Search Results (45)

Background/Objectives: Pathogenic recessive GJB2 variants are the main genetic cause of non-syndromic sensorineural hearing loss. However, following GJB2 testing, a significant proportion of deaf patients are only found to be heterozygous carriers of pathogenic GJB2 alleles. Five large deletions not affecting GJB2 but encompassing a minimal common 62 kb region within the neighbouring CRYL1 gene have been described to cause loss of cis GJB2 expression and, as a result, produce hearing loss when in trans with pathogenic GJB2 variants. We describe the identification and characterization of a novel deletion of this type in deaf patients from northwestern Spain. Methods: We used panel NGS sequencing to detect the deletion, MLPA to validate it, whole-genome sequencing to map its breakpoints, PCR + Sanger sequencing to finely characterize it and triple-primer PCR to screen for it. Results: We identified a novel 200 kb deletion spanning the whole CRYL1 gene in two unrelated deaf patients from Asturias (in northwestern Spain) who were heterozygous for the pathogenic GJB2 c.35delG variant. Although the large deletion was absent from gnomAD v4.1.0 and 2052 local control alleles, screening for it in 20 additional deaf carriers of monoallelic pathogenic GJB2 variants detected it in another patient from Galicia (also in northwestern Spain). The novel deletion, termed del(200 kb)insATTATA, explained hearing loss in 3/43 (7%) deaf patients from our cohort that were otherwise heterozygous for pathogenic GJB2 variants. Conclusions: This work highlights the importance of comprehensively testing all genomic regions known to be clinically relevant for a given genetic condition, including thorough CRYL1 CNV screening for DFNB1A diagnostics. Full article

Background/Objectives: Oxazolidinones are novel antimicrobial agents used to combat bacterial infections, particularly multidrug-resistant strains. However, the synthesis of oxazolidinone derivatives, such as linezolid, often involves the use of 3,4-difluoronitrobenzene (DFNB) as an initiator. Despite its effectiveness, residual DFNB in drug products raises significant health concerns due to its structural similarity to toxic and carcinogenic nitrobenzenes. This contamination is particularly concerning in pharmaceutical formulations, where it poses potential patient safety hazards. Therefore, strict concentration limits for this impurity are necessary. Methods: To ensure tight control of DFNB concentrations, this study established an 8.3 µg/g target limit. An advanced high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to overcome current limitations in detecting trace DFNB. Under negative atmospheric pressure chemical ionization (APCI) conditions, DFNB exhibited characteristic ion formations, including [M]^•− through electron capture and [M − F + O]⁻ via substitution reactions. The quantitative method utilizes MS/MS ion transitions of the substitution product while optimizing chromatographic and spectrometric parameters to enhance both sensitivity and specificity. Conclusions: Validation tests confirm the efficiency, precision, and accuracy of this method, with a low limit of quantification (LOQ) of 5 ng/mL (0.83 µg/g). This technique enables accurate detection and quantification of DFNB in linezolid active pharmaceutical ingredient (API) and various formulations, providing a reliable tool for quality control. This method ensures the safe use of linezolid by effectively monitoring and minimizing the risks associated with DFNB contamination. Full article

Background/Objectives: Hearing loss is one of the most common sensorineural impairments, and approximately 60% of early-onset cases are due to genetic variations. The otogelin-like protein, encoded by the OTOGL gene, is a component of the acellular membranes of the inner ear, such as the tectorial membrane, and is thought to play an important role in cochlear amplification. OTOGL gene variants are a rare cause of hearing loss such as DFNB84B, a mild-to-moderate sensorineural hearing loss presenting in early childhood with autosomal recessive inheritance. In this study, we aim to enhance our comprehension of the phenotypes of hearing loss caused by OTOGL variants. Methods: A total of 7056 Japanese patients with hearing loss were recruited, and based on massively parallel DNA sequencing on 158 target genes, we selected patients with biallelic OTOGL variants. Results: Ten affected individuals with OTOGL gene variants were detected, the largest group of patients yet to be reported, and eight of the eleven variants were novel. Our results showed that variations in this gene led to mild-to-moderate non-progressive hearing loss, and the accompanying symptoms, mainly vestibular symptoms, were speculated to present in adulthood. Conclusions: Determination of the phenotypes of genes causative of hearing loss is expected to greatly benefit patients with hearing loss as it can assist in predicting outcomes and lead to appropriate intervention, which, in OTOGL-associated hearing loss cases, is based around the fact that the patients need not be concerned with deterioration in hearing, but require careful follow-up for vestibular symptoms. Full article

