Case report of early-onset epileptic encephalopathy caused by compound heterozygous mutation of the WWOX gene / 中华实用儿科临床杂志

Clinical data of a case with early-onset epileptic encephalopathy admitted in the Department of Neuroendocrinology, Jinan Children′s Hospital in April 2020 were retrospectively analyzed.A 1-month-old male patient was hospitalized for convulsion for 4 days.The child had repeated seizures in the form of tonic and tonic-spasm seizures, accompanied by feeding difficulties, slow weight gain, and overall developmental delay.Electroencephalogram showed multifocal discharge, atypical hypsarrhythmia, and brain magnetic resonance imaging showed delayed myelination.The whole exome sequencing showed compound heterozygous mutation of the WWOX gene.Topiramate, Levetiracetam and Valporate were ineffective to this case.Genetic testing should be performed timely in patients with early-onset epileptic encephalopathy and overall developmental delay to make a clear etiology and prognosis, thus guiding prenatal diagnostics and genetic counseling.

Effects of trichostatin A on FHIT and WWOX genes expression, cell growth inhibition and apoptosis induction in hepatocellular carcinoma WCH 17 cell line

Previously, we evaluated the effect of trichostatin A (TSA) on the expression of DNA methyltransferase 1 (DNMT1) in Hepatocellular Carcinoma (HCC). Fragile histidine triad (FHIT) and WW domain-containing oxidoreductase (WWOX) are two of the most common down-regulated genes in many cancers located on chromosome 3p14.2 and 16q23.3-24.1 respectively. The aim of the current study was to assess the effect of TSA on these genes expression, cell growth, and apoptosis in HCC WCH 17 cell. The cells were seeded and treated with TSA at different times. Then, MTT assay, flow cytometry, and qRT-PCR were achieved to determine viability, apoptosis and gene expression respectively. Cell growth was significantly inhibited, 92 to 36% after 24 h, 86 to 28% after 48 h, and 78 to 24% after 72 h. The results of flow cytometry confirmed that TSA increased apoptosis compared to the control group, the apoptosis percentage increased to 12%, 16%, and 18% in comparison to control groups (2%). Significant up-regulation of the genes was observed in all treated groups. We concluded that re-expression of silenced WWOX and FHIT genes could be achieved by TSA resulting in cell growth inhibition and apoptosis induction in WCH 17 cell.

Immunohistochemical WWOX expression and association with angiogenesis, p53 expression, cell proliferation and clinicopathological parameters in cervical cancer / Avaliação da expressão do gene WWOX por avaliação imunohistoquímica, sua associação com marcador de angiogênese, expressão do p53, proliferação celular e parâmetros clinicopatológicos no câncer de colo uterino

Abstract Objective The current study evaluated the expression of WW domain-containing oxidoreductase (WWOX), its association with clinicopathological features and with p53, Ki-67 (cell proliferation) and CD31 (angiogenesis) expression in patients with invasive cervical squamous cell carcinoma (ICSCC). To the best of our knowledge, no other study has evaluated this association. Methods Women with IB stage-ICSCC (n = 20) and women with uterine leiomyoma (n = 20) were prospectively evaluated. Patients with ICSCC were submitted to type BC1 radical hysterectomy and pelvic lymphadenectomy. Patients in the control group underwent vaginal hysterectomy. Tissue samples were stained with hematoxylin and eosin for histological evaluation and protein expression was detected by immunohistochemistry studies. Results The WWOX expression was significantly lower in the tumor compared with the expression in thebenign cervix (p = 0.019). TheWWOXexpressionwas inversely associated with the CD31 expression in the tumor samples (p = 0.018). There was no association betweentheWWOXexpression with the p53 expression (p = 0.464)or the Ki-67expression (p = 0.360) in the samples of invasive carcinoma of the cervix. There was no association between the WWOX expression and tumor size (p = 0.156), grade of differentiation (p = 0.914), presence of lymphatic vascular invasion (p = 0.155), parametrium involvement (p = 0.421) or pelvic lymph node metastasis (p = 0.310) in ICSCC tissue samples. Conclusion The results suggested that WWOX may be involved in ICSCC carcinogenesis, and this marker was associated with tumor angiogenesis.

