Publications by authors named "Christelle Michiels"

26 Publications

  • Page 1 of 1

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

Int J Mol Sci 2021 Apr 23;22(9). Epub 2021 Apr 23.

Institut de la Vision, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Sorbonne Université, F-75012 Paris, France.

Mutations in 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 knock-out mouse model was genetically and functionally characterized. We confirmed that the insertion of a neo/lac Z cassette in intron 1 of disrupts the same gene. Spectral domain optical coherence tomography reveals no obvious retinal structure abnormalities. knock-out mice exhibit a so-called no-b-wave () 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 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.
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http://dx.doi.org/10.3390/ijms22094424DOI Listing
April 2021

Restoration of mGluR6 Localization Following AAV-Mediated Delivery in a Mouse Model of Congenital Stationary Night Blindness.

Invest Ophthalmol Vis Sci 2021 Mar;62(3):24

Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France.

Purpose: Complete congenital stationary night blindness (cCSNB) is an incurable inherited retinal disorder characterized by an ON-bipolar cell (ON-BC) defect. GRM6 mutations are the third most prevalent cause of cCSNB. The Grm6-/- mouse model mimics the human phenotype, showing no b-wave in the electroretinogram (ERG) and a loss of mGluR6 and other proteins of the same cascade at the outer plexiform layer (OPL). Our aim was to restore protein localization and function in Grm6-/- adult mice targeting specifically ON-BCs or the whole retina.

Methods: Adeno-associated virus-encoding Grm6 under two different promoters (GRM6-Grm6 and CAG-Grm6) were injected intravitreally in P15 Grm6-/- mice. ERG recordings at 2 and 4 months were performed in Grm6+/+, untreated and treated Grm6-/- mice. Similarly, immunolocalization studies were performed on retinal slices before or after treatment using antibodies against mGluR6, TRPM1, GPR179, RGS7, RGS11, Gβ5, and dystrophin.

Results: Following treatment, mGluR6 was localized to the dendritic tips of ON-BCs when expressed with either promoter. The relocalization efficiency in mGluR6-transduced retinas at the OPL was 2.5% versus 11% when the GRM6-Grm6 and CAG-Grm6 were used, respectively. Albeit no functional rescue was seen in ERGs, relocalization of TRPM1, GPR179, and Gβ5 was also noted using both constructs. The restoration of the localization of RGS7, RGS11, and dystrophin was more obvious in retinas treated with GRM6-Grm6 than in retinas treated with CAG-Grm6.

Conclusions: Our findings show the potential of treating cCSNB with GRM6 mutations; however, it appears that the transduction rate must be improved to restore visual function.
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http://dx.doi.org/10.1167/iovs.62.3.24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980044PMC
March 2021

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

Clin Genet 2021 Feb 9;99(2):298-302. Epub 2020 Nov 9.

Department of Genetics, Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France.

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.
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http://dx.doi.org/10.1111/cge.13872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049445PMC
February 2021

DEEP PHENOTYPING AND FURTHER INSIGHTS INTO ITM2B-RELATED RETINAL DYSTROPHY.

Retina 2021 Apr;41(4):872-881

Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France.

Purpose: To reappraise the presentation and the course of ITM2B-related retinal dystrophy and give further insights into ITM2B expression in the retina.

Methods: The clinical data of nine subjects with ITM2B-related retinal dystrophy were retrospectively reviewed. The genetic mutation was assessed for its influence on splicing in cultured fibroblasts. The cellular expression of ITM2B within the inner retina was investigated in wild-type mice through mRNA in situ hybridization.

Results: All patients complained of decreased vision and mild photophobia around their twenties-thirties. The peculiar feature was the hyperreflective material on optical coherence tomography within the inner retina and the central outer nuclear layer with thinning of the retinal nerve fiber layer. Although retinal imaging revealed very mild or no changes over the years, the visual acuity slowly decreased with about one Early Treatment Diabetic Retinopathy Study letter per year. Finally, full-field electroretinography showed a mildly progressive inner retinal and cone dysfunction. ITM2B mRNA is expressed in all cellular types of the inner retina. Disease mechanism most likely involves mutant protein misfolding and/or modified protein interaction rather than misplicing.

