New mitochondrial DNA homoplasmic mutations associated with Japanese patients with type 2 diabetes.

Epidemiological studies have shown that patients with type 2 diabetes are more likely to have affected mothers than fathers and that the disease is often transmitted in a mode of maternal inheritance (1,2). Because transmission of mitochondria is exclusively maternal (3), mitochondrial DNA (mtDNA) mutations have been implicated in the maternal inheritance of diabetes (4). However, mtDNA mutation at 3243 has been reported in only 1-1.5% of patients with type 2 diabetes (5,6). Therefore we postulated that other mtDNA mutations may be associated with type 2 diabetes. Recently, using polymerase chain reaction (PCR)-restriction fragment (RF)-single-strand conformation polymorphism (SSCP) analysis (7), we confirmed 56 mtDNA mutations in Japanese subjects (8). In this study, we investigated the prevalence of 5 mtDNA mutations, including 3 mutations that we reported (8) in Japanese subjects. The study was performed in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all subjects, and the study design was approved by the Ethical Committee of Yamanashi Medical University. Peripheral blood was obtained from 253 unrelated patients (mean age: 55.7 ± 1.8 years) with type 2 diabetes in the order of their visits to outpatient Clinic of Yamanashi Medical University Hospital. We also recruited 345 healthy control subjects (mean age: 43.6 ± 1.6 years) with no family history of diabetes and normal oral glucose tolerance or HbAlc, who visited the Isawa Curehouse or Koseiren Health Center for medical checkups. Genomic DNA was obtained from peripheral leukocytes using a DNAQuick kit (Dainippon Pharmaceutical, Osaka, Japan). mtDNA mutations were detected either by PCR-SSCP or PCR-restriction fragment length polymorphism (RFLP) analyses. Two primer pairs were used: primer pair A, for-