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Abstract Several TBC1D24 variants are causally involved in the development of profound, prelingual hearing loss (HL) and different epilepsy syndromes inherited in an autosomal recessive manner. Only two TBC1D24 pathogenic variants have been linked with postlingual progressive autosomal dominant HL (ADHL). To determine the role of TBC1D24 in the development of ADHL and to characterize the TBC1D24-related ADHL, clinical exome sequencing or targeted multigene (n = 237) panel were performed for probands (n = 102) from multigenerational ADHL families. In four families, TBC1D24-related HL was found based on the identification of three novel, likely pathogenic (c.553G>A, p.Asp185Asn; c.1460A>T, p. His487Leu or c.1461C>G, p.His487Gln) and one known (c.533C>T, p.Ser178Leu) TBC1D24 variant. Functional consequences of these variants were characterized by analyzing the proposed homology models of the human TBC1D24 protein. Variants not only in the TBC (p.Ser178Leu, p.Asp185Asn) but also in the TLDc domain (p.His487Gln, p.His487Leu) are involved in ADHL development, the latter two mutations probably affecting interactions between the domains. Clinically, progressive HL involving mainly mid and high frequencies was observed in the patients (n = 29). The progression of HL was calculated by constructing age-related typical audiograms. TBC1D24-related ADHL originates from the cochlear component of the auditory system, becomes apparent usually in the second decade of life and accounts for approximately 4% of ADHL cases. Given the high genetic heterogeneity of ADHL, TBC1D24 emerges as an important contributor to this type of HL.

Abstract Background Biallelic PTPRQ pathogenic variants have been previously reported as causative for autosomal recessive non-syndromic hearing loss. In 2018 the first heterozygous PTPRQ variant has been implicated in the development of autosomal dominant non-syndromic hearing loss (ADNSHL) in a German family. The study presented the only, so far known, PTPRQ pathogenic variant (c.6881G>A) in ADNSHL. It is located in the last PTPRQ coding exon and introduces a premature stop codon (p.Trp2294*). Methods A five-generation Polish family with ADNSHL was recruited for the study (n = 14). Thorough audiological, neurotological and imaging studies were carried out to precisely define the phenotype. Genomic DNA was isolated from peripheral blood samples or buccal swabs of available family members. Clinical exome sequencing was conducted for the proband. Family segregation analysis of the identified variants was performed using Sanger sequencing. Single nucleotide polymorphism array on DNA samples from the Polish and the original German family was used for genome-wide linkage analysis. Results Combining clinical exome sequencing and family segregation analysis, we have identified the same (NM_001145026.2:c.6881G>A, NP_001138498.1:p.Trp2294*) PTPRQ alteration in the Polish ADNSHL family. Using genome-wide linkage analysis, we found that the studied family and the original German family derive from a common ancestor. Deep phenotyping of the affected individuals showed that in contrast to the recessive form, the PTPRQ-related ADNSHL is not associated with vestibular dysfunction. In both families ADNSHL was progressive, affected mainly high frequencies and had a variable age of onset. Conclusion Our data provide the first confirmation of PTPRQ involvement in ADNSHL. The finding strongly reinforces the inclusion of PTPRQ to the small set of genes leading to both autosomal recessive and dominant hearing loss.