Conclusion: ITM2B-related retinal dystrophy is a peculiar, rare, slowly progressive retinal degeneration. Functional examinations (full-field electroretinography and visual acuity) seem more accurate in monitoring the progression in these patients because imaging tends to be stable over the years.
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http://dx.doi.org/10.1097/IAE.0000000000002953DOI Listing
April 2021

Identification and characterization of novel TRPM1 autoantibodies from serum of patients with melanoma-associated retinopathy.

PLoS One 2020 23;15(4):e0231750. Epub 2020 Apr 23.

Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231750PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7179873PMC
July 2020

Generation of human induced pluripotent stem cell lines from a patient with ITM2B-related retinal dystrophy and a non mutated brother.

Stem Cell Res 2019 12 5;41:101625. Epub 2019 Nov 5.

INSERM, CNRS, Institut de la Vision, Sorbonne Université, 17 rue Moreau, Paris, F-75012, France; CHNO des Quinze-Vingts, INSERM-DGOS CIC 1423, 28 rue de Charenton, Paris, F-75012, France. Electronic address:

Human induced pluripotent stem cell (iPSC) lines were generated from fibroblasts of a patient affected with an autosomal dominant retinal dystrophy carrying the mutation c.782A>C, p.Glu261Ala in ITM2B and from an unaffected brother. Three different iPSC lines were generated and characterized from primary dermal fibroblasts of the affected subject and two from the unaffected brother. All iPSC lines expressed the pluripotency markers, were able to differentiate into the three germ layers and presented normal karyotypes. This cellular model will provide a powerful tool to study this retinal dystrophy and better understand the role of ITM2B.
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http://dx.doi.org/10.1016/j.scr.2019.101625DOI Listing
December 2019

Phenotype Analysis of Retinal Dystrophies in Light of the Underlying Genetic Defects: Application to Cone and Cone-Rod Dystrophies.

Int J Mol Sci 2019 Sep 30;20(19). Epub 2019 Sep 30.

Institut de la Vision, Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, 17 rue Moreau, 75012 Paris, France.

Phenotypes observed in a large cohort of patients with cone and cone-rod dystrophies (COD/CORDs) are described based on multimodal retinal imaging features in order to help in analyzing massive next-generation sequencing data. Structural abnormalities of 58 subjects with molecular diagnosis of COD/CORDs were analyzed through specific retinal imaging including spectral-domain optical coherence tomography (SD-OCT) and fundus autofluorescence (BAF/IRAF). Findings were analyzed with the underlying genetic defects. A ring of increased autofluorescence was mainly observed in patients with and mutations (33% and 22% of cases respectively). "Speckled" autofluorescence was observed with mutations in three different genes ( 64%; and , 18% each). Peripapillary sparing was only found in association with mutations in , although only present in 40% of such genotypes. Regarding SD-OCT, specific outer retinal abnormalities were more commonly observed in particular genotypes: focal retrofoveal interruption and mutations (50%), foveal sparing and mutations (50%), and outer retinal atrophy associated with hyperreflective dots and mutations (69%). This study outlines the phenotypic heterogeneity of COD/CORDs hampering statistical correlations. A larger study correlating retinal imaging with genetic results is necessary to identify specific clinical features that may help in selecting pathogenic variants generated by high-throughput sequencing.
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http://dx.doi.org/10.3390/ijms20194854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6801687PMC
September 2019

Where are the missing gene defects in inherited retinal disorders? Intronic and synonymous variants contribute at least to 4% of CACNA1F-mediated inherited retinal disorders.

