PAX3 mutation suppress otic progenitors proliferation and induce apoptosis by inhibiting WNT1/ß-catenin signaling pathway in WS1 patient iPSC-derived inner ear organoids.

Waardenburg syndrome type 1 (WS1) is a hereditary disease mainly characterized by sensorineural hearing loss, dystopia canthorum, and pigmentary defects. To elucidate molecular mechanisms underlying PAX3-associated hearing loss, we developed inner ear organoids model using induced pluripotent stem cells (iPSCs) derived from WS1 patient and healthy individual. Our results revealed a significant reduction in the size of inner ear organoids, accompanied by an increased level of apoptosis in organoids derived from WS1 patient-iPSCs carrying PAX3 c.214A > G. Transcriptome profiling analysis by RNA-seq indicated that inner ear organoids from WS1 patients were associated with suppression of inner ear development and WNT signaling pathway. Furthermore, the upregulation of the WNT1/ß-catenin pathway which was achieved through the correction of PAX3 isogenic mutant iPSCs using CRISPR/Cas9, contributed to an increased size of inner ear organoids and a reduction in apoptosis. Together, our results provide insight into the underlying mechanisms of hearing loss in WS.

Low-intensity noise exposure takes an essential part in the mechanism of late-onset hereditary hearing loss caused by Abcc1 mutation.

With the development of the social economy, we are exposed to increasing noise in our daily lives. Our previous work found an ABCC1(NM_004996.3:c.A1769G, NP_004987.2:p.N590S) variant which cosegregated with the patients in an autosomal dominant non-syndromic hearing loss family. At present, the specific mechanism of deafness caused by ABCC1 mutation is still not clear. Using the knock-in mouse model simulating human ABCC1 mutation, we found that the occurrence of family-related phenotypes was likely attributed to the combination of the mouse genotype and low-intensity noise. GSH and GSSG are important physiological substrates of ABCC1. The destruction of GSH-GSSG balance in the cochleae of both Abcc1N591S/+ mice and Abcc1N591S/N591S mice during low-intensity noise exposure may result in irreversible damage to the hair cells of the cochleae, consequently leading to hearing loss in mice. The findings offered a potential novel idea for the prevention and management of hereditary hearing loss within this family.

A nonsense TMEM43 variant leads to disruption of connexin-linked function and autosomal dominant auditory neuropathy spectrum disorder.

Genes that are primarily expressed in cochlear glia-like supporting cells (GLSs) have not been clearly associated with progressive deafness. Herein, we present a deafness locus mapped to chromosome 3p25.1 and an auditory neuropathy spectrum disorder (ANSD) gene, TMEM43, mainly expressed in GLSs. We identify p.(Arg372Ter) of TMEM43 by linkage analysis and exome sequencing in two large Asian families segregating ANSD, which is characterized by inability to discriminate speech despite preserved sensitivity to sound. The knock-in mouse with the p.(Arg372Ter) variant recapitulates a progressive hearing loss with histological abnormalities in GLSs. Mechanistically, TMEM43 interacts with the Connexin26 and Connexin30 gap junction channels, disrupting the passive conductance current in GLSs in a dominant-negative fashion when the p.(Arg372Ter) variant is introduced. Based on these mechanistic insights, cochlear implant was performed on three subjects, and speech discrimination was successfully restored. Our study highlights a pathological role of cochlear GLSs by identifying a deafness gene and its causal relationship with ANSD.

Research and application progress in deep learning in otology. / æ·±åº¦å­¦ä¹ åœ¨è€³ç§‘å­¦ä¸­çš„ç ”ç©¶ä¸Žåº”ç”¨è¿›å±•.

With the optimization of deep learning algorithms and the accumulation of medical big data, deep learning technology has been widely applied in research in various fields of otology in recent years. At present, research on deep learning in otology is combined with a variety of data such as endoscopy, temporal bone images, audiograms, and intraoperative images, which involves diagnosis of otologic diseases (including auricular malformations, external auditory canal diseases, middle ear diseases, and inner ear diseases), treatment (guiding medication and surgical planning), and prognosis prediction (involving hearing regression and speech learning). According to the type of data and the purpose of the study (disease diagnosis, treatment and prognosis), the different neural network models can be used to take advantage of their algorithms, and the deep learning can be a good aid in treating otologic diseases. The deep learning has a good applicable prospect in the clinical diagnosis and treatment of otologic diseases, which can play a certain role in promoting the development of deep learning combined with intelligent medicine.