Background/Objectives: The OTOF gene is reported to be the causative gene for non-syndromic recessive sensorineural hearing loss and auditory neuropathy spectrum disorder. About 300 variants have been reported, but there have been no reports to date on copy gain variants. Methods: We identified a copy gain variant in the OTOF gene through short-read next-generation sequencing analysis from one patient with auditory neuropathy. We also performed long-read next-generation sequencing analysis using the Oxford Nanopore Technologies adaptive sampling procedure. Results: The four-year-old male carried a duplication of chr2: 26,477,852 to 26,483,106 (a 5254-base duplication including exon 14 to exon 18 of the OTOF gene NM_001287489) and a c.5385C>A single nucleotide variant. We also confirmed that these two variants were located in the trans configuration based on haplotype phasing results using the long-read next-generation sequencing data. Conclusions: This is the first report of an auditory neuropathy patient with a large duplication variant in the OTOF gene. The identified variants were novel, but based on the clinical phenotype of the patient, these variants seem to be the genetic cause of this patient’s phenotype. Oxford Nanopore Technologies adaptive sampling is a powerful tool for the analysis of structural variants (particularly for determining the breakpoint and direction) and haplotype phasing. Full article

Background/Objectives: MYO3A belongs to the unconventional myosin superfamily, and the myosin IIIa protein localizes on the tip of the stereocilia of vestibular and cochlear hair cells. Deficiencies in MYO3A have been reported to cause the deformation of hair cells into abnormally long stereocilia with an increase in spacing. MYO3A is a rare causative gene of autosomal recessive sensorineural hearing loss (DFNB30), with only 13 cases reported to date. In this study, we aimed to elucidate the phenotypes caused by MYO3A variations. Methods: Massively parallel DNA sequencing was performed on 15,684 Japanese hearing loss patients (mean age 27.5 ± 23.1 years old, 6574 male, 8612 female and 498 patients for whom information was unavailable), identifying nine candidate patients with MYO3A variants. Results: We identified eight causative MYO3A variants by massively parallel DNA sequencing, including six novel variants, and reported nine individuals possessing MYO3A gene variants, which is the largest group of non-related patients yet to be detected. Our findings confirmed that MYO3A variants cause progressive hearing loss, with its onset varying from birth to the second decade, eventually leading to severe-to-profound hearing loss. Conclusions: We clarified that patients with MYO3A gene variants present with late-onset, progressive hearing loss. Our findings have enabled us to predict the outcomes of hearing loss in patients with candidate MYO3A gene variants and to provide intervention in a timely manner. Full article

Background/Objectives: The OTOG gene is responsible for autosomal recessive non-syndromic sensorineural hearing loss and is assigned as DFNB18B. To date, 44 causative OTOG variants have been reported to cause non-syndromic hearing loss. However, the detailed clinical features for OTOG-associated hearing loss remain unclear. Methods: In this study, we analyzed 7065 patients with non-syndromic hearing loss (mean age 26.4 ± 22.9 years, 2988 male, 3855 female, and 222 without gender information) using massively parallel DNA sequencing for 158 target deafness genes. We identified the patients with biallelic OTOG variants and summarized the clinical characteristics. Results: Among the 7065 patients, we identified 14 possibly disease-causing OTOG variants in 26 probands, with 13 of the 14 variants regarded as novel. Patients with OTOG-associated hearing loss mostly showed congenital or childhood-onset hearing loss. They were considered to show non-progressive, mild-to-moderate hearing loss. There were no symptoms that accompanied the hearing loss in OTOG-associated hearing loss patients. Conclusions: We confirmed non-progressive, mild-to-moderate hearing loss as the clinical characteristics of OTOG-associated hearing loss. These findings will contribute to a better understanding of the clinical features of OTOG-associated HL and will be useful in clinical practice. Full article

