The Study Of Tumor MtDNA Mutations Based On The Next Generation Sequencing Technology

BACKGROUND: Mitochondria are the core of intracellular energy metabolism and are the only organelles in multicellular animals that have genetic material out of cell nucleus.Mitochondrial DNA(mtDNA)is a covalently attached,double-stranded circle DNA that is present in multiple copies in cell and encodes the products to assemble oxidative respiratory chain.Mitochondrial DNA mutations have significant effects on cellular energy metabolism.Since Warburg proposed the Warburg effect in 1956,mitochondrial DNA mutations in tumor have attracted considerable attention.On the one hand,somatic mutations of mitochondrial DNA have been found in almost all kinds of malignancies,and the proportion of missense mutations and high-risk mutations is much higher than that in the normal population.Therefore,many researchers believe that the somatic mutations of mitochondrial DNA in tumor tissues are positively selected and participate in the development of tumors,and some point mutations that affect the proliferation and metastasis of tumor cells were identified.On the other hand however,recent studies haveshown that the somatic mutations of mitochondrial DNA in tumors are caused by incorporation error of polymerase γ during mtDNA replication,and these mutations are neutral rather than the result of evolutionary selection.OBJECTIVE: 1.To establish a method for detecting somatic mutations of mitochondrial DNA in tumor patients based on the next generation sequencing technology;2.To explore the selection model of somatic mutations of mtDNA in tumor tissues;3.To observe the mtDNA mutation patterns of inflammation and tumor from HBV-HCC patients,to explore the possible role of mtDNA mutations in HBV-related hepatocarcinogenesis.METHODS: 1.Prepare the whole genome sequencing library with double barcodes;capture mitochondrial DNA from whole genome sequencing libraries with the homemade mtDNA capture probe;detect tumor mitochondrial DNA somatic mutations with homemade bioinformatics analysis pipeline.2.Tumor mitochondrial DNA somatic mutations were extracted from the literatures and tumor mutation databases(TCGA,ICGC).Mutations from HBV-HCC patients were combined with public data,to analyze the selective pattern of mtDNA mutations in tumor.3.Use above methods to complete the sequencing of the mtDNA in blood,hepatitis tissues and tumor tissues from 156HBV-HCC patients and analyze the mutation patterns in hepatitis tissues and tumor tissues.RESULTS: 1.The mutation detecting method can detect the mitochondrial DNA somatic mutations in tumor patients accurately and effectively;2.The negative selection on mitochondrial DNA somatic mutations in tumor were generally released,but selective pressure on the regions coding complex V(ATP6,ATP8)and tRNA were significantly higher than that on other regions,especially on the ATP8 gene and the loop and variable regions of tRNA.3.Mutations that generating Thr may be under positive selection,while the mutations generating terminator may be negatively selected.4.In hepatitis tissues,the mitochondrial DNA somatic mutations with high-risk have low heterogeneity levels,vice versa;but this phenomenon was not observed in tumor tissues;5.The T>C mutation in np72 presented in hepatitis tissues of multiple patients,while were rare in tumor tissues,implied the suppression role of this mutation in tumor development.CONCLUSION: Mitochondrial DNA somatic mutations in tumor are under site-specific selection.Mutations in tumor tissues have a greater effect on gene function than mutations in hepatitis tissues;np72 mutation may suppress the development of tumor.