Hum Mutat 2019 06 28;40(6):765-787. Epub 2019 Mar 28.

Center for Medical Genetics, Ghent University and Ghent University Hospital, Ghent, Belgium.

Inherited retinal disorders (IRD) represent clinically and genetically heterogeneous diseases. To date, pathogenic variants have been identified in ~260 genes. Albeit that many genes are implicated in IRD, for 30-50% of the cases, the gene defect is unknown. These cases may be explained by novel gene defects, by overlooked structural variants, by variants in intronic, promoter or more distant regulatory regions, and represent synonymous variants of known genes contributing to the dysfunction of the respective proteins. Patients with one subgroup of IRD, namely incomplete congenital stationary night blindness (icCSNB), show a very specific phenotype. The major cause of this condition is the presence of a hemizygous pathogenic variant in CACNA1F. A comprehensive study applying direct Sanger sequencing of the gene-coding regions, exome and genome sequencing applied to a large cohort of patients with a clinical diagnosis of icCSNB revealed indeed that seven of the 189 CACNA1F-related cases have intronic and synonymous disease-causing variants leading to missplicing as validated by minigene approaches. These findings highlight that gene-locus sequencing may be a very efficient method in detecting disease-causing variants in clinically well-characterized patients with a diagnosis of IRD, like icCSNB.
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http://dx.doi.org/10.1002/humu.23735DOI Listing
June 2019

A Novel Heterozygous Missense Mutation in Leads to Autosomal Dominant Riggs Type of Congenital Stationary Night Blindness.

Biomed Res Int 2018 23;2018:7694801. Epub 2018 Apr 23.

Department of Ophthalmology and Byers Eye Institute, Stanford University School of Medicine, Palo Alto, CA, USA.

Autosomal dominant congenital stationary night blindness (adCSNB) is rare and results from altered phototransduction giving a Riggs type of electroretinogram (ERG) with loss of the rod a-wave and small b-waves. These patients usually have normal vision in light. Only few mutations in genes coding for proteins of the phototransduction cascade lead to this condition; most of these gene defects cause progressive rod-cone dystrophy. Mutation analysis of an adCSNB family with a Riggs-type ERG revealed a novel variant (c.155T>A p.Ile52Asn) in coding for the -subunit of transducin, cosegregating with the phenotype. Domain predictions and 3D-modelling suggest that the variant does not affect the GTP-binding site as other adCSNB mutations do. It affects a predicted nuclear localization signal and a part of the first -helix, which is distant from the GTP-binding site. The subcellular protein localization of this and other mutant GNAT1 proteins implicated in CSNB are unaltered in mammalian GNAT1 overexpressing cells. Our findings add a third mutation causing adCSNB and suggest that different pathogenic mechanisms may cause this condition.
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http://dx.doi.org/10.1155/2018/7694801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937575PMC
October 2018

Further Insights into the Ciliary Gene and Protein KIZ and Its Murine Ortholog PLK1S1 Mutated in Rod-Cone Dystrophy.

Genes (Basel) 2017 Oct 18;8(10). Epub 2017 Oct 18.

Sorbonne Universités, UPMC University Paris 06, INSERM U968, CNRS UMR 7210, Institut de la Vision, 75012 Paris, France.