Dysfunction of some mitochondrial aminoacyl-tRNA synthetases (encoded by the KARS1, HARS2, LARS2 and NARS2 genes) results in a great variety of phenotypes ranging from non-syndromic hearing impairment (NSHI) to very complex syndromes, with a predominance of neurological signs. The diversity of roles that are played by these moonlighting enzymes and the fact that most pathogenic variants are missense and affect different domains of these proteins in diverse compound heterozygous combinations make it difficult to establish genotype–phenotype correlations. We used a targeted gene-sequencing panel to investigate the presence of pathogenic variants in those four genes in cohorts of 175 Spanish and 18 Colombian familial cases with non-DFNB1 autosomal recessive NSHI. Disease-associated variants were found in five cases. Five mutations were novel as follows: c.766C>T in KARS1, c.475C>T, c.728A>C and c.1012G>A in HARS2, and c.795A>G in LARS2. We provide audiograms from patients at different ages to document the evolution of the hearing loss, which is mostly prelingual and progresses from moderate/severe to profound, the middle frequencies being more severely affected. No additional clinical sign was observed in any affected subject. Our results confirm the involvement of KARS1 in DFNB89 NSHI, for which until now there was limited evidence. Full article

Deafness in vertebrates is associated with variants of hundreds of genes. Yet, many mutant genes causing rare forms of deafness remain to be discovered. A consanguineous Pakistani family segregating nonsyndromic deafness in two sibships were studied using microarrays and exome sequencing. A 1.2 Mb locus (DFNB128) on chromosome 5q11.2 encompassing six genes was identified. In one of the two sibships of this family, a novel homozygous recessive variant NM_005921.2:c.4460G>A p.(Arg1487His) in the kinase domain of MAP3K1 co-segregated with nonsyndromic deafness. There are two previously reported Map3k1-kinase-deficient mouse models that are associated with recessively inherited syndromic deafness. MAP3K1 phosphorylates serine and threonine and functions in a signaling pathway where pathogenic variants of HGF, MET, and GAB1 were previously reported to be associated with human deafness DFNB39, DFNB97, and DFNB26, respectively. Our single-cell transcriptome data of mouse cochlea mRNA show expression of Map3k1 and its signaling partners in several inner ear cell types suggesting a requirement of wild-type MAP3K1 for normal hearing. In contrast to dominant variants of MAP3K1 associated with Disorders of Sex Development 46,XY sex-reversal, our computational modeling of the recessive substitution p.(Arg1487His) predicts a subtle structural alteration in MAP3K1, consistent with the limited phenotype of nonsyndromic deafness. Full article

The universal newborn hearing screening (NHS) program was implemented in Russia in 2008 to replace the high-risk newborn hearing screening. More than 95% coverage and significant improvement in early detection and intervention is achieved. Meanwhile, it was shown that current OAE-based hearing screening missed 13% of newborns with genetically ascertained hereditary sensorineural hearing loss (SNHL). The aim of the study is to assess the results of genetic investigation and NHS in a large cohort of Russian children with bilateral SNHL and to study the feasibility of implementation of combined hearing and genetic screening in Russia. Genetic, audiological and NHS data of 1292 pediatric patients with bilateral SNHL born in 2008–2021 were analyzed. GJB2 sequencing was performed for all subjects, 644 patients had pathological GJB2 genotype, 406 of them were homozygous for c.35delG variant. The group of 155 GJB2-negative patients were searched for other SNHL genes, The pathological genotypes were identified at 87 patients. The most frequent genes were STRC (21.8%), USH2A (16.1%), OTOF (8%) and SLC26A4 (6.9%). Children with confirmed genetic etiology passed NHS in 21% of cases. The perspectives of implementation of national comprehensive newborn hearing and genetic screening including whole exome sequencing technologies are discussed. Full article

