家族性急性髓系白血病相关新基因TGM6及其致病突变的功能学研究

Further study of familial leukemia will shed lights on the mechanism of leukemogenesis. By the positional cloning strategies ,a causative point mutation of TGM6 in an acute myeloid leukemia(AML) predisposition family has been previously identified, which was proved to be co-segregated with the phenotype and maybe the culprit mutation. However, its role in leukemogenesis remains obscure. For the next investigation, bioinformatics methodologies will be employed to analyze the mutation's influence on the structure and function of TGM6. Screening and validation of TGM6 mutations in sporadic patients and other AML pedigrees aims to delineate its mutation map and define its relationship and significance of AML. Moreover, a comprehensive research of TGM6 and its mutation will be performed in three separate aspects, including the detection of their influence on the activity of transglutaminase, GTPase and protein kinase at the molecular level;the monitoring of their influence on cell proliferation, differentiation, apoptosis and intracellular position at the cellular level;the observation of their influence on survival time and bone marrow cell phenotypes of the mice as well as tumorigenesis of the nude mice at the animal levels, through constructing bone marrow transplantation murine model of wild-tyoe TGM6 over-expression and mutative TGM6 expression respectively. Thus,the functional study of TGM6 and its mutation at the molecular, cellular and animal levels will reveal their roles in leukemogenesis, and meanwhile provides the necessary theoretical basis for targed drugs development.

对家族性白血病的深入研究有助于揭示白血病发病机制。本项目采用定位克隆策略，在一个急性髓系白血病（AML）高发家系中发现了TGM6基因点突变，该基因突变与疾病表型共分离，为该家系AML的致病突变，但其在白血病发病中的作用尚不清楚。将用生物信息学预测TGM6突变对其结构、功能的影响；检测与验证散发及家系AML中TGM6的突变情况，绘制TGM6突变图谱，明确TGM6突变与AML的关系及意义；在分子水平上，检测TGM6突变对其转谷氨酰胺酶、GTP酶及蛋白激酶活性的影响；细胞水平上，观测TGM6突变对细胞增殖、分化、凋亡等的影响；动物水平上，构建过表达野生型与突变型TGM6的小鼠骨髓移植模型，观察TGM6过表达及突变对小鼠生存期、骨髓细胞表型等的影响及裸鼠中的致瘤性。从分子、细胞、动物水平研究TGM6基因及其突变体的功能，以揭示TGM6基因突变在白血病发生发展中的作用机制，为开发靶向药物奠定理论基础

前期通过定位克隆、捕获测序等技术在一个急性髓系白血病(AML)高发家系中发现TGM6基因点突变，该基因突变与疾病表型呈共分离，可能为该家系AML发生的致病基因变异。为探索TGM6基因及突变在AML发生中的作用及机制：首先，在190例散发AML患者及7个家族性MDS/AML易患家系患者中进行TGM6基因全外显子测序，均未发现TGM6非同义突变，结合多个肿瘤突变数据库分析，提示该基因突变为罕见变异；其次，在散发AML细胞及白血病细胞株中检测TGM6基因表达时，发现TGM6基因表达丰度低或不表达，结合多个表达谱芯片数据库分析，提示TGM6在白血病中无特异性的高/低表达。综上推测TGM6变异参与散发AML发病机制的可能性较小。因此，在后期研究中将重点转移到该家系患者体细胞突变的挖掘及功能研究上，取得一些重大进展：①该家系患者体细胞突变，DHX15基因突变，在散发AML患者中为重现性突变（4/220）；②DHX15基因在AML患者中明显高表达，患者OS及RFS显著下降；③DHX15基因在AML各亚型中的表达水平与细胞分化程度负相关，应用全反式维甲酸诱导NB4、HL60细胞分化成熟后，DHX15基因表达明显下降；在斑马鱼模型中敲除DHX15基因，引起斑马鱼早期原始造血受阻，提示DHX15基因可能参与造血分化调控；④利用shRNA下调NB4和Jurkat细胞中DHX15基因表达，发现细胞增殖明显受抑、凋亡显著增加、细胞周期阻滞在G1期；回复实验发现，DHX15基因表达部分恢复后，细胞增殖活性升高、细胞凋亡率下降；亚砷酸诱导NB4细胞凋亡中DHX15基因表达下降；DHX15基因过表达使NB4细胞对亚砷酸诱导的凋亡敏感性下降，提示DHX15基因参与细胞增殖、凋亡调控；⑤下调DHX15基因表达导致Bcl2表达下调、回复DHX15基因表达使Bcl2表达亦回复，DHX15基因过表达可活化NF-kB转录活性，提示DHX15基因可能通过调控NF-Kb/Bcl2等参与凋亡、增殖调控。综上，本项目发现了一个新的参与白血病发生的基因，DHX15基因，可调控白血病细胞的增殖、凋亡、分化，与白血病患者预后相关，在白血病发生发展中起着重要作用。本项目研究对于优化白血病的分层治疗，开发新的分子靶标等具有重要的研究与临床应用价值。