The Association Study Between Mtdna Variations And Lung Cancer Of Yunnan Province

Objectives:Association study between mtDNA variations and lung cancer from Yunnan provinceMethods:To investigate the somatic mutation spectrums in lung cancer patients from Yunnan province and test the potential relationship between the mtDNA variations, we screened the mtDNA somatic mutation spectrums in lung cancer patients by analyzing mtDNA control region variations and the entire mtDNA genomes of the cancer patients. We extracted the genome DNA of the79Chinese lung cancer patients (including237samples) with the primary cancerous, matched paracancerous normal and distant normal tissues for the same patients by using the standard phenol/chloroform method. The whole mtDNA D-loop region (including its HVRI, â…¡ and â…¢) of each sample was amplified with one pair of primers and sequenced the2PCR primers and4inner primers. We compared the mtDNA sequences of Chinese lung cancer patients and revised Cambridge reference sequence. Ten patients (including30samples) were selected for sequencing the entire mtDNA genomes, which were amplified with2pairs, and sequenced with more than66PCR and inner primers. The entire mtDNA genomes were aligned with the revised Cambridge reference sequences (rCRS). The phylogenetic tree of the10lung cancer patients was constructed manually. The Germ line variations and somatic mutations were identified by comparing with published entire mtDNA genomes and rCRS. The frequencies of the somatic mutations were calculated, and the position of the somatic mutations on the entire mtDNA genomes was analyzed. In addition, its potential biological characters were speculated by comparing with somatic mutations from mitomap (http://www.mitomap.org/MITOMAP).Results:Based on the mtDNA control region sequences, all the mtDNAs for the237samples of79lung cancer patients were assigned to the specific haplogroups with the aid of the updated phylogenetic trees of East Asian, Southeast Asian and South Asian, including haplogroups A, B, C, D, F, G, H, M*, M31, M7, M71, M9, N9, R9, R11, U and Z. We calculated the haplogroup frequency of the lung cancer patients, and we found that the South of East Asia prevalent haplogroups accounted a higher frequency in Yunnan lung cancer populations, and our results indicated that the investigated lung cancer population have the obvious southern of China and Southeast Asian population character for sharing the southern of China and Southeast Asian prevalent mtDNA haplogroups, such as B, F, M7,M9,R9and R11. To further investigate whether there were lung cancer sensitive haplogorups, which may affect the maternal components of south Chinese lung cancer population, the principle components analysis were performed based on their mtDNA haplogroup frequency, our results indicated that the lung cancer population has the similar clustering pattern with the healthy populations from Yunnan, which implied that there would not have the significant sensitive haplogroups that have changed the maternal components of lung cancer population, which was further supported with the X2result by taking all the health populations as a group. Further, we identified the somatic mutations of the lung cancer patients by comparing the mutation spectra between tumor tissue, matched paracancerous normal tissue and peripheral blood. We found24somatic mutations of20patients in the79lung cancer patients, which accounted the25.03%(20/79) of the investigated patients, the C tandem repeat region detected in the mtDNA control region303-309, which accounted70%(14/20) of the somatic variations, and which accounted17.72%(14/79) of all investigated patients, which suggested that mtDNA control region mutations can be used as an potential diagnostic marker for Chinese lung cancer patient. However, it was still uncertain on the role for the somatic mutations detected in Chinese lung cancer patients, which need further study in future.To detect the somatic mutations and character its spectrum in Chinese lung cancer patients at the whole entire mtDNA genomes level, in this study, we sequenced the whole mitochondrial DNA (mtDNA) genomes for ten lung cancer patients including the primary cancerous, matched paracancerous normal and distant normal tissues. By analyzing the30whole mtDNA genomes, eight somatic mutations were identified from five patients investigated, which were confirmed with the cloning and sequencing of the somatic mutations. The totally eight somatic mutations were found among5individuals, including178,294,309+C,1149,8920,15635and16293, with the309+C appeared twice. By comparing the distribution patterns, we found that three individuals have the somatic mutations in the control region and two individuals have the somatic mutations in the coding regions, which located in the genes s-rRNA, ATP6and cytB; our results indicated that the mtDNA control region was much more sensitive than coding region. In addition, the heterogeneity was the main character of the somatic mutations in Chinese lung cancer patients. The phylogenetic analysis of the30entire mtDNA genomes of the10lung cancer patients was performed. We found that there was not any somatic mutation taken place at the positions on defining haplogroups. Further potential disease-related screening showed that, except the C deletion at position309showed AD-weakly associated, most of them were not disease-related. Although the role of aforementioned somatic mutations was unknown, however, considering the relative higher frequency of somatic mutations among the whole mtDNA genomes in our lung cancer patients, it hints that somatic mutations of the whole mtDNA genomes could be used as a diagnostic biomarker for detection of lung cancer to some extent.Conclusions:1) The population of lung cancer patients from Yunnan has significant southern China population characters;2) The lung cancer population from Yunnan did not specific clustering pattern with health ethnic groups from Yunnan province, and there was mtDNA haplogroups has significant difference between lung cancer group and health groups;3) We totally detected24somatic mutations from79Yunnan lung cancer patients according to the mtDNA control region sequences, and positions309has relative higher frequency than other positions, which implied that detecting the somatic mutations from the mtDNA control region can serve as a potential molecular marker on diagnosing the lung cancer;4) mtDNA control region have higher somatic mutation rates than mtDNA coding region;5) Heterogeneity was the main characters of Yunnan lung cancer patient;6) No somatic mutational events were detected at the haplogroup specific site;7) Comparing the mtDNA spectrum in lung cancer tissue and the blood can serve as a useful tool for the Chinese lung cancer diagnostic to some extent.