In head and neck squamous cell carcinoma (HNSCC), an immunosuppressive tumor microenvironment (TME) frequently leads to natural killer (NK) cell dysfunction, but the molecular mechanisms underlying this impairment remain unclear. Here, we combined flow cytometry and multiplex immunohistochemistry to evaluate NK cells infiltration and functional status in HNSCC tumors. Bulk RNA-seq datasets from patient cohorts and single-cell RNA-seq data were analyzed to identify transcriptional correlates of NK cells dysfunction. Mechanistic studies in tumor cell lines assessed the role of osteopontin (OPN)-integrin αvβ3/NF-κB signaling in modulating TGF-β1 production and its effects on NK cell activity. NK cells in HNSCC tumors exhibited reduced infiltration and diminished activation marker expression, coinciding with high OPN levels in the TME. Transcriptomic analyses demonstrated a strong inverse correlation between tumor SPP1 (encoding OPN) expression and NK cells abundance. Mechanistically, tumor-derived OPN engaged integrin αvβ3 to activate NF-κB signaling, inducing robust TGF-β1 secretion that suppressed NK cell activation and cytotoxic function. Single-cell RNA-seq analysis further confirmed the exhausted functional states of NK cells within the HNSCC TME. Tumor-derived OPN promotes immune evasion in HNSCC by driving TGF-β1-mediated suppression of NK cell activity. These findings highlight the OPN-TGF-β1 axis as a potential therapeutic target for restoring NK cell-mediated immunity in HNSCC.

Objective:This study aimed to evaluate the diagnostic efficacy, clinical feasibility, and performance of PCR-coupled Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry（PCR-MALDI-TOF MS） in screening 20 common mutations across four deafness-associated genes in patients with non-syndromic hearing loss（NSHL）. Furthermore, it investigated the correlation between specific gene mutation types and the severity of hearing loss（genotype-phenotype correlation） was investigated. Methods:A total of 298 patients with NSHL（141 males and 157 females; age range: 3 months to 72 years） presenting to the Department of Otolaryngology Head and Neck Surgery at Xiangya Hospital, Central South University, between July, 2022, and July, 2024, were enrolled. The PCR-MALDI-TOF MS assay was employed to detect 20 hotspot mutations in four major deafness genes（GJB2 , SLC26A4 , GJB3 , and mtDNA 12S rRNA）. Sanger sequencing was used as the "gold standard" for bidirectional validation of all samples to assess performance metrics, including sensitivity, specificity, overall concordance rate, and the Kappa coefficient. Additionally, the audiometric phenotypes of patients carrying different pathogenic mutations were statistically analyzed. Results:Among the 298 subjects, pathogenic mutations were identified in 84 cases by PCR-MALDI-TOF MS, yielding a total detection rate of 28.19%. The mutation spectrum included 13 cases with homozygous variants, 42 with heterozygous variants, 16 with compound heterozygous variants, and 13 with homoplasmic mitochondrial DNA variants. The assay showed complete concordance with Sanger sequencing, with overall sensitivity, specificity, and concordance all reaching 100%. Among the 84 positive carriers, the distribution of hearing loss severity was as follows: mild（n=11）, moderate（n=20）, moderately severe（n=15）, severe（n=13）, and profound（n=25）. Notably, patients with profound hearing loss predominantly carried SLC26A4 mutations（13/25） and GJB2 mutations（8/25）, while those with severe hearing loss were primarily associated with SLC26A4 mutations（7/13）. Conclusion:The PCR-MALDI-TOF MS technology demonstrates high accuracy, sensitivity, and specificity, making it an ideal tool for rapid first-line screening of common deafness gene hotspots in the Chinese population. This study reveals a significant association between GJB2 truncating mutations and SLC26A4 splice site mutations with severe-to-profound hearing loss phenotypes. These findings suggest that early genetic diagnosis is crucial for predicting auditory prognosis and guiding timely clinical intervention for patients with these specific genotypes.