We identified herein additional patients with rod-cone dystrophy (RCD) displaying mutations in , encoding the ciliary centrosomal protein kizuna and performed functional characterization of the respective protein in human fibroblasts and of its mouse ortholog PLK1S1 in the retina. Mutation screening was done by targeted next generation sequencing and subsequent Sanger sequencing validation. mRNA levels were assessed on blood and serum-deprived human fibroblasts from a control individual and a patient, compound heterozygous for the c.52G>T (p.Glu18*) and c.119_122del (p.Lys40Ilefs*14) mutations in . KIZ localization, documentation of cilium length and immunoblotting were performed in these two fibroblast cell lines. In addition, PLK1S1 immunolocalization was conducted in mouse retinal cryosections and isolated rod photoreceptors. Analyses of additional RCD patients enabled the identification of two homozygous mutations in , the known c.226C>T (p.Arg76*) mutation and a novel variant, the c.3G>A (p.Met1?) mutation. Albeit the expression levels of were three-times lower in the patient than controls in whole blood cells, further analyses in control- and mutant patient-derived fibroblasts unexpectedly revealed no significant difference between the two genotypes. Furthermore, the averaged monocilia length in the two fibroblast cell lines was similar, consistent with the preserved immunolocalization of KIZ at the basal body of the primary cilia. Analyses in mouse retina and isolated rod photoreceptors showed PLK1S1 localization at the base of the photoreceptor connecting cilium. In conclusion, two additional patients with mutations in were identified, further supporting that defects in KIZ/PLK1S1, detected at the basal body of the primary cilia in fibroblasts, and the photoreceptor connecting cilium in mouse, respectively, are involved in RCD. However, albeit the mutations were predicted to lead to nonsense mediated mRNA decay, we could not detect changes upon expression levels, protein localization or cilia length in -mutated fibroblast cells. Together, our findings unveil the limitations of fibroblasts as a cellular model for RCD and call for other models such as induced pluripotent stem cells to shed light on retinal pathogenic mechanisms of mutations.
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http://dx.doi.org/10.3390/genes8100277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664127PMC
October 2017

Identification of a Novel Homozygous Nonsense Mutation Confirms the Implication of GNAT1 in Rod-Cone Dystrophy.

PLoS One 2016 15;11(12):e0168271. Epub 2016 Dec 15.

Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institut de la Vision, Paris, France.

GNAT1, encoding the transducin subunit Gα, is an important element of the phototransduction cascade. Mutations in this gene have been associated with autosomal dominant and autosomal recessive congenital stationary night blindness. Recently, a homozygous truncating 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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0168271PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5158031PMC
July 2017

Biallelic Mutations in GNB3 Cause a Unique Form of Autosomal-Recessive Congenital Stationary Night Blindness.

Am J Hum Genet 2016 05 7;98(5):1011-1019. Epub 2016 Apr 7.

Department of Ophthalmology, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada; Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; Department of Ophthalmology, University of Toronto, 340 College Street, Toronto, ON M5T 3A9, Canada. Electronic address:

Congenital stationary night blindness (CSNB) is a heterogeneous group of non-progressive inherited retinal disorders with characteristic electroretinogram (ERG) abnormalities. Riggs and Schubert-Bornschein are subtypes of CSNB and demonstrate distinct ERG features. Riggs CSNB demonstrates selective rod photoreceptor dysfunction and occurs due to mutations in genes encoding proteins involved in rod phototransduction cascade; night blindness is the only symptom and eye examination is otherwise normal. Schubert-Bornschein CSNB is a consequence of impaired signal transmission between the photoreceptors and bipolar cells. Schubert-Bornschein CSNB is subdivided into complete CSNB with an ON bipolar signaling defect and incomplete CSNB with both ON and OFF pathway involvement. Both subtypes are associated with variable degrees of night blindness or photophobia, reduced visual acuity, high myopia, and nystagmus. Whole-exome sequencing of a family screened negative for mutations in genes associated with CSNB identified biallelic mutations in the guanine nucleotide-binding protein subunit beta-3 gene (GNB3). Two siblings were compound heterozygous for a deletion (c.170_172delAGA [p.Lys57del]) and a nonsense mutation (c.1017G>A [p.Trp339(∗)]). The maternal aunt was homozygous for the nonsense mutation (c.1017G>A [p.Trp339(∗)]). Mutational analysis of GNB3 in a cohort of 58 subjects with CSNB identified a sporadic case individual with a homozygous GNB3 mutation (c.200C>T [p.Ser67Phe]). GNB3 encodes the β subunit of G protein heterotrimer (Gαβγ) and is known to modulate ON bipolar cell signaling and cone transducin function in mice. Affected human subjects showed an unusual CSNB phenotype with variable degrees of ON bipolar dysfunction and reduced cone sensitivity. This unique retinal disorder with dual anomaly in visual processing expands our knowledge about retinal signaling.
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http://dx.doi.org/10.1016/j.ajhg.2016.03.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867910PMC
May 2016

Next-generation sequencing applied to a large French cone and cone-rod dystrophy cohort: mutation spectrum and new genotype-phenotype correlation.

