应用高分辨率熔解曲线分析和焦磷酸测序快速筛查线粒体糖尿病MT3243处〉G突变  

Rapid screening of MT3243A〉G mutation in mitochondrial diabetes with high resolution melting curve analysisand pyrosequencing

作　　者：危新俊 杜秀娟[4] 王从容[4] 颜景斌[1,2,3] 

机构地区：[1]温州医科大学检验医学院、生命科学学院,浙江325035 [2]上海交通大学附属上海市儿童医院、上海医学遗传研究所,200040 [3]上海市胚胎与生殖工程重点实验室、卫生部医学胚胎分子生物学重点实验室,200040 [4]上海交通大学附属上海市第六人民医院、上海市糖尿病研究所,200040

出　　处：《中华医学遗传学杂志》2016年第4期447-451,共5页Chinese Journal of Medical Genetics

基　　金：国家科技支撑计划（2012BA109804）;国家自然科学基金面上项目（81570808）;卫生行业科研专项（201402018）;上海市卫生系统重要疾病联合攻关项目（2013ZYJB0015）;上海市糖尿病重点实验室开放课题（SHKLI）IKF_1101）

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。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.

关 键 词：MT3243A〉G突变 线粒体糖尿病 尿沉渣 高分辨率熔解曲线 焦磷酸测序 快速筛查 

分 类 号：R587.2[医药卫生—内分泌]