Clinical characteristics and surgical strategy of glomus tympanicum tumors. / 鼓室体瘤的临床特征及手术策略.

OBJECTIVES: To summarize the clinical characteristics of glomus tympanicum tumors, and to explore the surgical methods and the strategy for auditory protection. METHODS: Ten cases (ears) of glomus tympanicum tumors were collected from the Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital, Central South University from August 2014 to February 2022. All patients underwent endoscopic or microscopic surgery to achieve total removal of the tumor, followed up for 3 months to 8 years. We summarized and analyzed its clinical characteristics, compared the preoperative and postoperative hearing levels of patients, and made a retrospective summary of the surgical methods and the strategy for auditory protection. RESULTS: Ten patients were all female at (49.50±8.00) years old. Their medical history ranged from 15 days to 6 years. Seven patients complained of pulsatile tinnitus, and 80% (8/10) of the affected ears suffered different degrees of hearing loss. According to the modified Fisch & Mattox classification of glomus tympanicum tumors, 3 ears (30%) of 10 ears were A1, 2 ears (20%) were A2 and 5 ears (50%) were B1. In all 10 cases (ears), hearing was improved in 3 cases, bone gas conductance was maintained in 6 cases, and hearing was slightly decreased in 1 case. The difference of bone gas conductance was 0-10 dB in 7 cases (ears) after operation, and 10-20 dB in 3 cases (ears). There was no significant difference in the average air conduction hearing threshold, bone conduction hearing threshold and air-bone conduction difference between before and after operation (all P>0.05). All cases had no postoperative complications, and the external auditory canal and the incision behind the ear healed well. There was no recurrence after follow-up. CONCLUSIONS: Glomus tympanicum tumor is easy to bleed, so it is a challenge for total tumor resection and hearing function protection during operation. For type A and type B1 tumors, they can be completely removed under the condition of keeping the tympanic membrane and the ossicular chain. At the same time, the postoperative hearing function can be preserved, and even the hearing can be improved.

Genetic insights, disease mechanisms, and biological therapeutics for Waardenburg syndrome.

Waardenburg syndrome (WS), also known as auditory-pigmentary syndrome, is the most common cause of syndromic hearing loss (HL), which accounts for approximately 2-5% of all patients with congenital hearing loss. WS is classified into four subtypes depending on the clinical phenotypes. Currently, pathogenic mutations of PAX3, MITF, SOX10, EDN3, EDNRB or SNAI2 are associated with different subtypes of WS. Although supportive techniques like hearing aids, cochlear implants, or other assistive listening devices can alleviate the HL symptom, there is no cure for WS to date. Recently major progress has been achieved in preclinical studies of genetic HL in animal models, including gene delivery and stem cell replacement therapies. This review focuses on the current understandings of pathogenic mechanisms and potential biological therapeutic approaches for HL in WS, providing strategies and directions for implementing WS biological therapies, as well as possible problems to be faced, in the future.

Establishment of a risk index for early complications after trans-canal endoscopic ear surgery.

PURPOSE: This study aimed to determine the risk factors associated with early postoperative complications of trans-canal endoscopic ear surgery (TEES), then to develop a risk index. MATERIALS AND METHODS: This single-institution retrospective study reviewed TEESs from January 1, 2017, to December 31, 2019 in a tertiary hospital. In the derivation cohort, univariable and multivariable logistic regression were performed to identify factors significantly associated with early postoperative complications of TEES. Then these parameters were integrated into a trans-canal endoscopic ear surgery risk index (TEESRI). The performance of TEESRI was compared with that of the American Society of Anesthesiologists (ASA) classification using the validation cohort. RESULTS: 932 TEESs were enrolled in total and 151 (16.2%) developed early postoperative complications. In the derivation set, 8 factors including state of the opposite ear and presence of nasal or pharyngeal diseases were found to be independently associated with the occurrence of early postoperative complications on multivariable regression analysis [area under the curve (AUC), 0.806; 95% confidence interval (CI), 0.765-0.848]. Using the validation cohort, the AUC of the TEESRI was 0.776 [95%CI, 0.711-0.842], with a sensitivity of 82.2% and specificity of 65.5%, while the AUC of the ASA classification was 0.512 (95%CI, 0.421-0.603). The TEESRI outperformed the ASA classification when evaluating the risk for early postoperative complications of TEES. CONCLUSIONS: Based on the 8 risk factors, the TEESRI was established with satisfactory predicting capacity. Surgeons should pay extra attention to the risk factors in the TEESRI, when treating patients.