The PTPRQ gene has been identified as one of the genes responsible for non-syndromic sensorineural hearing loss (SNHL), and assigned as DFNA73 and DFNB84. To date, about 30 causative PTPRQ variants have been reported to cause SNHL. However, the detailed clinical features of PTPRQ-associated hearing loss (HL) remain unclear. In this study, 15,684 patients with SNHL were enrolled and genetic analysis was performed using massively parallel DNA sequencing (MPS) for 63 target deafness genes. We identified 17 possibly disease-causing PTPRQ variants in 13 Japanese patients, with 15 of the 17 variants regarded as novel. The majority of variants identified in this study were loss of function. Patients with PTPRQ-associated HL mostly showed congenital or childhood onset. Their hearing levels at high frequency deteriorated earlier than that at low frequency. The severity of HL progressed from moderate to severe or profound HL. Five patients with profound or severe HL received cochlear implantation, and the postoperative sound field threshold levels and discrimination scores were favorable. These findings will contribute to a greater understanding of the clinical features of PTPRQ-associated HL and may be relevant in clinical practice. Full article

Pendrin and prestin are evolutionary-conserved membrane proteins that are essential for normal hearing. Dysfunction of these proteins results in hearing loss in humans, and numerous deafness-associated pendrin and prestin variants have been identified in patients. However, the pathogenic impacts of many of these variants are ambiguous. Here, we report results from our ongoing efforts to experimentally characterize pendrin and prestin variants using in vitro functional assays. With previously established fluorometric anion transport assays, we determined that many of the pendrin variants identified on transmembrane (TM) 10, which contains the essential anion binding site, and on the neighboring TM9 within the core domain resulted in impaired anion transport activity. We also determined the range of functional impairment in three deafness-associated prestin variants by measuring nonlinear capacitance (NLC), a proxy for motor function. Using the results from our functional analyses, we also evaluated the performance of AlphaMissense (AM), a computational tool for predicting the pathogenicity of missense variants. AM prediction scores correlated well with our experimental results; however, some variants were misclassified, underscoring the necessity of experimentally assessing the effects of variants. Together, our experimental efforts provide invaluable information regarding the pathogenicity of deafness-associated pendrin and prestin variants. Full article

The etiology of sensorineural hearing loss is heavily influenced by genetic mutations, with approximately 80% of cases attributed to genetic causes and only 20% to environmental factors. Over 100 non-syndromic deafness genes have been identified in humans thus far. In non-syndromic sensorineural hearing impairment, around 75–85% of cases follow an autosomal recessive inheritance pattern. In recent years, groundbreaking advancements in molecular gene therapy for inner-ear disorders have shown promising results. Experimental studies have demonstrated improvements in hearing following a single local injection of adeno-associated virus-derived vectors carrying an additional normal gene or using ribozymes to modify the genome. These pioneering approaches have opened new possibilities for potential therapeutic interventions. Following the PRISMA criteria, we summarized the AAV gene therapy experiments showing hearing improvement in the preclinical phases of development in different animal models of DFNB deafness and the AAV gene therapy programs currently in clinical phases targeting autosomal recessive non syndromic hearing loss. A total of 17 preclinical studies and 3 clinical studies were found and listed. Despite the hurdles, there have been significant breakthroughs in the path of HL gene therapy, holding great potential for providing patients with novel and effective treatment. Full article

