Search Results (10)

Auditory neuropathy spectrum disorder (ANSD) associated with mutations of the OTOF gene is one of the common types of sensorineural hearing loss of a hereditary nature. Due to its high genetic heterogeneity, ANSD is considered one of the most difficult hearing disorders to diagnose. The dataset from 270 known annotated single amino acid substitutions (SAV) related to ANSD was created. It was used to estimate the accuracy of pathogenicity prediction using the known (from dbNSFP4.4) method and a new one. The new method (ConStruct) for the creation of the protein-centric classification model is based on the use of Random Forest for the analysis of missense variants in exons of the OTOF gene. A system of predictor variables was developed based on the modern understanding of the structure and function of the otoferlin protein and reflecting the location of changes in the tertiary structure of the protein due to mutations in the OTOF gene. The conservation values of nucleotide substitutions in genomes of 100 vertebrates and 30 primates were also used as variables. The average prediction of balanced accuracy and the AUC value calculated by the 5-fold cross-validation procedure were 0.866 and 0.903, respectively. The model shows good results for interpreting data from the targeted sequencing of the OTOF gene and can be implemented as an auxiliary tool for the diagnosis of ANSD in the early stages of ontogenesis. The created model, together with the results of the pathogenicity prediction of SAVs via other known accurate methods, were used for the evaluation of a manually created set of 1302 VUS related to ANSD. Based on the analysis of predicted results, 16 SAVs were selected as the new most probable pathogenic variants. Full article

Otoferlin mRNA expression is increased in JDM patients’ PBMCs and muscle compared to healthy controls. This study aims to evaluate the role of otoferlin in JDM disease pathophysiology and its association with disease activity in untreated children with JDM. A total of 26 untreated JDM (88.5% female, 92.3% white, non-Hispanic) and 15 healthy controls were included in this study. Otoferlin mRNA expression was determined by qRT-PCR before and a few months after therapy. Detailed flow cytometry of various cell surface markers and cytoplasmic otoferlin was performed to identify cells expressing otoferlin. In addition, muscle otoferlin expression was evaluated in situ in six untreated JDM patients and three healthy controls. There was a significant increase in otoferlin expression in JDM children compared to controls (Median 67.5 vs. 2.1; p = 0.001). There was a positive correlation between mRNA otoferlin expression and the following disease activity markers: disease activity scores (DAS)-total (r_s = 0.62, p < 0.001); childhood myositis assessment scale (CMAS) (r_s = −0.61, p = 0.002); neopterin (r_s = 0.57, p = 0.004) and von Willebrand factor antigen (vWF: Ag) (r_s = 0.60, p = 0.004). Most of the otoferlin-positive cells were unswitched B cells (63–99.4%), with 65–75% of them expressing plasmablast markers (CD19⁺, IgM⁺, CD38^hi, CD24⁻). The findings of this pilot study suggest that otoferlin expression is associated with muscle weakness, making it a possible biomarker of disease activity. Additionally, B cells and plasmablasts were the primary cells expressing otoferlin. Full article

Quantitative surface plasmon resonance (SPR) was utilized to determine binding strength and calcium dependence of direct interactions between dysferlin and proteins likely to mediate skeletal muscle repair, interrupted in limb girdle muscular dystrophy type 2B/R2. Dysferlin canonical C2A (cC2A) and C2F/G domains directly interacted with annexin A1, calpain-3, caveolin-3, affixin, AHNAK1, syntaxin-4, and mitsugumin-53, with cC2A the primary target and C2F lesser involved, overall demonstrating positive calcium dependence. Dysferlin C2 pairings alone showed negative calcium dependence in almost all cases. Like otoferlin, dysferlin directly interacted via its carboxy terminus with FKBP8, an anti-apoptotic outer mitochondrial membrane protein, and via its C2DE domain with apoptosis-linked gene (ALG-2/PDCD6), linking anti-apoptosis with apoptosis. Confocal Z-stack immunofluorescence confirmed co-compartmentalization of PDCD6 and FKBP8 at the sarcolemmal membrane. Our evidence supports the hypothesis that prior to injury, dysferlin C2 domains self-interact and give rise to a folded, compact structure as indicated for otoferlin. With elevation of intracellular Ca²⁺ in injury, dysferlin would unfold and expose the cC2A domain for interaction with annexin A1, calpain-3, mitsugumin 53, affixin, and caveolin-3, and dysferlin would realign from its interactions with PDCD6 at basal calcium levels to interact strongly with FKBP8, an intramolecular rearrangement facilitating membrane repair. Full article

