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Gene expression mechanism in normal and red cell membrane disorders

正常和红细胞膜疾病中的基因表达机制

基本信息

批准号：
11670151
负责人：
YAMADA Osamu
金额：
$ 0.77万
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：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670151/
关键词：
red cell membrane abnormality Hereditary spherocytosis gene expression b-spectrin band3 protein 4.2 5'-CpG-3' sites methylation 5'-CpG-3'sites methylation promoter β-spectrin gene AE1 gene protein 4.2 gene bisulfite method re

项目摘要

It is known that major membrane proteins of the erythroid cells are orderly expressed in a cascade fashion. Therefore, the control mechanism of the gene expresson of these proteins should exist. A possible abnormality of this control mechanism is assumed to be one of the the factors pathognomonic for hereditary spherocytosis (HS). From our results by previous investigations, it became evident that some of HS patients do not manifest the deficiency of membrane proteins, and that the abnormal expression of membrane protein genes may be crucial for disease states. The states of methylation of the 5'-CpG-3' sites are now known to regulate promoter functions by modifying the extent of gene expression. Therefore, the metylation profiles were examined by the bisulfite genomic sequencing method in the genomic DNAs of the human erythroid membrane proteins ; protein 4.2 gene, band 3 gene, and β-spectrin gene.1) The 5'-CpG-3' sites at the promoter regions of protein 4.2 and band 3 genes obtained from normal human peripheral blood mononuclear cells were extensively methylated, contrary to the fact that the promoter of β-spectrin gene was totally umethylated. 2) During erythroid differentiation, protein 4.2 gene was unmethylated in DNAs from the cell line UT-7/EPO, but became methylated in cultured erythroblasts from peripheral BFU-E.3) We also performed gene analyses for the ankyrin gene mutations in HS patients. As the result, we found 4 frameshifts, 2 nonsense mutations and 3 abnormal splicings as the etiologic abnormal gene mutations. Characteristically, missense mutations were rare, and the hot spots of these mutations were not present in the ankyrn gene.

众所周知，红系细胞的主要膜蛋白以级联方式有序表达。因此，这些蛋白的基因表达应该存在调控机制。这种控制机制的可能异常被认为是遗传性球形红细胞增多症（HS）的致病因素之一。从我们之前的研究结果来看，很明显，一些HS患者并没有表现出膜蛋白的缺乏，膜蛋白基因的异常表达可能是疾病状态的关键。目前已知5‘-CpG-3’位点的甲基化状态通过改变基因表达的程度来调节启动子功能。因此，我们采用亚硫酸盐基因组测序方法检测了人红细胞膜蛋白基因组dna的甲基化谱；蛋白4.2基因，带3基因，β-spectrin基因。1)正常人外周血单核细胞蛋白4.2和带3基因启动子区域的5′-CpG-3′位点被广泛甲基化，与β-spectrin基因启动子完全未甲基化相反。2)在红细胞分化过程中，蛋白4.2基因在UT-7/EPO细胞系dna中未甲基化，但在外周血BFU-E培养的红母细胞中甲基化。3)我们还对HS患者锚蛋白基因突变进行了基因分析。结果发现4个帧移、2个无义突变和3个异常剪接为病因异常基因突变。典型地，错义突变是罕见的，这些突变的热点不存在于锚定基因。