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Introduction: Next generation sequencing (NGS) is a method which is becoming increasingly available in clinical setting, especially in genetically heterogenous diseases as hereditary hearing loss. For each patient there are thousands of variants, including benign polymorphisms, pathogenic variants and variants of unknown clinical significance. The last ones are difficult to interpret, especially in case of silent variants and variants in non-coding parts of the gene - their mode of pathogenicity might be more elusive, i.e. alteration of splicing. Such variants require functional studies to properly assess their impact and pathogenic potential. Aim: To assess the pathogenicity of 11 novel variants with possible effect on splicing using minigene assay. Material and methods: Selected variants were: ATP2B2 c.941-7C>G, EYA1 c.1475+1G>T, EYA4 c.1282-12T>A, GSDME c.991delT, GSDME c.1127A>G, MYO6 c.816+1G>A, MYO6 c.1984-1G>A, MYO6 c.3281-13A>G, MYO7A c.2829G>A, MYO15A c.9230-4 A>T, SLC26A4 c.1001+1G>A. Each variant was detected in the custom HL gene panel performed for patient of Department of Genetics, Institute of Physiology and Pathology of Hearing. Fragments of genes of interest encompassing closest introns and exons were introduced into expression vector pDEST pCI-Neo RHO using Gateway cloning system. Cell cultures of HEK293T line were transfected with expression vectors for each gene, containing either wild type sequence or sequence with studied variant. After 48 h of incubation cell lysis and RNA isolation were performed. Transcripts were analyzed by subsequent RT-PCR, gel elec-trophoresis and Sanger sequencing. Results: The majority of studied variants displayed their effect on splicing (9 out of11, 82%). The most common aberrations were exon skipping and incorporation of intron fragment to the transcript, which usually resulted in frameshift and introduction of premature stop codon. In case of variants ATP2B2 c.941-7C>G and MYO15A c.9230-4 A>T there were no observable signs of splicing alteration. Conclusions: Genetic variants affecting splicing emerge as an important contributor to HL. The performed minigene assays allowed for better variant interpretation, which in turn allowed for the correct genetic diagnosis. The study demonstrates the significance of functional testing especially when it comes to the silent variants and intronic variants. [ABSTRACT FROM AUTHOR]

Congenital hearing loss can be caused by genetic abnormalities leading to inner ear malformations (IEM). SLC26A4 (pendrin) is an anion exchanger expressed in the inner ear, thyroid and β-intercalated cells in the kidney. Mutations in SLC26A4 cause a common form of IEMS: enlarged vestibular aqueduct (EVA), often accompanied by incomplete partition type 2 (EVA/IP2). However, only 25% of patients have confirmed mutations in SLC26A4. It has been reported that a group of polymorphisms upstream of the SLC26A4 gene, the CEVA haplotype, is frequently found in patients with monoallelic SLC26A4 mutations. Since the publication of the zebrafish genome in 2001, the zebrafish has become an increasingly popular animal model for studying human diseases. The transparency of the larvae, the large number of offspring and the accessibility of various methods of genetic, chemical and physical manipulation make it a very useful model for studying inner ear development. During early development, the inner ear undergoes dynamic changes from an otic placode and otic vesicle to a labyrinth of semicircular canals. Since data on the expression of genes associated with hearing are incomplete, we present here a detailed account of expression during early development. Secondly, we plan to create a zebrafish model for human SLC26A4 gene expression by introducing human upstream regulatory elements into the zebrafish genome. [ABSTRACT FROM AUTHOR]

The NLRP3 gene mutations are the cause of autosomal dominant autoinflammatory disorders (NLRP3-AID). Recently, hearing loss (HL) has been found to be the sole or major manifestation of NLRP3-AID. Here, we tested 110 autosomal dominant HL families with a custom panel of 237 HL genes and found one family carrying the NLRP3 c.1872C>G, p.Ser624Arg mutation. Functional studies revealed that this novel variant is a gain of function mutation, leading to increased activity of caspase-1 and subsequent oversecretion of proinflammatory interleukin-lb. Clinical reanalysis of the affected individuals, together with serological evidence of inflammation and pathological cochlear enhancement on FLAIR-MRI images, guided our diagnosis to atypical NLRP3-AID. The study highlights the role of genetic analysis in patients with progressive postlingual HL. This can help to identify individuals with hereditary HL as a consequence of NLRP3-AID and allow timely and effective treatment with interleukin-l-receptor antagonist. [ABSTRACT FROM AUTHOR]

Introduction: Each year, approximately 1-6 out of every 1000 children are born with severe to profound hearing loss (HL). In the majority of them HL is genetically determined and usually two pathogenic variants are detected in the DFNB1 locus. The aim of the study was to dissect the genetic background of non-DFNB1 HL in CI patients and to analyze their auditory development. Material and methods: The study group (n = 51) was recruited from patients with isolated profound prelingual deafness who received CI before the age of 24 months. All patients were negative for DFNB1 locus pathogenic variants. Genomic DNA was isolated from blood samples. In probands whole exome sequencing (WES) was performed. Validation of selected variants and family segregation analysis were performed using standard Sanger sequencing. Identified copy number variants were examined with aCGH and qPCR. Evaluation of patients auditory development was performed with the LittlEARS questionnaire (LEAQ) in three subsequent intervals - at the time of cochlear implant activation as well as in 5th and 9th month after CI. Results: Causative variants were identified in 74.5% of patients (38/51). The majority of them are localized in the MYO15A (n = 7) and PAX3 (n = 5) genes. Among the detected genetic variants, 28% (15/54) were inherited in an autosomal dominant manner and eight of them occurred de novo. A syndromic form of HL was diagnosed in 27% (14/51) of patients. The auditory development of the studied children was the most dynamic in the first 5 months after CI and slowed down between the 5 and 9 months of using the device. No differences were observed between the auditory development of patients with an identified and unknown genetic causes of HL. Conclusions: Obtained results show a high heterogeneity of genetic HL causes in the population of Polish DFNB1-negative cochlear-implanted patients. All tested children were good candidates for CI as their HL causative genetic variants are localized in genes preferentially expressed in the cochlea. In a group of patients without an identified genetic cause, the tested area should be expanded and more advanced technologies enabling full genome analysis (WGS) should be used. [ABSTRACT FROM AUTHOR]

