Assessing Noncoding Sequence Variants of GJB2 for Hearing Loss Association.

Involvement of GJB2 noncoding regions in hearing loss (HL) has not been extensively investigated. However, three noncoding mutations, c.-259C>T, c.-23G>T, and c.-23+1G>A, were reported. Also, c.-684 -675del, of uncertain pathogenicity, was found upstream of the basal promoter. We performed a detailed analysis of GJB2 noncoding regions in Portuguese HL patients (previously screened for GJB2 coding mutations and the common GJB6 deletions) and in control subjects, by sequencing the basal promoter and flanking upstream region, exon 1, and 3’UTR. All individuals were genotyped for c.-684 -675del and 14 SNPs. Novel variants (c.-731C>T, c.-26G>T, c.∗45G>A, and c.∗985A>T) were found in controls. A hearing individual homozygous for c.-684 - 675del was for the first time identified, supporting the nonpathogenicity of this deletion. Our data indicate linkage disequilibrium (LD) between SNPs rs55704559 (c.∗168A>G) and rs5030700 (c.∗931C>T) and suggest the association of c.[∗168G;∗931T] allele with HL. The c.∗168A>G change, predicted to alter mRNA folding, might be involved in HL.info:eu-repo/semantics/publishedVersio

Early inflammation precedes cardiac fibrosis and heart failure in desmoglein 2 murine model of arrhythmogenic cardiomyopathy.

The study of a desmoglein 2 murine model of arrhythmogenic cardiomyopathy revealed cardiac inflammation as a key early event leading to fibrosis. Arrhythmogenic cardiomyopathy (AC) is an inherited heart muscle disorder leading to ventricular arrhythmias and heart failure due to abnormalities in the cardiac desmosome. We examined how loss of desmoglein 2 (Dsg2) in the young murine heart leads to development of AC. Apoptosis was an early cellular phenotype, and RNA sequencing analysis revealed early activation of inflammatory-associated pathways in Dsg2-null (Dsg2-/-) hearts at postnatal day 14 (2 weeks) that were absent in the fibrotic heart of adult mice (10 weeks). This included upregulation of iRhom2/ADAM17 and its associated pro-inflammatory cytokines and receptors such as TNFα, IL6R and IL-6. Furthermore, genes linked to specific macrophage populations were also upregulated. This suggests cardiomyocyte stress triggers an early immune response to clear apoptotic cells allowing tissue remodelling later on in the fibrotic heart. Our analysis at the early disease stage suggests cardiac inflammation is an important response and may be one of the mechanisms responsible for AC disease progression

Defining the Cellular Environment in the Organ of Corti following Extensive Hair Cell Loss: A Basis for Future Sensory Cell Replacement in the Cochlea

Background: Following the loss of hair cells from the mammalian cochlea, the sensory epithelium repairs to close the lesions but no new hair cells arise and hearing impairment ensues. For any cell replacement strategy to be successful, the cellular environment of the injured tissue has to be able to nurture new hair cells. This study defines characteristics of the auditory sensory epithelium after hair cell loss. Methodology/Principal Findings: Studies were conducted in C57BL/6 and CBA/Ca mice. Treatment with an aminoglycoside-diuretic combination produced loss of all outer hair cells within 48 hours in both strains. The subsequent progressive tissue re-organisation was examined using immunohistochemistry and electron microscopy. There was no evidence of significant de-differentiation of the specialised columnar supporting cells. Kir4.1 was down regulated but KCC4, GLAST, microtubule bundles, connexin expression patterns and pathways of intercellular communication were retained. The columnar supporting cells became covered with non-specialised cells migrating from the outermost region of the organ of Corti. Eventually non-specialised, flat cells replaced the columnar epithelium. Flat epithelium developed in distributed patches interrupting regions of columnar epithelium formed of differentiated supporting cells. Formation of the flat epithelium was initiated within a few weeks post-treatment in C57BL/6 mice but not for several months in CBA/Ca’s, suggesting genetic background influences the rate of re-organisation

Molecular epidemiology of DFNB1 deafness in France

BACKGROUND: Mutations in the GJB2 gene have been established as a major cause of inherited non syndromic deafness in different populations. A high number of sequence variations have been described in the GJB2 gene and the associated pathogenic effects are not always clearly established. The prevalence of a number of mutations is known to be population specific, and therefore population specific testing should be a prerequisite step when molecular diagnosis is offered. Moreover, population studies are needed to determine the contribution of GJB2 variants to deafness. We present our findings from the molecular diagnostic screening of the GJB2 and GJB6 genes over a three year period, together with a population-based study of GJB2 variants. METHODS AND RESULTS: Molecular studies were performed using denaturing High Performance Liquid Chromatograghy (DHPLC) and sequencing of the GJB2 gene. Over the last 3 years we have studied 159 families presenting sensorineural hearing loss, including 84 with non syndromic, stable, bilateral deafness. Thirty families were genotyped with causative mutations. In parallel, we have performed a molecular epidemiology study on more than 3000 dried blood spots and established the frequency of the GJB2 variants in our population. Finally, we have compared the prevalence of the variants in the hearing impaired population with the general population. CONCLUSION: Although a high heterogeneity of sequence variation was observed in patients and controls, the 35delG mutation remains the most common pathogenic mutation in our population. Genetic counseling is dependent on the knowledge of the pathogenicity of the mutations and remains difficult in a number of cases. By comparing the sequence variations observed in hearing impaired patients with those sequence variants observed in general population, from the same ethnic background, we show that the M34T, V37I and R127H variants can not be responsible for profound or severe deafness

