WDR34, a candidate gene for non-syndromic rod-cone dystrophy

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 Fightin

Functional expression of complement factor I following AAV-mediated gene delivery in the retina of mice and human cells.

Funder: NIHR Oxford Biomedical Research CentreDry age-related macular degeneration (AMD) is characterised by loss of central vision and currently has no approved medical treatment. Dysregulation of the complement system is thought to play an important role in disease pathology and supplementation of Complement Factor I (CFI), a key regulator of the complement system, has the potential to provide a treatment option for AMD. In this study, we demonstrate the generation of AAV constructs carrying the human CFI sequence and expression of CFI in cell lines and in the retina of C57BL/6 J mice. Four codon optimised constructs were compared to the most common human CFI sequence. All constructs expressed CFI protein; however, most codon optimised sequences resulted in significantly reduced CFI secretion compared to the non-optimised CFI sequence. In vivo expression analysis showed that CFI was predominantly expressed in the RPE and photoreceptors. Secreted protein in vitreous humour was demonstrated to be functionally active. The findings presented here have led to the formulation of an AAV-vectored gene therapy product currently being tested in a first-in-human clinical trial in subjects with geographic atrophy secondary to dry AMD (NCT03846193)

Genotypic and Phenotypic Characterization of P23H Line 1 Rat Model

The authors are grateful to Manuel Simonutti, Julie Dégardin, Jennifer Da Silva, Samantha Beck and Caroline Carvalho for their valuable help in phenotyping (platform of Institut de la Vision) and to Isabelle Renault, Léa Biedermann and André Tiffoche for animal care (platform of Institut de la Vision). The authors thank Stéphane Fouquet for his support in developing a custom-made Image J macro to measure thickness of retinal layers.This work was supported by Fondation Valentin Hauy (IA, EO), Retina France (IA, EO), e-rare RHORCOD (IA), Fondation de l’Oeil—Fondation de France (IA), Foundation Voir et Entendre (CZ), Foundation Fighting Blindness (FFB) (CD-CL-0808-0466-CHNO) (IA), and the FFB center grant (CD-CL-0808-0466-CHNO), 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), the Regional Council of Ile de France (I09–1727/R) (EO), the National Institute of Health grants EY10609 (MIN), EY001919 (MML) and EY006842 (MML) and the Foundation Fighting Blindness (MIN and MML).Rod-cone dystrophy, also known as retinitis pigmentosa (RP), is the most common inherited degenerative photoreceptor disease, for which no therapy is currently available. The P23H rat is one of the most commonly used autosomal dominant RP models. It has been created by incorporation of a mutated mouse rhodopsin (Rho) transgene in the wild-type (WT) Sprague Dawley rat. Detailed genetic characterization of this transgenic animal has however never been fully reported. Here we filled this knowledge gap on P23H Line 1 rat (P23H-1) and provide additional phenotypic information applying non-invasive and state-of-the-art in vivo techniques that are relevant for preclinical therapeutic evaluations. Transgene sequence was analyzed by Sanger sequencing. Using quantitative PCR, transgene copy number was calculated and its expression measured in retinal tissue. Full field electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) were performed at 1-, 2-, 3- and 6-months of age. Sanger sequencing revealed that P23H-1 rat carries the mutated mouse genomic Rho sequence from the promoter to the 3’ UTR. Transgene copy numbers were estimated at 9 and 18 copies in the hemizygous and homozygous rats respectively. In 1-month-old hemizygous P23H-1 rats, transgene expression represented 43% of all Rho expressed alleles. ERG showed a progressive rod-cone dysfunction peaking at 6 months-of-age. SD-OCT confirmed a progressive thinning of the photoreceptor cell layer leading to the disappearance of the outer retina by 6 months with additional morphological changes in the inner retinal cell layers in hemizygous P23H-1 rats. These results provide precise genotypic information of the P23H-1 rat with additional phenotypic characterization that will serve basis for therapeutic interventions, especially for those aiming at gene editing.Yeshttp://www.plosone.org/static/editorial#pee

De l’identification de gènes candidats et leur caractérisation fonctionnelle à l’apport d’une preuve de concept dans le cas d’une thérapie génique par édition génomique dans les maladies génétiques rétiniennes stationnaires ou progressives

