IGSF4甲基化致珠蛋白合成障碍在地中海贫血中的作用及机制

There are greater differences in the clinical manifestations for thalassemia, oftenly showing the Mild or asymptomatic after the birth of homozygous children in the clinical,which brings a tremendous challenge on whether to terminate the pregnancy.It's also suggested a non-classical genetic pathway involved in the regulation for the clinical phenotype in thalassemia.It had been shown that hypermethylation of IGSF4 gene with its CpG islands and there was the positive correlation between its methylation differences and clinical severity in thalassemia. Study shows high expression of the key regulator CD45 in erythroid differentiation pathway can inhibit the production of globins, and IGSF4 can regulate the expression of CD45 negatively.Combined with bioinformatics, we speculate hypermethylation of gene IGSF4 in its promoter may affact its expression,which can promote the expression of CD45. Eventually lead to different severity of clinical manifestations in thalassemia.To evaluate the regulation of transcription and translation for gene IGSF4、CD45 and globins by collecting the peripheral blood of pregnant women and to cultivate various types of erythroid precursor cells in thalassemia.And meanwhile, using the method of interference suppression with CD45-siRNA to observe the expression of CD45 to explore the molecular mechanisms with hypermethylation of IGSF4 gene in different phenotypes for thalassemia.Our study is expected to clarify the pathogenesis in terms of epigenetics, and provide a novel idea for noninvasive prenatal diagnosis and early intervention in thalassemia

地中海贫血（地贫）临床表现差异较大，常有纯合子型患儿出生后临床表现为轻型或无症状，对是否终止妊娠带来极大挑战，亦提示有非经典遗传途径参与调控地贫的临床表型。项目组前期研究中发现，细胞表面粘附因子IGSF4基因启动子区存在CpG岛甲基化，且其甲基化差异与地贫临床严重程度呈正相关。研究表明红细胞分化途径关键调节因子CD45的高表达可导致珠蛋白合成障碍，且IGSF4负调控CD45的表达。结合生物信息学分析，我们推测：IGSF4基因甲基化后致自身表达下调促CD45过表达，使珠蛋白不同程度生成障碍，进而引起地贫临床表现不同。本项目拟通过采集孕妇外周血及培养各型地贫红系前体细胞，探讨甲基化对IGSF4、CD45及珠蛋白的调节作用；同时利用RNAi干扰技术，观察CD45的表达情况，探讨IGSF4基因甲基化在各地贫表型中的分子机制，从表观遗传学角度阐明其发病机制，为无创性产前诊断及早期干预提供新思路。

项目背景：地中海贫血临床表现差异较大，常有纯合子型患儿出生后临床表现为轻型或无症状，对是否终止妊娠带来极大挑战，亦提示有非经典遗传途径参与调控地贫的临床表型。项目组前期研究中发现，细胞表面粘附因子IGSF4基因启动子区存在CpG岛甲基化，且其甲基化差异与地贫临床严重程度呈正相关。本课题以IGSF4基因启动子甲基化在地贫不同临床表型中的作用为研究切入点，探讨其甲基化导致珠蛋白生成障碍的机制。.主要研究内容和结果：1、利用已建立的基于寡核苷酸芯片的地中海贫血DNA甲基化芯片平台，探讨了IGSF4基因在地中海贫血中甲基化的情况及意义研究，整体水平上表明该基因在重型到轻型地中海贫血中呈逐渐表达降低，且有显著性差异 (ratio=0.18，ratio<0.50)；2、利用Sequenom DNA甲基化质谱测序，分别在DNA及RNA水平上研究了IGSF4基因启动子甲基化对IGSF4的调节作用，初步探讨地贫各临床表型中IGSF4基因甲基化作用对其自身表达和翻译的调控机制，在蛋白水平进一步验证IGSF4基因在重型地中海贫血中相对于轻型而言呈低表达；3、利用293 细胞株，然后采用甲基转移酶抑制剂（5-氮杂-2’脱氧胞苷）干预培养，分别比较去甲基化培养前后IGS4及珠蛋白表达差异，即在蛋白水平上深入探讨导致地贫临床表现严重程度不同的分子机制，证实甲基化后珠蛋白及IGSF基因均呈低表达，且珠蛋白较IGSF4表达降低。已完成计划任务书绝大部分内容，相关实验结果已撰写成文正在投稿。.科学意义：本课题探讨IGSF4基因甲基化在各地贫表型中的分子机制，从表观遗传学角度阐明其发病机制，为无创性产前诊断及早期干预提供新思路。

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