Background and objectives: Otoferlin is a multi-C2 domain protein implicated in neurotransmitter-containing vesicle release and replenishment of the cochlear inner hair cell (IHC) synapses. Mutations in the OTOF gene have been associated with two different clinical phenotypes: a prelingual severe-to-profound sensorineural hearing loss (ANSD-DFNB9); and the peculiar temperature-sensitive auditory neuropathy (TS-ANSD), characterized by a baseline mild-to-moderate hearing threshold that worsens to severe-to-profound when the body temperature rises that returns to a baseline a few hours after the temperature has fallen again. The latter clinical phenotype has been described only with a few OTOF variants with an autosomal recessive biallelic pattern of inheritance. Case report: A 7-year-old boy presented a picture compatible with TS-ANSD exacerbated by febrile states or physical exercise with mild-to-moderate hearing loss at low and medium frequencies and a decrease in speech discrimination that worsened with an unfavorable speech-to-noise ratio. Otoacoustic emissions (OAEs) were present whereas auditory brainstem responses (ABRs) evoked by a click or tone-burst were generally absent. No inner ear malformations were described from the CT scan or MRI. Next-generation sequencing (NGS) of the known deafness genes and multi-phasic bioinformatic analyses of the data detected in OTOF a c.2521G>A missense variant and the deletion of 7.4 Kb, which was confirmed by array-comparative genomic hybridization (array-CGH). The proband’s parents, who were asymptomatic, were tested by Sanger sequencing and the father presented the c.2521G>A missense variant. Conclusions: The picture presented by the patient was compatible with OTOF-induced TS-ANSD. OTOF has been generally associated with an autosomal recessive biallelic pattern of inheritance; in this clinical report, two pathogenic variants never previously associated with TS-ANSD were described. Full article

Background. Our study describes the neoexpression (Juno) and suppression (catsperD, dysferlin, Fer1L5 and otoferlin) of selected genes in oral squamous cell carcinomas (OSCCs). As the expression pattern of these genes allows a “yes” or “no” statement by exhibiting an inverse expression pattern in malignant versus benign tissues, they represent potential biomarkers for the characterization of oral malignancies, particularly OSCCs. Methods. Differential expression analyses of selected genes of interest were examined by quantitative PCR of oral cancer tissues compared to normal. Results. Five candidates out of initially nine genes were examined, demonstrating Juno as a putative new tumor marker selectively expressed in OSCCs. Interestingly, the expression of four other genes in benign tissues was completely repressed in tumor tissues with a specificity and sensitivity of 100%. No correlation was observed regarding patients’ sex, tumor staging and grading, and tumor site. Conclusion. The present study shows novel candidates that might be useful tools for oral cancer diagnosis. The neoexpression of Juno in cancerous tissues makes it a promising target molecule regarding its potential in diagnosis as well a therapeutic tool. Moreover, our observations suggest that also the repression of gene expression can be used for diagnosing—at least—OSCCs. Full article

The OTOF gene encodes otoferlin, a critical protein at the synapse of auditory sensory cells, the inner hair cells (IHCs). In the absence of otoferlin, signal transmission of IHCs fails due to impaired release of synaptic vesicles at the IHC synapse. Biallelic pathogenic and likely pathogenic variants in OTOF predominantly cause autosomal recessive profound prelingual deafness, DFNB9. Due to the isolated defect of synaptic transmission and initially preserved otoacoustic emissions (OAEs), the clinical characteristics have been termed “auditory synaptopathy”. We review the broad phenotypic spectrum reported in patients with variants in OTOF that includes milder hearing loss, as well as progressive and temperature-sensitive hearing loss. We highlight several challenges that must be addressed for rapid clinical and genetic diagnosis. Importantly, we call for changes in newborn hearing screening protocols, since OAE tests fail to diagnose deafness in this case. Continued research appears to be needed to complete otoferlin isoform expression characterization to enhance genetic diagnostics. This timely review is meant to sensitize the field to clinical characteristics of DFNB9 and current limitations in preparation for clinical trials for OTOF gene therapies that are projected to start in 2021. Full article