Introduction: GRHL2 is one from over 50 genes causative of autosomal dominant hearing loss (ADHL); it is also implicated in other disorders, including cancers. GRHL2 encodes a transcription factor but up to now only a handful of ADHL-related GRHL2 pathogenic variants have been reported. Their mode of action leading to ADHL development remains unknown. The aim of the study was to identify the genetic basis of ADHL in a multigeneration family with postlingual, progressive HL and to gain insight into the molecular mechanism of the ADHL-related (DFNA28) GRHL2 mutations. Material and methods: Genomic DNA was isolated from the peripheral blood samples of the proband and other family members (n = 8). Next-generation sequencing was performed using a multi-gene panel with 237 HL-related genes. Segregation analysis of the selected GRHL2 variant with HL in the family was performed by Sanger sequencing. For four different ADHL-related GRHL2 variants expression vectors were prepared and luciferase reporter gene assay was conducted in HEK293T cells. Results: In the family a novel heterozygous GRHL2 variant (NM_024915.4: c.1061C>T; NP_079191.2: p.(Ala354Val)) segregating with HL was identified. It localizes in the region corresponding to the DNA binding domain. The functional effect of the variant as well as of the other two GRHL2 variants located in the DNA-binding domain (i.e. c.1258-1G>A, p.(Gly420Glufs*111) and c.1276C>T, p.(Arg426*)) was a reduction in GRHL2 transcriptional activity. In contrast, the c.1609-1610insC (p.(Arg537Profs*11)) variant affecting the DNA dimerization domain of the GRHL2 protein acted in a different way leading to a strong activation of the GRHL-responsive promoter. Conclusions: Our data show that only truncating GRHL2 mutations can cause ADHL. The pathogenicity of the novel missense ADHL-related GRHL2 variant was strengthened by the results of functional assays. GRHL2 mutations causing ADHL demonstrated both suppression and activation of GRHL2 transcriptional activity and the effect seems to depend on where the variant is located. While the variants located in the DNA-binding domain showed haploinsufficiency, the variant located in the DNA dimerization domain presented a gain of function effect. Our study sheds new light on the mechanism of GRHL2 mutations leading to hearing loss. [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]

Introduction: Inner ear liquid biopsy would help in understanding the molecular environment of the diseased organ. In cases of prelingual deafness it is postulated that cochlea' insufficiency, both congenital and acquired in early postnatal period is connected with remarkable changes in the perilymph proteome and the protein pathways. In cases in which deafness results from genetic change of the cochlea structures, like GJB2-related deafness, it is probably expected that proteins encoded by the defected gene should not be detected in the perilymph or should be detected at much different levels. Analogically, in cases of deafness acquired due to ototoxic agents exposure one could expect that proteome characteristics would be altered, among others towards inflammatory products and proteins involved in oxidative stress reactions. Having known the proteome status of the perilymph we could approach to more precise prognosis of the disease, especially in cases of residual hearing. Material and methods: We have collected perilymph samples using glass capillaries from preliminary group of prelingually deaf children during the procedure of cochlear implantation in Institute of Physiology and Pathology of Hearing, Warsaw. Each sample was no less than 2 | and was harvested via round window approach. Method: using mass spectrometry (MS) protein quality analysis was performed in each sample. Results: Number of detected proteins across samples varied from 1324 to 2103. Further samples taken from patients with different kinds of deafness are needed to continue the analyses. [ABSTRACT FROM AUTHOR]

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]