Resumo Objetivo O presente estudo avaliou a expressão do WWOX, sua associação com características clinicopatológicas e com a expressão do p53, ki-67 (proliferação celular) e CD31 (angiogênese) em pacientes com carcinoma invasivo de células escamosas do colo uterino, ou simplesmente câncer do colo uterino (CCE). Métodos Foram avaliadas prospectivamente pacientes com CCE no estágio IB (n = 20) e mulheres com mioma uterino, no grupo controle (n = 20). As pacientes com CCE foram submetidas à histerectomia radical e à linfadenectomia pélvica do tipo B-C1. As mulheres no grupo-controle foram submetidas à histerectomia vaginal. As amostras de tecido foramcoradas comhematoxilina e eosina para avaliação histológica e a expressão das proteínas foi detectada por imuno-histoquímico. Resultados A expressão do WWOX foi significativamente menor no tumor quando comparada com sua expressão no colo do útero benigno (p = 0,019). A expressão tumoral de CD31 foi inversamente associada à expressão de WWOX (p = 0,018). Sua expressão não foi associada à expressão tumoral de p53 e Ki-67 em pacientes com CCE (p = 0,464 e p = 0,360, respectivamente). Não houve associação entre a expressão de WWOX e o tamanho do tumor (p = 0,156), grau de diferenciação (p = 0,914), presença de invasão vascular linfática (p = 0,155), comprometimento do paramétrio (p = 0,421) ou metástase dos linfonodos pélvicos (p = 0,310) em pacientes com CCE. Conclusão Os resultados sugeriram que o WWOX pode estar envolvido na carcinogênese do CICECU e esse marcador foi associado à angiogênese tumoral.

Expression and Clinical Significance of WWOX Protein and BCL-2 Protein in Primary Lung Cancer / 现代检验医学杂志

Objective Toexplore expression and clinical significance of WWOX protein and the Bcl-2 protein in the organiza-tion of bronchial lung cancer (primary lung cancer).Methods Chose 76 lung cancer patients with clear pathological diagno-sis who were hospitalized in the Shaanxi Provincial People’s Hospital from 2010 and 2015(including 29 cases of adenocarci-noma,27 cases of squamous cell carcinoma,and 20 cases of small cell carcinoma)and 7 cases of normal lung tissue,8 cases of lung tuberculosis.The expressions of WWOX protein,Bcl-2 protein and more that 5 cm normal lung tissue adjacent to carci-noma were measured by immunohistochemistry SP method.The expression difference between patients and normal control group and the influence of sex,age,pathological type,differentiation degree,clinical stage,lymph node metastasis,smoking index on the expression of WWOX protein and Bcl-2 protein in lung cancer were analyzed.Results ①The positive expres-sion rate of WWOX protein in lung cancer group (35.52%)was significantly lower than that in normal lung tissue (73.33%,P<0.05).The positive expression rate of Bcl-2 protein in lung cancer group (78.06%)was significantly higher than that in control group (23.75%,P<0.05 ).②The positive expression rate of WWOX protein in male patients (21.43%)was significantly lower than that in female patients (52.94%),and the difference was statistically significant (χ2=8.146,P=0.04).The positive expression rate of Bcl-2 protein in male patients (71.43%)was significantly higher than that in female patients (35.29%),the difference was statistically significant (χ2=9.923,P=0.002).③In lung cancer with lymph node metastasis,the positive rate of WWOX protein (17.07%)was significantly lower than that in non-lymph node metastasis (48.57%),and the difference was statistically significant (χ2=8.67,P=0.003).In lung cancer with lymph node metastasis,the positive rate of Bcl-2 protein expression (68.29%)was significantly higher than that in non-lymph node me-tastasis (34.28%),and the difference was statistically significant (χ2=8.758,P=0.003).④The positive rate of expression of WWOX protein in patients whose smoking index≥400 and in patients that<400 was 15.63% and 47.73%,respectively, the differences were significant (χ2=8.48,P=0.003).The positive rate of expression of Bcl-2 protein in patients whose smoking index≥400 and in patients that<400 was 56.25% and 22.73%,respectively,the differences were significant (χ2=8.947,P=0.003).⑤WWOX and Bcl-2 protein expressions had no obvious relationship with ages,pathological type,degree of differentiation and clinical stage.⑥WWOX protein expression had negative correlation with Bcl-2 protein expression in lung cancr tissues.Conclusion WWOX protein expression in lung cancer was lower than that in adjacent normal lung tis-sue,Bcl-2 protein expression in lung cancer tissues was higher than that in adjacent normal lung tissue.WWOX protein ex-pression had negative correlation with Bcl-2 protein expression in lung cancer tissues.

