Connexins (Cxs) are transmembrane proteins that assemble into gap junction channels (GJCs) and hemichannels (HCs). Previous researches support the involvement of Rho GTPases and actin microfilaments in the trafficking of Cxs, formation of GJCs plaques, and regulation of channel activity. Nonetheless, it remains uncertain whether distinct types of Cxs HCs and GJCs respond differently to Rho GTPases or changes in actin polymerization/depolymerization dynamics. Our investigation revealed that inhibiting RhoA, a small GTPase that controls actin polymerization, or disrupting actin microfilaments with cytochalasin B (Cyto-B), resulted in reduced GJCs plaque size at appositional membranes and increased transport of HCs to non-appositional plasma membrane regions. Notably, these effects were consistent across different Cx types, since Cx26 and Cx43 exhibited similar responses, despite having distinct trafficking routes to the plasma membrane. Functional assessments showed that RhoA inhibition and actin depolymerization decreased the activity of Cx43 GJCs while significantly increasing HC activity. However, the functional status of GJCs and HCs composed of Cx26 remained unaffected. These results support the hypothesis that RhoA, through its control of the actin cytoskeleton, facilitates the transport of HCs to appositional cell membranes for GJCs formation while simultaneously limiting the positioning of free HCs at non-appositional cell membranes, independently of Cx type. This dynamic regulation promotes intercellular communications and reduces non-selective plasma membrane permeability through a Cx-type dependent mechanism, whereby the activity of Cx43 HCs and GJCs are differentially affected but Cx26 channels remain unchanged.

OBJECTIVE: To investigate the epidemiological characteristics and mutation spectrum of monogenic diseases in Chinese population through a large-scale, multicenter carrier screening.
METHODS: This study was conducted among a total of 33 104 participants (16 610 females) from 12 clinical centers across China.Carrier status for 223 genes was analyzed using high-throughput sequencing and different PCR methods.
RESULTS: The overall combined carrier frequency was 55.58% for 197 autosomal genes and 1.84% for 26 X-linked genes in these participants.Among the 16 669 families, 874 at-risk couples (5.24%) were identified.Specifically, 584 couples (3.50%) were at risk for autosomal genes, 306(1.84%) for X-linked genes, and 16 for both autosomal and X-linked genes.The most frequently detected autosomal at-risk genes included GJB2(autosomal recessive deafness type 1A, 393 couples), HBA1/HBA2(α-thalassemia, 36 couples), PAH (phenylketonuria, 14 couples), and SMN1(spinal muscular atrophy, 14 couples).The most frequently detected X-linked at-risk genes were G6PD (G6PD deficiency, 236 couples), DMD (Duchenne muscular dystrophy, 23 couples), and FMR1(fragile X syndrome, 17 couples).After excluding GJB2 c.109G>A, the detection rate of at-risk couples was 3.91%(651/16 669), which was lowered to 1.72%(287/16 669) after further excluding G6PD.The theoretical incidence rate of severe monogenic birth defects was approximately 4.35‰(72.5/16 669).Screening for a battery of the top 22 most frequent genes in the at-risk couples could detect over 95% of at-risk couples, while screening for the top 54 genes further increased the detection rate to over 99%.
CONCLUSIONS: This study reveals the carrier frequencies of 223 monogenic genetic disorders in the Chinese population and provides evidence for carrier screening strategy development and panel design tailored to the Chinese population.In carrier testing, genetic counseling for specific genes or gene variants can be challenging, and the couples need to be informed of these difficulties before testing and provided with options for not screening these genes or gene variants.

