OBJECTIVE: To investigate the clinical phenotype and genotype features of children with Epileptic encephalopathy caused by PCDH19 mutations. METHODS: Four children with epilepsy caused by PCDH19 gene mutations who were treated at Shanghai Children's Medical Center from August 2015 to May 2024 were selected as study subjects. A retrospective study method was used to collect the clinical data of the patients. Peripheral venous blood samples (2 mL each) were collected from the patients and their parents. Genomic DNA was extracted, and whole exome sequencing (WES) was performed, followed by family verification of candidate variants by Sanger sequencing. Pathogenicity of the candidate variants was classified according to the "Genetic Variation Classification Standards and Guidelines" established by the American College of Medical Genetics and Genomics (ACMG). This study was approved by the Medical Ethics Committee of Shanghai Children's Medical Center (Approval No. SCMCIRB-K2020060-1). RESULTS: The patients comprised of 3 females and 1 male, all presenting symptoms before the age of 3. Patients 1-3 exhibited generalized tonic-clonic seizures, while patient 4 manifested focal seizures accompanied by impaired consciousness. In addition to epilepsy, patient 2 showed language delay and patient 3 had frequent panic attacks. WES results identified four pathogenic PCDH19 variants these patients, including 2 previously unreported frameshifting mutations,1 hotspot missense mutation, and 1 mosaic missense mutation with a 32.4% mutation rate. The pathogenic mutation in patient 2 was inherited from her father, while the remaining 3 patients had de novo pathogenic mutations. CONCLUSION Children with PCDH19 gene mutations may exhibit early-onset refractory epilepsy, cognitive impairment, and developmental delay. Females are predominantly affected by the PCDH19 mutations, although males with mosaic mutations can also be affected. The genetic and clinical heterogeneity observed among patients 1-4 indicated the diverse nature of epilepsy related to the PCDH19 gene mutations. PCDH19 gene mutations may be the genetic cause of epilepsy in these affected children, which also enriched the mutational spectrum of the PCDH19 gene.
Child, Preschool ; Female ; Humans ; Infant ; Male ; Cadherins/genetics* ; Epilepsy/genetics* ; Exome Sequencing ; Mutation ; Phenotype ; Protocadherins ; Retrospective Studies

OBJECTIVE: To analyze the carrier rates and profiles of pathogenic and likely pathogenic variants for hearing loss-related genes MYO7A, PCDH15, and CDH23 among neonates in Nanjing city through targeted next-generation sequencing (NGS). METHODS: Heel-prick blood samples were collected from 30 043 newborns delivered at Nanjing Women and Children's Health Care Hospital between March 2022 and April 2024. Dried blood spots were prepared, and genomic DNA was extracted. Targeted NGS was applied to detect variants across the full coding regions of the MYO7A, PCDH15, and CDH23 genes. The carrier rates and profiles of pathogenic and likely pathogenic variants of the three genes were analyzed. This study was approved by the Medical Ethics Committee of Nanjing Maternal and Child Health Care Hospital (Ethics No.: 2021KY-071). RESULTS: The carrier rates of pathogenic and likely pathogenic variants (with ≥ 1 variant site) for the MYO7A, PCDH15, and CDH23 genes were 0.340%, 0.226%, and 0.156%, respectively. A total of 65, 49, and 30 variant types were detected in the MYO7A, PCDH15, and CDH23 genes, respectively. For MYO7A, single base variants were predominant, with the most common variant being c.5581C>T, followed by c.1343+1G>A, c.2837T>G, and c.5660C>T, with allelic frequencies of 0.013% (8/60 086), 0.007% (4/60 086), 0.007% (4/60 086), and 0.007% (4/60 086), respectively. PCDH15 variants were mainly deletions, with the most common variant site being c.4699_4715dupAGAGAAAAGATTCAGAG, followed by c.3441delA, c.440T>G, and c.4733_4736delTCAG, with allelic frequencies of 0.015% (9/60 086), 0.005% (3/60 086), 0.005% (3/60 086), and 0.005% (3/60 086), respectively. For CDH23, single base variants were predominant, with c.6604G>A being the most common, followed by c.6085C>T, c.6050+9G>A, and c.6253+1G>A, with allelic frequencies of 0.013% (8/60 086), 0.012% (7/60 086), 0.005% (3/60 086), and 0.005% (3/60 086). CONCLUSION This study analyzed the carrier rates and profiles of pathogenic and likely pathogenic variants of the MYO7A, PCDH15, and CDH23 genes, which can provide more evidence for the prevention and management of deafness in the region.