The first steps in vision occur in the retina when rod and cone photoreceptors transform light into a biochemical signal, which gets processed by bipolar cells, ganglion cells and finally by the brain. Our group investigates genetic causes and mechanisms involved in inherited stationary and progressive retinal diseases as congenital stationary night blindness (CSNB), and rod-cone dystrophy (RCD), also called retinitis pigmentosa. This thesis gives several insights on the retinal physiology. On one hand, we identified GPR179, a new gene mutated in complete CSNB, studied the localization and the physiopathology of missense and splice-site mutations. We also delivered a new knock-out mouse model which we functionally characterized, and studied GPR179 partners to provide a better understanding of the first visual synapse between photoreceptors and ON-bipolar cells. On the other hand, we genotypically and phenotypically characterized one of the most popular RCD model, the P23H rat model. There is currently no treatment for RCD and different therapeutic strategies are under investigation. We wanted to deliver the basis for a genome editing approach for RHO mutations, acting as a dominant negative effect, which cannot be addressed by current gene replacement strategies. We opened the field by performing in vitro, ex vivo and in vivo genome editing experiments using meganucleases, TALEN (Transcription Activator-Like Effector Nuclease) and finally CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) and revealed how challenging the setting of genome editing strategies was.La rétine est un tissu spécialisé dans le traitement de l'information visuelle par l'intermédiaire des photorécepteurs, cônes et bâtonnets, et des neurones de deuxième ordre, les cellules bipolaires et les cellules ganglionnaires dont les axones forment le nerf optique. Notre groupe s'intéresse à élucider les mécanismes génétiques impliqués dans les maladies rares stationnaires, comme dans la cécité nocturne congénitale stationnaire (CNCS), ou progressives comme dans la dystrophie de type bâtonnet-cône (DBC). Cette thèse apporte de nombreuses connaissances sur la physiologie rétinienne. D'une part, nous avons identifié GPR179, un nouveau gène impliqué dans la CNCS complète, étudié la localisation de la protéine et la physiopathologie des protéines mutantes. Nous avons également créé et caractérisé fonctionnellement un nouveau modèle souris invalidé pour GPR179 qui nous a permis de mieux approcher la première synapse rétinienne entre les photorécepteurs et les cellules bipolaires adjacentes. D'autre part, nous avons caractérisé le génotype et le phénotype de l'un des modèles les plus utilisés de la DBC, le rat P23H. Nous avons ensuite développé une approche d'édition génomique pour invalider les mutants RHO ayant un effet dominant négatif en testant in vitro, ex vivo et in vivo les meganucleases, TALEN (Transcription Activator-Like Effector Nuclease) puis le système CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9)

Further insights into GPR179: Expression, localization, and associated pathogenic mechanisms leading to complete congenital stationary night blindness

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)Peer Reviewe

RP1 and autosomal dominant rod-cone dystrophy: Novel mutations, a review of published variants, and genotype-phenotype correlation

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.)Peer Reviewe

Lrit3 deficient mouse (nob6): a novel model of complete congenital stationary night blindness (cCSNB).

Mutations in LRIT3, coding for a Leucine-Rich Repeat, immunoglobulin-like and transmembrane domains 3 protein lead to autosomal recessive complete congenital stationary night blindness (cCSNB). The role of the corresponding protein in the ON-bipolar cell signaling cascade remains to be elucidated. Here we genetically and functionally characterize a commercially available Lrit3 knock-out mouse, a model to study the function and the pathogenic mechanism of LRIT3. We confirm that the insertion of a Bgeo/Puro cassette in the knock-out allele introduces a premature stop codon, which presumably codes for a non-functional protein. The mouse line does not harbor other mutations present in common laboratory mouse strains or in other known cCSNB genes. Lrit3 mutant mice exhibit a so-called no b-wave (nob) phenotype with lacking or severely reduced b-wave amplitudes in the scotopic and photopic electroretinogram (ERG), respectively. Optomotor tests reveal strongly decreased optomotor responses in scotopic conditions. No obvious fundus auto-fluorescence or histological retinal structure abnormalities are observed. However, spectral domain optical coherence tomography (SD-OCT) reveals thinned inner nuclear layer and part of the retina containing inner plexiform layer, ganglion cell layer and nerve fiber layer in these mice. To our knowledge, this is the first time that SD-OCT technology is used to characterize an animal model for CSNB. This phenotype is noted at 6 weeks and at 6 months. The stationary nob phenotype of mice lacking Lrit3, which we named nob6, confirms the findings previously reported in patients carrying LRIT3 mutations and is similar to other cCSNB mouse models. This novel mouse model will be useful for investigating the pathogenic mechanism(s) associated with LRIT3 mutations and clarifying the role of LRIT3 in the ON-bipolar cell signaling cascade

RP1 and autosomal dominant rod-cone dystrophy: novel mutations, a review of published variants, and genotype-phenotype correlation.: RP1mutations in French adRP patients

International audienceRod-cone dystrophies (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 RP. Herein we review all previously reported and several novel RP1 mutations in relation to the associated phenotype in 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 UK and USA. The majority of mutations represent truncating mutations which 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 43 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. ©2011 Wiley Periodicals, Inc

A New Mouse Model for Complete Congenital Stationary Night Blindness Due to Gpr179 Deficiency

International audienceMutations in GPR179 lead to autosomal recessive complete congenital stationary night blindness (cCSNB). This condition represents a signal transmission defect from the photoreceptors to the ON-bipolar cells. To confirm the phenotype, better understand the pathogenic mechanism in vivo, and provide a model for therapeutic approaches, a Gpr179 knock-out mouse model was genetically and functionally characterized. We confirmed that the insertion of a neo/lac Z cassette in intron 1 of Gpr179 disrupts the same gene. Spectral domain optical coherence tomography reveals no obvious retinal structure abnormalities. Gpr179 knock-out mice exhibit a so-called no-b-wave (nob) phenotype with severely reduced b-wave amplitudes in the electroretinogram. Optomotor tests reveal decreased optomotor responses under scotopic conditions. Consistent with the genetic disruption of Gpr179, GPR179 is absent at the dendritic tips of ON-bipolar cells. While proteins of the same signal transmission cascade (GRM6, LRIT3, and TRPM1) are correctly localized, other proteins (RGS7, RGS11, and GNB5) known to regulate GRM6 are absent at the dendritic tips of ON-bipolar cells. These results add a new model of cCSNB, which is important to better understand the role of GPR179, its implication in patients with cCSNB, and its use for the development of therapies