中文摘要：

目的建立快速、准确、无创、低成本的线粒体糖尿病MT3243A：〉G位点突变的筛查方法。方法采集上海市儿童医院和上海市第六人民医院确诊的6例线粒体糖尿病患者及50名正常对照的血液、唾液及尿沉渣样本，用焦磷酸测序法进行检测，并比较不同来源样本中MT3243A〉G突变位点的杂合度水平；应用高分辨率熔解曲线（highresolutionmeltingcurveanalysis，HRM）对来自上海市4个社区的1070例糖尿病患者尿沉渣样本中的MT3243A〉G突变位点进行快速筛查，阳性样本进一步采用焦磷酸测序技术进行验证，并定量检测突变位点的杂合度水平。结果对比实验发现，6例线粒体糖尿病患者尿沉渣中MT3243A〉G突变位点的杂合度均高于唾液和血液2～7倍，唾液中突变的杂合度略高于血液；在50名正常对照的血液、唾液及尿沉渣样本中均未检出MT3243A〉G位点突变。在1070例糖尿病尿沉渣样本的筛查实验中，用HRM筛查出2例疑似MT3243A〉G位点突变的样本，经焦磷酸测序验证后确认为MT3243A〉G位点突变，两例患者尿沉渣样本中MT3243A〉G位点杂合度分别为33．32％和14．67％。结论尿沉渣样本取材方便、无创、突变杂合度高，可作为人群中MT3243A〉G位点突变筛查的首选样本。HRM技术使用成本低，可实现点突变的快速筛查。焦磷酸测序技术检测灵敏度高、定量精确。我们将两者的优势相结合，建立了用两步法快速筛查检测线粒体糖尿病MT3243A〉G位点突变的新方法。

英文摘要：

Objective To establish a rapid, accurate, noninvasive and low cost method for screening MT3243A〉G mutation in mitochondrial diabetes. Methods Blood, saliva, and urine sediment samples were collected from 6 patients with confirmed mitochondrial diabetes and 50 healthy controls from ShanghaiChildren＇s Hospital and Shanghai Sixth People＇s Hospital. The heterozygosity levels of MT3243A〉G mutation in above samples were detected with pyrosequencing, and the data were compared. MT3243A〉G mutations were rapidly screened with high resolution melting curve analysis （HRM） in the urine sediment samples of 1070 diabetic patients from 4 communities in Shanghai. Furthermore, pyrosequencing was used to validate the suspected positive samples, and the heterozygosity levels were also quantified. Results Comparative experiments found that heterozygosity of MT3243A〉G mutation were 2 to 7 times higher in urine sediment than in saliva and blood samples from the 6 patients with confirmed mitochondrial diabetes. However, the heterozygosity was slightly higher in saliva than blood samples. MT3243A〉G mutation was not detected in the 50 healthy controls. Two samples with suspected MT3243A〉G mutation were identified in the 1070 urine sediment samples of diabetes patients with HRM screening, which were validated by pyrosequencing. The heterozygosity of MT3243A〉G mutation were 33. 32% and 14. 67% in the urine sediment samples, respectively. Conclusion Urine sediment samples can be used for rapid screening of MT3243A〉G mutation for its ease to collect, noninvasiveness and higher level of heterozygosity. HRM is suitable for rapid screening for mitoehondrian mutations for its low cost, while such mutations could be detected with sensitivity and accuracy by pyrosequencing.