Elmod3 knockout leads to progressive hearing loss and abnormalities in cochlear hair cell stereocilia.

ELMOD3, an ARL2 GTPase-activating protein, is implicated in causing hearing impairment in humans. However, the specific role of ELMOD3 in auditory function is still far from being elucidated. In the present study, we used the CRISPR/Cas9 technology to establish an Elmod3 knockout mice line in the C57BL/6 background (hereinafter referred to as Elmod3-/- mice) and investigated the role of Elmod3 in the cochlea and auditory function. Elmod3-/- mice started to exhibit hearing loss from 2 months of age, and the deafness progressed with aging, while the vestibular function of Elmod3-/- mice was normal. We also observed that Elmod3-/- mice showed thinning and receding hair cells in the organ of Corti and much lower expression of F-actin cytoskeleton in the cochlea compared with wild-type mice. The deafness associated with the mutation may be caused by cochlear hair cells dysfunction, which manifests with shortening and fusion of inner hair cells stereocilia and progressive degeneration of outer hair cells stereocilia. Our finding associates Elmod3 deficiencies with stereocilia dysmorphologies and reveals that they might play roles in the actin cytoskeleton dynamics in cochlear hair cells, and thus relate to hearing impairment.

Otological manifestations in branchiootorenal spectrum disorder: A systematic review and meta-analysis.

Branchiootorenal spectrum disorder (BORSD) is a group of rare autosomal dominant entities characterized by branchiogenic malformations, hearing loss (HL) and renal anomalies. It comprises branchiootorenal syndrome and branchiootic syndrome, distinguished by the presence or absence of renal abnormalities. Pathogenic variants have been discovered in the following genes: EYA1, SIX5, SIX1 and SALL1. As the otological phenotype in BORSD is inconsistently reported, we performed a systematic review to provide an up-to-date overview, correlated with the genotype. Forty publications were included, describing 295 individual patients. HL was diagnosed in 95%, usually bilateral and mixed-type, and differed among the different genes involved. Mixed moderate-to-severe HL was the predominant finding in patients with EYA1 involvement, regardless of the presence of renal abnormalities. The sensorineural HL of profound severity was more prevalent in patients with SIX1 mutations. No significant differences among different mutation types or location within the genes could be observed. Structural otological manifestations, ranging from periauricular to inner ear anomalies, were common in both genes. Especially periauricular anomalies were more common and more severe in EYA1. In summary, otological differences among the different genes involved in BORSD are observed, so the molecular analysis is strongly advised.

Identification and functional analysis of a novel missense mutation of PAX3 associated with Waardenburg syndrome type I.

PURPOSE: Waardenburg syndrome type 1 (WS1) is a rare genetic disorder characterized by dystopia canthorum, abnormal iris pigmentation, and congenital hearing loss with variable penetrance.WS1 is caused by mutations in paired box gene 3 (PAX3). The current study aimed to investigate the genetic cause of hearing loss in a four-generation Chinese WS1 family. METHODS: The phenotype of the study family was characterized using clinical evaluation and pedigree analysis. Target region high-throughput sequencing system was designed to screen the all coding exons and flanking intronic sequences of the six WS-associated genes. Sanger sequencing was used to identify the causative nucleotide changes and perform the co-segregating analysis. The expression, subcellular distribution, and transcriptional activity of the mutant PAX3 protein were analysis to reveal the functional consequences of the mutation. RESULTS: Based on diagnostic criteria, the proband of this pedigree classified as WS1. We identified a novel missense mutation (c.117 C > A, p. Asn39Lys) in exon 2 of the PAX3 gene. In vitro, the Asn39Lys PAX3 retained nuclear distribution ability. However, it failed to activate the melanocyte inducing transcription factor (MITF) promoter and impaired the function of WT PAX3. CONCLUSIONS: Our study reports a novel missense PAX3 mutation in a Chinese family and shows haploinsufficiency may be the underlying mechanism for the WS1 phenotype.

