Study On Mutations Of Mitochondrial DNA From Human Osteosarcoma Tissues

Background and objective: Mitochondrial genomes (mt-genomes) are, with the exception of viruses, the most economically packed forms of DNA in the whole biosphere. The human mtDNA is a double-stranded circular molecule of 16569bp and contains 37 genes coding for 2 rRNAs, 22 tRNAs and 13 polypeptides. All these genes are organized continuously with no non-coding intervening sequences except for a small segment called displacement-loop (D-loop). The D-Loop region is the major congrol site for mtDNA expression because it contains the leading-strand for origin of replication and major promoters for transcription. Furthermore, mtDNA is known for having high acquired mutation rates. The mutation rate for mtDNA is around 10 times higher than that of nuclear genomic DNA. It is generally accepted that high mutation rates of mtDNA are caused by a lack of protective histones, inefficient DNA repair systems, and continuous exposure to mutagenic effects of oxygen radicals generated by oxidative phosphorylation. Nucleotide positions 16024-16324 and 63-322 located in the D-Loop are termed first hypervariable region(HV1) and second hypervariable region(HVⅡ), respectively. It is known that for the D-loop hypervariable region I (HVI), mutation rate may reach 100–200-fold higher levels than that of nuclear DNA due to two structural properties of the D-loop: First, it is the point of attachment of mtDNA to the mt-membrane and therefore, is heavily exposed to lipid peroxydes; Second, it has a triple strand DNA architecture generated by the synthesis of a short piece of the H strand which is highly sensitive to oxidative damage due to its single strand condition. Over the last decade there has been recognition that defects of the mitochondrial genome are important causes of diseases including ageing, cancer, neurodegeneration, myopathy, cardiomyopathy and diabetes. Tumor cells generally carry a large number of DNA changes. Investigations until recently have concentrated mainly on the alteration s of nuclear DNA in various cancers. However, the alterations of mtDNA in tumors have received much less attention, despite the fact that the mutational rate of mtDNA is at least 10 times higher than that of nuclear DNA. The recently recognized involvement of mitochondria in apoptosis has stimulated interest in examining the potential role of mtDNA mutation in the development and progression of cancers. The occurrence of somatic mitochondrial DNA mutations in various human cancers has recently been reported. Among primary bone tumors, osteosarcoma, mostly found in the adolescent age group, has the highest incidence with the incidence of 20%. It has been found that the amplification, rearrangement and point mutations of oncogenes and anti-oncogenes were closely related with the development and progression of osteosarcoma. However, there have been few reports about variations of mt-genomes in osteosarcoma. In this study, we analyzed the sequences of mtDNA D-loop, ATPase6 gene and Cytb gene and aimed to know the variations of mtDNA in osteosarcoma tissues and to inquire into the relationship between mutations of mitochondrial genome and carcinogenesis.Methods: In this study ,we utilized PCR-RFLP and DNA sequencing as well as light microscopy and electronic microscopy to explore the mutations of D-loop, ATPase6 gene and Cytb gene of mtDNA in human osteosarcoma tissues.Results:mtDNA D-loop mutations were detected in 100%(20/20) osteosarcoma tissues. 59 sequence variants, which were 51.6% of the total sequence variants in D-loop region, were found in HVⅠ located in nucleotide positions 16024-16324 of D-loop; and 15 sequence variants, which were 31.5% of the total sequence variants in D-loop region, were found in HVⅡ located in nucleotide positions 63-322 of D-loop. That is to say, 83.1% sequence variants were located in the mtDNA D-loop hypervariable regions.mtDNA ATPase6 gene mutations were found in 65%(13/20) osteosarcoma tissues. 23 mutations were found, 4 of which were frameshift mutations and 19 of which were base substitu...