Genetic findings of Sanger and nanopore single-molecule sequencing in patients with X-linked hearing loss and incomplete partition type III.

BACKGROUND: POU3F4 is the causative gene for X-linked deafness-2 (DFNX2), characterized by incomplete partition type III (IP-III) malformation of the inner ear. The purpose of this study was to investigate the clinical characteristics and molecular findings in IP-III patients by Sanger or nanopore single-molecule sequencing. METHODS: Diagnosis of IP-III was mainly based on clinical characteristics including radiological and audiological findings. Sanger sequencing of POU3F4 was carried out for these IP-III patients. For those patients with negative results for POU3F4 Sanger sequencing, nanopore long-read single-molecule sequencing was used to identify the possible pathogenic variants. Hearing intervention outcomes of hearing aids (HAs) fitting and cochlear implantation (CI) were also analyzed. Aided pure tone average (PTA) was further compared between two groups of patients according to their different locations of POU3F4 variants: in the exon region or in the upstream region. RESULTS: In total, 18 male patients from 14 unrelated families were diagnosed with IP-III. 10 variants were identified in POU3F4 by Sanger sequencing and 6 of these were reported for the first time (p.Gln181*, p.Val215Gly, p.Arg282Gln, p.Gln316*, c.903_912 delins TGCCA and p.Arg205del). Four different deletions that varied from 80 to 486 kb were identified 876-1503 kb upstream of POU3F4 by nanopore long-read single-molecule sequencing. De novo genetic mutations occurred in 21.4% (3/14) of patients with POU3F4 mutations. Among these 18 patients, 7 had bilateral HAs and 10 patients received unilateral CI. The mean aided PTA for HAs and CI users were 41.1 ± 5.18 and 40.3 ± 7.59 dB HL respectively. The mean PTAs for patients with the variants located in the exon and upstream regions were 39.6 ± 6.31 versus 43.0 ± 7.10 dB HL, which presented no significant difference (p = 0.342). CONCLUSIONS: Among 14 unrelated IP-III patients, 28.6% (4/14) had no definite mutation in exon region of POU3F4. However, possible pathogenic deletions were identified in upstream region of this gene. De novo genetic mutations occurred in 21.4% (3/14) of patients with POU3F4 mutation. There was no significant difference of hearing intervention outcomes between the IP-III patients with variants located in the exon region and in the upstream region.

Notch signaling is active in normal mouse middle ear epithelial cells.

Mucous cell metaplasia/hyperplasia in the middle ear epithelium is associated with the occurrence of otitis media with effusion during infections. However, the mechanism by which Notch signaling regulates cell fate in the middle ear epithelium is unclear. The aim of the present study was to elucidate this mechanism by investigating the localization of Notch receptors, such as Notch1 and Notch2, and Notch ligands, such as Jagged1, in the normal mouse middle ear epithelium (NMMEE) using immunofluorescence. Furthermore, the mRNA expression levels of Notch receptors and ligands were evaluated using reverse transcription polymerase chain reaction (PCR). The effects of the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine tert-butyl ester (DAPT) on epithelial cell proliferation were determined using 5-ethynyl-2'-deoxyuridine (EdU) staining and immunofluorescence staining of the apoptosis marker caspase-3 and the epithelial proliferation marker pan-cytokeratine. In addition, the differentiation of the NMMEE cells was characterized by evaluating the mRNA expression levels of the mucous cell-associated genes Arg2, Muc2, Spdef, Spink4 and Tff1 using quantitative PCR. Notch1, Notch2 and Jagged1 were observed to be co-localized throughout the mouse middle ear epithelium. Furthermore, Notch1-4, Jagged1, Jagged2, Dll1 and Dll4 mRNAs were expressed in the NMMEE cells. The inhibition of Notch by DAPT resulted in fewer EdU-positive cells and the upregulation of the expression levels of various mucous cell-associated genes. The results indicate that DAPT suppresses the proliferation of NMMEE cells while promoting their differentiation into mucous cells. Therefore, DAPT may provide a specific therapeutic strategy for the reversal of multiple pathological processes that are associated with epithelium thickening in the middle ear.

Comparative Study on the Roles of the Number of Accelerations and Rotation Angle in the Treatment Maneuvers for Posterior Semicircular Canal Benign Paroxysmal Positional Vertigo.

AIMS: This study aims to investigate the roles of the number of accelerations and rotation angle in the treatment of posterior semicircular canal benign paroxysmal positional vertigo (PC-BPPV). METHODS: We enrolled 344 patients with unilateral PC-BPPV. Of these, 167 patients in the simple-step maneuver (SSM) group were accelerated twice and rotated 120° per step, whereas 177 patients in the multi-step maneuver (MSM) group were accelerated 4 times and rotated 60° per step. Dix-Hallpike (DH) tests were performed to categorize the treatment outcome as follows: 'symptom free' if the result was negative, 'symptom persistent' if the result remained positive after performing the maneuver 3 times or 'canal conversion' if horizontal nystagmus was evoked. RESULTS: Of the patients in the SSM and MSM groups, 78.4 and 91.5% became symptom free, respectively, while canal conversion occurred in 13.8 and 5.1%, respectively (p = 0.003, χ(2) test). The success rate after performing the maneuver once was 57.1% in the MSM and 32.3% in the SSM symptom-free patients (p = 0.001, χ(2) test). One month after the treatment, 22.0 and 9.6% of the SSM and MSM patients had symptom relapse, respectively (p = 0.007, χ(2) test). CONCLUSIONS: More accelerations and a smaller rotation angle improved the effectiveness and efficiency of the repositioning maneuvers and reduced canal conversion.

Atoh1 expression levels define the fate of rat cochlear nonsensory epithelial cells in vitro.

Atonal homolog 1 (Atoh1) is a basic helix­loop­helix transcription factor that is essential for inner ear hair cell differentiation. Previous studies have reported that Atoh1 gene transfer induces the production of ectopic hair cell­like cells (EHCLCs). In the present study, the effect of different Atoh1 expression levels and the duration of EHCLC formation on the lesser epithelial ridge (LER) of cochleae was examined using a human adenovirus serotype 5 (Ad5) vector encoding atoh1 and the reporter gene EGFP. Different Ad5­EGFP­atoh1/Ad5­EGFP virus titers were added to cultured cochlear explants and EHCLCs were detected in the LER at various time points. The results demonstrated that GFP alone did not induce EHCLCs. By contrast, Atoh1 expression induced EHCLCs as early as 2.5­5 days following EGFP­atoh1 infection in the LER and depending upon the viral titer, the number of EHCLCs increased with time. Higher Ad5­EGFP­atoh1 titers induced enhanced Atoh1 expression, resulting in an increase in EHCLCs. Lower Ad5­EGFP­atoh1 titers required more time for EHCLC formation and very low titers of Ad5­EGFP­atoh1 induced only weak Atoh1 expression and did not trigger EHCLC formation. In conclusion, the present study utilized an appropriate Ad5­EGFP­atoh1 titer range to induce Atoh1 expression and the subsequent production of EHCLCs. The results revealed that the Atoh1 expression level defined the fate of LER cells as either EHCLCs or nonsensory epithelial cells. This evidence may provide an important guideline for future studies into gene therapy strategies for the treatment of deafness.