidence for a correlation between mutation type and presence of the characteristic low-molecular-weight proteinuria.

International audience; Mutations in the myosin VIIa gene cause Usher syndrome type IB (USH1B), characterized by deaf-blindness. A delay of opsin trafficking has been observed in the retinal photoreceptor cells of myosin VIIa-deficient mice. We identified spectrin bV, the mammalian b-heavy spectrin, as a myosin VIIa-and rhodopsin-interacting partner in photoreceptor cells. Spectrin bV displays a polarized distribution from the Golgi apparatus to the base of the outer segment, which, unlike that of other b spectrins, matches the trafficking route of opsin and other phototransduction proteins. Formation of spectrin bV-rhodopsin complex could be detected in the differentiating photoreceptors as soon as their outer segment emerges. A failure of the spectrin bV-mediated coupling between myosin VIIa and opsin molecules thus probably accounts for the opsin transport delay in myosin VIIa-deficient mice. We showed that spectrin bV also associates with two USH1 proteins, sans (USH1G) and harmonin (USH1C). Spectrins are supposed to function as heteromers of a and b subunits, but fluorescence resonance energy transfer and in vitro binding experiments indicated that spectrin bV can also form homodimers, which likely supports its aII-independent bV functions. Finally, consistent with its distribution along the connecting cilia axonemes, spectrin bV binds to several subunits of the microtubule-based motor proteins, kinesin II and the dynein complex. We therefore suggest that spectrin bV homomers couple some USH1 proteins, opsin and other phototransduction proteins to both actin-and microtubule-based motors, thereby contributing to their transport towards the photoreceptor outer disks.

Doumanov, Jordan A. et al., Mutations in BEST1 gene, encoding the bestrophin-1 (Best1) protein are associated with macular dystrophies. Best1 is predominantly expressed in the retinal pigment epithelium (RPE), and is inserted in its basolateral membrane. We investigated the cellular localization in polarized MDCKII cells of disease-associated Best1 mutant proteins to study specific sorting motifs of Best1. Real-time PCR and western blots for endogenous expression of BEST1 in MDCK cells were performed. Best1 mutant constructs were generated using site-directed mutagenesis and transfected in MDCK cells. For protein sorting, confocal microscopy studies, biotinylation assays and statistical methods for quantification of mislocalization were used. Analysis of endogenous expression of BEST1 in MDCK cells revealed the presence of BEST1 transcript but no protein. Confocal microscopy and quantitative analyses indicate that transfected normal human Best1 displays a basolateral localization in MDCK cells, while cell sorting of several Best1 mutants (Y85H, Q96R, L100R, Y227N, Y227E) was altered. In contrast to constitutively active Y227E, constitutively inactive Y227F Best1 mutant localized basolaterally similar to the normal Best1 protein. Our data suggest that at least three basolateral sorting motifs might be implicated in proper Best1 basolateral localization. In addition, non-phosphorylated tyrosine 227 could play a role for basolateral delivery. © 2013 by the authors; licensee MDPI, Basel, Switzerland., This work was supported by Agence Nationale de la Recherche (Chaire d’Excellence to Shomi S. Bhattacharya), Fondation Voir et Entendre (Young Investigator Grants to Christina Zeitz and Emeline F. Nandrot), Centre National de la Recherche Scientifique (CNRS) and Fondation Bettencourt Schueller (to Emeline F. Nandrot), Université Pierre et Marie Curie-Paris6 (Bonus Qualité Recherche to Christina Zeitz), Foundation Fighting Blindness (grant number CD-CL-0808-0466-CHNO to Isabelle Audo), Centre d’Investigation Clinique 503 recognized as a Foundation Fighting Blindness Center (grant number C-CMM-0907-0428-INSERM04), Fundacion Progreso y Salud (to Shomi S. Bhattacharya, Maria Luz Bellido Diaz, Abhay Krishna and Paloma Dominguez Gimenez), Instituto de Salud Carlos III (grant number CM06/00183 to Maria Luz Bellido Diaz) and Bulgarian National Science Fund (grant number DDVU 02/10). Additionally, the Institut de la Vision is funded by Institut National de la Santé et de la Recherche Médicale, Université Pierre et Marie Curie-Paris 6, Centre National de la Recherche Scientifique and Départment de Paris.

