β-地中海贫血相同基因型个体间表型差异的分子机制研究

探索β-地中海贫血(β-地贫)相同基因型个体间表型差异的遗传学机制是本领域面临的新挑战，本课题聚焦于影响β-地贫患者表型变异的胎儿到成人血红蛋白转换失调机制这一关键科学问题，采用基于疾病表型的单细胞测序策略研究β-地贫患者骨髓来源的造血干细胞及其分化细胞，通过整合分析胎儿血红蛋白高/低极值患者组的红系分化单细胞转录组和全基因组DNA甲基化测序数据，鉴定红系分化的细胞类型，重建细胞分化轨迹及基因调控网络，以及疾病表型相关的DNA甲基化差异基因及其表观调控相关的基因表达谱，从而发现血红蛋白转换调控的潜在关键基因。通过体外细胞学和体内小鼠功能验证，揭示新基因的作用及其调控机制；并开展新基因的遗传突变和DNA甲基化变异的临床病例队列评价研究。本研究可提供解释β-地贫临床表型个体差异的遗传和表观遗传学新证据，为发展β-地贫的人群防控及临床精准诊治新技术提供原创性新靶点。

It is a new challenge for us to explore the genetic mechanisms underlying phenotypic differences between genetically identical individuals with β-thalassemia. The aim of this study is mainly focused on dysregulation mechanism of the fetal-to-adult hemoglobin switch in disease states that is the key point of impacting on phenotypic variation of β-thalassemia. We will applicate the phenotype-based single-cell sequencing approaches to analyze the bone marrow (BM)-derived hematopoietic stem cells (HSCs) and its erythroid cell sub-populations from these patients with β-thalassemia. We intend to perform a systematic and integrative analysis of the single-cell transcriptomic and whole-genome DNA methylation data between two groups with extreme high and low levels of fetal hemoglobin (HbF), which enable the identification of cell types and subtypes during erythroid differentiation, the modeling and inference of gene regulatory networks, and the reconstruction of differentiation trajectories of fetal-to-adult hematopoietic stem cells. In addition, we can obtain the expected results of differential methylation genes (DMGs) and epigenetic-related gene expression profile, which could be involved in γ-globin gene regulation. The systematic investigation on the role in regulating HbF levels can enable us to look for the potential key regulators involved in the hemoglobin switching pathway. Further functional validations are performed to reveal the potential target gene’s role as an HbF modulator and its molecular mechanisms in the regulation of reactivating γ-globin expression by both the in-vitro cellular and in-vivo mouse experiments; Furthermore, clinical cohort studies are carried out to evaluate the contribution of the gene mutations and DNA methylation alterations to the phenotypes in β-thalassemia. Therefore, this study will hopefully provide novel genetic and epigenetic evidence on the interpretation of the clinical diversity of β-thalassemia. Our findings elucidate the potential novel targets in the development of precise genetic testing for population-based molecular screening in thalassemia-prevention programs, as well as in application of molecular therapies for treatment of hemoglobinopathies.