Humans ; Cadherins/genetics* ; High-Throughput Nucleotide Sequencing/methods* ; Infant, Newborn ; Female ; Myosin VIIa/genetics* ; Cadherin Related Proteins ; Male ; Hearing Loss/genetics* ; Myosins/genetics* ; Heterozygote

OBJECTIVE: To analyze a Chinese pedigree affected with Non-syndromic autosomal recessive deafness type 12 (NFNB12), validate the function of candidate variants, and explore the underlying mechanisms. METHODS: A NFNB12 pedigree presented at Henan Provincial People's Hospital in February 2023 was selected as the study subject. Whole exome sequencing (WES) was carried out, and candidate variant was verified by Sanger sequencing of the pedigree members. Reverse transcription polymerase chain reaction (RT-PCR) was used to determine the level of mRNA transcription in the peripheral blood samples from the pedigree members, and protein expression was evaluated with Western blotting assay. This study was approved by Medical Ethics Committee of Henan Provincial People's Hospital (Ethics No.: 2019-134). RESULTS: WES analysis revealed that the proband has harbored homozygous c.6688delG (p.Ala2230Profs*4) variant of the CDH23 gene, for which both parents were identified as heterozygous carriers. RT-PCR analysis demonstrated the sole presence of the variant mRNA in the proband, and both the variant and wild-type mRNAs in both parents. Furthermore, Western blotting analysis indicated that the proband had exclusively expressed the truncated CDH23 protein, while both the normal and truncated forms of the protein were noted in her parents. CONCLUSION The c.6688delG (p.Ala2230Profs*4) variant of the CDH23 gene probably underlay the pathogenesis of NFNB12 in this pedigree. The loss of function of the CDH23 gene resulting from this variant is not related with nonsense-mediated mRNA decay, but rather production of a truncated protein. Above finding has not only enriched the mutational spectrum of the CDH23 gene and offered a method for investigating the function of its variants using peripheral blood samples, but also delineated the molecular basis for the loss of function, which has provided crucial evidence for genetic counseling and prenatal diagnosis for this family.
Humans ; Pedigree ; Male ; Female ; Asian People/genetics* ; Cadherins/genetics* ; Exome Sequencing ; Deafness/genetics* ; Mutation ; China ; Adult ; Cadherin Related Proteins ; Hearing Loss, Sensorineural/genetics* ; East Asian People

OBJECTIVE: To investigate the effect of TBL1XR1 mutation on cell biological characteristics of diffuse large B-cell lymphoma (DLBCL). METHODS: The TBL1XR1 overexpression vector was constructed and DNA sequencing was performed to determine the mutation status. The effect of TBL1XR1 mutation on apoptosis of DLBCL cell line was detected by flow cytometry and TUNEL fluorescence assay; CCK-8 assay was used to detect the effect of TBL1XR1 mutation on cell proliferation; Transwell assay was used to detect the effect of TBL1XR1 mutation on cell migration and invasion; Western blot was used to detect the effect of TBL1XR1 mutation on the expression level of epithelial-mesenchymal transition (EMT) related proteins. RESULTS: The TBL1XR1 overexpression plasmid was successfully constructed. The in vitro experimental results showed that TBL1XR1 mutation had no significant effect on apoptosis of DLBCL cells. Compared with the control group, TBL1XR1 mutation enhanced cell proliferation, migration and invasion of DLBCL cells. TBL1XR1 gene mutation significantly increased the expression of N-cadherin protein, while the expression of E-cadherin protein decreased. CONCLUSION TBL1XR1 mutation plays a role in promoting tumor cell proliferation, migration and invasion in DLBCL. TBL1XR1 could be considered as a potential target for DLBCL therapy in future research.