TBC1D24 genetic variants are causally involved in the development of both autosomal recessive hearing loss and epilepsy syndromes, and autosomal dominant hearing loss (ADHL). So far, our group published four novel ADHL-causative TBC1D24 probably pathogenic variants by performing high-throughput genetic testing in families with ADHL, and more variants are yet to be revealed. In the light of current discoveries, variants in TBC1D24 emerge as a more significant cause of ADHL. The molecular mechanism behind the TBC1D24-associated ADHL is unknown. Using a zebrafish model, we investigated involvement of TBC1D24 in hearing and the functional effects of the associated ADHL-causing genetic variants. Different methodological approaches were used in the study, including (i) expression studies by whole mount in situ hybridization (WISH), qPCR on different developmental stages and cryosections, (ii) assessment of the zebrafish ear and neuromast hair cell morphology by high-resolution imaging and (iii) behavioral studies in a developed tbc1d24-deficient zebrafish models (by knock-down or knock-out of tbc1d24) and in overexpression and rescue tbc1d24 models. We show that the morpholino-mediated knockdown of Tbc1d24 resulted in defective ear kinocilia structure and reduced locomotor activity of the embryos. The observed phe-notypes were rescued by a wild-type TBC1D24 mRNA but not by a mutant mRNA carrying the ADHL-causing variant c.553G>A (p.Asp185Asn), supporting its pathogenic potential. CRISPR-Cas9-mediated knockout of tbc1d24 led to mechanosensory deficiency of lateral line neuromasts. Overexpression of TBC1D24 mRNA resulted in developmental abnormalities associated with ciliary dysfunction and mesen-dodermal mispatterning. We observed that the ADHL-causing TBC1D24 variants: c.553G>A (p.Asp185Asn); c.1460A>T (p.His487Leu), c.1461C>G (p.His487Gln) or a novel variant c.905T>G (p.Leu302Arg) alleviated the effect of overexpression, indicating that these variants disrupt the TBC1D24 function. Furthermore, the zebrafish phenotypes correspond to the severity of ADHL. Specific changes in ear structures upon TBC1D24 overexpression further highlighted its tissue-specific role in ciliary function and inner ear development. Our findings provide functional evidence for the pathogenic potential of the ADHL-causing TBC1D24 variants and lead to new insights into the function of TBC1D24 in cilia morphogenesis. [ABSTRACT FROM AUTHOR]

Introduction: Autosomal dominant hearing loss (ADHL) is the second most common form of inherited HL with an onset usually after the first decade of life. It affects mainly high frequencies and progresses over time. Autosomal-dominant genes are responsible for about 20% of cases of hereditary non-syndromic deafness, with 63 different genes identified to date. Material and methods: In this study, 105 families with a vertical inheritance pattern of hearing impairment were recruited. Genomic DNA was isolated from peripheral blood samples or buccal swabs of available family members. In all probands targeted next-generation sequencing (NGS) using a targeted multi-gene panel (237 genes) was performed. In 6 largest unsolved families linkage analysis and whole genome sequencing (WGS) were performed. Presence of the selected probably pathogenic variants and their segregation with HL within the family were confirmed by standard Sanger sequencing. Results: Genetic cause of ADHL was identified in 43.8% (46/105) of the examined families. Among the 46 identified HL variants only 26% (12/46) have been previously reported and the remaining 74% are novel (34/46). We identified missense variants (27/46; 58.7%), splice site variant (9/46; 19.5%), stop-gain variants (5/46; 10.9%) as well as frameshift variants (5/46; 10.9%). Among the most common causative genes were MYO6 (n = 8), TBC1D24 (n = 5), KCNQ4 (n = 4), GSDME (n = 4), POU4F3 (n = 4) and WFS1 (n = 4). Pathogenic variants causative of HL in the NLRP3, LMX1A, FGFR3, CD164, GRHL2, TMC1, COCH, ATP2B2 and CEACAM16 genes were detected in single families. Implementation of linkage analysis and WGS resulted in the identification of the non-coding variants in the EYA4 and ATP11A genes and two novel candidate genes. Conclusions: Our custom multigene panel has demonstrated good diagnostic performance. Considering frequent identification of novel genetic variants it is necessary to perform thorough clinical examination and variant segregation analysis with ADHL in all available family members. The use of linkage analysis and WGS increases the detection rate of causative variants, especially located in the non-coding regions, and provides the opportunity to identify novel genes. [ABSTRACT FROM AUTHOR]

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Background: Genetically determined prelingual hearing loss (HL) may occur in an isolated or syndromic form. Objective: The aim of the study was to unravel the genetic cause of medical problems in a 21-year-old woman, whose phenotypic presentation extended beyond Stickler syndrome and included enlarged vestibular aqueduct (EVA) and persistent microhematuria. Methods and Results: After sequencing of clinical exome, a known de novo COL2A1 pathogenic variant (c.1833+1G>A, p.?) causative for Stickler syndrome and one paternally inherited pathogenic change in COL4A5 (c.1871G>A, p.Gly624Asp) causative for X-linked Alport syndrome were found. No pathogenic variants, including those within the SLC26A4 5′ region (Caucasian EVA haplotype), explaining the development of EVA, were identified. Conclusions: The study reveals a multilocus genomic variation in one individual and provides a molecular diagnosis of two HL syndromes that co-occur in the proband independent of each other. For the third entity, EVA, no etiological factor was identified. Our data emphasize the relevance of detailed clinical phenotyping for accurate genotype interpretation. Focus on broadening the phenotypic spectrum of known genetic syndromes may actually obscure patients with multiple molecular diagnoses. [ABSTRACT FROM AUTHOR]