Objectives Branchio-oto syndrome (BOS) is an autosomal dominant disorder characterized by multiple system anomalies, typically sparing the kidneys. BOS exhibits considerable clinical heterogeneity and ethnic variability; most studies have been conducted in European populations rather than in Asian populations, and its prevalence is approximately 1 in 40,000. EYA1 is the most commonly implicated gene in BOS, with mutations ranging from missense to frameshift, splicing, and nonsense variants. Although splicing mutations are important contributors to the disease, previous research has paid less attention to novel mutations that cause aberrant RNA splicing and their pathogenic mechanisms. Furthermore, reproductive interventions aimed at preventing disease transmission have not been reported. Methods We collected samples from a three-generation Chinese family affected by BOS. Whole exome sequencing was used to screen for candidate causative genes. A minigene assay was performed to identify aberrant splicing products, and additional molecular biology techniques were employed to analyze the pathogenicity of the resulting mistranslated proteins. Preimplantation genetic testing (PGT), based on single-nucleotide polymorphism analysis, was utilized to prevent hearing loss in this family. Results Whole-exome sequencing identified a novel mutation, EYA1:c.1598-2AG>TA, which was classified as pathogenic according to American College of Medical Genetics and Genomics criteria. The minigene assay verified aberrant RNA splicing that is predicted to result in premature termination of EYA1 protein translation. Cytological experiments demonstrated that the truncated EYA1 protein is unstable, exhibits impaired nuclear translocation, and fails to interact properly with SIX1. PGT enabled the proband to give birth to a healthy boy. Conclusion We identified a novel splicing variant in the EYA1 gene and elucidated its potential molecular pathogenic mechanism through several functional experiments. Based on these findings, we successfully implemented PGT to ensure the birth of a healthy offspring free from BOS.

Waardenburg syndrome (WS) is distinguished by depigmented patches of hair and skin, striking blue eyes and sensorineural hearing loss. Studies on alternative splicing (AS) in the abnormal pigmentation in skin in WS are currently poorly understood. In this study, we conducted comprehensive full-length transcriptome sequencing analysis on abdominal skin tissues from mice harboring the Mitf p.R324del mutation and wild-type controls. Our analysis revealed 3619 differentially expressed genes (DEGs), with 1916 upregulated and 1703 downregulated. Notably, pigment-associated genes and related signaling pathways were significantly enriched. Additionally, we identified extensive AS events in the skin tissue of Mitf mutant mice, including 97 A3, 85 A5, 101 AF, 42 AL, 8 MX, 44 RI, and 184 SE events (P < 0.05), suggesting AS events may contribute to the pigment distribution patterns in Mitf p.R324del mutation mice. Furthermore, we observed downregulation of Dct, Mlana, and Snai2, which indicates that Mitf mutations disrupt neural crest cells into melanocytes, melanosome structure, and melanin biosynthesis. Overall, our results support that variation in gene expression and AS are important and complementary mechanisms governing pigmentation defects in WS.

OBJECTIVE:This study reports a novel heterozygous, likely truncating mutation in the diaphanous homolog 1 (DIAPH1) gene associated with non-syndromic hearing loss. METHODS:Family members underwent audiological and imaging assessments, whole-exome sequencing (WES), and Sanger sequencing. RESULTS:Sensorineural hearing loss was observed in all five individuals, with severity ranging from mild to severe. None of the affected patients reported vestibular complaints, and routine blood tests showed normal platelet counts. Whole-exome sequencing (WES) revealed a novel frameshift variation, c.3555delA (p.Gln1185Hisfs*3), in exon 26 of the DIAPH1 gene. This variation co-segregated with the hearing-impaired phenotype in the family. The data collected support the classification of c.3555delA as a genetic etiology of hereditary hearing loss according to the American College of Medical Genetics and Genomics guidelines. CONCLUSION:We identified a novel pathogenic mutation in the DIAPH1 gene, thereby expanding the mutation spectrum associated with hearing loss.

