Pyrosequencing is an accurate and reliable method for the analysis of heteroplasmy of the A3243G mutation in patients with mitochondrial diabetes.

Human mitochondrial DNA is a circular DNA molecule that encodes some of the proteins required for oxidative phosphorylation. Different mitochondrial DNA genotypes may coexist within a single cell, a condition known as heteroplasmy. An A-to-G transition at position 3243 of mitochondrial DNA (A3243G) can result in maternally inherited diabetes and deafness (mitochondrial diabetes). However, the commonly used methods of PCR restriction fragment length polymorphism and Sanger sequencing are neither sensitive nor reliable enough to detect this low level of heteroplasmy. Here, we developed a quantitative method based on pyrosequencing to analyze the heteroplasmy of the A3243G mutation in leukocyte DNA obtained from 83 persons of 15 unrelated pedigrees with mitochondrial diabetes. The accuracy and reliability of this method were also measured by comparing the results with those from high-resolution melting analysis, Sanger sequencing, and PCR restriction fragment length polymorphism with artificial heteroplasmy standard samples. The results showed that the accuracy of pyrosequencing was much higher than that of the other methods, and the limitation of heteroplasmy detection with this method reached 2%, based on our artificial control studies. An inverse correlation was found between the level of heteroplasmy and the age of the onset in our patients. This result suggested that the heteroplasmy of the A3243G mutation could become a significant prediction index for the onset of mitochondrial diabetes.