Explore the expression of FHIT,WWOX and MDR1 gene in nasopharyngeal carcinoma / 国际检验医学杂志

Objective To investigate the expression of FHIT,WWOX and MDR1 gene in nasopharyngeal carcinoma and FHIT and WWOX mechanism of inactivation.Methods Real-time PCR was used to test FHIT,WWOX and MDR1 gene′s mRNA expression in 89 nasopharyngeal carcinoma patients (experimental group) and 61 inflammatory patients (control group).Q-MSP was used to test the FHIT and WWOX promoter methylation status.Denatured polyacrylamide gel electrophoresis was used to test the LOH of FHIT and WWOX gene.Results (1)The three genes′ mRNA expression were different between experimental group and control group (P<0.05).After grouped according to the histological type and clinical stages,the expression of FHIT and WWOX mRNA between the patients with serious illness or poorly differentiated squamous cell carcinoma and mild cases or highly differentiated squamous cell carcinoma were significantly different in the experimental group,the difference is statistically significant (P<0.05)Meanwhile,the FHIT and WWOX mRNA expression had statistical association with the clinical stage and histological type(r=-0.731,P=0.000;r=-0.816,P=0.000;r=-0.626,P=0.000;r=-0.536,P=0.001).The MDR1 mRNA expression was different between poorly and highly differentiated squamous cell carcinoma (P=0.021),which was statistical associated with the histological type (r=-0.697,P<0.001).(2)The degrees of FHIT and WWOX promoter methylation in the experimental group was higher than those in the control group,the difference was statistically significant (P<0.05);Also,the expression of FHIT and WWOX mRNA were closely related to the degree of promoter methylation(r=-0.689,P=0.000;r=-0.594,P=0.000).(3) In the experimental group,there were 39 cases (43.8%) of LOH in the FHIT gene,and 42 cases (47.2%)of the WWOX genes were significantly higher than those in the control group (4.9% and 3.3%),the difference was statistically significant (P<0.05).The FHIT and WWOX gene mRNA were negatively correlated with the loss of heterozygosity(r=-0.239,P=0.049;r=-0.364,P=0.013).Conclusion Promoter methylation is the main reason for the down-regulation of FHIT gene and WWOX gene expression in nasopharyngeal carcinoma patients,which may be the main reason for the occurrence and development of nasopharyngeal carcinoma.The higher expression of MDR 1 mRNA is statistical association with the histological type.

Effect of Fragile Site WWOX Gene on Regulating Proliferation of Human Gallbladder Cancer Cells in Vitro / 昆明医科大学学报

Objective To explore the effect and mechanism of fragile site WWOX gene on regulating proliferation of gallbladder cancer cells in vitro. Methods The pcDNA3.0 - WWOX recombinant plasmid which was previous successfully built was transfected to GBC-SD cells and empty carrier by liposome medium. Liposome and GBC-SD were served as the negative control and the blank control,respectively. After 48 hours transfection, inverted microscope was used to observe the changes of gallbladder cancer cells' morphology,MTT and BrdU were used to detect the proliferation level of gallbladder cancer cells,and flow cytometry instrument was used to detect the change of the cell proliferation cycle. Results The results of inverted microscope shown: the number of GBC-SD cells in pcDNA3.0-WWOX group decreased significantly,the suspension cells and cell debris increased,while cells in the vector control,NC and Mock groups were in normal proliferation state. MTT test showed the proliferation levels of GBC-SD cells in pcDNA3.0-WWOX group was lower than those in the control group in 24 h,48 h,72 h,96 h and 120 h,and the differences were statistically significant(P 0.05). Conclusion The overexpression of WWOX gene in vitro could effectively inhibit the proliferation activity of gallbladder cancer cells. WWOX might participate in the development of the malignant biological behavior of gallbladder cancer cells. It is expected to become a new potential target for the gene therapy to gallbladder cancer.

RUNX2 and WWOX genes as molecular biomarkers and candidates for targeted therapy in Egyptian patients with primary conventional osteosarcoma

Background: The conventional osteosarcoma (OS) is the commonest primary malignant, bone tumor with complex genomic profiles and poor survival. Runt-related transcription factor 2 (RUNX2) and WW domain containing oxidoreductase (WWOX) genes are implicated in normal osteogenesis as well as in the development of primary conventional OS. Methods: We retrospectively assessed protein and RNA expression of the RUNX2 and WWOX genes by quantitative real time PCR (qPCR) and immunohistochemistry (IHC) in 80 cases of primary OS and 20 normal control (NC) subjects. Proteins and RNA expression levels of both genes were correlated to clinico-pathological features of the patients, progression free and overall survival (PFS& OS) rates. Results: In OS, RUNX2 protein was detected in 72/80 (90%) cases compared to 4/20 (20%) NC samples (p. < 0.001) and RUNX2-RNA was up regulated (up to 103.2 folds) in 60/80 (75%) (p = 0.01). WWOX protein and RNA (up to 7.2 folds) were detected in all NC samples but in 24/80 (30%) and 20/80 (20%) OS cases; respectively (p. < 0.001 for each). The concordance between the RNA and protein expressions for RUNX2 and WWOX was significantly high (X_trend

