Screening Of Genetic Variant Genes In Malignant Ovarian Germ Cell Tumors

Malignant ovarian germ cell tumors are rare but aggressive gynecological malignancy,accounting for about 3%of ovarian malignant tumors.It usually occurs in children and young women,which has a serious impact on the reproductive function and living quality of patients.Despite the cure rate of early malignant ovarian germ cell tumors is high,and the cure rate of advanced patients can also reach 75%,but the treatment effect is not good after recurrence,and the mortality rate is high.At present,the pathogenesis of malignant ovarian germ cell tumors is largely unknown.In recent years,studies on gene mutations in malignant ovarian germ cell tumors have shown that germline mutations may be an early molecular event in the occurrence of the disease.Therefore,further study of germline mutation in malignant ovarian germ cell tumors may helpful to understand the cause of this disease and clarify its pathogenesis,and provide a new basis for the diagnosis and treatment of malignant ovarian germ cell tumors.In this study,we used Illumina Nova Seq 6000 platform to performed whole exome sequencing in peripheral blood of patients with malignant ovarian germ cell tumors.The copy number variation located gene OCT4which screened by our sequencing was detected by real-time fluorescence quantitative PCR.We analyzed the relationship between OCT4 protein expression and clinicopathological features of malignant ovarian germ cell tumors.Our study provides a novel insight for the genomic characteristics of this rare malignancy of ovary,and provides a theoretical basis for further research,diagnosis and treatment of malignant ovarian germ cell tumors in the future.Part one Whole exome sequencing reveals potential germline mutations and bioinformatic analysis in malignant ovarian germ cell tu-morsObjective:The next-generation sequencing technology was used to analyze the potential germline mutations in the peripheral blood DNA of patients with malignant ovarian germ cell tumors,and to find genetic susceptibility genes related to the occurrence of malignant ovarian germ cell tumors and explore the possible genetic mechanisms preliminarily.Methods:Using the Illumina Nova Seq 6000 platform,whole exome sequencing was performed on the peripheral blood DNA of 60 patients with malignant ovarian germ cell tumors.Germline mutation were screened and analyzed by preliminary bioinformatic analysis,including GO and KEGG pathway.Results:A total of 243 mutation sites and copy number variations in malignant ovarian germ cell tumors were detected.The functional analysis of the genes where the copy number variation regions located showed that the enriched GO entries mainly focus on positive and negative regulation of gene expression,negative regulation of cell differentiation,T cell activation of immune response,G protein coupled receptor signaling pathway,positive regulation of cell proliferation,negative regulation of apoptosis process,transcription negative regulation,T cell aggregation and other biological processes.In terms of pathway mainly focuses on signaling pathways regulating pluripotency of stem cells,RIG-I-like receptor signaling pathways,Toll-like receptor signaling pathways,NF-kappa B signaling pathway,natural killer cell-mediated cytotoxicity,autoimmune diseases,graft versus host disease and other pathway.Conclusion:There were 243 potential germline mutation sites and copy number variations in malignant ovarian germ cell tumors.Bioinformatic analysis of the genes with copy number variation regions found that copy number variations may be related to the pathogenesis of malignant ovarian germ cell tumors.Part two Detection of OCT4 gene copy number variation in malignant ovarian germ cell tumorsObjective:We selected the gene OCT4 whose copy number deletion and amplification region frequency were greater than 10%,which was screened out by whole exome sequencing,indirectly confirmed the results of whole exome sequencing by detecting the copy number variation of its exon region.Methods:Real-time fluorescent quantitative PCR was used to detect the copy number variation in exon of OCT4 gene in peripheral blood DNA of 62malignant ovarian germ cell tumors patients.Results:Real-time fluorescent quantitative PCR technology was used to detect the copy number variation of the OCT4 gene exon.The results showed that the OCT4 gene exon copy number of malignant ovarian germ cell tumors was not significantly different from control group.The stratified analysis showed that the copy number in dysgerminoma is lower than that of non-dysgerminoma.Conclusion:Real-time fluorescent quantitative PCR technology was used to detect the copy number variation of OCT4 gene,and there was no copy number variation in exon regions of OCT4 was found in patients with malignant ovarian germ cell tumors.Part Three The expression of OCT4 in malignant ovarian germ cell tumors and the relationship between OCT4 and clinico-pathological characteristicsObjective:Study the expression of OCT4 gene in patients with malignant ovarian germ cell tumors and analyze the relationship between gene expression and clinical pathological characteristics of patients.Methods:Immunohistochemistry was used to assess the protein expression level of OCT4 in 48 patients with malignant ovarian germ cell tumors.The relationship between OCT4 protein expression and clinicopa-thological characteristics was analyzed based on the clinical data of the patients.Results:The results of immunohistochemical assay showed that 9 of 11dysgerminoma were positive for OCT4,the positive rate was 81.82%,but only16 of 37 non-dysgerminoma were positive,positive rate was 43.24%.Statistical analysis showed that compared with non-dysgerminoma,the expression level of OCT4 protein in dysgerminoma was significantly higher(?~2=5.06,P<0.05).The expression level of OCT4 protein in malignant ovarian germ cell tumors was closely related to histological type and the difference was statistically significant(P<0.05),but there was no relationship to the age,FIGO stage,histological grade and tumors size(P>0.05).Conclusion:Compared with non-dysgerminoma,the expression of OCT4 protein was higher in dysgerminoma.The expression level of OCT4protein in malignant ovarian germ cell tumors was related to histological type,but there was no relationship to age,FIGO stage,histological grade and tumor size.In summary,this study performed whole-exome sequencing and bioin-formatic analysis in malignant ovarian germ cell tumors.Verified the copy number variation of OCT4 gene screened out by sequencing.Analyzed the expression OCT4 protein in malignant ovarian germ cell tumors and the relationship between OCT4 protein expression and the clinicopathological characteristics.The present study revealed that:1)There were 243 potential germline mutation sites and copy number variations in malignant ovarian germ cell tumors.Bioinformatic analysis of the genes with copy number variation regions found that copy number variations may be related to the pathogenesis of malignant ovarian germ cell tumors.2)There was no copy number variation of OCT4 gene exons was found in patients with malignant ovarian germ cell tumors.3)The expression of OCT4 protein was higher in dysgerminoma and the expression level of OCT4 protein in malignant ovarian germ cell tumors was related to histological type,but there was no relationship with age,FIGO stage,histological grade and tumor size.