Objectives: There are different genetic variants located in the RMND1 gene that are causally involved in the development of a mitochondrial disease with complex phenotype including sensorineural hearing loss (HL), hypotonia, developmental delay, lactic academia and renal dysfunction. In 2018 the first and so far the only report on association of RMND1 pathogenic variants with Perrault-like syndrome has been published. Material and methods: Patient diagnosed with progressive HL at 4 y.o., hypergonadotropic hypogonadism at 17 y.o. and chronic kidney disease at 33 y.o. and her family members were consulted at the Department of Genetics, Institute of Physiology and Pathology of Hearing. Genomic DNA was isolated from whole blood and buccal swabs samples using a standard salting-out procedure and an automatic method (Maxwell RSC Instrument, Promega, Germany), respectively. Our custom multigene panel (SeqCap EZ choice, Roche, Switzerland) encompassing 237 HL-related genes was used to prepare proband's sample library that was further sequenced on the MiSeq platform (Illumina Inc, USA). All bioinformatics and expert analysis were done and probable pathogenic variants were selected based on their population frequencies (<1%), functional consequences (e.g.: missense, frameshift, splice-site) and pathogenic predictions (CADD, LRT, FATHHM, MutationTaster, PolyPhen-2, SIFT). Results: Genetic testing revealed two novel probably pathogenic c.583G>A (p.Gly195Arg) and c.818A>C (p.Tyr273Ser) RMND1 variants, that were inherited in an autosomal recessive manner from the proband's parents. Identified variants were also present in the DNA sample from proband's sister who has also been diagnosed with HL and primary amenorrhea. Clinical reanalysis revealed that patients have no neurological or intellectual impairment, and nephrological evaluation predicts a benign course of kidney disease. Conclusions: Our results represent the first independent confirmation of RMND1 involvement in the development of Perrault-like syndrome and broaden our knowledge on phenotypic presentation of the disease. Considering the probability of mild presentation of renal phenotype in patients with RMND1 pathogenic variant, we strongly recommend inclusion of the RMND1 gene in standard genetic diagnostic process for Perrault syndrome. [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: STRC is commonly associated with mild-to-moderate HL, and it is frequently part of a large deletion within chromosome 15q15.3 at the DFNB16 locus. Among individuals with deafness, STRC deletions are detected in more than 1% of cases but there is a limited number of studies on STRC variation. Material: For this project we recruited patients with mild-to-moderate HL (n=200) based on the analysis of pure tone audiometry results, who were lacking HL environmental risk factors and pathogenic variants in the DFNB1 locus (GJB2 and GJB6 genes). Methods: DNA was extracted from blood samples using a standard salting-out method. Afterwards each sample was tested against the CNVs presence in the STRC gene using the Quantitative Fluorescence-Polymerase Chain Reaction (QF-PCR) method with specifically designed primers flanking the desired region of the STRC gene as well as its challenging pseudogene. In the next step the DNA samples were tested with multiplex-ligation probe amplification (MLPA) in order to validate the results and check the QF-PCR accuracy by using an independent method. Results: The STRC gene mutation profile will be presented and the contribution of STRC gene variants to the development of mild-to-moderate hearing loss will be estimated. Conclusions: Variants in STRC are frequently found in patients with mild-to-moderate HL. That makes STRC a matter of concern when deliberating over major causes of this type of HL. Further research concerning STRC point mutations in gene is needed to get a better insight into the spectrum of its pathogenic variants. [ABSTRACT FROM AUTHOR]

Introduction: Incomplete partition type 2 (IP2) encompasses enlarged vestibular aqueduct (EVA), the most frequently observed congenital inner ear malformation, cochlear hypoplasia and enlarged vestibule. In most EVA/IP2 cases congenital hearing impairment is observed. EVA/IP2 can occur in an isolated manner or in genetically determined syndromes such as Pendred syndrome, branchio-oto-renal syndrome (BOR), Waardenburg syndrome, CHARGE, distal renal tubular acidosis (RTA). Around 25% of EVA/IP2 patients carry two pathogenic SLC26A4 variants, while another 25% have only one variant. In 2017, the CEVA haplotype was identified in the 5' region of SLC26A4. CEVA's function remains unknown, and its frequency has not been assessed in Polish patients. Objective: The aim of the study was to identify genetic variants that cause hearing loss in patients with IP2. Material and methods: The genetic background of 20 probands with bilateral IP2 was analyzed by next generation sequencing (NGS) using a custom hearing loss gene panel. Presence and segregation of selected variants and CEVA haplotype was verified by Sanger sequencing. Results: Genetic testing identified the cause of IP2 in 11/20 (55%) individuals. Two pathogenic variants of the SLC26A4 gene were identified as the cause of IP2 in 5 out of 20 (20%). CEVA haplotype may be the cause of IP2 in 5/8 (63%) patients, who had only one SLC26A4 genetic variant detected in NGS. In one case IP2 were caused by pathogenic variant of the EYA1 gene, related to BOR. Conclusions: In our study 10/20 patients carry pathogenic variants in SLC26A4 which also confirms the association of IP2 occurrence with variants in this gene. The identification of CEVA may explain hearing loss caused by IP2 in a large group of patients with only one pathogenic variant found in the SLC26A4 gene. Studies on CEVA allows better understanding of its role in the phenotype. CEVA is a likely pathogenic allele linked to a milder phenotype than pathogenic SLC26A4 variants. Finding a pathogenic variant of the EYA1 gene in a patient with a phenotype matching BOR allows to consider this variant as the cause of IP2. [ABSTRACT FROM AUTHOR]

