| Breast cancer is the most common malignant tumor among females worldwide. Most of breast cancer originates from the epithelial cells of lobules, while a few of which originates from non-epithelial tissue of the breast. However, the tumor of breast carcinosarcoma occasionally could been found. According to the data from Globocan, newly diagnosed cases of breast cancer in women worldwide was up to 1.38 million in 2008, which accounted for 23% of overall female malignant tumor incidence and threaten to women’s health severely. In China, more than 1.2 million people die of cancer every year. Meanwhile the breast cancer has become the highest rate in women, ranking sixth in the cause of cancer death. Breast cancer in China accounts for 12.2% and 9.6% in newly diagnosed cases and cancer deaths in the world, respectively. The new breast cancer cases have an equal number in China and American, although the number of cases is reduced by half in Europe. Over the past 30 to 40 years, the cancer mortality rate in urban was much higher than that in rural areas in China. If that rise continues, the new breast cancer cases may reach 2.5 million and the incidence rate will be doubled in 2012. Therefore, Chinese government carried out the project of cancer early detection, diagnosis and treatment in city. It was planed to investigating the risk factor and the high-risk population, screening cancers, and evaluating health economics of the top five types of high incidence (lung cancer, colorectal cancer, upper gastrointestinal cancer, breast cancer and hepatocellular carcinoma) in the 14 provinces of nine areas.Breast cancer is a systemic disease, which can widespread at early stages. The recurrences and metastasis often lead to treatment failure. The majority mode of tumor metastasis is hematogenous metastasis, which can generate metastases in all tissues and organs under certain conditions. Doctors may overlook the tumor when it is less than 1 cm. But Some reports suggested the circulating breast cancer cells can be found in the blood although the tumor was less than 1 mm. It is significant to predict the risk factors and prevent against breast cancer by early diagnosis.Engaged in the study of breast cancer, many scientists try to find the pathogenesis of breast cancer with a variety of methods. Some states with high incidence of breast cancer have made huge progress in the risk factors. According to the investigation and analysis of the specific population, researchers have determined the risk factors for breast cancer. As a result, physicians could predict the risk of developing breast cancer by individual and quantized information. It has become an important part of cancer screenings.Breast is the target organs of multiple endocrine hormones, such as estrogen, progesterone and prolactin. Estrogen is the main stimulant to breast cell proliferation, while progesterone can increase breast cell proliferation rate. Estrone and estradiol are directly related with breast cancer incidence. The breast cancer incidence rate varies by age. It seldom happens among women before the age 20, while it increases rapidly after that. Women have a high incidence rate when they reaches age 45-50, but the rate continues to rise after menopause for the increasing level of estrone in the elderly. Breast cancer is often associated with early menarche, late menopause, infertility and first labor of full-term age. The incidence risk is 2-3 times of the general population if the first-degree relatives have a history of breast cancer. It is a controversial issue for the relationship of benign breast disease and breast cancer. Most researchers believe the intraepithelial hyperplasia or atypical hyperplasia in the breasr lobular correlate with potential breast cancer. In addition, nutrition, obesity and fat diet can enhance or extend estrogen stimulatant to breast epithelial cells, thus increasing the breast cancer incidence. Breast cancer incidence in North America and the Nordic regions is about four times of that in Asia, Africa and Latin America. The second and third generation of immigrants who migrated from low-incidence to high-incidence countries acquired a high breast cancer incidence, indicating that environmental factors as well as lifestyle are also related to breast cancer.With the breakthroughs of gene research, it is increasingly recognized that mutations correlate well with cancer. Researchers have conducted a lot of research on the mechanism at genetic levels and achieved a lot of results which aregradually used in the diagnosis and prevention of cancer.With the completion of the Human Genome Project and HapMap program and with the application of various high-throughput sequencing technology and gene chips, the breast cancer susceptibility genes can be discovered by genome-wide association study. In the 1980s, epidemiologist Mary-Claire King found that a gene on the chromosome 17 may be