Orphanet J Rare Dis 2015 Jun 24;10:85. Epub 2015 Jun 24.

INSERM, U968, Paris, F-75012, France.

Background: Cone and cone-rod dystrophies are clinically and genetically heterogeneous inherited retinal disorders with predominant cone impairment. They should be distinguished from the more common group of rod-cone dystrophies (retinitis pigmentosa) due to their more severe visual prognosis with early central vision loss. The purpose of our study was to document mutation spectrum of a large French cohort of cone and cone-rod dystrophies.

Methods: We applied Next-Generation Sequencing targeting a panel of 123 genes implicated in retinal diseases to 96 patients. A systematic filtering approach was used to identify likely disease causing variants, subsequently confirmed by Sanger sequencing and co-segregation analysis when possible.

Results: Overall, the likely causative mutations were detected in 62.1 % of cases, revealing 33 known and 35 novel mutations. This rate was higher for autosomal dominant (100 %) than autosomal recessive cases (53.8 %). Mutations in ABCA4 and GUCY2D were responsible for 19.2 % and 29.4 % of resolved cases with recessive and dominant inheritance, respectively. Furthermore, unexpected genotype-phenotype correlations were identified, confirming the complexity of inherited retinal disorders with phenotypic overlap between cone-rod dystrophies and other retinal diseases.

Conclusions: In summary, this time-efficient approach allowed mutation detection in the most important cohort of cone-rod dystrophies investigated so far covering the largest number of genes. Association of known gene defects with novel phenotypes and mode of inheritance were established.
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http://dx.doi.org/10.1186/s13023-015-0300-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4566196PMC
June 2015

Genotypic and phenotypic characterization of P23H line 1 rat model.

PLoS One 2015 26;10(5):e0127319. Epub 2015 May 26.

INSERM, U968, Paris, France; CNRS, UMR_7210, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris, France; Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, DHU ViewMaintain, INSERM-DHOS CIC 1423, Paris, France; Institute of Ophthalmology, University College of London, London, United Kingdom.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127319PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4444340PMC
April 2016

LRIT3 is essential to localize TRPM1 to the dendritic tips of depolarizing bipolar cells and may play a role in cone synapse formation.

Eur J Neurosci 2015 Aug 4;42(3):1966-75. Epub 2015 Jul 4.

INSERM, U968, Paris, F-75012, France.

Mutations in LRIT3 lead to complete congenital stationary night blindness (cCSNB). The exact role of LRIT3 in ON-bipolar cell signaling cascade remains to be elucidated. Recently, we have characterized a novel mouse model lacking Lrit3 [no b-wave 6, (Lrit3(nob6/nob6) )], which displays similar abnormalities to patients with cCSNB with LRIT3 mutations. Here we compare the localization of components of the ON-bipolar cell signaling cascade in wild-type and Lrit3(nob6/nob6) retinal sections by immunofluorescence confocal microscopy. An anti-LRIT3 antibody was generated. Immunofluorescent staining of LRIT3 in wild-type mice revealed a specific punctate labeling in the outer plexiform layer (OPL), which was absent in Lrit3(nob6/nob6) mice. LRIT3 did not co-localize with ribeye or calbindin but co-localized with mGluR6. TRPM1 staining was severely decreased at the dendritic tips of all depolarizing bipolar cells in Lrit3(nob6/nob6) mice. mGluR6, GPR179, RGS7, RGS11 and Gβ5 immunofluorescence was absent at the dendritic tips of cone ON-bipolar cells in Lrit3(nob6/nob6) mice, while it was present at the dendritic tips of rod bipolar cells. Furthermore, peanut agglutinin (PNA) labeling was severely reduced in the OPL in Lrit3(nob6/nob6) mice. This study confirmed the localization of LRIT3 at the dendritic tips of depolarizing bipolar cells in mouse retina and demonstrated the dependence of TRPM1 localization on the presence of LRIT3. As tested components of the ON-bipolar cell signaling cascade and PNA revealed disrupted localization, an additional function of LRIT3 in cone synapse formation is suggested. These results point to a possibly different regulation of the mGluR6 signaling cascade between rod and cone ON-bipolar cells.
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http://dx.doi.org/10.1111/ejn.12959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4733627PMC
August 2015