Objectives: Hearing loss (HL) is one of the most common sensory disorders that affects almost 466 mln people worldwide. Each year, approximately 1--6 out of every 1000 children are born with severe to profound HL. In the majority of deaf children HL is genetically determined and usually two pathogenic variants are detected in the GJB2/GJB6 genes (DFNB1 locus). The aim of the study was to dissect the genetic background of non-DFNB1 HL in CI patients. Material: The study group (n=51) was recruited from patients with isolated profound prelingual deafness who received CI before the age of 24 months. All patients were negative for DFNB1 locus pathogenic variants and had no environmental HL risk factors. Detailed medical history including temporal bone imaging data was analyzed. Methods: Genomic DNA was isolated from blood samples of deaf probands and available family members. In 51 patients whole exome sequencing (WES) was performed. After bioinformatics analysis, all pathogenic and probably pathogenic variants involved in HL development were selected based on their population frequencies (<1%), functional consequences (e.g.: missense, frameshift, splice-site) and pathogenic predictions (CADD, LRT, FATHHM, MutationTaster, PolyPhen-2, SIFT). Validation of selected variants and family segregation analysis were performed using standard Sanger sequencing. Identified copy number variants were additionally examined with array comparative genomic hybridization (aCGH) and qPCR. Results: Causative variants were identified in almost 73% of patients (37/51). The majority of them are localized in the MYO15A (n=7) and PAX3 (n=5) genes. Among the detected genetic variants, 26% (14/54) were inherited in an autosomal dominant manner and as many as eight of them occurred de novo. A syndromic form of HL was diagnosed in 25% (13/51) of patients. More than half (28/54) of the identified HL causative variants have not been associated with HL before. Genes for which pathogenic variants have been detected are expressed primarily in the cochlea; their main role is to maintain the proper function of the hair cells, supporting cells and stria vascularis. Conclusions: Obtained results show a high heterogeneity of genetic HL causes in the population of Polish DFNB1-negative cochlear-implanted patients. All tested children were good candidates for CI as their HL causative genetic variants are localized in genes preferentially expressed in the cochlea. In a group of patients without an identified genetic cause, the tested area should be expanded and more advanced technologies enabling full genome analysis (WGS) should be used. [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]

The GRHL2 gene, encoding the Grainyhead-like 2 transcription factor, is essential for various biological processes. While GRHL2 has a complex role in cancer biology, its genetic variants have been also implicated in different forms of hearing loss (HL), including autosomal dominant non-syndromic hearing loss (DFNA28). Here, we report a novel c.1061C>T, p.(Ala354Val) mutation within the DNA binding domain (DBD) of GRHL2 that was identified in a three-generation HL family using a targeted multi-gene panel covering 237 HL-related genes. Unlike the previously reported DFNA28-causing variants that result in protein truncation, the impact of the p.(Ala354Val) missense change cannot be attributed to GRHL2 transcript level or composition, but to an alteration in protein function. Molecular dynamics simulations revealed destabilization of the p.(Ala354Val) mutant GRHL2 dimer interface and an altered DNA binding dynamics, leading to chaotic interaction patterns despite increased binding affinity to DNA. Functional assays demonstrated that the p.(Ala354Val) mutation and other DFNA28-related mutations in the DBD lead to loss of GRHL2 transcriptional transactivation activity, while the p.(Arg537Profs*11) mutation in the dimerization domain results in a gain-of-function effect. The findings indicate that both GRHL2 haploinsufficiency and gain-of-function contribute to HL and underscore the complex regulatory role of GRHL2 in maintaining proper function of the auditory system. Our study emphasizes the need to consider structural and functional aspects of gene variants to better understand their pathogenic potential. As GRHL2 is involved in a multitude of cellular processes, the data gathered here can be also applicable to other conditions., (© The Author(s) 2025. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Purpose: If before cochlear implantation it was possible to assay biomarkers of neuroplasticity, we might be able to identify those children with congenital deafness who, later on, were at risk of poor speech and language rehabilitation outcomes., Methods: A group of 40 children aged up to 2 years with DFNB1-related congenital deafness was observed in this prospective cohort study over three follow-up intervals (0, 8, and 18 months) after cochlear implant (CI) activation. Children were assessed for auditory development using the LittlEARS Questionnaire (LEAQ) score, and at the same time, measurements were made of matrix metalloproteinase-9 (MMP-9) plasma levels., Results: There were significant negative correlations between plasma levels of MMP-9 at 8-month follow-up and LEAQ score at cochlear implantation (p = 0.04) and LEAQ score at 18-month follow-up (p = 0.02) and between MMP-9 plasma levels at 18-month follow-up and LEAQ score at cochlear implantation (p = 0.04). As already reported, we confirmed a significant negative correlation between MMP-9 plasma level at cochlear implantation and LEAQ score at 18-month follow-up (p = 0.005). Based on this latter correlation, two clusters of good and poor CI performers could be isolated., Conclusions: The study shows that children born deaf who have an MMP-9 plasma level of less than 150 ng/ml at cochlear implantation have a good chance of attaining a high LEAQ score after 18 months of speech and language rehabilitation. This indicates that MMP-9 plasma level at cochlear implantation is a good prognostic marker for CI outcome., (© 2023. The Author(s).)

Congenitally deaf children who undergo cochlear implantation before 1 year of age develop their auditory skills faster than children who are implanted later. In this longitudinal study, a cohort of 59 implanted children were divided into two subgroups according to their ages at implantation-below or above 1 year old-and the plasma levels of matrix metalloproteinase-9 (MMP-9), brain-derived neurotrophic factor (BDNF), and pro-BDNF were measured at 0, 8, and 18 months after cochlear implant activation, while auditory development was simultaneously evaluated using the LittlEARs Questionnaire (LEAQ). A control group consisted of 49 age-matched healthy children. We identified statistically higher BDNF levels at 0 months and at the 18-month follow-ups in the younger subgroup compared to the older one and lower LEAQ scores at 0 months in the younger subgroup. Between the subgroups, there were significant differences in the changes in BDNF levels from 0 to 8 months and in LEAQ scores from 0 to 18 months. The MMP-9 levels significantly decreased from 0 to 18 months and from 0 to 8 months in both subgroups and from 8 to 18 months only in the older one. For all measured protein concentrations, significant differences were identified between the older study subgroup and the age-matched control group.