Expression of WWOX and C-JUN in keloid / 中华医学美学美容杂志

Objective To study the expression of WWOX and C-JUN in keloid and to approach their role and mechanism in the pathogenesis of keloid.Methods Immunohistochemical SP methods were used with computer pathological image analysis.Western blot and reverse transcription polymerase chain reaction (RT-RCR) were performed to detect the expression of WWOX and C-JUN in keloid and normal skin with statistical analysis.Results In keloid,the expression of WWOX protein was located in the cytoplasm of fibroblasts,and the expression of WWOX protein and its mRNA decreased,with significantly statistical difference (P＜0.05) compared to normal skin in the control group; the expression of C-JUN protein was located in the cell nucleus and cytoplasm of fibroblasts,with increased expression of C-JUN protein and its mRNA,with significantly statistical difference (P＜0.05) in comparison to normal skin in the control group.The expression of both was negative correlation (r=-0.626,P＜0.01).Conclusions Both WWOX with low expression and C-JUN with high expression are keloid-related genes,having significantly negative correlation between them,which may be one of the mechanisms for the keloid formation.It indicates that the WWOX protein may be an inhibitory factor to the expression of C-JUN protein,and the genes may play a major role in the pathogenesis of keloiod through fibroblasts.

Abnormal Expression of Tumor Suppressor Gene WWOX in Human Benign and Malignant Pleural Effusion / 中国肿瘤临床

Objective: To detect the abnormal expression of WWOX (WW domain containing oxidoreduc-tase) exons 6-8 at mRNA level in human benign and malignant pleural effusion and to investigate the role of loss of WWOX exons 6-8 in the development of malignant pleural effusion. Methods; Deletion of WWOX ex-ons 6-8 mRNA transcript was analyzed by reverse transcriptase-PCR technology. Results: Of the 56 malig-nant pleural effusion samples, 39 showed loss of WWOX exons 6-8 mRNA transcript (69.6%). This deletion was not detected in the 20 benign pieural effusion samples. Conclusion: WWOX gene may play an important role in the develepment of malignant Neural effusion, Detection of WWOX exons 6-8 mRNA may serve as a candidate molecular target for treatment of malignant pleural effusion and can be a promising index in differen-tial diagnosis of benign and malignant pleural effusion.

WWOX gene expression and aberrant CpG island hypermethylation of WWOX gene in breast cancer / 中华普通外科杂志

Objective To evaluate the expression of WWOX gene in breast cancer and its relation to hypermethylation of WWOX gene CpG island. Methods The expression of WWOX protein was evaluated by immunohistochemical staining in breast cancer cell line and breast cancer tissues.Methylation specific PCR(MSP)was used to check whether it Was methylated in the promoter and exon 1 of WWOX.Results Deletion in the WWOX expression was observed more frequently in invasive breast tumors,(32.2%)than normal breasttissues(5.4%)(P＜0.01).20.3% of premenopause eases were completely negative for WWOX expression compared to 57.2% of postmenopause breast carcinomas(P＜0.01).Furthermore,23.1% of Stage Ⅰ(6/26),28.6%of Stage Ⅱ(10/35),46.2%of Stage Ⅲ(12/26)tunlors showed negative WWOX protein expression.In breast cancer specimens.methylation rate of promoter and exon 1 CpG island of WWOX gene was 55%and 45%respectively.Whereas.WWOX CpG islands of normal mammary tissues were completely unmethylated in all cases.CpG islands of WWOX promoter and exon 1 were methylated in MDA-MB-231 cell line but not MCF-7 cell.Conclusions Some deletion in WWOX expression is common in breast cancer and methylation of WWOX DNA CpG islands plays a crucial role in the silence of WWOX.WWOX may play a role in the carcinogenesis and development of breast cancer.

