Gene expression mechanism in normal and red cell membrane disorders

已知红系细胞的主要膜蛋白以级联方式有序表达。因此，这些蛋白基因表达的调控机制应该是存在的。这种控制机制的可能异常被认为是遗传性球形细胞增多症(HS)的致病因素之一。从我们以往的研究结果可以看出，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在疾病状态中起着至关重要的作用。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的功能。1)从正常人外周血单核细胞中获得的蛋白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-光谱蛋白基因启动子完全非甲基化的事实相反。2)在红系分化过程中，UT-7/EPO细胞DNA中的蛋白4.2基因未发生甲基化，而外周血红细胞中的蛋白4.2基因发生甲基化。3)我们还对HS患者的Ankyrin基因突变进行了基因分析。结果发现4例移码、2例无义突变和3例异常剪接为致病基因异常突变。典型的是，错义突变很少见，这些突变的热点不存在于ankyrn基因中。

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.