The most frequently observed congenital inner ear malformation is enlarged vestibular aqueduct (EVA). It is often accompanied with incomplete partition type 2 (IP2) of the cochlea and a dilated vestibule, which together constitute Mondini malformation. Pathogenic SLC26A4 variants are considered the major cause of inner ear malformation but the genetics still needs clarification. The aim of this study was to identify the cause of EVA in patients with hearing loss (HL). Genomic DNA was isolated from HL patients with radiologically confirmed bilateral EVA ( n = 23) and analyzed by next generation sequencing using a custom HL gene panel encompassing 237 HL-related genes or a clinical exome. The presence and segregation of selected variants and the CEVA haplotype (in the 5' region of SLC26A4 ) was verified by Sanger sequencing. Minigene assay was used to evaluate the impact of novel synonymous variant on splicing. Genetic testing identified the cause of EVA in 17/23 individuals (74%). Two pathogenic variants in the SLC26A4 gene were identified as the cause of EVA in 8 of them (35%), and a CEVA haplotype was regarded as the cause of EVA in 6 of 7 patients (86%) who carried only one SLC26A4 genetic variant. In two individuals with a phenotype matching branchio-oto-renal (BOR) spectrum disorder, cochlear hypoplasia resulted from EYA1 pathogenic variants. In one patient, a novel variant in CHD7 was detected. Our study shows that SLC26A4 , together with the CEVA haplotype, accounts for more than half of EVA cases. Syndromic forms of HL should also be considered in patients with EVA. We conclude that to better understand inner ear development and the pathogenesis of its malformations, there is a need to look for pathogenic variants in noncoding regions of known HL genes or to link them with novel candidate HL genes.

Background: Low-frequency non-syndromic hearing loss (LFNSHL) is a rare form of hearing loss (HL). It is defined as HL at low frequencies (≤2,000 Hz) resulting in a characteristic ascending audiogram. LFNSHL is usually diagnosed postlingually and is progressive, leading to HL affecting other frequencies as well. Sometimes it occurs with tinnitus. Around half of the diagnosed prelingual HL cases have a genetic cause and it is usually inherited in an autosomal recessive mode. Postlingual HL caused by genetic changes generally has an autosomal dominant pattern of inheritance and its incidence remains unknown., Summary: To date, only a handful of genes have been found as causing LFNSHL: well-established WFS1 and, reported in some cases, DIAPH1, MYO7A, TNC, and CCDC50 (respectively, responsible for DFNA6/14/38, DFNA1, DFNA11, DFNA56, and DFNA44). In this review, we set out audiological phenotypes, causative genetic changes, and molecular mechanisms leading to the development of LFNSHL., Key Messages: LFNSHL is most commonly caused by pathogenic variants in the WFS1 gene, but it is also important to consider changes in other HL genes, which may result in similar audiological phenotype., (© 2023 S. Karger AG, Basel.)

Hearing is an important human sense for communicating and connecting with others. Partial deafness (PD) is a common hearing problem, in which there is a down-sloping audiogram. In this study, we apply a practical system for classifying PD patients, used for treatment purposes, to distinguish two groups of patients: one with almost normal hearing thresholds at low frequencies (PDT-EC, n = 20), and a second group with poorer thresholds at those same low frequencies (PDT-EAS, n = 20). After performing comprehensive genetic testing with a panel of 237 genes, we found that genetic factors can explain a significant proportion of both PDT-EC and PDT-EAS hearing losses, accounting, respectively, for approx. one-fifth and one-half of all the cases in our cohort. Most of the causative variants were located in dominant and recessive genes previously linked to PD, but more than half of the variants were novel. Among the contributors to PDT-EC we identified OSBPL2 and SYNE4 , two relatively new hereditary hearing loss genes with a low publication profile. Our study revealed that, for all PD patients, a postlingual hearing loss more severe in the low-frequency range is associated with a higher detection rate of causative variants. Isolating a genetic cause of PD is important in terms of prognosis, therapeutic effectiveness, and risk of recurrence.

The NLRP3 gene mutations are the cause of autosomal dominant autoinflammatory disorders (NLRP3-AID). Recently, hearing loss (HL) has been found to be the sole or major manifestation of NLRP3-AID. Here, we tested 110 autosomal dominant HL families with a custom panel of 237 HL genes and found one family carrying the NLRP3 c.1872C>G, p.Ser624Arg mutation. Functional studies revealed that this novel variant is a gain of function mutation, leading to increased activity of caspase-1 and subsequent oversecretion of proinflammatory interleukin-1β. Clinical reanalysis of the affected individuals, together with serological evidence of inflammation and pathological cochlear enhancement on FLAIR-MRI images, guided our diagnosis to atypical NLRP3-AID. The study highlights the role of genetic analysis in patients with progressive postlingual HL. This can help to identify individuals with hereditary HL as a consequence of NLRP3-AID and allow timely and effective treatment with interleukin-1-receptor antagonist., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Oziębło, Leja, Jeznach, Orzechowska, Skirecki, Więsik-Szewczyk, Furmanek, Bałdyga, Skarżyński and Ołdak.)