Purpose: Schnyder corneal dystrophy (SCD) is a rare autosomal dominant disorder characterized by corneal lipid accumulation and caused by UBIAD1 pathogenic variants. UBIAD1 encodes a vitamin K (VK) biosynthetic enzyme. To assess the corneal and vascular VK status in SCD patients, we focused on matrix Gla protein (MGP), a VK‐dependent protein. Methods: Conformation‐specific immunostainings of different MGP maturation forms were performed on corneal sections and primary keratocytes from corneal buttons of two SCD patients with UBIAD1 p.Asp112Asn and p.Asn102Ser pathogenic variants and unrelated donors. Native or UBIAD1‐transfected keratocytes were used for gene expression analysis. Plasma samples from SCD patients (n = 12) and control individuals (n = 117) were subjected for inactive desphospho‐uncarboxylated MGP level measurements with an ELISA assay. Results: Substantial amounts of MGP were identified in human cornea and most of it in its fully matured and active form. The level of mature MGP did not differ between SCD and control corneas. In primary keratocytes from SCD patients, a highly increased MGP expression and presence of immature MGP forms were detected. Significantly elevated plasma concentration of inactive MGP was found in SCD patients. Conclusion: High amount of MGP and the predominance of mature MGP forms in human cornea indicate that VK metabolism is active in the visual system. Availability of MGP seems of vital importance for a healthy cornea and may be related to protection against corneal calcification. Systemic MGP findings reveal a poor vascular VK status in SCD patients and indicate that SCD may lead to cardiovascular consequences. [ABSTRACT FROM AUTHOR]

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) is a transmembrane enzyme that plays an essential physiological role in the human body. The most important functions of the UBIAD1 protein include (i) the synthesis of endogenous vitamin K, (ii) direct and indirect participation in the cholesterol synthesis pathway and (iii) synthesis of the non-mitochondrial ubiquinone Q10 (CoQ10), a cellular antioxidant [27, 28, 30]. UBIAD1 is the only protein in the human body that is known to convert derivatives of the plant form of vitamin K1 to the endogenous vitamin K2 (MK-4).///Mutations in the UBIAD1 gene and/or dysfunction of the UBIAD1 protein have severe consequences on cellular metabolism and are causative of many diseases, including Schnyder corneal dystrophy (SCD). It is an inherited disease that leads to gradual vision loss by the deposition of lipids in the cornea. The mechanism of the disease remains largely unknown. Importantly, dysfunction of UBIAD1 was also found in other diseases, such as (i) Parkinson's disease, (ii) cardiovascular diseases and (iii) prostate and bladder cancer.///In this paper we attempt to present a wide characterization of UBIAD1 metabolic functions at the cellular and tissue level. We also show the mechanisms that may lead to pathological changes observed in SCD. Based on previous research, we demonstrate the complexity of processes in which the UBIAD1 protein is involved. The aim of this review is to analyze possible causes and to present a probable explanation for the occurrence of various diseases related to the dysfunction of UBIAD1. [ABSTRACT FROM AUTHOR]

Introduction: Genetically determined hearing loss may occur in an isolated or syndromic form. There is a wide range of syndromes for which hearing loss represents one of the characteristic features, such as Stickler and Alport syndrome. One of the most frequently observed inner ear malformation identified during a radiological workup of hearing loss patients is large vestibular aqueduct syndrome (LVAS). Objective: The aim of the study was to identify genetic variants that cause hearing loss in an adult female patient who was diagnosed with bilateral hearing loss, LVAS, high myopia, osteoarthritis and microhematuria. Material and methods: Next-generation sequencing of clinical exome was performed using the TruSight One sequencing kit (Illumina) on DNA isolated from the proband's blood sample. Analysis of the results focused on variants located in the genes related to hearing loss. To test for the presence of a haplotype located in the 5' region of the SLC26A4 gene (CEVA) in the proband and to confirm the presence and segregation of the identified pathogenic variants in the proband and her family members Sanger sequencing was performed. Results: Nextgeneration sequencing revealed the presence of a known COL2A1 pathogenic variant (NM_001844.4: c.1833+1G>A) causative for Stickler syndrome and one pathogenic change in COL4A5 (NM_000495.4: p.Gly624Asp/c.1871G>A) causative for Alport syndrome. In SLC26A4 (NM_000441.1: p.Leu597Ser/c.1790T>C) only one known pathogenic variant was found and the CEVA haplotype was not detected. Conclusions: Simultaneous testing of many genes using nextgeneration sequencing followed by family analysis identified a de novo pathogenic variant in the COL2A1 gene and after clinical reanalysis, the patient was diagnosed with Stickler syndrome, which is inherited in an autosomal dominant manner. The patient was found to be a carrier of a COL4A5 variant for the X-linked Alport syndrome, which explains the occurrence of microhematuria and may account for some degree of her hearing loss. The cause of LVAS still remains unknown as only one SLC26A4 pathogenic variant and no CEVA haplotype were found, and the disorder is inherited in an autosomal recessive manner. Our study identifies the presence of three rare known hearing loss syndromes in one patient and emphasizes the important role of genetic testing in guiding clinical diagnosis. [ABSTRACT FROM AUTHOR]

