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Development of the rapid diagnosis method of hemoglobinopathies, thalssemias and abnormal hemoglobins.

开发血红蛋白病、地中海贫血和血红蛋白异常的快速诊断方法。

基本信息

批准号：
12672259
负责人：
HARANO Teruo
金额：
$ 2.43万
依托单位：
KAWASAKI MEDICAL SCHOOL
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12672259/
关键词：
Japanese Myanmar β-thalassemia α-thalassemia PCR Mnl I ARMS sequencing multiplex PCR シーケンシング血色素異常症 PCR-RFLP ミャンマー

项目摘要

Blood samples from patients expected to have hemoglobinopathy from the whole area of Japan and from transfusion dependent patients from Myanmar with severe anemia were analysed for the purpose of development of the rapid diagnosis method of hemoglobinopathies by the technique of molecular engineering.To the patients expected to p-thalassemia from Hb analysis, the ARMS (Amplification Refractory MutationSystem) method is useful for the detection of 5 mutations found commonly in Japan (-31CapA -> G, CD41/42TTCTTT→TT, CD90GAG→TAG, IVS II-1G→A, IVS II-654C→T) and those found in Myanmar (CD17 →T, IVS I-1G→T, IVS I-5G→C, CD41/42TTCTTT→TT, IVS II-654C→T), being discovered in about 70 percentof patients of both countries. However, this is difficult to adopt as a screening method, it is necessary to set up areaction condition to detect every mutation at the same ARMS. Additionally, the detection of an unknown or raremutation is required the PCR/direct sequencing.Major of abnormal Hb found in Myanmar is Hb E, that is easily found by agarose gel electrophoresis of PCRproduct digested with Mnl I. In the present study, the carriers of rare Hbs of Hb S and Hb Monroe had been foundIn Japan where found the various kind of Hb variants the PCR-sequencing method is necessary for the diagnosis.On the other hand, a-thalassemia mutations found in both countries were only two mutations of -α3.7 and - -SEA, and the mutations of -α4.2, - -MED, - -Fil and - -Thai were not detected. It is considered that the detection ofa-thalassemia mutations must be enough to take a technique of multiplex PCR-agarose gel electrophoresis.

为了利用分子工程技术开发血红蛋白病的快速诊断方法，分析了来自日本整个地区的血红蛋白病患者和来自缅甸的严重贫血的输血依赖患者的血样。武器（扩增难治性突变系统）方法可用于检测日本常见的5种突变（-31CapA -> G，CD 41/42 TTCTTT →TT，CD 90 GAG →TAG，IVS II-1G→A，IVS II-654 C →T）和在缅甸发现的那些（CD 17 →T，IVS I-1G→T，IVS I-5G→C，CD 41/42 TTCTTT →TT，IVS II-654 C →T），在这两个国家约70%的患者中发现。但是，这很难作为一种筛选方法，需要设置反应条件以在同一ARMS上检测每个突变。在缅甸发现的异常Hb主要是Hb E，这很容易通过Mnl I消化的PCR产物的琼脂糖凝胶电泳发现。本研究发现了罕见Hb S和Hb门罗的携带者，在日本发现了多种Hb变异，PCR-测序方法是诊断所必需的，而在这两个国家发现的α-地中海贫血突变只有-α3.7和- -SEA两种，-α4.2、- -MED、- -Fil和- -Thai未检出。认为应用多重聚合酶链反应-琼脂糖凝胶电泳技术检测α-地中海贫血基因突变已足够。