Because of vast variability of cochlear implantation outcomes in prelingual deafness treatment, identification of good and poor performers remains a challenging task. To address this issue, we investigated genetic variants of matrix metalloproteinase 9 (MMP9) and brain-derived neurotrophic factor (BDNF) and plasma levels of MMP-9, BDNF, and pro-BDNF that have all been implicated in neuroplasticity after sensory deprivation in the auditory pathway. We recruited a cohort of prelingually deaf children, all implanted before the age of 2, and carried out a prospective observation (N = 61). Next, we analyzed the association between (i) functional MMP9 (rs20544, rs3918242, rs2234681) and BDNF (rs6265) gene variants (and their respective protein levels) and (ii) the child's auditory development as measured with the LittlEARS Questionnaire (LEAQ) before cochlear implant (CI) activation and at 8 and 18 months post-CI activation. Statistical analyses revealed that the plasma level of MMP-9 measured at implantation in prelingually deaf children was significantly correlated with the LEAQ score 18 months after CI activation. In the subgroup of DFNB1-related deafness (N = 40), rs3918242 of MMP9 was significantly associated with LEAQ score at 18 months after CI activation; also, according to a multiple regression model, the ratio of plasma levels of pro-BDNF/BDNF measured at implantation was a significant predictor of overall LEAQ score at follow-up. In the subgroup with DFNB1-related deafness, who had CI activation after 1 year old (N = 22), a multiple regression model showed that rs3918242 of MMP9 was a significant predictor of overall LEAQ score at follow-up., (© 2022. The Author(s).)

Novel hearing loss (HL) genes are constantly being discovered, and evidence from independent studies is essential to strengthen their position as causes of hereditary HL. To address this issue, we searched our genetic data of families with autosomal dominant HL (ADHL) who had been tested with high-throughput DNA sequencing methods. For CD164, only one pathogenic variant in one family has so far been reported. For LMX1A, just two previous studies have revealed its involvement in ADHL. In this study we found two families with the same pathogenic variant in CD164 and one family with a novel variant in LMX1A (c.686C>A; p.(Ala229Asp)) that impairs its transcriptional activity. Our data show recurrence of the same CD164 variant in two HL families of different geographic origin, which strongly suggests it is a mutational hotspot. We also provide further evidence for haploinsufficiency as the pathogenic mechanism underlying LMX1A-related ADHL., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A . Seven unexplained cases were selected for future analysis with whole genome sequencing.

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.

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., (© 2020 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

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., (© 2020 S. Karger AG, Basel.)

Genetic biomarkers of neuroplasticity in deaf children treated with cochlear implantation (CI) might facilitate their clinical management, especially giving them better chances of developing proficient spoken language. We investigated whether carrying certain variants of the genes encoding matrix metalloproteinase MMP9 and neurotrophin brain-derived neurotrophic factor ( BDNF ), involved in synaptic plasticity, can be taken as prognostic markers of how well auditory skills might be acquired. Association analysis of functional MMP9 rs3918242 and BDNF rs6265 variants and the child's auditory development measured at CI activation and 1, 5, 9, 14, and 24 months post CI activation with LittlEARS Questionnaire (LEAQ) was conducted in a group of 100 children diagnosed with DFNB1-related deafness, unilaterally implanted before the age of 2 years. Statistical analysis in the subgroup implanted after 1 year of life ( n  = 53) showed significant association between MMP9 rs3918242 and LEAQ scores at 1 month ( p  = .01), at 5 months ( p  = .01), at 9 months ( p  = .01), and at 24 months ( p  = .01) after CI activation. No significant associations in the subgroup implanted before 1 year of life were observed. No significant associations between the BDNF rs6265 and LEAQ score were found. Multiple regression analysis ( R 2  = .73) in the subgroup implanted after 1 year of life revealed that MMP9 rs3918242 was a significant predictor of treatment outcome. In conclusion, C/C rs3918242 MMP9 predisposes their deaf carriers to better CI outcomes, especially when implanted after the first birthday, than carriers of C/T rs3918242 MMP9.

Introduction: Otosclerosis (OTSC) is one of the most common causes of progressive adult-onset hearing loss in the Caucasian population, with a female preponderance. The etiology of OTSC is complex and there are a number of genetic variants reported to be associated with OTSC susceptibility, but no data on the genetic background of OTSC in patients originating from the central-eastern part of Europe have been available. The purpose of our study was to investigate in Polish patients the frequency of genetic variants previously reported to be most strongly associated with OTSC., Material and Methods: Genomic DNA was isolated from blood samples or buccal swabs. Variants in TGFB1 (rs1800472) and RELN (rs39335, rs39350, rs39374) were genotyped in surgically confirmed OTSC patients ( n = 94) and a control group ( n = 198) using custom TaqMan SNP genotyping assays and real-time PCR. Allele and genotype frequencies were compared between the groups in statistical analysis and the odds ratios with 95% confidence intervals were calculated to estimate the risk., Results: For all of the tested variants the distributions of alleles and genotypes were not statistically significantly different between OTCS patients and the control group. There were also no statistically significant differences in relation to gender of the tested subjects., Conclusions: Despite multiple confirmatory studies on TGFB1 and RELN association with OTSC development in some populations, no significant association between the studied variants and OTSC was found in Polish patients. Our results indicate the presence of inter-population differences in OTSC susceptibility factors and confirm the large genetic heterogeneity of this disorder., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2020 Termedia & Banach.)