[New deafness gene: Progress of research on ABCC1 in biological barriers].

ABCC1 gene is expressed in various tissues and organs of the human body, and can transport substrates including drugs, heavy metals, toxic substances and organic anions. Previous research on ABCC1 gene has mostly focused on tumor multidrug resistance. Recently, ABCC1 has been proposed as a candidate gene for hereditary hearing impairment, which has attracted much attention. ABCC1-associated deafness may be related to its role in biological barriers. This article has summarized recent progress in the study of the role of ABCC1 in the blood-testis barrier, placental barrier, blood-brain barrier, blood-labyrinth barrier, which may provide insight into its biological functions.

Next-generation sequencing-based mutation analysis of genes associated with enlarged vestibular aqueduct in Chinese families.

OBJECTIVES: The identification of gene mutations enables more appropriate genetic counseling and proper medical management for EVA patients. The purpose of this study was to validate the accuracy and sensitivity of our method for comprehensive mutation detection in EVA, and summarize these data to explore a more accurate and convenient genetic diagnosis method. METHODS: A multiplex PCR sequencing panel was designed to capture the exons of three known EVA-associated genes (SLC26A4, KCNJ10, and FOXI1), and NGS was conducted in 17 Chinese families with EVA. RESULTS: A total of 16 SLC26A4 variants were found in 21 probands with bilateral EVA, including three novel variants (c.416G>A, c.823G>A and c.1027G>C), which were not reported in the dbSNP, gnomAD database, and ClinVar databases. One patient carried a FOXI1 variant (heterozygous, c.214C>A) and one patient carried a KCNJ10 variant (heterozygous, c.1054C>A), both of which were novel variants. Biallelic potential pathogenic variants were detected in 21/21patient samples, leading to a purported diagnostic rate of 100%. All results were verified by Sanger sequencing. CONCLUSION: This result supplemented the mutation spectrum of EVA, and supports that combined multiple PCR-targeted enrichment, and NGS is a valuable molecular diagnostic tool for EVA, and is suitable for clinical application.

Extrusion pump ABCC1 was first linked with nonsyndromic hearing loss in humans by stepwise genetic analysis.

PURPOSE: To determine the genetic etiology of deafness in a family (HN-SD01) with autosomal dominant nonsyndromic hearing loss (NSHL). METHODS: Stepwise genetic analysis was performed on family HN-SD01, including hotspot variant screening, exome sequencing, virtual hearing loss gene panel, and genome-wide linkage analysis. Targeted region sequencing was used to screen ABCC1 in additional cases. Cochlear expression of Abcc1 was evaluated by messenger RNA (mRNA) and protein levels. Computational prediction, immunofluorescence, real-time quantitative polymerase chain reaction, and flow cytometry were conducted to uncover functional consequences of candidate variants. RESULTS: Stepwise genetic analysis identified a heterozygous missense variant, ABCC1:c.1769A>G (p.Asn590Ser), cosegregating with phenotype in HN-SD01. Screening of ABCC1 in an additional 217 cases identified candidate pathogenic variants c.692G>A (p.Gly231Asp) in a sporadic case and c.887A>T (p.Glu296Val) in a familial proband. Abcc1 expressed in stria vascularis and auditory nerve of mouse cochlea. Immunofluorescence showed p.Asn590Ser distributed in cytomembrane and cytoplasm, while wild type was shown only in cytomembrane. Besides, it generated unstable mRNA and decreased efflux capacity of ABCC1. CONCLUSION: Stepwise genetic analysis is efficient to analyze the genetic etiology of NSHL. Variants in ABCC1 are linked with NSHL and suggest an important role of extruding pumps in maintaining cochlea function.

New Genotypes and Phenotypes in Patients with 3 Subtypes of Waardenburg Syndrome Identified by Diagnostic Next-Generation Sequencing.

