The Significance Of Mitochondrial DNA D-loop Mutations In Human Oncocytoma

Objective:1. The paraffin embedded tissues (PET) which were fixed by formalin are the most common sample in pathological subject. Extracting DNA from PET, in one hand, could augment case resource; in the other hand, it could contribute to combine molecular biology with clinical medicine by integrating gene detection and retrospective analysis. The destination of this research was to explore the most optimal method of DNA extracting from PET, and to improve the amplification of long fragments from it.2. As plenty of abnormal mitochondria, oncocytoma was the best intermediary agent in the research of the relation between mitochondrial DNA (mtDNA) and tumorigenesis and tumor development. For further understanding the relationship between mtDNA and tumorigenesis, we investigated the mutations in mtDNA displacement loop (mtDNA D-loop) region of oncocytomas, providing a molecular diagnostic criterion for oncocytomas as well.Methods:1. Twenty normal thyroid tissues of 1999-2009 years which were fixed by formalin and embedded by paraffin were selected from the pathological department of Tianjin general hospital. Three methods that are one step method,phenol-chloroform extraction method and genomic DNA purification kit were employed to extract DNA from samples. Then a segment of mtDNA D-loop region was amplified by traditional PCR and nest PCR respectively.2. In the first part of this experiment, the best method of extracting DNA and amplifying long fragment PCR products from formalin fixed PET had been obtained. In this part, the mtDNA D-Loop region of 20 thyroid or renal oncocytomas and the adjacent normal tissues that were collected from the Tianjin General Hospital were amplified by nest PCR, and then sequenced. In order to find the mutation sites and polymorphisms of mtDNA D-loop region.Results: 1. The quality of DNA extracted from PET by three different methods were as following:the highest average proportionality of OD260/OD280 in them was 1.703±0.086 which were obtained by phenol-chloroform extraction method; the highest average concentration was 23.456±1.854ng/μl which were extracted by one step method.2. To amplifying 644bp of mtDNA D-loop region, the achievement ratio of one step method,phenol-chloroform extraction method and genomic DNA purification kit were 0%,5%,10% by traditional PCR method, but 0%,95%,85% by nest PCR, respectively. The products of nest PCR could be used in sequential experiments and have smooth sequencing peaks.3. Among the twenty oncocytomas and corresponding normal tissues,7(35%) tumor cases and 1(5%) normal case had been detected gene mutations. The difference had statistics significance (P<0.05 by corrected X2 analysis).4. There were 21 heterogeneous mutation sites in the twenty oncocytomas tissues and corresponding normal tissues. Most of the mutations,94.12%, were focus on hypervariable segmentⅠ(HVⅠ,np16024-npl6383). Besides one base deletion mutation, both mutations were base substitution.5. There were no obviously difference between thyroid oncocytomas and renal oncocytomas about gene mutation.6. Simultaneously,191 polymorphisms have been found in the 20 cases (100%), 7-11 per sample medially, and most of the polymorphisms were homogeneous. Every specimen both existed polymorphisms of 73A-G,146T-C,150C-T,263A-G,303-309 C insert,311-315C insert,16223C-T.7. In the 20 oncocytoma cases, the mtDNA mutations had no relation with the age,sex of patient and tumor size via statistical analysis.Conclusions:1. It is the best method to extracte DNA from PET that adopting phenol-chloroform extraction method based on increasing the concentration and active time of protease K. And it is effective to amplify long DNA segments from PET by nest PCR. 2. Through sequencing the mtDNA D-loop region of 20 oncocytomas, we found that the mutation rate in tumor tissues was 35%, in contrast, only 5% in normal tissues. We can come to a conclusion that the mtDNA mutation was closely involved in the abnormal mitochondrial formation and tumorigenesis of oncocytomas.3. On the above, HVⅠwas more important in the tumorigenesis of oncocytoma, on account of the mutations were centralized in HVⅠother than HVⅡor HVⅢ.4. In this experiment, it was further confirmed that tumor progression was the process of homogeneous position convert to heterogeneity, because both mutations were heterogeneous, but most of the polymorphisms were homogenous.5. As the mtDNA mutations had no relations with age,sex and tumor size, which point out that the mtDNA variation would not influenced by objective conditions such as age,sex et al. The mtDNA mutation is a prophase and continuous event in the tumor development.6. The site locating in np 303-309 was not only the replicative promoter of mtDNA high strand, but also the combining site of mitochondrial translation factor A (mtTFA) that was necessary in mtDNA specific transcription. Both samples had one base insert polymorphism in this site, which could induce disorder transcription.7. MtDNA D-loop region, especially HVⅠ, was the predilection site of mutation in oncocytomas. The variations in mtDNA D-loop region could influence the replicative and transcriptive ability of mtDNA; then, induce the mistake of copy number and encoding; finally, result in abnormal mitochondria and tumorigenesis by interact with nuclear DNA.8. The sequence and single nucleotide polymorphism of mtDNA are related with race and area. Every specimen in the experiment both existed polymorphisms of 73A-G,146T-C,150C-T,263A-G,309-310 C insert,315-316C insert,16223C-T. The result could assist to human gene evolution.