the main cause of breast cancer with the analysis of familial breast cancer. In 1994, the US company Myriad Genetics successfully found BRCA1 gene on this basis. The same year the company took advantage of the samples of high incidence of familial breast cancer from two families in Iceland and found the BRCA2 gene on the chromosome 13. As so far, hundreds of BRCA gene mutations have been disclosed. However, it is limited to population with a family history of breast cancer, including first-degree relatives with breast cancer before the age 40, first-degree relatives with bilateral breast cancer before the age 50, and two or two more first-degree relatives with breast cancer. The involving methods include high-frequency mutations screening, exon 11 screening and all exons and adjacent introns sequencing monitor. However, breast cancer is a disease involving multiple factors and BRCA can only explain parts of the connection. Therefore, a comprehensive analysis must be applied in jugdment. Meanwhile, it is necessary to discover other related molecular mechanisms of breast cancer, to screen and assess the high-risk population for predicting and protection from breast cancer.A comprehensive analysis of genome-wide association study with a huge sample size and high-throughput sequencing still costs a lot, although the cost of high-throughput sequencing becomes cheaper than ever. Therefore, this study aims at the mitochondrial genome.Human mitochondrial DNA (mtDNA) is a double loop chain of 16.6kb, with light and heavy chains. mtDNA is the only genetic material outside the nucleus in eukaryotic cells, correlating with oxidative phosphorylation. mtDNA contains 13 subunits coding genes for the oxidative phosphorylation system (OXPHOS), including seven subunits in complex Ⅰ (ND1, ND2, ND3, ND4, ND4L, ND5, ND6), a subunit in complex Ⅲ(CytB), three subunits in complex IV (COX1, COX2, COX3) and two subunits in complex V (ATPase6, ATPase8), resptively. mtDNA can also encoded 22 ATP tRNAs and 2 rRNAs for the protein and ATP synthesis. mtDNA has no introns. The only non-coding region is D-loop region (displacement-region), which has been identified as regulatory region of mtDNA replication and transcription, containing replication origin and promoter region. Although mtDNA can self-replicate, most protein in energy production encoded by the nuclear genome (nuclear DNA, nDNA). Therefore, mitochondria are semi-autonomous replication organelles.Over the past ten years, researchers have found mtDNA mutation rate is several times higher than nDNA mutation rate. The mutation types include gene deletions, missense mutations, frameshift mutations and insertions. The structural and functional features lead to the mtDNA becomes more susceptible to carcinogenic exposure. mtDNA mutations can impair normal respiratory function and release high levels of ROS, increasing the risk of tumorigenesis. From the bove data, it is believed mtDNA correlates well with tumorigenesis.Many scholars have confirmed that detection of mtDNA mutations in tumor cells is more simple and reliable than detection of nDNA. Nuclear genetic damage often failed to detection and did not match the expectation because of the interference of the wild-type nDNA. However, it is more reliable to detect mtDNA mutations, because the number of copies of mtDNA are 200 times higher than those of p53 gene.Our study developed the mtDNA genome-wide association analysis to screen high risk germline mutations with the peripheral blood of breast cancer patients. Differing from previous studies using breast tissue for analysis of somatic mutations, germline mutations was adopt, which has a great significance in prediction of breast cancer in high-risk groups, the prevention and early non-invasive diagnosis of breast cancer.Patients with first diagnosed of breast cancer and untreated were assigned to the case group. The control group conducted a comprehensive physical examination and imaging studies to exclude various tumors.118 and 137 peripheral blood samples were obtained respectively from the case group and the control group. Total DNA was extracted and mtDNA was enriched using two stage process and high-throughput sequencing libraries of 200bp in length were constructed sucessfully. The quality of libraries was assessed by determining the concentration and fragment size with Qubit 2.0 and Agilent 2100, then the amount of templates for sequencing was measured. Templates were prepared for high-throughput sequencing with Ion 316 chip in Ion torrent PGM sequencer.Fast QC-3.4.1.1, Assembler-3.4.2.0, Alignment 2.0, coverage Analysis 3.0 software of Torrent Suite4.2 in the Ion Torrent PGM server system were used for quality control and results analysis. First of all, the sequencing data was process with preliminary analysis, and removing of the junction, low quality and polyclonal sequence. Second of all, the original sequence was assembled