Non-syndromic hearing impairment (NSHI) is a very heterogeneous genetic condition, involving over 130 genes. Mutations in GJB2, encoding connexin-26, are a major cause of NSHI (the DFNB1 type), but few other genes have significant epidemiological contributions. Mutations in the STRC gene result in the DFNB16 type of autosomal recessive NSHI, a common cause of moderate hearing loss. STRC is located in a tandem duplicated region that includes the STRCP1 pseudogene, and so it is prone to rearrangements causing structural variations. Firstly, we screened a cohort of 122 Spanish familial cases of non-DFNB1 NSHI with at least two affected siblings and unaffected parents, and with different degrees of hearing loss (mild to profound). Secondly, we screened a cohort of 64 Spanish sporadic non-DFNB1 cases, and a cohort of 35 Argentinean non-DFNB1 cases, all of them with moderate hearing loss. Amplification of marker D15S784, massively parallel DNA sequencing, multiplex ligation-dependent probe amplification and long-range gene-specific PCR followed by Sanger sequencing were used to search and confirm single-nucleotide variants (SNVs) and deletions involving STRC. Causative variants were found in 13 Spanish familial cases (10.7%), 5 Spanish simplex cases (7.8%) and 2 Argentinean cases (5.7%). In all, 34 deleted alleles and 6 SNVs, 5 of which are novel. All affected subjects had moderate hearing impairment. Our results further support this strong genotype–phenotype correlation and highlight the significant contribution of STRC mutations to moderate NSHI in the Spanish population. Full article

One of the most common forms of genetic deafness has been predominantly associated with pathogenic variants in the GJB2 gene, encoding transmembrane protein connexin 26 (Cx26). The Cx26 molecule consists of an N-terminal domain (NT), four transmembrane domains (TM1–TM4), two extracellular loops (EL1 and EL2), a cytoplasmic loop, and a C-terminus (CT). Pathogenic variants in the GJB2 gene, resulting in amino acid substitutions scattered across the Cx26 domains, lead to a variety of clinical outcomes, including the most common non-syndromic autosomal recessive deafness (DFNB1A), autosomal dominant deafness (DFNA3A), as well as syndromic forms combining hearing loss and skin disorders. However, for rare and poorly documented variants, information on the mode of inheritance is often lacking. Numerous in vitro studies have been conducted to elucidate the functional consequences of pathogenic GJB2 variants leading to amino acid substitutions in different domains of Cx26 protein. In this work, we summarized all available data on a mode of inheritance of pathogenic GJB2 variants leading to amino acid substitutions and reviewed published information on their functional effects, with an emphasis on their localization in certain Cx26 domains. Full article

Progressive corneal opacification can result from multiple etiologies, including corneal dystrophies or systemic and genetic diseases. We describe a novel syndrome featuring progressive epithelial and anterior stromal opacification in a brother and sister and their mildly affected father, with all three family members having sensorineural hearing loss and two also with tracheomalacia/laryngomalacia. All carried a 1.2 Mb deletion at chromosome 13q12.11, with no other noteworthy co-segregating variants identified on clinical exome or chromosomal microarray. RNAseq analysis from an affected corneal epithelial sample from the proband’s brother revealed downregulation of XPO4, IFT88, ZDHHC20, LATS2, SAP18, and EEF1AKMT1 within the microdeletion interval, with no notable effect on the expression of nearby genes. Pathway analysis showed upregulation of collagen metabolism and extracellular matrix (ECM) formation/maintenance, with no significantly down-regulated pathways. Analysis of overlapping deletions/variants demonstrated that deleterious variants in XPO4 were found in patients with laryngomalacia and sensorineural hearing loss, with the latter phenotype also being a feature of variants in the partially overlapping DFNB1 locus, yet none of these had reported corneal phenotypes. Together, these data define a novel microdeletion-associated syndromic progressive corneal opacification and suggest that a combination of genes within the microdeletion may contribute to ECM dysregulation leading to pathogenesis. Full article