Making new genetic diagnoses with old data:iterative reanalysis and reporting from genome-wide data in 1,133 families with developmental disorders

Purpose Given the rapid pace of discovery in rare disease genomics, it is likely that improvements in diagnostic yield can be made by systematically reanalyzing previously generated genomic sequence data in light of new knowledge. Methods We tested this hypothesis in the United Kingdom–wide Deciphering Developmental Disorders study, where in 2014 we reported a diagnostic yield of 27% through whole-exome sequencing of 1,133 children with severe developmental disorders and their parents. We reanalyzed existing data using improved variant calling methodologies, novel variant detection algorithms, updated variant annotation, evidence-based filtering strategies, and newly discovered disease-associated genes. Results We are now able to diagnose an additional 182 individuals, taking our overall diagnostic yield to 454/1,133 (40%), and another 43 (4%) have a finding of uncertain clinical significance. The majority of these new diagnoses are due to novel developmental disorder–associated genes discovered since our original publication. Conclusion This study highlights the importance of coupling large-scale research with clinical practice, and of discussing the possibility of iterative reanalysis and recontact with patients and health professionals at an early stage. We estimate that implementing parent–offspring whole-exome sequencing as a first-line diagnostic test for developmental disorders would diagnose >50% of patients.</p

BAAV Mediated GJB2 Gene Transfer Restores Gap Junction Coupling in Cochlear Organotypic Cultures from Deaf Cx26Sox10Cre Mice

The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26Sox10Cre mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10–Cre line. Cx26Sox10Cre mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26Sox10Cre mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans

Protection from Intracellular Oxidative Stress by Cytoglobin in Normal and Cancerous Oesophageal Cells

Cytoglobin is an intracellular globin of unknown function that is expressed mostly in cells of a myofibroblast lineage. Possible functions of cytoglobin include buffering of intracellular oxygen and detoxification of reactive oxygen species. Previous work in our laboratory has demonstrated that cytoglobin affords protection from oxidant-induced DNA damage when over expressed in vitro, but the importance of this in more physiologically relevant models of disease is unknown. Cytoglobin is a candidate for the tylosis with oesophageal cancer gene, and its expression is strongly down-regulated in non-cancerous oesophageal biopsies from patients with TOC compared with normal biopsies. Therefore, oesophageal cells provide an ideal experimental model to test our hypothesis that downregulation of cytoglobin expression sensitises cells to the damaging effects of reactive oxygen species, particularly oxidative DNA damage, and that this could potentially contribute to the TOC phenotype. In the current study, we tested this hypothesis by manipulating cytoglobin expression in both normal and oesophageal cancer cell lines, which have normal physiological and no expression of cytoglobin respectively. Our results show that, in agreement with previous findings, over expression of cytoglobin in cancer cell lines afforded protection from chemically-induced oxidative stress but this was only observed at non-physiological concentrations of cytoglobin. In addition, down regulation of cytoglobin in normal oesophageal cells had no effect on their sensitivity to oxidative stress as assessed by a number of end points. We therefore conclude that normal physiological concentrations of cytoglobin do not offer cytoprotection from reactive oxygen species, at least in the current experimental model

Bax-Induced Apoptosis in Leber's Congenital Amaurosis: A Dual Role in Rod and Cone Degeneration

Pathogenesis in the Rpe65−/− mouse model of Leber's congenital amaurosis (LCA) is characterized by a slow and progressive degeneration of the rod photoreceptors. On the opposite, cones degenerate rapidly at early ages. Retinal degeneration in Rpe65−/− mice, showing a null mutation in the gene encoding the retinal pigment epithelium 65-kDa protein (Rpe65), was previously reported to depend on continuous activation of a residual transduction cascade by unliganded opsin. However, the mechanisms of apoptotic signals triggered by abnormal phototransduction remain elusive. We previously reported that activation of a Bcl-2-dependent pathway was associated with apoptosis of rod photoreceptors in Rpe65−/− mice during the course of the disease. In this study we first assessed whether activation of Bcl-2-mediated apoptotic pathway was dependent on constitutive activation of the visual cascade through opsin apoprotein. We then challenged the direct role of pro-apoptotic Bax protein in triggering apoptosis of rod and cone photoreceptors