Background: Waardenburg syndrome (WS) is one of the most common forms of syndromic deafness with heterogeneity of loci and alleles and variable expressivity of clinical features. Methods: The technology of single-nucleotide variants (SNV) and copy number variation (CNV) detection was developed to investigate the genotype spectrum of WS in a Chinese population. Results: Ninety WS patients and 24 additional family members were recruited for the study. Fourteen mutations had not been previously reported, including c.808C>G, c.117C>A, c.152T>G, c.803G>T, c.793-3T >G, and c.801delT on PAX3; c.642_650delAAG on MITF; c.122G>T and c.127C>T on SOX10; c.230C>G and c.365C>T on SNAI2; and c.481A>G, c.1018C>G, and c.1015C>T on EDNRB. Three CNVs were de novo and first reported in our study. Five EDNRB variants were associated with WS type 1 in the heterozygous state for the first time, with a detection rate of 22.2%. Freckles occur only in WS type 2. Yellow hair, amblyopia, congenital ptosis, narrow palpebral fissures, and pigmentation spots are rare and unique symptoms in WS patients from China. Conclusions: EDNRB should be considered as another prevalent pathogenic gene in WS type 1. Our study expanded the genotype and phenotype spectrum of WS, and diagnostic next-generation sequencing is promising for WS.

[Giant cell reparative granuloma of the temporal bone: A case report].

Giant cell reparative granuloma (GCRG) is a type of non-neoplastic lesion that can be rarely found in clinical practices. Due to the lack of specificity in symptoms, signs and auxiliary examinations, it is likely to be misdiagnosed, and thereby affecting the treatment and prognosis. In July 2018, a GCRG patient who was described with "4 years of hearing loss in the left ear, accompanied by 2 months of preauricular swelling" as the first symptom was admitted in our hospital. Both the HRCT and MRI scans for the temporal bone suggested the presence of tumor at the left lateral skull base, but the nature still needed further examination. Intraoperatively, the tumor was completely removed and repaired locally. Pathological examination confirmed the symptoms as GCRG. Immunohistochemistry showed the expression of CD68 and CD163 in the tumor cells. Postoperatively, the patient recovered well without complications, and had the stitches removed before being discharged on schedule.

ELMOD3, a novel causative gene, associated with human autosomal dominant nonsyndromic and progressive hearing loss.

Autosomal dominant nonsyndromic hearing loss (ADNSHL) is a highly genetically heterogeneous disorder. Up to date only approximately 37 ADNSHL-causing genes have been identified. The goal of this study was to determine the causative gene in a five-generation Chinese family with ADNSHL. A Chinese family was ascertained. Simultaneously, two affected individuals and one normal hearing control from the family were analyzed by whole exome capture sequencing. To assess the functional effect of the identified variant, in-vitro studies were performed. novel missense variant, c.512A>G (p.His171Arg) in exon 8 of the ELMO domain-containing 3 (ELMOD3) gene, was identified as a causative variant in this family affected by late-onset and progressive ADNSHL. The variant was validated by Sanger sequencing and found to co-segregate with the phenotype within the pedigree and was absent in 500 ethnically matched unrelated normal hearing control subjects. To our knowledge, this is the first report of a family with ADNSHL caused by ELMOD3 mutation. Western blots and immunofluorescence staining demonstrated that p.His171Arg resulted in abnormal expression levels of ELMOD3 and abnormal subcellular localization. Furthermore, the analysis of the stability of the wild-type (WT) and mutant ELMOD3 protein shows that the decay of p.His171Arg is faster than that of the WT, suggesting a shorter halflife of the c.512A > G variant. A novel variant in the ELMOD3 gene, encoding a member of the engulfment and cell motility (ELMO) family of GTPase-activating proteins, was identified for the first time as responsible for ADNSHL.

Wnt signaling pathway involvement in genotypic and phenotypic variations in Waardenburg syndrome type 2 with MITF mutations.

