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Mutations in GPR179 are one of the most common causes of autosomal recessive complete congenital stationary night blindness (cCSNB). This retinal disease is characterized in patients by impaired dim and night vision, associated with other ocular symptoms, including high myopia. cCSNB is caused by a complete loss of signal transmission from photoreceptors to ON-bipolar cells. In this study, we hypothesized that the lack of Gpr179 and the subsequent impaired ON-pathway could lead to myopic features in a mouse model of cCSNB. Using ultra performance liquid chromatography, we show that adult Gpr179 -/- mice have a significant decrease in both retinal dopamine and 3,4-dihydroxyphenylacetic acid, compared to Gpr179 +/+ mice. This alteration of the dopaminergic system is thought to be correlated with an increased susceptibility to lens-induced myopia but does not affect the natural refractive development. Altogether, our data added a novel myopia model, which could be used to identify therapeutic interventions.

Variants in the X-linked retinitis pigmentosa GTPase regulator gene ( RPGR) and, specifically, in its retinal opening reading frame-15 isoform ( RPGR ORF15 ) may cause rod-cone (RCD), cone, and cone-rod dystrophies (CDs and CRDs). While RPGR -related RCDs have been frequently evaluated, the characteristics and progression of RPGR -related CD/CRDs are largely unknown. Therefore, the goal of our work was to perform genotype-phenotype correlations specifically in RPGR ORF15 -related CD/CRDs. This retrospective longitudinal study included 34 index patients and two affected relatives with a molecular diagnosis of RPGR -related CD/CRDs. Patients were recruited at the "Quinze-Vingts" Hospital, Paris, France and screened for mutations in RPGR ORF15 at the Institut de la Vision, Paris, France. We identified 29 distinct variants, of which 27 were truncating. All were located in the 3' half of the RPGR ORF15 transcript. Twenty of them were novel. Fifteen subjects were affected by CD, the remaining had CRD. When analyzing the longitudinal data, a progressive decline in visual acuity (VA) was noted, with more than 60% of the patients reaching VA ≥ 1 LogMar in the best eye after the fifth decade of life. To our knowledge, this is the largest described study of a cohort of CD/CRD patients affected by RPGR ORF15 variants. Longitudinal data showed a rapidly progressive disease, possibly locating an optimal window of intervention for future therapies in younger ages.

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.

Variants of the TTLL5 gene, which encodes tubulin tyrosine ligase-like family member five, are a rare cause of cone dystrophy (COD) or cone-rod dystrophy (CORD). To date, only a few TTLL5 patients have been clinically and genetically described. In this study, we report five patients harbouring biallelic variants of TTLL5. Four adult patients presented either COD or CORD with onset in the late teenage years. The youngest patient had a phenotype of early onset severe retinal dystrophy (EOSRD). Genetic analysis was performed by targeted next generation sequencing of gene panels and assessment of copy number variants (CNV). We identified eight variants, of which six were novel, including two large multiexon deletions in patients with COD or CORD, while the EOSRD patient harboured the novel homozygous p.(Trp640*) variant and three distinct USH2A variants, which might explain the observed rod involvement. Our study highlights the role of TTLL5 in COD/CORD and the importance of large deletions. These findings suggest that COD or CORD patients lacking variants in known genes may harbour CNVs to be discovered in TTLL5, previously undetected by classical sequencing methods. In addition, variable phenotypes in TTLL5 -associated patients might be due to the presence of additional gene defects.

Mutations 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.

We investigated the prevalence of reported deep-intronic variants in a French cohort of 70 patients with Stargardt disease harboring a monoallelic pathogenic variant on the exonic regions of ABCA4 . Direct Sanger sequencing of selected intronic regions of ABCA4 was conducted. Complete phenotypic analysis and correlation with the genotype was performed in case a known intronic pathogenic variant was identified. All other variants found on the analyzed sequences were queried for minor allele frequency and possible pathogenicity by in silico predictions. The second mutated allele was found in 14 (20%) subjects. The three known deep-intronic variants found were c.5196+1137G>A in intron 36 (6 subjects), c.4539+2064C>T in intron 30 (4 subjects) and c.4253+43G>A in intron 28 (4 subjects). Even though the phenotype depends on the compound effect of the biallelic variants, a genotype-phenotype correlation suggests that the c.5196+1137G>A was mostly associated with a mild phenotype and the c.4539+2064C>T with a more severe one. A variable effect was instead associated with the variant c.4253+43G>A. In addition, two novel variants, c.768+508A>G and c.859-245_859-243delinsTGA never associated with Stargardt disease before, were identified and a possible splice defect was predicted in silico. Our study calls for a larger cohort analysis including targeted locus sequencing and 3D protein modeling to better understand phenotype-genotype correlations associated with deep-intronic changes and patients' selection for clinical trials., Competing Interests: The authors declare no conflict of interest.

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 CRX and GUCY2D mutations (33% and 22% of cases respectively). "Speckled" autofluorescence was observed with mutations in three different genes ( ABCA4 64%; C2Orf71 and PRPH2 , 18% each). Peripapillary sparing was only found in association with mutations in ABCA4 , 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 GUCY2D mutations (50%), foveal sparing and CRX mutations (50%), and outer retinal atrophy associated with hyperreflective dots and ABCA4 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.

Here we report novel mutations in ABCA4 with the underlying phenotype in a large French cohort with autosomal recessive Stargardt disease. The DNA samples of 397 index subjects were analyzed in exons and flanking intronic regions of ABCA4 (NM_000350.2) by microarray analysis and direct Sanger sequencing. At the end of the screening, at least two likely pathogenic mutations were found in 302 patients (76.1%) while 95 remained unsolved: 40 (10.1%) with no variants identified, 52 (13.1%) with one heterozygous mutation, and 3 (0.7%) with at least one variant of uncertain significance (VUS). Sixty-three novel variants were identified in the cohort. Three of them were variants of uncertain significance. The other 60 mutations were classified as likely pathogenic or pathogenic, and were identified in 61 patients (15.4%). The majority of those were missense (55%) followed by frameshift and nonsense (30%), intronic (11.7%) variants, and in-frame deletions (3.3%). Only patients with variants never reported in literature were further analyzed herein. Recruited subjects underwent complete ophthalmic examination including best corrected visual acuity, kinetic and static perimetry, color vision test, full-field and multifocal electroretinography, color fundus photography, short-wavelength and near-infrared fundus autofluorescence imaging, and spectral domain optical coherence tomography. Clinical evaluation of each subject confirms the tendency that truncating mutations lead to a more severe phenotype with electroretinogram (ERG) impairment ( p = 0.002) and an earlier age of onset ( p = 0.037). Our study further expands the mutation spectrum in the exonic and flanking regions of ABCA4 underlying Stargardt disease.

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.