Humans ; Lymphoma, Large B-Cell, Diffuse/pathology* ; Cell Proliferation ; Mutation ; Receptors, Cytoplasmic and Nuclear/genetics* ; Apoptosis ; Cell Line, Tumor ; Epithelial-Mesenchymal Transition ; Cell Movement ; Repressor Proteins/genetics* ; Nuclear Proteins/genetics* ; Cadherins/metabolism*

OBJECTIVES: Esophageal squamous cell carcinoma (ESCC) is characterized by complex pathogenesis and poor prognosis. In recent years, epithelial-mesenchymal transition (EMT) in tumor initiation and progression has attracted increasing attention. Enhancer of zeste homolog 2 (EZH2), which is aberrantly expressed in various tumors, may be closely related to the EMT process. This study aims to examine the expression and correlation of EZH2 and EMT markers in ESCC cells and tissues, evaluate the effects of EZH2 knockdown on ESCC cell proliferation, invasion, and migration, and explore how EZH2 contributes to the malignant biological behavior of ESCC. METHODS: Bioinformatics analyses were used to assess EZH2 expression levels in ESCC. Small interfering RNA was used to knock down EZH2 in ESCC cell lines EC109 and EC9706. Cell proliferation, invasion, and migration were evaluated using cell counting kit-8 (CCK-8), wound healing, and Transwell assays. Protein and mRNA expression levels of EZH2, E-cadherin (E-cad), and vimentin (Vim) were detected by Western blotting and real time fluorogenic quantitative PCR (RT-qPCR), respectively. Immunohistochemical (IHC) staining was performed on 70 ESCC tissue samples and 40 paired adjacent normal tissues collected from the First Affiliated Hospital of Shihezi University between 2010 and 2016 to assess the expression of EZH2, E-cad, and Vim, and to analyze their associations with clinicopathological feature and patient prognosis. RESULTS: Bioinformatics analysis showed that EZH2 was highly expressed in ESCC (P<0.001), and high EZH2 expression was associated with worse prognosis (P<0.001). CCK-8, wound healing, and Transwell assays demonstrated that EZH2 knockdown significantly suppressed the proliferation, invasion, and migration of ESCC cells (P<0.001). In addition, Vim expression was significantly reduced, while E-cad expression was significantly increased at both protein and mRNA levels in EZH2-silenced cells (all P<0.05). IHC staining analysis revealed higher expression of EZH2 and Vim and lower expression of E-cad in ESCC tissues compared to adjacent normal tissues. Kaplan-Meier survival analysis showed that low expression of EZH2 and Vim and high expression of E-cad were associated with longer survival (all P<0.05). CONCLUSIONS EZH2 promotes malignant biological behavior in ESCC by mediating EMT. Elevated EZH2 expression is associated with poor prognosis in ESCC patients.
