title: Pyrosequencing is an accurate and reliable method for the analysis of heteroplasmy of the A3243G mutation in patients with mitochondrial diabetes.
abstract: 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.
authors.affiliation: Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai Jiaotong University, Shanghai; the Key Laboratory of Embryo Molecular Biology,y Ministry of Health of China & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai; and the Department of Endocrinology and Metabolism
authors.forename: Jing-bin
authors.initials: JB
authors.lastname: Yan
authors.affiliation: Key Laboratory of Embryo Molecular Biology, Ministry of Health of China & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai, Shanghai, China.
authors.forename: Rong
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiaotong University, Shanghai, Shanghai, China; Department of Endocrinology and Metabolism, Shanghai Jiaotong University Affiliated Sixth People's Hospital, Shanghai Diabetes Institute, Shanghai Clinical Center of Diabetes, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai, China.
authors.forename: Can
authors.initials: C
authors.lastname: Xiong
authors.affiliation: Key Laboratory of Embryo Molecular Biology, Ministry of Health of China & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai, Shanghai, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Hu
authors.affiliation: Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiaotong University, Shanghai, Shanghai, China; Department of Endocrinology and Metabolism, Shanghai Jiaotong University Affiliated Sixth People's Hospital, Shanghai Diabetes Institute, Shanghai Clinical Center of Diabetes, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Lv
authors.affiliation: Key Laboratory of Embryo Molecular Biology, Ministry of Health of China & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai, Shanghai, China.
authors.forename: Cong-rong
authors.initials: CR
authors.lastname: Wang
authors.affiliation: Key Laboratory of Embryo Molecular Biology, Ministry of Health of China & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai, Shanghai, China.
authors.forename: Wei-ping
authors.initials: WP
authors.lastname: Jia
authors.affiliation: Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiaotong University, Shanghai, Shanghai, China; Department of Endocrinology and Metabolism, Shanghai Jiaotong University Affiliated Sixth People's Hospital, Shanghai Diabetes Institute, Shanghai Clinical Center of Diabetes, Shanghai Key Laboratory of Diabetes Mellitus, Shanghai, China. Electronic address: fzeng@sjtu.edu.cn.
authors.forename: Fanyi
authors.initials: F
authors.lastname: Zeng
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D004272:DNA, Mitochondrial / Q000235:genetics*; D003638:Deafness / Q000235:genetics*; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D028361:Mitochondrial Diseases; D010375:Pedigree; D017354:Point Mutation; D012680:Sensitivity and Specificity
chemical_list: D004272:DNA, Mitochondrial