Orhan, Elise et al., Purpose. Mutations in GPR179, which encodes the G protein-coupled receptor 179, lead to autosomal recessive complete (c) congenital stationary night blindness (CSNB), which is characterized by an ON-bipolar retinal cell dysfunction. This study further defined the exact site of Gpr179 expression and its protein localization in human retina and elucidated the pathogenic mechanism of the reported missense and splice site mutations. Methods. RNA in situ hybridization was performed with mouse retinal sections. A commercially available antibody was validated with GPR179-overexpressing COS-1 cells and applied to human retinal sections. Live-cell extracellular staining along with subsequent intracellular immunolocalization and ELISA studies were performed using mammalian cells overexpressing wild-type or missense mutated GPR179. Wild-type and splice site-mutated mini-gene constructs were transiently transfected, and RNA was extracted. RT-PCR-amplified products were cloned, and Sanger sequenced. Results. Mouse Gpr179 transcript was expressed in the upper part of the inner nuclear layer, and the respective human protein localized at the dendritic tips of bipolar cells in human retina. The missense mutations p.Tyr220Cys, p.Gly455Asp, and p.His603Tyr led to severely reduced cell surface localization, whereas p.Asp126His did not. The mutated splice donor site altered GPR179 splicing. Conclusions. Our findings indicate that the site of expression and protein localization of human and mouse GPR179 is similar to that of other proteins implicated in cCSNB. For most of the mutations identified so far, loss of the GPR179 protein function seems to be the underlying pathogenic mechanism leading to this form of cCSNB. © 2013 The Association for Research in Vision and Ophthalmology, Inc., Supported by Agence Nationale de la Recherche (ANR-12-BSVS1-0012-01_GPR179) (CZ), Foundation Voir et Entendre (CZ), Prix Dalloz for la recherche en ophtalmologie (CZ), Foundation Fighting Blindness (FFB) (CD-CL-0808-0466-CHNO) (IA), and the CIC503, recognized as an FFB center (FFB Grant C-CMM-0907-0428-INSERM04), Ville de Paris and Region Ile de France, Labex Lifesenses (reference ANR-10-LABX-65) supported by French state funds managed by the ANR within the Investissements d'Avenir programme (ANR-11-IDEX-0004-0), and the Regional Council of Ile-de-France (I09 - 1727/R) (EO)

International audience; The mechanisms underlying retinal dystrophy in Usher syndrome type I (USH1) remain unknown because mutant mice lacking any of the USH1 proteins-myosin VIIa, harmonin, cadherin-23, protocadherin-15, sans-do not display retinal degeneration. We found here that, in macaque photoreceptor cells, all USH1 proteins colocalized at membrane interfaces (i) between the inner and outer segments in rods and (ii) between the microvillus-like calyceal processes and the outer segment basolateral region in rods and cones. This pattern, conserved in humans and frogs, was mediated by the formation of an USH1 protein network, which was associated with the calyceal processes from the early embryonic stages of outer segment growth onwards. By contrast, mouse photoreceptors lacked calyceal processes and had no USH1 proteins at the inner-outer segment interface. We suggest that USH1 proteins form an adhesion belt around the basolateral region of the photoreceptor outer segment in humans, and that defects in this structure cause the retinal degeneration in USH1 patients.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et. al., [Background]: Inherited retinal disorders are clinically and genetically heterogeneous with more than 150 gene defects accounting for the diversity of disease phenotypes. So far, mutation detection was mainly performed by APEX technology and direct Sanger sequencing of known genes. However, these methods are time consuming, expensive and unable to provide a result if the patient carries a new gene mutation. In addition, multiplicity of phenotypes associated with the same gene defect may be overlooked., [Methods]: To overcome these challenges, we designed an exon sequencing array to target 254 known and candidate genes using Agilent capture. Subsequently, 20 DNA samples from 17 different families, including four patients with known mutations were sequenced using Illumina Genome Analyzer IIx next-generation-sequencing (NGS) platform. Different filtering approaches were applied to identify the genetic defect. The most likely disease causing variants were analyzed by Sanger sequencing. Co-segregation and sequencing analysis of control samples validated the pathogenicity of the observed variants., [Results]: The phenotype of the patients included retinitis pigmentosa, congenital stationary night blindness, Best disease, early-onset cone dystrophy and Stargardt disease. In three of four control samples with known genotypes NGS detected the expected mutations. Three known and five novel mutations were identified in NR2E3, PRPF3, EYS, PRPF8, CRB1, TRPM1 and CACNA1F. One of the control samples with a known genotype belongs to a family withtwo clinical phenotypes (Best and CSNB), where a novel mutation was identified for CSNB. In six families the disease associated mutations were not found, indicating that novel gene defects remain to be identified., The project was financially supported by GIS-maladies rares (CZ), Agence Nationale de la Recherche (ANR, SSB), Foundation Voir et Entendre and BQR, Foundation Fighting Blindness (IA, FFB Grant # CD-CL-0808-0466-CHNO and the CIC503 recognized as an FFB center, FFB Grant # C-CMM-0907-0428-INSERM04), Ville de Paris and region Ile de France.