Background:Spatial transcriptome (ST) provides molecular profiles of tumor cells at the spatial level, which brings new progress to the research of tumors and the tumor microenvironment. This study summarizes the experiences and lessons learned in the spatial section preparation of two different pathological types of nose and skull base tumors at our institution, with the aim of offering guidelines to researchers to avoid wasting precious samples and provide a basis for the application of ST in clinical practice. Methods:Frozen tissue blocks from patients with squamous cell carcinoma and adenocarcinoma of the nose and skull base diagnosed at our institution were prepared. The effects of different procedures and pathological tissue types on slide quality were explored and evaluated using RNA integrity number (RIN) and HE scores as criteria. The effects of different RIN values on ST sequencing data were explored. Results:A total of 43 samples were obtained from 26 patients, including 22 with squamous carcinomas and 21 with adenocarcinomas. Thirteen samples with satisfactory RNA quality control and good histological morphology were sequenced for ST. Sample isolation time <15 min and abandonment of snap-frozen isopentane significantly improved RNA quality (p = 0.004, p < 0.0001) and histomorphological integrity (p = 0.02, p = 0.02). Selection of a suitable tissue RNA extraction kit was critical for RNA quality (p < 0.0001). No difference between 6 ≤ RIN <7 and RIN >7 in ST sequencing results was found, indicating that RIN ≥6 can be used as a criterion for qualified RNA quality control. Therefore, fresh tissues washed as soon as possible with cold PBS and then dried using OCT for snap freezing are currently the best method for preparing spatial sections of nose and skull base tumor tissues of different pathological types. Conclusion:This study is the first to investigate the feasibility of applying ST to different pathological types of nose and skull base tumors and to demonstrate the widespread application of ST in tumors. Rational optimization of spatial slide preparation procedures and exploration of individualized pre-sequencing protocols are used as the first stage to ensure the quality of spatial sequencing and lay the foundation for subsequent spatial analysis.

Hereditary hearing loss has a genetic and phenotypic heterogeneity. However, it is still difficult to explain this heterogeneity perfectly with known deafness genes. Here, we report a novel causative gene EPHA10 as well as its non-coding variant in 5' untranslated region identified in a family with post-lingual autosomal dominant non-syndromic hearing loss from southern China. One affected member of this family had an ideal hearing restoration after cochlear implantation. We speculated that there were probable deafness-causing abnormalities in the cochlea according to clinical imaging and auditory evaluations. A heterozygous variant c.-81_-73delinsAGC was found co-segregating with hearing loss. Epha10 was expressed in mouse cochlea at both transcription and translation levels. The variant caused upregulation of EPHA10 which may result from promoter activity enhancement after sequence change. Overexpression of Eph (the homolog of human EPHA10) exerted effects on the structure and function of chordotonal organ in fly model. In summary, our study linked pseudo-kinase EPHA10 to hearing loss in humans for the first time.

Adenoid cystic carcinoma (ACC) is a rare malignant tumor derived mainly from the salivary glands, representing approximately 1% of all headandneck carcinomasand 10% of all salivary gland neoplasms. ACC displays a paradoxical behavioral combination of an indolent growth pattern but an aggressive progression, with local recurrence and distant metastasis. The propensity of ACC of the head and neck (ACCHN) for perineural invasion and its anatomical location, especially if it extends to the nasal cavity and paranasal sinuses, facilitates tumor involvement in the surrounding structures, such as the orbit, pterygopalatine fossa, Meckel'scave, and cavernous sinus, which can lead to skull base involvement and intracranial extension. Despite advances in molecular mechanisms and diagnostic imaging, ACC treatment remainschallenging due to the lack ofconsensuson treatment patterns. In this review, we aimed toprovideanupdatedinsight intothe understanding of ACCHN by focusing on clinical behavior, imaging diagnosis, pathological features, and therapeutic strategies. We reviewed the molecular mechanisms, especially in ACCHN with perineural invasion, and elaborated on treatment options, including chemotherapy, targeted therapies, and immunotherapy, to establish a comprehensive understanding of ACC to arrive at a policy for proper diagnosis, preoperative evaluation, and therapeutic strategies.

To evaluate the efficacy of non-invasive prenatal screening (NIPT) for detecting fetal sex chromosome abnormalities, a total of 639 women carrying sex chromosome abnormalities were selected from 222,107 pregnant women who participated in free NIPT from April 2018 to December 2020. The clinical data, prenatal diagnosis results, and follow-up pregnancy outcomes of participants were collected. The positive predictive value (PPV) was used to analyze the performance of NIPT. Around 235 cases were confirmed with sex chromosome abnormalities, including 229 cases with sex chromosome aneuploidy (45, X (n = 37), 47, XXX (n = 37), 47, XXY (n = 110), 47, XYY (n = 42)) and 6 cases with structural abnormalities. The total incidence rate was 0.11% (235/222,107). The PPV of NIPT was 45.37% (235/518). NIPT accuracy for detecting sex chromosome polysomes was higher than that for sex chromosome monomers. The termination of pregnancy rate for fetal diagnosis of 45, X, and 47, XXY was higher than that of 47, XXX, and 47, XYY. The detection rate of fetal sex chromosome abnormalities was higher in 2018–2020 than in 2010–2012 (χ2 = 69.708, P < 2.2 × 10−16), indicating that NIPT is greatly efficient to detect fetal sex chromosome abnormalities.