RMND1 (required for meiotic nuclear division 1 homolog) pathogenic variants are known to cause combined oxidative phosphorylation deficiency (COXPD11), a severe multisystem disorder. In one patient, a homozygous RMND1 pathogenic variant, with an established role in COXPD11, was associated with a Perrault-like syndrome. We performed a thorough clinical investigation and applied a targeted multigene hearing loss panel to reveal the cause of hearing loss, ovarian dysfunction (two cardinal features of Perrault syndrome) and chronic kidney disease in two adult female siblings. Two compound heterozygous missense variants, c.583G>A (p.Gly195Arg) and c.818A>C (p.Tyr273Ser), not previously associated with disease, were identified in RMND1 in both patients, and their segregation with disease was confirmed in family members. The patients have no neurological or intellectual impairment, and nephrological evaluation predicts a benign course of kidney disease. Our study presents the mildest, so far reported, RMND1 -related phenotype and delivers the first independent confirmation that RMND1 is causally involved in the development of Perrault syndrome with renal involvement. This highlights the importance of including RMND1 to the list of Perrault syndrome causative factors and provides new insight into the clinical manifestation of RMND1 deficiency.

Background: Despite its established association with chronic kidney disease (CKD) the role of myosin-9 (MYH9) gene variation on transplanted kidney function remains unknown. This study aimed at evaluating the effect of donor MYH9 nephrogenic variants on renal allograft function within the first post transplantation year., Methods: In the longitudinal kidney transplant study 207 deceased donors were genotyped for previously known risk MYH9 single nucleotide polymorphisms (SNPs). The predictor was MYH9 high-risk variants status. The primary outcome was mean eGFR found in low vs. high risk MYH9 genotypes between third and twelfth post-transplant month, the secondary outcome was the risk of proteinuria., Results: Distribution of genotypes remained in Hardy-Weinberg equilibrium. The T allele of rs3752462 (dominant model, TT or TC vs. CC) was associated with higher filtration rate (P = 0.05) in a multivariate analysis after adjusting for delayed graft function and donor sex. Two G alleles of rs136211 (recessive model, GG vs. GA or AA) resulted in doubling the risk of proteinuria (OR = 2.22; 95% CI = 1.18-4.37, P = 0.017) after adjusting for donor and recipient sex., Conclusion: Deceased donor kidneys of European descent harboring MYH9 SNPs rs3752462 T allele show significantly superior estimated filtration rate while those of rs136211 GG genotype excessive risk of proteinuria. These findings, if replicated, may further inform and improve individualization of allocation and treatment policies.

Almost 60% of children with profound prelingual hearing loss (HL) have a genetic determinant of deafness, most frequently two DFNB1 locus ( GJB2/GJB6 genes) recessive pathogenic variants. Only few studies combine HL etiology with cochlear implantation (CI) outcome. Patients with profound prelingual HL who received a cochlear implant before 24 months of age and had completed DFNB1 genetic testing were enrolled in the study (n = 196). LittlEARS questionnaire scores were used to assess auditory development. Our data show that children with DFNB1-related HL (n = 149) had good outcome from the CI (6.85, 22.24, and 28 scores at 0, 5, and 9 months post-CI, respectively). A better auditory development was achieved in patients who receive cochlear implants before 12 months of age. Children without residual hearing presented a higher rate of auditory development than children with responses in hearing aids over a wide frequency range prior to CI, but both groups reached a similar level of auditory development after 9 months post-CI. Our data shed light upon the benefits of CI in the homogenous group of patients with HL due to DFNB1 locus pathogenic variants and clearly demonstrate that very early CI is the most effective treatment method in this group of patients., Competing Interests: The authors declare no conflict of interest.

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&#95;001145026.2:c.6881G>A, NP&#95;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.

Background: Hearing loss (HL) is the most common disability of human senses characterized by a great allelic heterogeneity. GJB2 and TMPRSS3 are two well-known HL genes typically underlying its monogenic form. Recently, TMPRSS3/GJB2 digenic inheritance has been proposed. As results of genetic testing can be easily overinterpreted, we aimed to verify the hypothesis., Methods: From genetic database of HL patients with at least one TMPRSS3 pathogenic variants we have selected individuals with additional GJB2 pathogenic variants. All of the available family members were recruited for the study. Segregation analysis of the respective TMPRSS3 and GJB2 pathogenic variants was performed within the families., Results: The strategy has allowed to identify four individuals who were double heterozygous for known pathogenic TMPRSS3 and GJB2 variants. Two individuals from different families had GJB2 c.35delG and TMPRSS3 c.208delC and in two other individuals from one family GJB2 c.35delG together with TMPRSS3 c.1343T>C variants were found. None of these subjects has ever reported hearing problems and their hearing status was normal., Conclusions: Our data provide evidence against TMPRSS3/GJB2 digenic inheritance of HL. As high throughput sequencing is increasingly used for genetic testing, particular caution should be taken to provide the patients with accurate genetic counseling.

Purpose: Schnyder corneal dystrophy (SCD) is a rare inherited disease that leads to gradual vision loss by the deposition of lipids in the corneal stroma. The aim of this study is to report a novel pathogenic variant in the UBIAD1 gene and present clinical and molecular findings in Polish patients with SCD., Methods: Individuals (n = 37) originating from four Polish SCD families were subjected for a complete ophthalmological check-up and genetic testing. Corneal changes were visualized by slit-lamp examination, anterior segment optical coherent tomography (AS-OCT), and in vivo confocal microscopy (IVCM)., Results: In a proband with primarily mild SCD that progressed rapidly at the end of the fifth decade of life, a novel missense pathogenic variant in UBIAD1 (p.Thr120Arg) was identified. The other studied SCD family represents the second family reported worldwide with the UBIAD1 p.Asp112Asn variant. SCD in the remaining two families resulted from a frequently identified p.Asn102Ser pathogenic variant. All affected subjects presented a crystalline form of SCD. The severity of corneal changes was age-dependent, and their morphology and localization are described in detail., Conclusion: The novel p.Thr120Arg is the fourth SCD-causing variant lying within the FARM motif of the UBIAD1 protein, which underlines a high importance of this motif for SCD pathogenesis. The current study provides independent evidence for the pathogenic potential of UBIAD1 p.Asp112Asn and new information useful for clinicians.

