三个铁调节基因变异对β中间型地中海贫血性状严重性的修饰作用及其机理研究

The anemia level of intermediate beta thalassemia is between moderate and serious, and the phenotypes is various, but the mechanism to these characters is still unclear. In the former studies, we found that in addition to the HbF level, the mutation of iron metabolism related gene also affect the phenotype of intermediate beta thalassemia patients. In this application, we assumed that iron relative regulator gene is a kind of modifier gene which lead to the variety of intermediate beta thalassemia phenotypes. Therefore, we will design with case-only method and careful use our big population thalassemia samples and genetic resource, to detect their HbF and Hepicidin level, TF-FE saturation, iron concentration and CBC, and analyze the mutation of TMPRSS6, BMP6 and HFE genes using DNA sequencing. By genotyping targeted gene and analyzing the genotype-phenotype with bioinformatics, so we could find the evidences of relationship between intermediate thalassemia and iron metabolism relative genes. Where after the over expression of siRNA of the iron metabolism relative gene of mutation/wild type and HbF are invested in cells, and the mutation genes expression level affecting on HbF level was observed, so as to illuminate the mechanism of iron metabolism relative genes mutation affect the intermediate thalassemia anemia level variety, and its potential value in clinical application.

β中间型地中海贫血（地贫）的贫血程度介于轻型和重型之间，表型差异大，其发病机制一直不清。本课题在前期研究中发现中间型地贫患者与HbF有关外还与铁代谢相关基因的变异有关。本申请假设：铁相关调节基因是中间型地贫表型多样性的修饰基因。为此，拟在人群水平上利用本组前期积累的大样本地贫患者遗传和临床信息资源，采用case-only设计探讨受试者血浆中HbF 、Hepicidin和铁浓度等指标，进一步采用测序检测TMPRSS6、BMP6和HFE基因的结构变异，经大样本病例群体的基因分型，生物信息学功能和遗传统计学分析，比较基因型-表型的关联，获得中间型地贫与铁代谢相关基因的变异有关的证据。并在细胞水平上考察铁代谢相关基因的变异型/野生型与HbF的超表达/SiRNA，观察相关基因变异型产物的表达对HbF表达的修饰作用，以阐明铁代谢相关基因变异与β中间型地贫贫血程度差异的机理。

在前一个自然科学基金（81260093）的资助下，我们发现HBS1L-MYB和 BCL11A等基因相关位点变异影响了HbF的表达。本课题为了进一步探讨HbF水平修饰基因变化与β-中间型地贫血红蛋白HbF表达的关系以及铁调节基因位点变异与β-中间型地贫性状的关系。我们立项进行研究。通过对广西β-中间型地贫人群研究，（1）证明了在广西β中间型人群中HBG2，BCL11A及HBS1L-MYB 是HbF 数量性状位点相关基因，这三个基因通过协同效应促进HbF水平高表达；（2）阐明HBS1L-MYB基因上的rs4895441 (G) 修饰位点与HbF水平高表达显示正相关；（3）首次报道了广西壮族正常人群HFE基因位点突变和H63D等位基因突变发生情况。（4）证明了HbF 与铁调素Hepcindin、血清铁SF和转铁蛋白STfR之间无相关性；（5）揭示β-中间型地贫人群HFE基因H63D位点突变增加了铁过载的风险：（6）发现和鉴定、世界首报3种新发地贫突变基因；（7）发表科研论文11篇，其中SCI收录10篇，获得发明专利1项。这些成果对今后开展中间型地贫及重型地贫患者治疗提供了新的思路；另外本课题研究中首次发现3例新发地贫突变基因类型，不仅丰富和完善了广西地区地贫突变类型, 而且为今后地贫防治基础研究和地贫防治计划进一步实施提供了理论基础和科学依据。