Whole-exome sequencing identifies KIZ as a ciliary gene associated with autosomal-recessive rod-cone dystrophy.

Am J Hum Genet 2014 Apr 27;94(4):625-33. Epub 2014 Mar 27.

Institut National de la Santé et de la Recherche Médicale U968, Paris 75012, France; Centre National de la Recherche Scientifique UMR_7210, Paris 75012, France; Institut de la Vision UMR_S 968, Université Pierre et Marie Curie (Paris 6), Sorbonne Universités, Paris 75012, France; Institut National de la Santé et de la Recherche Médicale and Direction de l'Hospitalisation et de l'Organisation des Soins Centre d'Investigation Clinique 1423, Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Paris 75012, France; University College London Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK. Electronic address:

Rod-cone dystrophy (RCD), also known as retinitis pigmentosa, is a progressive inherited retinal disorder characterized by photoreceptor cell death and genetic heterogeneity. Mutations in many genes have been implicated in the pathophysiology of RCD, but several others remain to be identified. Herein, we applied whole-exome sequencing to a consanguineous family with one subject affected with RCD and identified a homozygous nonsense mutation, c.226C>T (p.Arg76(∗)), in KIZ, which encodes centrosomal protein kizuna. Subsequent Sanger sequencing of 340 unrelated individuals with sporadic and autosomal-recessive RCD identified two other subjects carrying pathogenic variants in KIZ: one with the same homozygous nonsense mutation (c.226C>T [p.Arg76(∗)]) and another with compound-heterozygous mutations c.119_122delAACT (p.Lys40Ilefs(∗)14) and c.52G>T (p.Glu18(∗)). Transcriptomic analysis in mice detected mRNA levels of the mouse ortholog (Plk1s1) in rod photoreceptors, as well as its decreased expression when photoreceptors degenerated in rd1 mice. The presence of the human KIZ transcript was confirmed by quantitative RT-PCR in the retina, the retinal pigment epithelium, fibroblasts, and whole-blood cells (highest expression was in the retina). RNA in situ hybridization demonstrated the presence of Plk1s1 mRNA in the outer nuclear layer of the mouse retina. Immunohistology revealed KIZ localization at the basal body of the cilia in human fibroblasts, thus shedding light on another ciliary protein implicated in autosomal-recessive RCD.
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http://dx.doi.org/10.1016/j.ajhg.2014.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3980423PMC
April 2014

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

PLoS One 2014 5;9(3):e90342. Epub 2014 Mar 5.

INSERM, U968, Paris, France; CNRS, UMR_7210, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0090342PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943948PMC
January 2015

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

Invest Ophthalmol Vis Sci 2013 Dec 9;54(13):8041-50. Epub 2013 Dec 9.

Institut National de la Santé et de la Recherche Médicale (INSERM), U968, Paris, France.

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.
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http://dx.doi.org/10.1167/iovs.13-12610DOI Listing
December 2013

The familial dementia gene revisited: a missense mutation revealed by whole-exome sequencing identifies ITM2B as a candidate gene underlying a novel autosomal dominant retinal dystrophy in a large family.