Humans ; Enhancer of Zeste Homolog 2 Protein/physiology* ; Esophageal Squamous Cell Carcinoma/pathology* ; Epithelial-Mesenchymal Transition/genetics* ; Esophageal Neoplasms/metabolism* ; Cell Proliferation ; Cell Line, Tumor ; Cell Movement ; Cadherins/genetics* ; Vimentin/genetics* ; Male ; Female ; Middle Aged ; Neoplasm Invasiveness ; Prognosis ; RNA, Small Interfering/genetics* ; Gene Expression Regulation, Neoplastic

OBJECTIVES: Increasing detection of low-risk papillary thyroid carcinoma (PTC) is associated with overdiagnosis and overtreatment. N6-methyladenosine (m⁶A)-mediated microRNA (miRNA) dysregulation plays a critical role in tumor metastasis and progression. However, the functional role of m⁶A-miRNAs in PTC remains unclear. This study aims to elucidate the regulatory mechanism of m⁶A-miR-139-5p expression in PTC, determine its association with PTC metastasis, and evaluate its potential as a diagnostic biomarker for PTC metastasis, thereby providing experimental evidence for precision diagnosis and therapy. METHODS: Expression profiles of m⁶A-miRNAs were compared between the The Cancer Genome Atlas (TCGA) and GSE130512 cohorts to identify metastasis-associated candidates. Clinical specimens from 13 metastasis and 18 non-metastasis PTC patients were analyzed to assess m⁶A-miR-139-5p expression and its correlation with metastasis. Functional experiments were conducted to investigate the effect of fat mass and obesity-associated protein (FTO) on pri-miR-139 methylation and processing, clarifying its regulatory role in miR-139-5p expression. In TPC-1 cells, MTT assays were performed to evaluate whether miR-139-5p overexpression could counteract FTO-mediated cell proliferation. Transwell invasion assays were used to determine the impact of miR-139-5p on PTC cell invasion, exploring whether it functions through the ZEB1/E-cadherin axis. RESULTS: By comparing TCGA and GSE130512 cohorts, it was found that circulating m⁶A-miR-139-5p could serve as a biological indicator for detecting PTC metastasis. Detection of 13 metastatic and 18 non-metastatic clinical specimens showed that FTO inhibited the processing of pri-miR-139 by reducing its methylation level, leading to the dysregulation of miR-139-5p in PTC (P<0.05). In TPC-1 cells, MTT assay showed that overexpression of miR-139-5p could partially reverse FTO overexpression-mediated cell proliferation (P<0.05). In addition, miR-139-5p inhibited the invasive ability of PTC cells by targeting the ZEB1/E-cadherin axis, while FTO overexpression could partially weaken this inhibitory effect. CONCLUSIONS Circulating miR-139-5p can be a potential marker for evaluating PTC metastasis. FTO affects the expression and function of miR-139-5p by regulating m⁶A modification of pri-miR-139, but its clinical value needs further verification.
Humans ; MicroRNAs/metabolism* ; Thyroid Cancer, Papillary/metabolism* ; Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism* ; Thyroid Neoplasms/metabolism* ; Cell Line, Tumor ; Neoplasm Metastasis ; Adenosine/genetics* ; Gene Expression Regulation, Neoplastic ; Female ; Male ; Cadherins/metabolism* ; Cell Proliferation ; Zinc Finger E-box-Binding Homeobox 1/genetics*

Objective:To investigate the molecular characteristics and clinical heterogeneity of Usher syndrome（USH） -related gene variants in patients with hereditary hearing loss in southwest China, providing a basis for early diagnosis and clinical management. Methods:Thirteen patients from twelve families with hearing loss who attended the Affiliated Children's Hospital of Kunming Medical University between January 2017 and March 2021 were enrolled. All patients were identified as carrying USH-related gene variants through next-generation sequencing. Sanger sequencing was performed for all patients and their parents to validate the pathogenic variants. Comprehensive clinical evaluations, including medical history collection, otologic and ophthalmologic examinations, and vestibular function assessments, were conducted. Results:Among the 13 patients, 4 were diagnosed with USH type 1 and 2 with USH type 2. A total of 19 pathogenic or likely pathogenic variants were detected in USH-related genes, including MYO7A,CDH23,USH1C, and USH2A. The causative gene was MYO7A in 3 probands, CDH23 in 5, USH1C in 3, and USH2Ain 2. All patients exhibited an autosomal recessive inheritance pattern. Vestibular dysfunction was observed in 4 patients, and retinitis pigmentosa（RP） in 3 patients. Based on the genotype-phenotype correlation, 6 patients were initially diagnosed with USH, while 7 were classified as having non-syndromic hearing loss（NSHL）. Conclusion:This study revealed the clinical heterogeneity of USH-related gene variants in patients with hereditary deafness in southwest China. Although the clinical manifestations of USH are complex and there are overlapping characteristics between different subtypes, genetic testing provides an important basis for early diagnosis and precise clinical management. Especially for those with typical hearing loss, early genetic diagnosis can provide a window of time for early detection and intervention of retinitis pigmentosa.