OBJECTIVE: To determine the spectrum and frequency of disease-causing variants in patients with non-syndromic hearing loss (NSHL) and to investigate the diagnostic yield of the applied genetic methods.
METHODS: The study enrolled 306 unrelated patients with childhood-onset, mild-to-profound NSHL referred to Children\'s Hospital Zagreb for genetic testing between March 2006 and October 2023. The GJB2 variants were analyzed with the multiplex ligation-dependent probe amplification method and Sanger sequencing of the coding region of the GJB2 gene. In 21 patients negative for GJB2 biallelic variants, clinical exome sequencing (CES) was performed.
RESULTS: Among 234 disease-associated GJB2 alleles detected, 19 were clinically relevant, of which 18 were reported as pathogenic/likely pathogenic. The c.35delG variant accounted for 73.5% of the mutated alleles. More than half of the patients with biallelic GJB2 variants (64/110, 58.2%) were 35delG homozygotes. Seventeen non-GJB2 variants were found in 10 genes (TECTA, NOG, SLC26A4, PCDH15, TMPRSS3, USH2A, GATA3, MYO15A, SOX10, COL2A1) in 11 participants, and 5 variants (in TECTA, NOG, PCDH15, and SOX10) were novel (29.4%).
CONCLUSIONS: We were able to elucidate the genetic cause of hearing loss in 121 patients, with an overall diagnostic rate of 39.5%. The c.35delG was the most common variant. CES allowed us to diagnose almost half of the patients with HL; to distinguish NSHL from the syndromic form of HL in cases where the phenotype was unclear or where symptoms were absent from an early age; and to discover novel variants.

BACKGROUND: Hereditary hearing loss is a rare hereditary condition that has a significant presence in consanguineous populations. Despite its prevalence, hearing loss is marked by substantial genetic diversity, which poses challenges for diagnosis and screening, particularly in cases with no clear family history or when the impact of the genetic variant requires functional analysis, such as in the case of missense mutations and UTR variants. The advent of next-generation sequencing (NGS) has transformed the identification of genes and variants linked to various conditions, including hearing loss. However, there remains a high proportion of undiagnosed patients, attributable to various factors, including limitations in sequencing coverage and gaps in our knowledge of the entire genome, among other factors. In this study, our objective was to comprehensively identify the spectrum of genes and variants associated with hearing loss in a cohort of 106 affected individuals from the UAE.
RESULTS: In this study, we investigated 106 sporadic cases of hearing impairment and performed genetic analyses to identify causative mutations. Screening of the GJB2 gene in these cases revealed its involvement in 24 affected individuals, with specific mutations identified. For individuals without GJB2 mutations, whole exome sequencing (WES) was conducted. WES revealed 33 genetic variants, including 6 homozygous and 27 heterozygous DNA changes, two of which were previously implicated in hearing loss, while 25 variants were novel. We also observed multiple potential pathogenic heterozygous variants across different genes in some cases. Notably, a significant proportion of cases remained without potential pathogenic variants.
CONCLUSIONS: Our findings confirm the complex genetic landscape of hearing loss and the limitations of WES in achieving a 100% diagnostic rate, especially in conditions characterized by genetic heterogeneity. These results contribute to our understanding of the genetic basis of hearing loss and emphasize the need for further research and comprehensive genetic analyses to elucidate the underlying causes of this condition.

UNASSIGNED: To explore the functional consequences of two common variants, p.V37I and c.299-300delAT in hearing loss associated gene GJB2.
UNASSIGNED: Connexin 26 expression and gap junctional permeability were studied in HEK 293T cells transfected with plasmids expressing GJB2 wild-type, p.V37I, or c.299-300delAT CX26 proteins with fluorescent tags. Functional analyses of different GJB2 haplotypes were performed to fully assess the alteration of ionic and small-molecule coupling.
UNASSIGNED: The p.V37I protein was localized at the plasma membrane, but failed to effectively transport intercellular propidium iodide or Ca2+ efficiently, indicating impairment of both biochemical and ionic coupling. The presence of GJB2 p.V37I appeared to increase the sensitivity of cells to H2O2 treatment. In contrast, the known variant c.299-300delAT protein was not transported to the cell membrane and could not form gap junctions, instead being confined to the cytoplasm. Ionic and biochemical coupling was defective in c.299-300delAT-transfected cells.
UNASSIGNED: The p.V37I and c.299-300delAT GJB2 mutations resulted in deficient gap junction-mediated coupling. Environmental factors may impact the functional consequences of GJB2 p.V37I. These results may inspire the development of molecular therapies targeting GJB2 mutations for hearing loss.

Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO2. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E mutation pushes a conformational equilibrium towards the channel having a constricted pore entrance, similar to effects seen on raising the partial pressure of CO2. In previous structures of connexins, the cytoplasmic loop, important in regulation and where K125 is located, is disordered. Through further cryo-EM studies we trap distinct states of Cx26 and observe density for the cytoplasmic loop. The interplay between the position of this loop, the conformations of the transmembrane helices and the position of the N-terminal helix, which controls the aperture to the pore, provides a mechanism for regulation.