Hum Mol Genet 2014 Jan 10;23(2):491-501. Epub 2013 Sep 10.

INSERM, U968, Paris F-75012, France.

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 β 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.
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http://dx.doi.org/10.1093/hmg/ddt439DOI Listing
January 2014

Whole-exome sequencing identifies LRIT3 mutations as a cause of autosomal-recessive complete congenital stationary night blindness.

Am J Hum Genet 2013 Jan 13;92(1):67-75. Epub 2012 Dec 13.

Unité Mixte de Recherche S968, Institut National de la Santé et de la Recherche Médicale, F-75012 Paris, France.

Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous retinal disorder. Two forms can be distinguished clinically: complete CSNB (cCSNB) and incomplete CSNB. Individuals with cCSNB have visual impairment under low-light conditions and show a characteristic electroretinogram (ERG). The b-wave amplitude is severely reduced in the dark-adapted state of the ERG, representing abnormal function of ON bipolar cells. Furthermore, individuals with cCSNB can show other ocular features such as nystagmus, myopia, and strabismus and can have reduced visual acuity and abnormalities of the cone ERG waveform. The mode of inheritance of this form can be X-linked or autosomal recessive, and the dysfunction of four genes (NYX, GRM6, TRPM1, and GPR179) has been described so far. Whole-exome sequencing in one simplex cCSNB case lacking mutations in the known genes led to the identification of a missense mutation (c.983G>A [p.Cys328Tyr]) and a nonsense mutation (c.1318C>T [p.Arg440(∗)]) in LRIT3, encoding leucine-rich-repeat (LRR), immunoglobulin-like, and transmembrane-domain 3 (LRIT3). Subsequent Sanger sequencing of 89 individuals with CSNB identified another cCSNB case harboring a nonsense mutation (c.1151C>G [p.Ser384(∗)]) and a deletion predicted to lead to a premature stop codon (c.1538_1539del [p.Ser513Cysfs(∗)59]) in the same gene. Human LRIT3 antibody staining revealed in the outer plexiform layer of the human retina a punctate-labeling pattern resembling the dendritic tips of bipolar cells; similar patterns have been observed for other proteins implicated in cCSNB. The exact role of this LRR protein in cCSNB remains to be elucidated.
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http://dx.doi.org/10.1016/j.ajhg.2012.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542465PMC
January 2013

Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness.

Am J Hum Genet 2012 Feb;90(2):321-30

Institut National de la Santé et de la Recherche Médicale, Paris, France.

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.Pro93Glnfs(∗)57]) 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.
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http://dx.doi.org/10.1016/j.ajhg.2011.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3276675PMC
February 2012

Development and application of a next-generation-sequencing (NGS) approach to detect known and novel gene defects underlying retinal diseases.

Orphanet J Rare Dis 2012 Jan 25;7. Epub 2012 Jan 25.

INSERM, U968, Paris, F-75012, France.

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 with two 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.

Conclusions: In summary, this unbiased and time-efficient NGS approach allowed mutation detection in 75% of control cases and in 57% of test cases. Furthermore, it has the possibility of associating known gene defects with novel phenotypes and mode of inheritance.
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http://dx.doi.org/10.1186/1750-1172-7-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3352121PMC
January 2012

A mutation in SLC24A1 implicated in autosomal-recessive congenital stationary night blindness.

Am J Hum Genet 2010 Oct 16;87(4):523-31. Epub 2010 Sep 16.

National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore 53700, Pakistan.

Congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder that can be associated with impaired night vision. The last decade has witnessed huge progress in ophthalmic genetics, including the identification of three genes implicated in the pathogenicity of autosomal-recessive CSNB. However, not all patients studied could be associated with mutations in these genes and thus other genes certainly underlie this disorder. Here, we report a large multigeneration family with five affected individuals manifesting symptoms of night blindness. A genome-wide scan localized the disease interval to chromosome 15q, and recombination events in affected individuals refined the critical interval to a 10.41 cM (6.53 Mb) region that harbors SLC24A1, a member of the solute carrier protein superfamily. Sequencing of all the coding exons identified a 2 bp deletion in exon 2: c.1613_1614del, which is predicted to result in a frame shift that leads to premature termination of SLC24A1 (p.F538CfsX23) and segregates with the disorder under an autosomal-recessive model. Expression analysis using mouse ocular tissues shows that Slc24a1 is expressed in the retina around postnatal day 7. In situ and immunohistological studies localized both SLC24A1 and Slc24a1 to the inner segment, outer and inner nuclear layers, and ganglion cells of the retina, respectively. Our data expand the genetic basis of CSNB and highlight the indispensible function of SLC24A1 in retinal function and/or maintenance in humans.
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http://dx.doi.org/10.1016/j.ajhg.2010.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2948789PMC
October 2010

Pharmacological inhibition of stearoyl-CoA desaturase 1 improves insulin sensitivity in insulin-resistant rat models.

Eur J Pharmacol 2009 Sep 17;618(1-3):28-36. Epub 2009 Jul 17.

GlaxoSmithKline, Les Ulis Cedex, France.

Stearoyl-CoA Desaturase 1 (SCD1) is a central enzyme that catalyzes the biosynthesis of monounsaturated fatty acids from saturated fatty acids. SCD1 is an emerging target in obesity and insulin resistance due to the improved metabolic profile obtained when the enzyme is genetically inactivated. Here, we have investigated if the pharmacological inhibition of SCD1 could elicit the same profile. We have identified a small molecule, GSK993 and characterized it as a potent and orally available SCD1 inhibitor. In Zucker(fa/fa) rats, GSK993 exerted a marked reduction in hepatic lipids as well as a significant improvement of glucose tolerance. Furthermore, in a diet-induced insulin resistant rat model, GSK993 induced a very strong reduction in Triton-induced hepatic Very Low Density Lipoprotein-Triglyceride production. In addition, following a hyperinsulinemic-euglycemic clamp in GSK993-treated animals, we observed an improvement in the whole body insulin sensitivity as reflected by an increase in the glucose infusion rate. Taken together, these findings demonstrate that the pharmacological inhibition of SCD1 translates into improved lipid and glucose metabolic profiles and raises the interest of SCD1 inhibitors as potential new drugs for the treatment of insulin resistance.
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http://dx.doi.org/10.1016/j.ejphar.2009.07.004DOI Listing
September 2009

Prevention of autoimmunity and control of recall response to exogenous antigen by Fas death receptor ligand expression on T cells.

Immunity 2008 Dec 13;29(6):922-33. Epub 2008 Nov 13.

INSERM U580, Université Paris Descartes, Hopital Necker, 161 rue de Sèvres 75015 Paris, France.

Mice with mutations in the gene encoding Fas ligand (FasL) develop lymphoproliferation and systemic autoimmune diseases. However, the cellular subset responsible for the prevention of autoimmunity in FasL-deficient mice remains undetermined. Here, we show that mice with FasL loss on either B or T cells had identical life span as littermates, and both genotypes developed signs of autoimmunity. In addition, we show that T cell-dependent death was vital for the elimination of aberrant T cells and for controlling the numbers of B cells and dendritic cells that dampen autoimmune responses. Furthermore, we show that the loss of FasL on T cells affected the follicular dentritic cell network in the germinal centers, leading to an impaired recall response to exogenous antigen. These results disclose the distinct roles of cellular subsets in FasL-dependent control of autoimmunity and provide further insight into the role of FasL in humoral immunity.
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http://dx.doi.org/10.1016/j.immuni.2008.10.007DOI Listing
December 2008