Identification of novel mechanisms of fetal-haemoglobin induction by common genetic variation in patients with sickle cell disease

我们的目标是发现BCL11A和HBS1L-MYB两个遗传位点的良性常见DNA变异影响成人胎儿血红蛋白水平的分子机制。胎儿血红蛋白（HbF）是在未出生的红细胞中占主导地位的携氧分子。它在出生时被关闭，取而代之的是成人形式的血红蛋白。由于镰状细胞病（SCD）患者的成人血红蛋白有缺陷，一些成年患者产生胎儿血红蛋白的能力将使疾病明显减轻。关于BCL11A和HBS1L-MYB位点的结构和功能已经积累了很多知识。影响hbf的遗传变异存在于被称为“超级增强子”的基因调节元件中，这种元件控制红细胞前体中邻近基因的活性，从而影响它们的发育和血红蛋白含量。对于自然发生的DNA变化如何影响这些“超级增强剂”的功能，人们知之甚少。在这里，我们的目标是发现新的调控途径，并确定与遗传可变增强子元件结合的转录因子。这将增加旨在重新激活患者HbF的新治疗方法的靶标库。除了指导新的基因治疗策略外，我们的研究结果将有助于为开发新的负担得起的药物奠定基础，使主要来自中低收入国家的SCD患者受益，特别是非洲，那里每年有超过20万名受影响的儿童出生。在英国，这种疾病主要是通过非洲侨民出现的，并且表现出显著的临床多样性，部分原因是由遗传决定的可变乙型肝炎病毒的存在。我们的实验策略将建立在通过合作产生的三个主要资源的基础上：(1)来自英国、坦桑尼亚和尼日利亚的四组不同种族的特征明确的患者（约3000人），我们将在那里收集广泛的遗传数据；(2)从TwinsUK队列中获得2000名遗传和血液学特征的受试者，在那里我们将能够获得来自16名具有BCL11A和HBS1L-MYB特定遗传谱的个体的祖细胞；(3)通过与红细胞生成细胞系BEL-A的创造者Jan Frayne教授合作对其进行基因组编辑而产生的红细胞生成细胞系携带个体关键DNA变体。我们的主要目标是：(1)从遗传和功能上解剖胎儿血红蛋白水平的两个主要数量性状位点（BCL11A和HBS1L-MYB）的常见遗传变异，以揭示遗传变异控制基因表达、决定HbF水平和影响红细胞生成的潜在新分子机制。从我们在坦桑尼亚或尼日利亚的合作者中招募的一名博士后研究员将研究转录因子结合（使用的技术将是EMSA -“电泳迁移转移试验”，ChIP -“染色质免疫沉淀”）和染色质环（在活性细胞中发生的DNA折叠，将调控元件定位在目标基因旁边，我们将使用的技术称为“4C-seq”）和基因活性与所研究细胞的基因型的关系。(2)通过遗传作图和功能研究相结合的方法，鉴定上述3个独立亚位点（HMIP-1、HMIP-2B、BCL11A-2）的DNA变化原因。从这些数据中，我们将创建一个可以为每个患者计算的遗传评分，旨在预测镰状细胞病的HbF水平和临床严重程度。该分数将成为基因和临床研究（包括药物试验）中确定的参数，有助于使此类研究更具信息性；(3)通过培训研究人员和为其患者群体建立广泛的遗传数据集，帮助坦桑尼亚和尼日利亚建立镰状细胞研究的能力和专业知识。

We aim to discover the molecular mechanisms by which benign common DNA variants at two genetic sites, BCL11A and HBS1L-MYB, influence fetal haemoglobin levels in adults. Fetal haemoglobin (HbF) is the oxygen-carrying molecule dominating red blood cells in the unborn. It is switched off around the time of birth and replaced with the adult form of haemoglobin. Since in patients with sickle cell disease (SCD) adult haemoglobin is defective, the ability of some adult patients to produce the fetal form will make the disease significantly milder. Much knowledge has been accumulated regarding the structure and function of the BCL11A and HBS1L-MYB sites. The HbF-affecting genetic variants reside in gene-regulating elements called 'super-enhancers' that control the activity of neighbouring genes in red blood cell precursors and thus affect their development and haemoglobin content. Little is known about how the naturally-occurring DNA changes affect the function of these 'super-enhancers'. Here, we aim to uncover novel regulatory pathways and identify transcription factors binding to genetically-variable enhancer elements. This will add to the arsenal of targets for new therapeutic approaches aiming at reactivating HbF in patients. In addition to guiding new gene therapy strategies, our results will help laying the groundwork for the development of new affordable drugs to benefit the patients suffering from SCD mainly in low-and-middle income countries, especially Africa, where more than 200,000 affected children are born annually. In the UK, the disease is present mostly through the African diaspora and shows significant clinical diversity, partially driven by the variable, genetically-determined presence of HbF. Our experimental strategy will build on three major resources generated through collaboration: (1) four ethnically-diverse groups of well-characterised patients (n > 3,000) from the UK, Tanzania and Nigeria, where we will assemble extensive genetic data; (2) access to 2,000 genetically and haematologically characterised subjects from the TwinsUK cohort, where we will be able to obtain progenitor cells from 16 individuals with specific genetic profiles at BCL11A and HBS1L-MYB and (3) red blood cell producing cell lines carrying individual critical DNA variants generated through genome editing of the red blood cell-producing cell line BEL-A in collaboration with its creator, Prof Jan Frayne.Our principal goals are:(1) to genetically and functionally dissect common genetic variability at the two major quantitative trait loci for fetal-haemoglobin levels, BCL11A and HBS1L-MYB in order to unravel potentially novel molecular mechanisms through which genetic variation controls gene expression, determines HbF levels and influences the generation of red blood cells. A post-doctoral researcher recruited from our collaborators in Tanzania or Nigeria will investigate transcription factor binding (the techniques used will be EMSA - 'electrophoretic mobility shift assays', ChIP - 'chromatin immunoprecipitation') and chromatin looping (a folding of the DNA that occurs in active cells to position regulatory elements next to their target genes, the technique we will use is called '4C-seq') and gene activity in relationship with the genotype of the cells studied.(2) to identify the causal DNA changes at three independent subloci (HMIP-1, HMIP-2B, BCL11A-2) of the above through a combination of genetic mapping and functional studies. From these we will create a genetic score that can be calculated for each patient, aimed at predicting HbF levels and clinical severity in sickle cell disease. This score will become a parameter ascertained in genetic and clinical studies, including drug trials, helping to make such studies more informative;(3) to help build capacity and expertise for sickle cell research in Tanzania and Nigeria through training of researchers and building of extensive genetic datasets for their patient cohorts.