Objectives: TBC1D24 genetic variants are causally involved in the development of both autosomal recessive hearing loss, epilepsy syndromes, and autosomal dominant hearing loss (ADHL). So far, our group published four novel ADHLcausative TBC1D24 probably pathogenic variants by performing high-throughput genetic testing in families with ADHL, and more variants are yet to be revealed. In the light of current discoveries, variants in TBC1D24 emerge as a more significant cause of ADHL. In this study we aimed to use zebrafish model for deciphering the pathogenicity of novel TBC1D24 variants. As a background, we investigated the function of tbc1d24 orthologue and its involvement in hearing in zebrafish. Material and methods: Different methodological approaches were used in the study, including (i) expression studies by whole mount in situ hybridization (WISH), qPCR on different developmental stages and cryosections, (ii) assessment of the zebrafish ear and neuromast hair cell morphology by high-resolution imaging and (iii) behavioral studies in a developed tbc1d24-deficient zebrafish models (by knock-down or knock-out of tbc1d24) and in overexpression and rescue tbc1d24 models. Results: The expression pattern of tbc1d24 revealed by WISH in 24 hours post fertilization (hpf) zebrafish embryos consists of clusters of primary neurons in the brain and spinal cord. Noteworthy is tbc1d24 expression in the sensory cranial ganglia. Interestingly, according to our qPCR data, the highest tbc1d24 expression levels are detected at the very early stage of embryo development (1--5 hpf), during the dynamic cell divisions and cell-fate programming. In the later development, in 3--5 days post fertilization embryos (dpf), tbc1d24 is expressed more broadly in the brain and neural retina. As this stage, the expression pattern revealed by WISH was in line with levels of mRNA expression measured by qPCR in different organs of dissected larvae. The qPCR data also revealed an expression of tbc1d24 in larval ear. The additional experiments are planned to clarify the details of tbc1d24 expression and function in the ear and adjacent neural ganglia. The morpholino-mediated knock-down and overexpression of tbc1d24 caused no significant ear-specific morphological changes in zebrafish larvae. However, the behavioral studies revealed changes in the locomotor activity upon tbc1d24 morpholino-mediated knock-down at 24 hpf and 5 dpf. Importantly, the detrimental effect of overexpression of TBC1D24 p.Asp185Asn variant on locomotor activity of 24 hpf larvae was detected in rescue experiments. Conclusions: Further studies are needed to understand the role of tbc1d24 in the zebrafish ear and nervous system and to decipher the pathogenicity of other TBC1D24 ADHL-causative variants. [ABSTRACT FROM AUTHOR]