Deletion and Mutation of WWOX Exons 6-8 in Human Non-small Cell Lung Cancer / 华中科技大学学报（医学）（英德文版）

To examine the deletion and point mutation of WWOX (WW domain containing oxidoreductase) exons 6-8 in human non-small cell lung cancer and their possible relationship with pathological stages, tumor tissues and the corresponding normal tissues were obtained from 44 Chinese patients who had undergone surgery for non-small cell lung cancer. RNA was extracted from each sample and deletion and mutation of WWOX exons 6-8 were analyzed by RT-PCR and DNA sequencing. Our results showed that 28 of 44 (63.6 %) lung cancer samples showed loss of WWOX exons 6-8 transcript and the deletion was detected in only 3 of 44 (6.8 %) corresponding adjacent normal tissues (P＜0.05). The transcript sequencing analyses of the 16 lung cancer samples without transcript loss of WWOX exons 6-8 revealed no difference from the sequence of GenBank. Moreover, the deletion of WWOX exons 6-8 was significantly higher in the smokers when compared with the non-smokers. It is also higher in the men and squamous carcinomas than in women and adenocarcinomas (P＜0.05). The deletion, however, was not found to be associated with pathological stages of the tumors. Our study documented a high incidence of deletion of WWOX exons 6-8 in non-small cell lung cancer in Chinese patients and suggested that the frequent loss of WWOX exons 6-8 might play an important role in the tumorigenesis of non-small cell lung cancer in Chinese. WWOX exons 6-8 may serves as a candidate molecular target of smoking carcinogenesis, and point mutation is not a predominant way of alteration of WWOX exons 6-8.

WWOX gene expression in non-small cell lung cancer and its clinical significance / 西安交通大学学报(医学版)

Objective To study the expression of WWOX gene in non-small cell lung cancer and its significance. Methods WWOX protein expression was evaluated by immunohistochemistry in 81 NSCLC patients(50 squamous cell carcinomas,31 adenocarcinomas and 20 adjacent normal lung tissues),and correlation with histopathologic(histotype,grade,tumor-node-metastasis,stage) and clinical characteristics was studied. Results WWOX expression was absent/reduced in 72.8% of NSCLC,whereas it was normal in 80.0% of adjacent normal lung tissues.WWOX expression was strongly associated with tumor histology and histologic grade(P

Expression of FHITmRNA and WWOXmRNA in human breast cancer and their clinical significance / 中国普通外科杂志

Objective To investigate the expression of FHITmRNA and WWOXmRNA in human breast cancer tissues and its relation to clinicopathological and other molecular parameters. Methods With reference to the expression of ?-actin,the expression of FHITmRNA and WWOXmRNA was determined by reverse (transcription)-polymerase chain reaction(RT-PCR) in 51 breast cancer and adjacent breast tissue, and (semi-quantitative) analysis of band densities was performed. The protein expression of estrogen receptor(ER), progesterone receptor (PR), Her-2 gene in the 51 breast cancer lesions was detected by (immunohistochemical) method. Results FHITmRNA and WWOXmRNA expression was significantly different in 54 breast cancer tissue compared to adjacent breast tissue (P0.05); of FHITmRNA and WWOX mRNA was related to axillary lymph node metastasis (P

A study on expression changes of a tumor suppressor WWOX in human lung adenocarcinoma cell line A549 / 中国癌症杂志

Purpose:To detect the abnormalities of WWOX(WW domain containing oxidoreductase) gene in human lung adenocarcinoma cell line A549. Methods:Deletion of WWOX exons 6-8 transcript was analyzed by reverse transcriptase-PCR technology; loss of heterozygosity (LOH) of WWOX gene was analyzed by PCR-based assays for dinucleotide repeat polymorphisms technology. Aberrant expression of WWOX protein was analyzed by western blot. Results:A549 cells samples showed loss of WWOX exons 6-8 transcript.This deletion was not detected in normal primary cultured human bronchial epithelial cells samples.Three microsatellites(D16S3029、D16S3096、D16S504)did not have LOH in the normal primary cultured human bronchial epithelial cells samples, but D16S2029 and D16S3096 were all found to have LOH in A549 Cells samples. We further observed that expression of WWOX protein was significantly lower in A549 cell samples compared to the normal primary cultured human bronchial epithelial cells samples. Conclusions:WWOX gene may be important during tumorigenesis in lung adenocarcinoma cancer.Deletion of exons 6-8,LOH and aberrant expression of protein are all modes of WWOX gene inactivity in lung adenocarcinoma cancer.

Study on modulation for immunosupperssion by WWOX in Lewis lung cancer cells / 中国免疫学杂志

Objective:To investigate the mechanism of WWOX regulating immunosuppression in Lewis lung cancer cells.Methods:Transfected pCDNA4.0/myc-WWOX plasmid was transfected into Lewis lung cancer cells to detected the effect of cell supernatant on generation of lymphocytes by MTT method,and mRNA expression of immunosuppression factors by semi-quantitative RT-PCR.Function change of NK,CTL to kill tumor and lymphocte proliferation were analyzed by MTT and tumor volume.Results:The suppression of the supernatant to generation of lymphocytes was present compared with control group(P