Fukuda M, Nakano S, Imaizumi N, Kitazawa M, Nishizawa M, Kigoshi T, Uchida K
Division of Endocrinology, Department of Internal Medicine, Kanazawa Medical University, Ishikawa, Japan.
J Diabetes Complications. 1999 Sep-Dec;13(5-6):277-83. doi: 10.1016/s1056-8727(99)00060-4.
To assess the roles of various mitochondrial (Mt) DNA mutations in diabetic and nondiabetic subjects, we screened Mt DNAs at the 3243 base pair (bp) and its adjacent portion in unrelated Japanese diabetic and nondiabetic subjects. Furthermore, to clarify the clinical features of diabetic subjects harboring a Mt DNA mutation, we evaluated the ability of insulin secretion and microvascular complications in diabetic subjects. Five hundred thirty-seven diabetic patients and 612 unrelated nondiabetic subjects were recruited into this study. In Mt DNA analyses, Mt DNA was isolated from peripheral leukocytes of the subjects, and then an Mt DNA fragment surrounding the tRNA(Leu(UUR)) site was amplified by the polymerase chain reaction (PCR) using two sets of primers. These fragments were further digested with three kinds of restriction endonucleases and were subjected to agarose gel electrophoresis. When a mutation was present, Mt DNA fragments were directly sequenced with an autosequencer. Baseline characteristics in all subjects were examined, and microvascular complications and insulin secretory capacity in diabetic subjects were newly evaluated. Eight kinds of Mt DNA mutations, which were point mutations, were found in 74 subjects. Each affected subject had only one mutation in the Mt DNA examined. Among them, the mutations at np 3316, 3394, 3593, and 3391 were accompanied by amino acid replacement. Thirty-eight diabetic patients were affected (7.1%), including two subjects with a point mutation at np 3243, and 26 nondiabetic subjects were affected (4.2%). Thus, there was a higher prevalence in diabetic subjects than in nondiabetic subjects. There was no significant difference in the prevalence of maternally inherited diabetes between these two groups. The mean level of urinary C-peptide excretion was lower in diabetic subjects with an Mt DNA mutation (DM+) than in those without it (DM-). Although the prevalence of hypertension in DM+ was higher than that in DM-, diabetic retinopathy and nephropathy in DM+ were problematic, in comparison with those in DM-, when statistical corrections were performed for the effect of hypertension. Furthermore, a strategy based on logistic regression analysis revealed that advanced retinopathy and decreased urinary C-peptide excretion in all diabetic subjects studied were strongly related to the presence of Mt DNA mutation. Our results suggest that Mt DNA mutations in Japanese diabetic subjects are related to the development of diabetes, and also that these mutations are associated with not only a decrease in insulin secretion but also advanced diabetic microvascular complications.
为评估各种线粒体（Mt）DNA突变在糖尿病和非糖尿病患者中的作用，我们在无关的日本糖尿病和非糖尿病患者中筛查了位于3243碱基对（bp）及其相邻区域的Mt DNA。此外，为阐明携带Mt DNA突变的糖尿病患者的临床特征，我们评估了糖尿病患者的胰岛素分泌能力和微血管并发症情况。本研究招募了537例糖尿病患者和612例无关的非糖尿病患者。在Mt DNA分析中，从受试者的外周血白细胞中分离出Mt DNA，然后使用两组引物通过聚合酶链反应（PCR）扩增围绕tRNA（Leu（UUR））位点的Mt DNA片段。这些片段进一步用三种限制性内切酶消化，并进行琼脂糖凝胶电泳。当存在突变时，用自动测序仪对Mt DNA片段进行直接测序。检查了所有受试者的基线特征，并重新评估了糖尿病患者的微血管并发症和胰岛素分泌能力。在74名受试者中发现了8种Mt DNA点突变。每个受影响的受试者在所检测的Mt DNA中仅有一种突变。其中，np 3316、3394、3593和3391位点的突变伴有氨基酸替换。38例糖尿病患者受影响（7.1%），包括2例np 3243位点有突变的患者，26例非糖尿病患者受影响（4.2%）。因此，糖尿病患者中的患病率高于非糖尿病患者。这两组之间母系遗传糖尿病的患病率没有显著差异。携带Mt DNA突变的糖尿病患者（DM +）的尿C肽排泄平均水平低于未携带突变的患者（DM -）。虽然DM +中高血压的患病率高于DM -，但在对高血压的影响进行统计学校正后，与DM -相比，DM +中的糖尿病视网膜病变和肾病问题更为突出。此外，基于逻辑回归分析的策略显示，在所有研究的糖尿病患者中，晚期视网膜病变和尿C肽排泄减少与Mt DNA突变的存在密切相关。我们的结果表明，日本糖尿病患者中的Mt DNA突变与糖尿病的发生有关，并且这些突变不仅与胰岛素分泌减少有关，还与晚期糖尿病微血管并发症有关。