Humans ; Usher Syndromes/genetics* ; Myosin VIIa ; Phenotype ; Male ; Female ; Myosins/genetics* ; Mutation ; Cadherins/genetics* ; Child ; Extracellular Matrix Proteins/genetics* ; Adolescent ; Pedigree ; High-Throughput Nucleotide Sequencing ; Cadherin Related Proteins ; Cytoskeletal Proteins ; Cell Cycle Proteins

OBJECTIVES: To investigate CEACAM6 expression in nasopharyngeal carcinoma (NPC) and its regulatory effects on tumor cell proliferation, migration, and epithelial-mesenchymal transition (EMT). METHODS: CEACAM6 expression in NPC was analyzed using GEO datasets and validated by immunohistochemistry in NPC tissues and by Western blotting and RT-qPCR in NPC cell lines (HNE1, C666-1, HK1, 5-8F and CNE2Z) and normal nasopharyngeal epithelial NP69 cells. In the NPC cell lines, the effects of lentivirus-mediated CEACAM6 overexpression and knockdown on cell proliferation, migration, invasion and cytoskeletal structures were evaluated using CCK-8 assay, Edu staining, wound healing assay, Transwell assay, and phalloidin staining. Western blotting was performed to determine the expressions of EMT-related proteins (FN1, ITGA5, ITGB1, E-cadherin, N-cadherin and vimentin) in the NPC cells and the effect of FN1 overexpression on ITGA5 and ITGB1 protein expressions. RESULTS: Analysis of the data from the GEO datasets suggested that CEACAM6 was significantly downregulated in NPC, which was associated with poor patient prognosis. Immunohistochemistry also showed low expressions of CEACAM6 in clinical NPC tissues (P<0.05). In NPC cells, CEACAM6 overexpression significantly suppressed cell proliferation, migration and invasion and reduced the fluorescence intensity of actin. CEACAM6 overexpression also resulted in significant downregulation of FN1, ITGA5, ITGB1, N-cadherin and vimentin expressions and upregulation of E-cadherin expression, and FN1 overexpression obviously attenuated the inhibitory effect of CEACAM6 overexpression on ITGA5 and ITGB1 expressions. CONCLUSIONS CEACAM6 inhibits NPC cell migration and invasion by inhibiting EMT via regulating FN1, ITGA5 and ITGB1 expressions.
Humans ; Epithelial-Mesenchymal Transition ; Cell Movement ; Cell Proliferation ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms/metabolism* ; Cell Line, Tumor ; Cell Adhesion Molecules/genetics* ; Antigens, CD/metabolism* ; GPI-Linked Proteins ; Integrin alpha5/metabolism* ; Integrin beta1/metabolism* ; Cadherins/metabolism* ; Fibronectins ; Integrins

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a prevalent type of cancer with a high mortality rate in its late stages. One of the major challenges in OSCC treatment is the resistance to epidermal growth factor receptor (EGFR) inhibitors. Therefore, it is imperative to elucidate the mechanism underlying drug resistance and develop appropriate precision therapy strategies to enhance clinical efficacy. METHODS: To evaluate the efficacy of the combination of the Ca 2+ /calmodulin-dependent protein kinase II (CAMK2) inhibitor KN93 and EGFR inhibitors, we performed in vitro and in vivo experiments using two FAT atypical cadherin 1 ( FAT1 )-deficient (SCC9 and SCC25) and two FAT1 wild-type (SCC47 and HN12) OSCC cell lines. We assessed the effects of EGFR inhibitors (afatinib or cetuximab), KN93, or their combination on the malignant phenotype of OSCC in vivo and in vitro . The alterations in protein expression levels of members of the EGFR signaling pathway and SRY-box transcription factor 2 (SOX2) were analyzed. Changes in the yes-associated protein 1 (YAP1) protein were characterized. Moreover, we analyzed mitochondrial dysfunction. Besides, the effects of combination therapy on mitochondrial dynamics were also evaluated. RESULTS: OSCC with FAT1 mutations exhibited resistance to EGFR inhibitors treatment. The combination of KN93 and EGFR inhibitors significantly inhibited the proliferation, survival, and migration of FAT1 -mutated OSCC cells and suppressed tumor growth in vivo . Mechanistically, combination therapy enhanced the therapeutic sensitivity of FAT1 -mutated OSCC cells to EGFR inhibitors by modulating the EGFR pathway and downregulated tumor stemness-related proteins. Furthermore, combination therapy induced reactive oxygen species (ROS)-mediated mitochondrial dysfunction and disrupted mitochondrial dynamics, ultimately resulting in tumor suppression. CONCLUSION Combination therapy with EGFR inhibitors and KN93 could be a novel precision therapeutic strategy and a potential clinical solution for EGFR-resistant OSCC patients with FAT1 mutations.