Objectives: In the hearing science, zebrafish has been long appreciated as a model organism to study ear development and function as well as the diverse effects induced by different ototoxic compounds. In zebrafish larvae, sensory hair cells are grouped into structures called neuromasts and located on the fish body surface, which makes them easily accessible for observation and manipulation. Neuromasts resemble in structure and function the sensory patches of the human inner ear. Studies on zebrafish morphology and behaviour help to asses ototoxic effects and reveal mechanism leading to hearing loss (HL) after administration of ototoxic drugs in humans. Aim: The aim of the study was to prepare a referent zebrafish HL model using a well-known highly ototoxic drug -- gentamicin (GNT) to cause GNT-induced hair cell death in the zebrafish lateral line as a representative model for the latter studies on HL in zebrafish. The study aimed to asses hearing defects by (i) evaluating the morphology of zebrafish auditory system by dimensional measurements of the inner ear structures; (ii) applying imaging techniques to asses structure and function of neuromasts -- small sensory patches containing hair cells, using a different vital dyes; (iii) use of behavioral studies for testing hearing responses -- different types of movements and reactions dependent on function of the hearing apparatus. Material and methods: To induce damage of neuromast sensory hair cells, 5 dpf (days post-fertilization) zebrafish larvae were incubated in embryo medium containing GNT for 6 h at a range of concentrations from 50 to 400 μM. After the treatment larvae were subjected for neuromast live cell staining with DASPEI and Yo-Pro1 dyes (Thermo Fisher Scientific, Massachusetts, USA). Neuromast imaging was performed using fluorescence stereomicroscopy and confocal microscopy. Locomotor activity of zebrafish larvae after GNT administration was measured with the ZebraBox System (ViewPoint, Civrieux, France) using tracking mode before and after acoustic stimulus. Results: In 5 dpf zebrafish larvae treated with GNT, vital dyes' fluorescent signals were gradually lost in neuromasts in a dose-dependent manner indicating a GNT-induced cell death. All GNT-treated groups showed a statistically significant reduction in locomotor activity also indicating a lower sensitivity to acoustic stimuli. The obtained phenotype was assessed to be related to GNT ototoxicity and a consequent hearing loss in zebrafish larvae. Conclusions: The developed GNT-induced HL model showed a range of features related to disrupted sound processing mechanisms and structures in zebrafish. The prepared model can be used in a subsequent analyses of HL mechanisms in zebrafish and in further studies on the GNT-induced HL in humans. [ABSTRACT FROM AUTHOR]

Introduction: With fast development in molecular biology, next-generation sequencing (NGS) became more accessible and less costly as a diagnostic tool, invaluable especially in diagnosing such heterogenic disorders as hearing loss. Vastness of data generated by this method allows speedy diagnosis but also creates a need for experimental verification of identified variants pathogenicity. Many of synonymous and intronic variants were thought of as having no impact on coded protein and as such as not pathogenic. Now it is known that they might influence transcript splicing. The goal of this study was to assess pathogenicity of 4 intronic variants in ATP2B2 (c.941-7C>G), MYO6 (c.1984-1G>A, c.2508-1G>A) and MYO15A (c.6178-1G>A) genes by using a "minigene" approach. The variants were identified in hearing loss patients from the Institute of Physiology and Pathology of Hearing.. In silico analysis predicted their impact on transcript splicing. Objective: Assessing influence on transcript splicing using a „minigene" model for variants identified in ATP2B2, MYO6 and MYO15A genes. Material: Genomic DNA used in this study was isolated from blood samples. Informed consent form was provided. Used plasmid vectors were pDONR221 (Thermo Scientific), pCS2+ and pDEST pCI-NEO RHO. Methods: In silico analysis of variants impact on transcript splicing was performed using NNSplice, MaxEntScan and SpliceSiteFinder. Sequences spanning one exon upstream and one exon downstream of the analyzed variant was cloned into pCS2+ expression vector or in case of ATP2B2 into pDEST pCI-NEO RHO using the Gateway cloning system. Expression vectors with wild type sequences underwent mutagenesis reactions, introducing the studied variants. NIH/3T3 and HEK-293 cells were transfected with both wild type and mutant vectors and harvested after 48h. RNA was isolated and used in RT-PCR reaction. Sanger sequencing of the reaction products was performed. Results: The c.1984-1G>A variant in MYO6 shortened the transcript by 1 bp in exon 20, as predicted in silico. The MYO6 c.2508-1G>A variant shortened exon 25 by 144 bp, more than bioinformatically predicted (19 bp). The MYO15A variant c.6178-1G>A led to the expected shortening of exon 29 by 1 bp and to skipping of exon 29 by deleting its acceptor site. The c.941-7C>G variant did not influence ATP2B2 transcript splicing, despite bioinformatic predictions. Conclusions: Variants in MYO6 and MYO15A could be classified as potentially pathogenic as they change transcript splicing and it is a known mechanism of pathogenicity in these genes. The c.941-7C>G variant in ATP2B2 could be classified as likely benign, since it does not affect transcript splicing. Gateway cloning system turned out to be a quick and easy alternative to cloning using restriction enzymes. [ABSTRACT FROM AUTHOR]