Study On The MtDNA Mutation In Diabetics With The Age Of Onset Younger Than 45a

Objectives: In recent years, rapidly increased incidence and more affected youngers have emerged in many diabetic epidemiologiacal studies. More researchers have taken great interest in the etiological studies on early onset diabetes(EOD), and it has been shown that defective mtDNA is implicated in the development of diabetes. Our main aim is to explore the relationship between mtDNA mutations (including np3243, np3316, np3394, np 3426, np 12026 ) and EOD in tianjin, and to improve the research methods.Methods: According to the diagnostic criteria of ADA in 1997, 348 non-related diabetes whose age-onset was and below 45a were randomly recruited, as well as 207 non-related and non-diabetes control subjects enrolled. The clinical biochemical indexes were collected. Total genome is extracted conventionally in peripheral leucocytes from all the participants, and plasma free DNA isolated similarly from mere 20 cases, half of which is positive mutant. PCR-RFLP techniques were performed to screen 5 mtDNA mutations in the hot spots including ND1, ND4 and tRNA Leu(UUR). Cloning technique was applied to differentiate the mutant mtDNA from a large excess w-t mtDNA since the low heteroplasmic level of A3243G mutation in peripheral leucocytes. The final identification of these mutations were depended on reliable directly sequencing of PCR production or plasmid DNA. Through great effort, 4 pedigrees were acquired to analyze the clinical characteristics.Results: In present study, we knew that in diabetic group we detected 17 cases with mtDNA ND4 A12026G mutation with the frequency of 4.9%, no significantly difference compared to control subjects; 5 cases with NDl np 3394mutation and 4 cases with np3316, one subject with np3426 ,the frequencies of which is 1.4%, 1.2%, 0.3%, respectively , much lower than others' results, but no significant differences between diabetes and control subjects; by the contrast, the frequency of A3243 G mutation is 0.6% in this population.On the other hand, plasma free DNA extracted successfully from 20 samples which had the identified mutation in the peripheral leucocytes; furthermore, mtDNA mutation can be detected by PCR-RFLP. Through gradually diluted plasmid DNA after cloning of A3243G mutation, and followed by its PCR-RFLP, we could detect the heteroplasmy of 12%, 8% by conventional agarose electrophoresis and page, respectively.Results from the analysis of pedigrees seemed to suggest that mtDNA ND4 mutation might be associated with autoimmune diseases such as hyperthyroidism; but mtDNA ND1 and ND4 mutations did not co-segregate with diabetes; the proband of mtDNA tRNA^Leu(UUR) A3243G had typical picture of MIDD, while the other carriers in his family had heterogeneous presentation-Conclusion: The special diabetic population with the age-onset of younger than 45 a haboured many mtDNA mutations, which may be involved in the pathogenesis of EOD. Our and others' results suggested that mtDNA ND1mutations were tightly correlated with aging instead of EOD. The famous tRNA Leu(uuR) A3 243 G mutation did exist in the population studied, and its low heteroplasmic level will decrease with aging in mitotic tissues. What's more, the plasma can be an alternative resource of sampling tissues with the advantages of its quickness and economy, compared to conventional method.