ABSTRACT Objective To explore the pregnancy outcomes of women who couldn't obtain effective results from noninvasive prenatal testing (NIPT) and examine the factors leading to test failure. Methods From April 2017 to December 2019, 120,041 pregnant women enrolled for voluntary NIPT. The case group comprised of 274 (274/120,041) women who failed to obtain effective NIPT results, and the control group (n = 540) was from the same population who obtained effective NIPT results and matched by age at a 1:2 ratio. Abnormal pregnancy rates between the two groups were analyzed using Chi-square analysis. NIPT failure risk factors were analyzed using logistic regression analysis. Results Logistic regression analysis showed that increased maternal age (OR = 0.988; 95% CI = 0.982–0.994), increased pregnancy age (OR = 0.989; 95%CI = 0.988–0.991), and decreased cell-free fetal DNA concentration (OR = 1.050; 95%CI = 1.043–1.058) were independent risk factors for NIPT failure. Fifteen cases showed fetus loss in cases of NIPT failure. There was a significant difference in abnormal pregnancy rate between the NIPT success and failure groups (χ2 = 50.943, P < 0.05). Expert commentary The specific interventions, guidance, and precautions are needed for pregnant women who have no effective NIPT results.

Objectives/HypothesisTracheal stenosis is an obstructive disease of the upper airway that commonly develops as a result of abnormal wound healing. We evaluated the anti‐inflammatory and antifibrotic properties of nintedanib on tracheal stenosis both in vitro and in vivo.Study DesignProspective controlled animal study and in vitro comparative study of human cells.MethodsAn animal model of tracheal stenosis was induced via tracheal trauma. Postsurgical rats were orally administered with nintedanib (10 or 20 mg/kg/d) or saline (negative control) for 2 weeks, and tracheal specimens were harvested after 3 weeks. Degree of stenosis, collagen deposition, fibrotic surrogate markers expression, and T‐lymphocytic infiltration were evaluated. Human fetal lung fibroblast‐1 (HFL‐1) cells were cultured to determine the effects of nintedanib on changes of cellular biological function induced by transforming growth factor‐β1 (TGF‐β1).ResultsRat tracheal stenotic tissues exhibited thickened lamina propria with irregular epithelium, characterized by significantly increased collagen deposition and elevated TGF‐β1, collagen I, α‐SMA and fibronectin expressions. Nintedanib markedly attenuated the tracheal stenotic lesions, reduced the collagen deposition and the expression of fibrotic marker proteins, and mitigated CD4+ T‐lymphocyte infiltration. Additionally, cellular proliferation and migration were decreased dose‐dependently in TGF‐β1‐stimulated HFL‐1 cells when treated with nintedanib. Furthermore, nintedanib inhibited TGF‐β1‐induced HFL‐1 differentiation and reduced the mRNA levels of the profibrotic genes. TGF‐β1‐activated phosphorylation of the TGF‐β/Smad2/3 and ERK1/2 pathways were also blocked by nintedanib.ConclusionNintedanib effectively prevented tracheal stenosis in rats by inhibiting fibrosis and inflammation. The antifibrotic effect of nintedanib may be achieved by inhibiting fibroblasts' proliferation, migration and differentiation and suppressing the TGF‐β1/Smad2/3 and ERK1/2 signaling pathways.Level of EvidenceNA Laryngoscope, 131:E2496–E2505, 2021

Waardenburg Syndrome (WS) is a common autosomal dominant syndrome associated with hearing loss. Its clinical manifestations include hearing impairment and pigmentation anomalies. In this study, we generated an induced pluripotent stem cell (iPSC) line from the Epstein-Barr virus-immortalized B lymphocytes of a 6-year-old boy affected with WS type I, caused by a heterozygous splice site mutation in the PAIRED BOX GENE 3 (PAX3) (NM_181457.3: c.452-2A > G). The patient-specific iPSC line (CSUXHi004-A) carrying the same PAX3 mutation showed a normal karyotype, expressed pluripotent markers, and presented differentiation capacity in vitro. This method may be a useful tool for the in vitro modeling of WS.