Background: Hearing loss and ovarian dysfunction are key features of Perrault syndrome (PRLTS) but the clinical and pathophysiological features of hearing impairment in PRLTS individuals have not been addressed. Mutations in one of five different genes HSD17B4, HARS2, LARS2, CLPP or TWNK (previous symbol C10orf2) cause the autosomal recessive disorder but they are found only in about half of the patients., Methods: We report on two siblings with a clinical picture resembling a severe, neurological type of PRLTS. For an exhaustive characterisation of the phenotype neuroimaging with volumetric measurements and objective measures of cochlear hair cell and auditory nerve function (otoacustic emissions and auditory brainstem responses) were used. Whole exome sequencing was applied to identify the genetic cause of the disorder. Co-segregation of the detected mutations with the phenotype was confirmed by Sanger sequencing. In silico analysis including 3D protein structure modelling was used to predict the deleterious effects of the detected variants on protein function., Results: We found two rare biallelic mutations in TWNK, encoding Twinkle, an essential mitochondrial helicase. Mutation c.1196A>G (p.Asn399Ser) recurred for the first time in a patient with PRLTS and the second mutation c.1802G>A (p.Arg601Gln) was novel for the disorder. In both patients neuroimaging studies showed diminished cervical enlargement of the spinal cord and for the first time in PRLTS partial atrophy of the vestibulocochlear nerves and decreased grey and increased white matter volumes of the cerebellum. Morphological changes in the auditory nerves, their desynchronized activity and partial cochlear dysfunction underlay the complex mechanism of hearing impairment in the patients., Conclusions: Our study unveils novel features on the phenotypic landscape of PRLTS and provides further evidence that the newly identified for PRLTS TWNK gene is involved in its pathogenesis.

Variation in the ABCA4 locus has emerged as the most prevalent cause of monogenic retinal diseases. The study aimed to discover causative ABCA4 mutations in a large but not previously investigated cohort with ABCA4-related diseases originating from Central Europe and to refine the genetic relevance of all identified variants based on population evidence. Comprehensive clinical studies were performed to identify patients with Stargardt disease (STGD, n = 76) and cone-rod dystrophy (CRD, n = 16). Next-generation sequencing targeting ABCA4 was applied for a widespread screening of the gene. The results were analyzed in the context of exome data from a corresponding population (n = 594) and other large genomic databases. Our data disprove the pathogenic status of p.V552I and provide more evidence against a causal role of four further ABCA4 variants as drivers of the phenotype under a recessive paradigm. The study identifies 12 novel potentially pathogenic mutations (four of them recurrent) and a novel complex allele p.[(R152*; V2050L)]. In one third (31/92) of our cohort we detected the p.[(L541P; A1038V)] complex allele, which represents an unusually high level of genetic homogeneity for ABCA4-related diseases. Causative ABCA4 mutations account for 79% of STGD and 31% of CRD cases. A combination of p.[(L541P; A1038V)] and/or a truncating ABCA4 mutation always resulted in an early disease onset. Identification of ABCA4 retinopathies provides a specific molecular diagnosis and justifies a prompt introduction of simple precautions that may slow disease progression. The comprehensive, population-specific study expands our knowledge on the genetic landscape of retinal diseases., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Fuchs endothelial corneal dystrophy is the most common genetically determined degenerative disease of the cornea. In Polish patients the dystrophy is a leading indication for lamellar posterior keratoplasty. The genetic background of Fuchs endothelial corneal dystrophy is complex and heterogeneous. A number of TCF4 gene variants have been strongly associated with the development of this disorder with the most important of them being the trinucleotide repeat expansion CTG18.1. The aim of the study is to present this novel and extraordinarily strong genetic association with Fuchs endothelial corneal dystrophy. Studies on the impact of CTG18.1 on corneal endothelial cells may help to explain the molecular mechanism involved in the pathogenesis of the corneal dystrophy. This could significantly improve diagnostics and therapy of Fuchs endothelial corneal dystrophy patients.

Fuchs endothelial corneal dystrophy (FECD) is a common corneal endotheliopathy with a complex and heterogeneous genetic background. Different variants in the TCF4 gene have been strongly associated with the development of FECD. TCF4 encodes the E2-2 transcription factor but the link between the strong susceptibility locus and disease mechanism remains elusive. Here, we confirm a strong positive association between TCF4 single nucleotide polymorphism rs613872 and FECD in Polish patients (OR = 12.95, 95% CI: 8.63-19.42, χ (2) = 189.5, p < 0.0001). We show that TCF4 expression at the mRNA level in corneal endothelium (n = 63) does not differ significantly between individuals with a particular TCF4 genotype. It is also not altered in FECD patients as compared to control samples. The data suggest that changes in the transcript level containing constitutive TCF4 exon encoding the amino-terminal part of the protein seem not to contribute to disease pathogenesis. However, considering the strong association of TCF4 allelic variants with FECD, genotyping of TCF4 risk alleles may be important in the clinical practice.