Mutation in the gene encoding microphthalmia-associated transcription factor (MITF) lead to Waardenburg syndrome 2 (WS2), an autosomal dominantly inherited syndrome with auditory-pigmentary abnormalities, which is clinically and genetically heterogeneous. Haploinsufficiency may be the underlying mechanism for WS2. However, the mechanisms explaining the genotypic and phenotypic variations in WS2 caused by MITF mutations are unclear. A previous study revealed that MITF interacts with LEF-1, an important factor in the Wnt signaling pathway, to regulate its own transcription through LEF-1-binding sites on the MITF promoter. In this study, four different WS2-associated MITF mutations (p.R217I, p.R217G, p.R255X, p.R217del) that are associated with highly variable clinical features were chosen. According to the results, LEF-1 can activate the expression of MITF on its own, but MITF proteins inhibited the activation. This inhibition weakens when the dosage of MITF is reduced. Except for p.R217I, p.R255X, p.R217G, and p.R217del lose the ability to activate TYR completely and do not inhibit the LEF-1-mediated activation of the MITF-M promoter, and the haploinsufficiency created by mutant MITF can be overcome; correspondingly, the mutants' associated phenotypes are less severe than that of p.R217I. The dominant negative of p.R217del made it have a second-most severe phenotype. This study's data imply that MITF has a negative feedback loop of regulation to stabilize MITF gene dosage that involves the Wnt signaling pathway and that the interaction of MITF mutants with this pathway drives the genotypic and phenotypic differences observed in Waardenburg syndrome type 2 associated with MITF mutations.

A novel mutation in the SMPX gene associated with X-linked nonsyndromic sensorineural hearing loss in a Chinese family.

X-linked inheritance is very rare and is estimated to account for only 1-5% of all nonsyndromic hearing loss cases. We found a multiplex family from China segregating with X-linked nonsyndromic hearing loss. After exclusive analysis of 10 common variations of three hearing loss-related genes, GJB2, mtDNA12srRNA and SLC26A4, a novel truncated variant of SMPX, c.87dupA (p.Gly30Argfs*12) (NCBI ClinVar Submission ID: SUB3136126), was identified by whole-exome sequencing. This variant was co-segregated with hearing loss in the entire family and was absent in 576 unrelated ethnically and geographically matched controls. We also detected a single nucleotide variation in two male controls with normal hearing, SMPX c.55A>G (p.Asn19Asp), which has been annotated as a rare variant in the Single Nucleotide Polymorphism (dbSNP) (rs759552778) and Exome Aggregation Consortium (ExAC) databases. This study has enriched the mutation spectrum of the SMPX gene.

Multivariate analysis of prognostic factors for idiopathic sudden sensorineural hearing loss treated with adjuvant hyperbaric oxygen therapy.

The objective of this study is to evaluate possible prognostic factors of idiopathic sudden sensorineural hearing loss (ISSNHL) treated with adjuvant hyperbaric oxygen therapy (HBOT) using univariate and multivariate analyses. From January 2008 to October 2016, records of 178 ISSNHL patients treated with auxiliary hyperbaric oxygen therapy were reviewed to assess hearing recovery and evaluate associated prognostic factors (gender, age, localization, initial hearing threshold, presence of tinnitus, vertigo, ear fullness, hypertension, diabetes, onset of HBOT, number of HBOT, and audiogram), by using univariate and multivariate analyses. The overall recovery rate was 37.1%, including complete recovery (19.7%) and partial recovery (17.4%). According to multivariate analysis, later onset of HBOT and higher initial hearing threshold were associated with a poor prognosis in ISSNHL patients treated with HBOT. HBOT is a safe and beneficial adjuvant therapy for ISSNHL patients. 20 sessions of HBOT is possibly enough to show its therapeutic effect. Earlier HBOT onset and lower initial hearing threshold is associated with favorable hearing recovery.

[Mechanism for synergistic effect of IRF4 and MITF on tyrosinase promoter].

OBJECTIVE: To investigate the mechanism for the synergistic effect of interferon regulatory factor 4 (IRF4) and microphthalmia-associated transcription factor (MITF) on tyrosinase (TYR) promoter.
 Methods: The synergistic transcriptional effect, subcellular localization, and protein-protein interaction for IRF4 and MITF were observed by luciferase assay, immunofluorescence, GST-pull down, and co-immunoprecipitation, respectively.
 Results: IRF4 and MITF proteins were co-expressed in the cell nucleus. IRF4 augmented the transcriptional function of MITF (but not the mutant MITF) to activate the expression of the TYR promoter, but with no effect on other MITF-specific target promoters. IRF4 alone did not affect TYR promoter significantly. No direct interaction between the two proteins was noted.
 Conclusion: IRF4 and MITF exert a specifically synergistic effect on activation of TYR promoter through IRF4-mediated upregulation of transcriptional function of MITF. This synergistic effect is mainly regulated by MITF; DNA might be involved in the interaction between the two proteins.