Sensory systems experience a period of intrinsically generated neural activity before maturation is complete and sensory transduction occurs. Here we review evidence describing the mechanisms and functions of this \'spontaneous\' activity in the auditory system. Both ex vivo and in vivo studies indicate that this correlated activity is initiated by non-sensory supporting cells within the developing cochlea, which induce depolarization and burst firing of groups of nearby hair cells in the sensory epithelium, activity that is conveyed to auditory neurons that will later process similar sound features. This stereotyped neural burst firing promotes cellular maturation, synaptic refinement, acoustic sensitivity, and establishment of sound-responsive domains in the brain. While sensitive to perturbation, the developing auditory system exhibits remarkable homeostatic mechanisms to preserve periodic burst firing in deaf mice. Preservation of this early spontaneous activity in the context of deafness may enhance the efficacy of later interventions to restore hearing.

BACKGROUND: Hearing loss (HL) is a common sensory impairment worldwide, with genetic and environmental factors contributing to its occurrence. Next Generation Sequencing (NGS) plays a crucial role in identifying the genetic factors involved in this heterogeneous disorder.
RESULTS: In this study, a total of 9 unrelated Iranian families, each having at least one affected individual who tested negative for mutations in GJB2, underwent screening using whole exome sequencing (WES). The pathogenicity and novelty of the identified variant was checked using various databases. Co-segregation study was also performed to confirm the presence of the candidate variants in parents. Plus, The pathogenicity of the detected variant was assessed through in silico analysis using a number of mutation prediction software tools. Among the 9 investigated families, hearing loss-causing genes were identified in 6 families. the mutations were observed in USH2A, CLRN1, BSND, SLC26A4, and MITF, with two of the identified mutations being novel.
CONCLUSIONS: Discovering additional variants and broadening the range of mutations associated with hearing impairment has the potential to enhance the diagnostic effectiveness of molecular testing in patient screening, and can also lead to improved counseling aimed at reducing the risk of affected offspring for high-risk couples.

BACKGROUND: Recessive GJB2 variants, the most common genetic cause of hearing loss, may contribute to progressive sensorineural hearing loss (SNHL). The aim of this study is to build a realistic predictive model for GJB2-related SNHL using machine learning to enable personalized medical planning for timely intervention.
METHODS: Patients with SNHL with confirmed biallelic GJB2 variants in a nationwide cohort between 2005 and 2022 were included. Different data preprocessing protocols and computational algorithms were combined to construct a prediction model. We randomly divided the dataset into training, validation, and test sets at a ratio of 72:8:20, and repeated this process ten times to obtain an average result. The performance of the models was evaluated using the mean absolute error (MAE), which refers to the discrepancy between the predicted and actual hearing thresholds.
RESULTS: We enrolled 449 patients with 2184 audiograms available for deep learning analysis. SNHL progression was identified in all models and was independent of age, sex, and genotype. The average hearing progression rate was 0.61 dB HL per year. The best MAE for linear regression, multilayer perceptron, long short-term memory, and attention model were 4.42, 4.38, 4.34, and 4.76 dB HL, respectively. The long short-term memory model performed best with an average MAE of 4.34 dB HL and acceptable accuracy for up to 4 years.
CONCLUSIONS: We have developed a prognostic model that uses machine learning to approximate realistic hearing progression in GJB2-related SNHL, allowing for the design of individualized medical plans, such as recommending the optimal follow-up interval for this population.

BACKGROUND: Mutations in the GJB2 gene, which encodes the protein connexin 26 and is involved in inner ear homeostasis, are identified in approximately 50% of patients with autosomal recessive nonsyndromic hearing loss, making it one of the primary causes of prelingual nonsyndromic hearing loss in various populations. The 35delG mutation, one of the most common mutations of the GJB2 gene, usually causes prelingual, bilateral mild to profound, nonprogressive sensorineural hearing loss.
METHODS: We present an unusual case of an 18-year-old Turkish female with heterozygous 35delG mutation and postlingual, profound-sloping, progressive and fluctuating unilateral sensorineural hearing loss. The phenotype is different from the usual findings.
CONCLUSIONS: The 35delG mutation causing hearing loss may not always be reflected in the phenotype as expected and therefore may have different audiologic manifestations.