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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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 <it>NR2E3, PRPF3, EYS, PRPF8, CRB1, TRPM1 </it>and <it>CACNA1F</it>. 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.</p> <p>Conclusions</p> <p>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.</p

Les influenceuses et expérimentatrices des comptabilités écologiques, Numéro spécial : Nouveau contrat écologique et social, comment passer à l’acte ?

International audienc

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

Targeted Next Generation Sequencing Identifies Novel Mutations in RP1 as a Relatively Common Cause of Autosomal Recessive Rod-Cone Dystrophy

We report ophthalmic and genetic findings in families with autosomal recessive rod-cone dystrophy (arRCD) and RP1 mutations. Detailed ophthalmic examination was performed in 242 sporadic and arRCD subjects. Genomic DNA was investigated using our customized next generation sequencing panel targeting up to 123 genes implicated in inherited retinal disorders. Stringent filtering coupled with Sanger sequencing and followed by cosegregation analysis was performed to confirm biallelism and the implication of the most likely disease causing variants. Sequencing identified 9 RP1 mutations in 7 index cases. Eight of the mutations were novel, and all cosegregated with severe arRCD phenotype, found associated with additional macular changes. Among the identified mutations, 4 belong to a region, previously associated with arRCD, and 5 others in a region previously associated with adRCD. Our prevalence studies showed that RP1 mutations account for up to 2.5% of arRCD. These results point out for the necessity of sequencing RP1 when genetically investigating sporadic and arRCD. It further highlights the interest of unbiased sequencing technique, which allows investigating the implication of the same gene in different modes of inheritance. Finally, it reports that different regions of RP1 can also lead to arRCD

CRB1 mutations in inherited retinal dystrophies.

International audienceMutations in the CRB1 gene are associated with variable phenotypes of severe retinal dystrophies, ranging from leber congenital amaurosis (LCA) to rod-cone dystrophy, also called retinitis pigmentosa (RP). Moreover, retinal dystrophies resulting from CRB1 mutations may be accompanied by specific fundus features: preservation of the para-arteriolar retinal pigment epithelium (PPRPE) and retinal telangiectasia with exudation (also referred to as Coats-like vasculopathy). In this publication, we report seven novel mutations and classify over 150 reported CRB1 sequence variants that were found in more that 240 patients. The data from previous reports were used to analyze a potential correlation between CRB1 variants and the clinical features of respective patients. This meta-analysis suggests that the differential phenotype of patients with CRB1 mutations is due to additional modifying factors rather than particular mutant allele combination

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

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