MERTK mutation update in inherited retinal diseases

International audienceMER tyrosine kinase (MERTK) encodes a surface receptor localized at the apical membrane of the retinal pigment epithelium. It plays a critical role in photoreceptor outer segment internalization prior to phagocytosis. Mutations in MERTK have been associated with severe autosomal recessive retinal dystrophies in the RCS rat and in humans. We present here a comprehensive review of all reported MERTK disease causing variants with the associated phenotype. In addition, we provide further data and insights of a large cohort of 1,195 inherited retinal dystrophies (IRD) index cases applying state-of-the-art genotyping techniques and summarize current knowledge. A total of 79 variants have now been identified underlying rod-cone dystrophy and cone-rod dystrophy including 11 novel variants reported here. The mutation spectrum in MERTK includes 33 missense, 12 nonsense, 12 splice defects, 12 small deletions, two small insertion-deletions, three small duplications, and two exonic and three gross deletions. Altogether, mutations in MERTK account for ∼2% of IRD cases with a severe retinal phenotype. These data are important for current and future therapeutic trials including gene replacement therapy or cell-based therapy

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

International audiencePhenotypes 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. View Full-Tex

Expanding the Mutation Spectrum in ABCA4: Sixty Novel Disease Causing Variants and Their Associated Phenotype in a Large French Stargardt Cohort

International audienceHere 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

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

International audienceBackground: 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.ResultsOverall, 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.ConclusionsIn 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

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

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

Whole-Exome Sequencing Identifies KIZ as a Ciliary Gene Associated with Autosomal-Recessive Rod-Cone Dystrophy

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

Clinical observations.

<p>(A) Color vision test with the Farnworth desaturated 15HUE shows a tritan axis defect. (B) Kinetic visual field tests demonstrate visual field constriction in both eyes. (C) Color fundus photographs reveal optic nerve pallor, narrowed retinal vessels, pigment clumping in retinal periphery some of which resembling more to coarse nummular pigments rather than classical bone spicules, as well as perifoveal atrophic changes. (D) Short-wavelength fundus autofluorescence shows hypo-autofluorescence in the periphery as well as in the perifoveal area. In (A, C, D), Ocula dextra (right eye; OD) is presented in the left part and ocular sinistra (left eye; OS) in the right part. (E) SD-OCT reveals thinning of the outer retinal layers. The two first SD-OCT correspond to OD results, the two next to OS results.</p

Validation and co-segregation of <i>GNAT1</i> variant in family F780.

<p>The pedigree and the respective electropherograms of each tested family member are depicted. Family F780 is composed of two unaffected parents (father: I.1, CIC01294; mother: I.2, CIC06690), one affected son (II.1; CIC01293) and one unaffected son (II.2). The nonsense variant c.963C>A p.(Cys321*) [M] in <i>GNAT1</i> (NM_144499.2; MIM *139330) was found homozygous in the affected boy (II.1, CIC01293), heterozygous in the unaffected mother (I.2, CIC06690) and absent in the unaffected father (I.1, CIC01294). Females and males are depicted by circles and squares, respectively. Filled and unfilled symbols indicate affected and unaffected status, respectively. The arrow indicates the nucleotide position 963 heterozygously and homozygously changed in the mother and index patient, respectively, and unchanged in the father.</p

Mutation and protein consequences in <i>GNAT1</i>.

<p>(A) Known and novel mutations leading to CSNB or RCD on the genomic structure of <i>GNAT1</i> (upper part) and the respective protein consequences (lower part). Different arrows indicate the mutation site and associated phenotype. C-terminal nonsense variants were associated with severe RCD (present study) or moderate RCD [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168271#pone.0168271.ref008" target="_blank">8</a>], while missense variants were associated with adCSNB and arCSNB affecting the nuclear localization signal (NLS) and/or GTP/GDP-binding site (GTP) (adCSNB) and an unknown domain of GNAT1 (arCSNB) (lower part) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168271#pone.0168271.ref002" target="_blank">2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168271#pone.0168271.ref004" target="_blank">4</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168271#pone.0168271.ref009" target="_blank">9</a>]. (B) The protein is highly conserved in metazoa from human to hydra (data not shown), with 99% of identity between bovine and human GNAT1. Amino acid sequences of the human normal (huGNAT1) and two mutants, (Cys321* and Gln302*) GNAT1, of the bovine GNAT1 (boGNAT1) and the bovine GNAT1 sequence used for crystallization of the protein (1TND) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168271#pone.0168271.ref009" target="_blank">9</a>]. This last sequence corresponds to the bovine GNAT1 sequence lacking 25 amino acids at the N-terminus and the last phenylalanine amino acid residues, at position 350 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168271#pone.0168271.ref009" target="_blank">9</a>]. In addition, known CSNB causing mutations are depicted. Human and bovine amino acid sequences are highly conserved. α-helices are represented in black rectangles and β sheets in black arrows (below amino acid sequences) and named as previously reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168271#pone.0168271.ref009" target="_blank">9</a>] except for the α-helices G, 4 and 5 which became here 4, 5 and 6, respectively. Specific binding sites are present at following amino acid residues: βγ transducin binding at 1 to 23 (Gtβγ; black dotted and gray shaded box, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168271#pone.0168271.ref009" target="_blank">9</a>]), NLS at 21–52 (NLS, gray unfilled box, predicted by a software, NLS Mapper), Magnesium binding sites at 43 and 177 (Mg, dark shaded box, predicted by Uniprot, GNAT1_HUMAN), GTP/GDP binding sites at 36–43, 171–177, 196–200, 265–268 and 321–323 (GTP, light grey shaded boxes, predicted by Uniprot, GNAT1_HUMAN and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168271#pone.0168271.ref009" target="_blank">9</a>]), PDE6γ inhibitory binding site at 306–310 (PDEγ, black dotted unfilled boxes, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168271#pone.0168271.ref009" target="_blank">9</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168271#pone.0168271.ref010" target="_blank">10</a>]) and activated-RHO binding sites at 311–328 and 340–350 (RHO, black filled boxes, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168271#pone.0168271.ref009" target="_blank">9</a>]).</p