PMCID: PMC3454532.-- et al., Leber congenital amaurosis (LCA) is an infantile-onset form of inherited retinal degeneration characterized by severe vision loss. Two-thirds of LCA cases are caused by mutations in 17 known disease-associated genes (Retinal Information Network (RetNet)). Using exome sequencing we identified a homozygous missense mutation (c.25G>A, p.Val9Met) in NMNAT1 that is likely to be disease causing in two siblings of a consanguineous Pakistani kindred affected by LCA. This mutation segregated with disease in the kindred, including in three other children with LCA. NMNAT1 resides in the previously identified LCA9 locus and encodes the nuclear isoform of nicotinamide mononucleotide adenylyltransferase, a rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD +) biosynthesis. Functional studies showed that the p.Val9Met alteration decreased NMNAT1 enzyme activity. Sequencing NMNAT1 in 284 unrelated families with LCA identified 14 rare mutations in 13 additional affected individuals. These results are the first to link an NMNAT isoform to disease in humans and indicate that NMNAT1 mutations cause LCA. © 2012 Nature America, Inc. All rights reserved., This work was supported by grants from the US National Institutes of Health (RO1-EY12910 (E.A.P.), R03-DK082446 (M.J.F.), R01-GM097409 (E.N.-O.), P30HD026979 (M.J.F. and R.X.) and P30EY014104 (Massachusetts Eye and Ear Infirmary core support)); the Foundation Fighting Blindness USA (I.A., A.D.B., E.L.B., S.S.B., Q.L., A.T.M., D.S.M., E.A.P., J.-A.S., S.M.-S. and A.R.W.); the Rosanne Silbermann Foundation (E.A.P.); the Penn Genome Frontiers Institute (E.A.P. and X.G.); the Institutional Fund to the Center for Biomedical Informatics by the Loyola University Stritch School of Medicine (X.G.); the Foerderer Award for Excellence from the Children’s Hospital of Philadelphia (M.J.F. and X.G.); the from the Division of Child Development and Metabolic Disease at the Children’s Hospital of Philadelphia (M.J.F.); the Clinical and Translational Research Center at the Children’s Hospital of Philadelphia (UL1-RR-024134) (M.J.F. and E.A.P.); the Department of Biotechnology, the Government of India and the Champalimaud Foundation, Portugal (C.K.); the Hyderabad Eye Research Foundation (C.K.); a senior research fellowship from the Council for Scientific and Industrial Research (R.S.); the Foundation Voir et Entendre, Ville de Paris and Région Ille de France (C.Z.); RP Fighting Blindness (UK) (A.R.W.); Fight For Sight (UK) (A.D.B., S.S.B., A.T.M., D.S.M. and A.R.W.); Moorfields Eye Hospital National Institute of Health Research (NIHR) British Research Council (BRC) for Ophthalmology (A.D.B., S.S.B., A.T.M., D.S.M. and A.R.W.); and the Special Trustees of Moorfields Eye Hospital (A.D.B., S.S.B., A.T.M., D.S.M. and A.R.W.).This project is funded, in part, by the Penn Genome Frontiers Institute under a grant with the Pennsylvania Department of Health, which disclaims responsibility for any analyses, interpretations or conclusions.

Otoferlin, a C2-domain-containing Ca2+binding protein, is required for synaptic exocytosis in auditory hair cells. However, its exact role remains essentially unknown. Intriguingly enough, no balance defect has been observed in otoferlin-deficient (Otof−/−) mice. Here, we show that the vestibular nerve compound action potentials evoked during transient linear acceleration ramps inOtof−/−mice display higher threshold, lower amplitude, and increased latency compared with wild-type mice. Using patch-clamp capacitance measurement in intact utricles, we show that type I and type II hair cells display a remarkable linear transfer function between Ca2+entry, flowing through voltage-activated Ca2+channels, and exocytosis. This linear Ca2+dependence was observed when changing the Ca2+channel open probability or the Ca2+flux per channel during various test potentials. InOtof−/−hair cells, exocytosis displays slower kinetics, reduced Ca2+sensitivity, and nonlinear Ca2+dependence, despite morphologically normal synapses and normal Ca2+currents. We conclude that otoferlin is essential for a high-affinity Ca2+sensor function that allows efficient and linear encoding of low-intensity stimuli at the vestibular hair cell synapse.