Humans ; ErbB Receptors/metabolism* ; Mouth Neoplasms/metabolism* ; Cell Line, Tumor ; Animals ; Drug Resistance, Neoplasm/genetics* ; Cadherins/metabolism* ; Carcinoma, Squamous Cell/metabolism* ; Mice ; Mutation/genetics* ; Mice, Nude ; Protein Kinase Inhibitors/therapeutic use* ; Cetuximab/pharmacology* ; Afatinib/therapeutic use* ; Cell Proliferation/drug effects* ; Signal Transduction/drug effects*

Objective To elucidate the molecular mechanism by which exosomes (Exo) derived from cancer-associated fibroblasts (CAF) carrying HOX transcript antisense intergenic RNA (lncRNA HOTAIR) promote the metastasis of esophageal squamous cell carcinoma (ESCC). Methods CAFs were collected from tumor tissues, and non-cancer associated fibroblasts (NFs) were obtained from adjacent normal tissues at least 5 cm away from the tumor. Exosomes (CAFs-Exo and NFs-Exo) were isolated from conditioned media collected from CAFs or NFs. CAFs-Exo and NFs-Exo were incubated with human ESCC cell line TE-1 for 24 hours, and CCK-8 was used to determine the cell proliferation ability. Scratch test and Transwell test were performed to determine the cell migration and invasion ability. TE-1 cells were divided into the following two groups: NC group and KD group. The NC group and KD group were transfected with control siRNAs or siRNAs targeting HOTAIR respectively. The effects of HOTAIR knock-down on cell proliferation, migration, invasion and glycolysis were determined. Results CAFs-Exo promoted the proliferation of TE-1 cells more significantly than NFs-Exo. Compared with NFs-Exo group, the migration and invasion ability of TE-1 cells treated with CAFs-Exo were improved significantly. In addition, CAFs-Exo treatment inhibited the expression of E-cadherin and enhanced the expression of N-cadherin. The expression of HOTAIR in CAFs was significantly higher than that in NFs. Compared with NFs-Exo, the expression level of HOTAIR in CAFs-Exo increased significantly. Compared with NC group, the proliferation, migration and invasion of TE-1 cells in KD group decreased significantly. Compared with NC group, hexokinase 2 (HK2), extracellular acidification rate (ECAR) and ATP/ADP ratio of TE-1 cells in KD group decreased significantly. Conclusion HOTAIR, an exosome derived from CAFs, may be involved in metastasis and EMT by regulating glycolysis in ESCC cells.
Humans ; RNA, Long Noncoding/metabolism* ; Esophageal Neoplasms/metabolism* ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Cell Line, Tumor ; Esophageal Squamous Cell Carcinoma ; Exosomes/genetics* ; Neoplasm Metastasis ; Neoplasm Invasiveness ; Gene Expression Regulation, Neoplastic ; Glycolysis/genetics* ; Cancer-Associated Fibroblasts/metabolism* ; Carcinoma, Squamous Cell/metabolism* ; Cadherins/genetics*