Fer1L5 is a dysferlin and myoferlin related protein, which has been predicted to have a role in vesicle trafficking and muscle membrane fusion events. Mutations in dysferlin and otoferlin genes cause heredity diseases: muscular dystrophy and deafness in humans, respectively. Dysferlin is implicated in membrane repair. Myoferlin has a role in myogenesis. In this study, we investigated the role of the Fer1L5 protein during myoblast fusion and membrane repair. To study the functions of Fer1L5 we used confocal microscopy, biochemical fractionation, Western blot analysis and multiphoton laser wounding assay. By immunolabelling, Fer1L5 was detected in vesicular structures. By biochemical fractionation Fer1L5 was observed in low density vesicles. Our studies show that the membranes of Fer1L5 vesicles are non-resistant to non-ionic detergent. Partial co-staining of Fer1L5 with other two ferlin vesicles, respectively, was observed. Fer1L5 expression was highly detected at the fusion sites of two apposed C2C12 myoblast membranes and its expression level gradually increased at D2 and reached a maximum at day 4 before decreasing during further differentiation. Our studies showed that Fer1L5 has fusion defects during myoblast fusion and impaired membrane repair when the C2C12 cultures were incubated with inhibitory Fer1L5 antibodies. In C2C12 cells Fer1L5 vesicles are involved in two stages, the fusion of myoblasts and the formation of large myotubes. Fer1L5 also plays a role in membrane repair. Full article

Anisotropic gap junctional coupling is a distinct feature of astrocytes in many brain regions. In the lateral superior olive (LSO), astrocytic networks are anisotropic and oriented orthogonally to the tonotopic axis. In Ca_V1.3 knock-out (KO) and otoferlin KO mice, where auditory brainstem nuclei are deprived from spontaneous cochlea-driven neuronal activity, neuronal circuitry is disturbed. So far it was unknown if this disturbance is also accompanied by an impaired topography of LSO astrocyte networks. To answer this question, we immunohistochemically analyzed the expression of astrocytic connexin (Cx) 43 and Cx30 in auditory brainstem nuclei. Furthermore, we loaded LSO astrocytes with the gap junction-permeable tracer neurobiotin and assessed the network shape and orientation. We found a strong elevation of Cx30 immunoreactivity in the LSO of Ca_V1.3 KO mice, while Cx43 levels were only slightly increased. In otoferlin KO mice, LSO showed a slight increase in Cx43 as well, whereas Cx30 levels were unchanged. The total number of tracer-coupled cells was unaltered and most networks were anisotropic in both KO strains. In contrast to the WTs, however, LSO networks were predominantly oriented parallel to the tonotopic axis and not orthogonal to it. Taken together, our data demonstrate that spontaneous cochlea-driven neuronal activity is not required per se for the formation of anisotropic LSO astrocyte networks. However, neuronal activity is required to establish the proper orientation of networks. Proper formation of LSO astrocyte networks thus necessitates neuronal input from the periphery, indicating a critical role of neuron-glia interaction during early postnatal development in the auditory brainstem. Full article

Ferlins are multiple-C2-domain proteins involved in Ca²⁺-triggered membrane dynamics within the secretory, endocytic and lysosomal pathways. In bony vertebrates there are six ferlin genes encoding, in humans, dysferlin, otoferlin, myoferlin, Fer1L5 and 6 and the long noncoding RNA Fer1L4. Mutations in DYSF (dysferlin) can cause a range of muscle diseases with various clinical manifestations collectively known as dysferlinopathies, including limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. A mutation in MYOF (myoferlin) was linked to a muscular dystrophy accompanied by cardiomyopathy. Mutations in OTOF (otoferlin) can be the cause of nonsyndromic deafness DFNB9. Dysregulated expression of any human ferlin may be associated with development of cancer. This review provides a detailed description of functions of the vertebrate ferlins with a focus on muscle ferlins and discusses the mechanisms leading to disease development. Full article

In mammal myocytes, endothelial cells and inner ear cells, ferlins are proteins involved in membrane processes such as fusion, recycling, endo- and exocytosis. They harbour several C2 domains allowing their interaction with phospholipids. The expression of several Ferlin genes was described as altered in several tumoural tissues. Intriguingly, beyond a simple alteration, myoferlin, otoferlin and Fer1L4 expressions were negatively correlated with patient survival in some cancer types. Therefore, it can be assumed that membrane biology is of extreme importance for cell survival and signalling, making Ferlin proteins core machinery indispensable for cancer cell adaptation to hostile environments. The evidences suggest that myoferlin, when overexpressed, enhances cancer cell proliferation, migration and metabolism by affecting various aspects of membrane biology. Targeting myoferlin using pharmacological compounds, gene transfer technology, or interfering RNA is now considered as an emerging therapeutic strategy. Full article