Background Head and neck squamous cell carcinoma (HNSCC) is characterised by a dismal prognosis; nonetheless, limited studies have unveiled the mechanisms underlying HNSCC relapse. Methods Next-generation sequencing was performed to identify the somatic mutations in 188 matched samples, including primary tumours, tumour-adjacent tissues (TATs), pre- and post-operative plasma, saliva and peripheral blood lymphocytes (PBLs) from 27 patients. The evolutionary relationship between TATs and tumours were analysed. The dynamic changes of tumour- and TAT-specific mutations in liquid biopsies were monitored together with survival analysis. Results Alterations were detected in 27 out of 27 and 19 out of 26 tumours and TATs, respectively. TP53 was the most prevalently mutated gene in TATs. Some TATs shared mutations with primary tumours, while some other TATs were evolutionarily unrelated to tumours. Notably, TP53 mutations in TATs are stringently associated with premalignant transformation and are indicative of worse survival (hazard ratio = 14.01). TAT-specific mutations were also detected in pre- and/or post-operative liquid biopsies and were indicative of disease relapse. Conclusions TATs might undergo the processes of premalignant transformation, tumorigenesis and eventually relapse by either inheriting tumorigenic mutations from ancestral clones where the tumour originated or gaining private mutations independent of primary tumours. Detection of tumour- and/or TAT-specific genetic alterations in post-operative biopsies shows profound potential in prognostic use.

Objective:To verify the accuracy and effectiveness of Goldengate high-throughput deafness gene chip in detecting the patients with enlarged vestibular aqueduct syndrome(EVAS), and to provide a reference for genetic detection strategy of EVAS.Methods:From August 2016 to February 2018, 15 patients with EVAS and 60 normal controls were detected by Goldengate high-throughput deafness detection chip developed by our team, and the results were verified by Sanger sequencing. SLC26A4 gene sequencing was carried out in all the patients with EVAS. Results:12/15 of patients with EVAS were detected mutations of SLC26A4 gene. Nine mutations were detected by chip detection and SLC26A4 gene direct sequencing, seven of which were detected by both methods. The chip could detect 93.33%(28/30) of the allele information provided by SLC26A4 gene direct sequencing. In addition to SLC26A4 gene, mutations of GJB2, PCDH15, TMC1, MYO6 and mitochondrial genes were detected in 15 patients with EVAS. These results were verified by Sanger sequencing. Conclusion:Goldengate high-throughput deafness gene chip possesses the traits of wide coverage and high accuracy, which can be used as a preliminary detection method for patients with EVAS.

Waardenburg syndrome (WS) is a genetic disorder characterized by sensorineural deafness. It has a variable presentation of pigmentation defects. Here, we generated an induced pluripotent stem cell (iPSC) line using episomal plasmid vectors from the fibroblasts of a 4-year-old boy affected with WS type II, caused by a novel mutation in microphthalmia-associated transcription factor (MITF) (NM_000248.3: exon6:c.626A>T). The patient-specific iPSC line (CSUXHi003-A) carrying the same MITF mutation showed normal karyotype, expressed pluripotent markers, and presented differentiation capacity in vitro. It may be a useful tool for in vitro modeling of WS.

