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

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.

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

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.

CRB1 mutations in inherited retinal dystrophies.

Mutations 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.

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

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.

A novel DFNB31 mutation associated with Usher type 2 syndrome showing variable degrees of auditory loss in a consanguineous Portuguese family.

PURPOSE: To identify the genetic defect of a consanguineous Portuguese family with rod-cone dystrophy and varying degrees of decreased audition. METHODS: A detailed ophthalmic and auditory examination was performed on a Portuguese patient with severe autosomal recessive rod-cone dystrophy. Known genetic defects were excluded by performing autosomal recessive retinitis pigmentosa (arRP) genotyping microarray analysis and by Sanger sequencing of the coding exons and flanking intronic regions of eyes shut homolog-drosophila (EYS) and chromosome 2 open reading frame 71 (C2orf71). Subsequently, genome-wide homozygosity mapping was performed in DNA samples from available family members using a 700K single nucleotide polymorphism (SNP) microarray. Candidate genes present in the significantly large homozygous regions were screened for mutations using Sanger sequencing. RESULTS: The largest homozygous region (~11 Mb) in the affected family members was mapped to chromosome 9, which harbors deafness, autosomal recessive 31 (DFNB31; a gene previously associated with Usher syndrome). Mutation analysis of DFNB31 in the index patient identified a novel one-base-pair deletion (c.737delC), which is predicted to lead to a truncated protein (p.Pro246HisfsX13) and co-segregated with the disease in the family. Ophthalmic examination of the index patient and the affected siblings showed severe rod-cone dystrophy. Pure tone audiometry revealed a moderate hearing loss in the index patient, whereas the affected siblings were reported with more profound and early onset hearing impairment. CONCLUSIONS: We report a novel truncating mutation in DFNB31 associated with severe rod-cone dystrophy and varying degrees of hearing impairment in a consanguineous family of Portuguese origin. This is the second report of DFNB31 implication in Usher type 2.

Novel C2orf71 mutations account for ∼1% of cases in a large French arRP cohort.

Autosomal-recessive retinitis pigmentosa (arRP) is a genetically heterogeneous group of disorders to which a novel gene, C2orf71, was recently associated. The purpose of our study was to establish the prevalence and nature of C2orf71 mutations in a clinically well-characterized cohort of 345 sporadic and arRP French cases. Direct sequencing of C2orf71 was performed in 209 subjects for whom mutations had previously been excluded by microarray technology and direct sequencing of EYS. Putative pathogenicity of the identified variants was evaluated through co-segregation analysis, screening of more than 188 control chromosomes and prediction programs. We identified two patients compound heterozygous for mutations predicted to lead to a premature stop codon, 3 of which are novel. In addition, 3 patients carried a single variant of likely pathogenicity. Furthermore a large number of novel putative non-disease causing variants were identified, highlighting the extremely polymorphic nature of C2orf71. To our knowledge, our study provides the first large scale screening of C2orf71 in a French arRP cohort through direct sequencing and suggests that it would account for approximately 1% of arRP cases.

A new regulation of IL-6 production in adult cardiomyocytes by beta-adrenergic and IL-1 beta receptors and induction of cellular hypertrophy by IL-6 trans-signalling.

The sympathetic nervous system and pro-inflammatory cytokines play key roles in numerous cardiovascular disorders. Chronic beta-adrenergic receptor (beta-AR) stimulation in myocardium induces expression of pro-inflammatory cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6), which contribute to cardiac hypertrophy and failure. To evaluate the relationship between beta-AR stimulation and pro-inflammatory cytokines, we studied the effects of the beta-AR agonist isoprenaline (ISO) on IL-1-induced IL-6 production in adult rat ventricular myocytes (ARVMs). We report that ISO and IL-1 synergistically enhanced IL-6 gene expression and secretion. The synergistic effect of ISO was mimicked by cAMP elevating agents and involved the G(s) protein/cAMP/PKA signalling pathway, but not the exchange factor EPAC. To evaluate the contribution of IL-6 to cellular hypertrophy, we examined the signalling pathways stimulated by the membrane-bound IL-6 receptor (IL-6R), and the IL-6 soluble receptor (sIL-6R) involved in the mechanism named IL-6 trans-signalling. The IL-6/sIL-6R complex promoted a rapid and persistent phosphorylation of STAT3(Tyr705) in ARVMs. Moreover, IL-6 trans-signalling increased protein synthesis, c-fos gene expression and B-type natriuretic peptide secretion, three markers of cardiac hypertrophy. IL-6 trans-signalling also increased cell size. In contrast, IL-6 alone had no significant effect on either cell size or STAT3 phosphorylation although it induced phosphorylation of ERK1/2, AKT and S6K, demonstrating the presence of a functional IL-6R in ARVMs. Taken together, these results demonstrate that beta-AR stimulation synergises with IL-1 for IL-6 secretion in adult ventricular myocytes and indicate that IL-6 induces cardiac hypertrophy only via IL-6 trans-signalling. The IL-6 soluble receptor may thus serve as a switch for IL-6 to activate STAT3 phosphorylation and hypertrophy.