to abtain, the overall data, the average depth of sequencing, quality information.Then, original data and sequencing report can be exported in fastq format. Last of all, with the cloud-based platform of Fanse2 and reference to Homo sapiens GRCh38 genome (NC012920) sequence alignment, mutation analysis and gene annotation were performed to get a mutation list of each blood sample.The numbers of mutation in each sample were counted. The mutation frequency was calculated, which was derived from mutational samples divided by total samples. Chi-square test was used to analyzed the significance test of difference between the control group and the case group with SPSS 19.0 software. With the OR (Odds Ratio),95% CI (95% Confident Internal) and p-value, the association between SNP and breast cancer was analysed to screen high-risk factor.The samples were eliminated when the filtration rate less than 40%, or mapping rate less than 80%, or the depth of coverage less than 100X or nonuniform coverage. The final valid data comprised 22 items of the control group and 83 items of the case group. The average depth of coverage was over 300X. Sequencing data showed 170 mtDNA mutations in the control group and 393 mtDNA mutations in the case group. 283 mtDNA mutations (232 in the control group and 88 in the case group) had not been described in academic papers. Correlation analysis showed that the 32 mtDNA mutations had a significant difference. They were located in the D-loop region (4/32), RNR2 coding region (1/32), COX1 (8/32), COX2 (3/32), ATP6 (1/32), COX3 (4/32), ND4 (3/32), ND5 (4/32) and CYTB (2/32). According to the relative risk analysis in mtDNA mutations, OR of 27 mutations was less than 1, illustrating these mutation may act as protective factors to breast cancer. The other 5 mtDNA mutations, RNR2-2463 indelA, COX1-6296 C>A, COX1-6298 indelT, ATP6-8860 A>G and ND5-13327 indelA, OR of which were 8.050,4.464,4.464,5.254 and 4.853, respectively, were regarded as risk factors to breast cancer.Encoded by mitochondrion, MT-RNR2 was a 16s rRNA and a member of the MT-RNR family. MT-RNR2 was involved in the assembly of ribosome in mitochondrion. RNR2-2463 indelA may change the structure of MT-RNR2 and affect its binding with other proteins or nucleic acids. As a part of mitochondrial transcription and translation system, MT-RNR2 afunction may directly affect the synthesis of other proteins and the entire mitochondrion.With catalytic function, MT-COX1 was one subunit of cytochrome oxidase, which was synthesized in mitochondrion. Although COX1-C6296T was a nonsense mutation, but we have found that point mutation at C6296T and deletion mutation of 6298T on COX1 always occur simultaneously, and the reasons were unknown. Deletion mutations at 6298T might cause shift mutations on the coding region frame, changing the entire amino acid sequence of the peptide chain. COX1 was an important catalytic subunit of cytochrome oxidase, soits structure modification may directly result in the loss of catalytic function of cytochrome oxidase.MT-ATP6 can encode a subunit of "biological molecular motors", that is ATP synthase. Point mutation at A8860G changed threonine into non polar alanine, inducing the alteration of the tertiary structureand reducing the catalytic activity of ATP.MT-ND5 can encode the fifth subunit of NADH dehydrogenase (complex Ⅰ). ND5-13327 indelA leaded to frameshift mutation in coding region. As a result, missense mutation occured in the entire amino acid sequence of a peptide chain. Changes in the protein secondary and tertiary structure affected the function of NADH dehydrogenase complex.All the 5 mtDNA mutations above were supposed to affect the synthesis of the respiratory chain. The mutual interaction of nDNA and mtDNA may increase the risk of nuclear gene mutation and induce apoptosis. The above-mentioned mutations may play a very important role in breast cancer development.To sum up, different from former researches, our studies showed the relationship between mtDNA germline mutations and breast cancer, five mutations may serve as an indicator in prevention of breast cancer. Adopting peripheral blood samples was easy to operate, which may be applied to non-invasive diagnostic techniques in the future.Although only 255 samples were collected in our study with time limited, high-throughput sequencing of mtDNA had not been reported in China. A complete technical process has been established in this project, but some of the sequencing quality is not ideal. For the relevance and reliability, only 22 items in the control group and 83 items in the case group were used for association analysis. Therefore, it is oblige to collect more samples throughout the country for the mtDNA genome-wide association analysis in further research,and the obtained 5 mutations in this study need further validate in large-scale studies. |
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