Waardenburg syndrome (WS) is an autosomal dominant inherited disorder that is characterized by sensorineural hearing loss and abnormal pigmentation. SOX10 is one of its main pathogenicity genes. The generation of patient-specific induced pluripotent stem cells (iPSCs) is an efficient means to investigate the mechanisms of inherited human disease. In our work, we set up an iPSC line derived from a WS patient with SOX10 mutation and differentiated into neural crest cells (NCCs), a key cell type involved in inner ear development. Compared with control-derived iPSCs, the SOX10 mutant iPSCs showed significantly decreased efficiency of development and differentiation potential at the stage of NCCs. After that, we carried out high-throughput RNA-seq and evaluated the transcriptional misregulation at every stage. Transcriptome analysis of differentiated NCCs showed widespread gene expression alterations, and the differentially expressed genes (DEGs) were enriched in gene ontology terms of neuron migration, skeletal system development, and multicellular organism development, indicating that SOX10 has a pivotal part in the differentiation of NCCs. It’s worth noting that, a significant enrichment among the nominal DEGs for genes implicated in inner ear development was found, as well as several genes connected to the inner ear morphogenesis. Based on the protein-protein interaction network, we chose four candidate genes that could be regulated by SOX10 in inner ear development, namely, BMP2, LGR5, GBX2, and GATA3. In conclusion, SOX10 deficiency in this WS subject had a significant impact on the gene expression patterns throughout NCC development in the iPSC model. The DEGs most significantly enriched in inner ear development and morphogenesis may assist in identifying the underlying basis for the inner ear malformation in subjects with WS.

Objective: To explore the application value of high-throughput gene detection method of copy number variations (CNV) in the diagnosis of enlarged vestibular aqueduct (EVA). Methods: A total of 46 nonsyndromic hearing loss patients with EVA were recruited between May 2014 and December 2016 from Department of Otolaryngology of Xiangya Hospital, Central South University. A high-throughput multiplex analysis method based on double ligation and multiple fluorescent PCR was designed and performed to detect CNV in the three EVA-related genes (SLC26A4, FOXI1 and KCNJ10). The data were analyzed by GeneMapper v4.1. Healthy volunteers (n=100) were selected as normal controls. Results: A total of 46 EVA patients were detected (32 males, 14 females, aged 1 to 26 years). In 4 EVA patients, deletions of exons 1-3 of SLC26A4 gene (4/46, 8.7%) were detected, which were not reported in the database of genomic variants (DGV), and were absent in 100 normal controls. There was no CNV detected in FOXI1 and KCNJ10 in the study. Conclusions: In the current study, three known EVA-related genes were designed as the target area for CNV detection by high-throughput ligation-dependent probe amplification (HLPA) analysis. This method can be used as a supplementary analysis of point mutation detection of hearing loss, which helps achieve the accurate genetic diagnosis of EVA.

NRXN1 is involved in synaptogenesis and have been implicated in Autism spectrum disorders. However, many rare inherited missense variants of NRXN1 have not been thoroughly evaluated. Here, functional analyses in vitro and in Drosophila of three NRXN1 missense mutations, Y282H, L893V, and I1135V identified in ASD patients in our previous study were performed. Our results showed these three mutations interfered protein degradation compared with NRXN1-WT protein. Expressing human NRXN1 in Drosophila could lead to abnormal circadian rhythm and sleep behavior, and three mutated proteins caused milder phenotypes, indicating the mutations may change the function of NRXN1 slightly. These findings highlight the functional role of rare NRXN1 missense variants identified in autism patients, and provide clues for us to better understand the pathogenesis of abnormal circadian rhythm and sleep behavior of other organisms, including humans.

Hypopharyngeal squamous cell carcinoma (HPSCC) is one of the most common malignant tumors in otolaryngology head and neck surgery and is one of the worst prognostic malignant tumors. Endogenous circular RNA (circRNA) is more stable than mRNA, microRNA (miRNA), and long non-coding RNA (LncRNA) in exosomes, plasma, and urine, and participates in gene expression regulation to perform different functions. Therefore, circRNA is expected to become a biomarker and therapy target for many tumors. However, the expression and function of circRNA regulated by N6-methyladenosine (m6A) are still unclear in HNSCC. In this study, we demonstrated that a specific circRNA, circCUX1, was upregulated in HPSCC patients who are resistant to radiotherapy and predicts poor survival outcome. We further found that methyltransferase like 3 (METTL3) mediated the m6A methylation of circCUX1 and stabilizes its expression. Knockdown circCUX1 promotes the sensitivity of hypopharyngeal cancer cells to radiotherapy. In addition, circCUX1 binds to Caspase1 and inhibits its expression, resulting in a decrease in the release of inflammatory factors, thereby developing tolerance to radiotherapy. Our findings indicate that circCUX1 is a potential therapeutic target for radiotherapy tolerance in HPSCC patients.