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

镰状细胞病患者常见遗传变异诱导胎儿血红蛋白的新机制的鉴定

• 批准号: MR/T013389/1
• 负责人: Stephan Menzel
• 金额: $ 94.79万
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FT013389%2F1
• 关键词: Identification; novel; mechanisms; fetal; haemoglobin

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.

我们的目标是发现两个遗传位点BCL 11 A和HBS 1 L-MYB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）是未出生的红细胞中占主导地位的携氧分子。它在出生时关闭，并被成人形式的血红蛋白所取代。由于镰状细胞病（SCD）患者的成人血红蛋白是有缺陷的，一些成年患者产生胎儿形式的能力将使疾病显着减轻。关于BCL 11 A和HBS 1 L-MYB位点的结构和功能已经积累了很多知识。影响HbF的遗传变异存在于称为“超级增强子”的基因调节元件中，这些元件控制红细胞前体中相邻基因的活性，从而影响它们的发育和血红蛋白含量。关于自然发生的DNA变化如何影响这些“超级增强子”的功能，人们知之甚少。在这里，我们的目标是发现新的调节途径，并确定转录因子结合到遗传可变增强子元件。这将为旨在重新激活患者HbF的新治疗方法增加靶点。除了指导新的基因治疗策略外，我们的研究结果还将有助于为开发新的负担得起的药物奠定基础，使主要在中低收入国家，特别是非洲的SCD患者受益，非洲每年有超过20万名受影响的儿童出生。在英国，该疾病主要通过非洲散居者存在，并显示出显著的临床多样性，部分原因是由HbF的可变遗传决定的存在。我们的实验策略将建立在通过合作产生的三个主要资源上：（1）四个种族多样化的患者群体，（n > 3,000）来自英国、坦桑尼亚和尼日利亚，我们将在那里收集广泛的遗传数据;（2）从TwinsUK队列中获得2,000名遗传学和血液学特征的受试者，其中我们将能够获得来自16个具有BCL 11 A和HBS 1 L-MYB特异性遗传谱的个体的祖细胞，以及（3）携带通过与其创造者合作对红细胞产生细胞系BEL-A进行基因组编辑而产生的个体关键DNA变体的红细胞产生细胞系，我们的主要目标是：（1）从遗传学和功能上剖析胎儿血红蛋白水平的两个主要数量性状基因座（BCL 11 A和HB S1 L-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 -“电泳迁移率变动分析”，ChIP -“染色质免疫沉淀”）和染色质成环（在活性细胞中发生的DNA折叠，以将调节元件定位在其靶基因旁边，我们将使用的技术称为“4C-seq”）和与所研究细胞的基因型相关的基因活性。(2)通过遗传作图和功能研究相结合，确定上述三个独立亚位点（HMIP-1，HMIP-2B，BCL 11 A-2）的致病DNA变化。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。该评分将成为遗传和临床研究（包括药物试验）中确定的参数，有助于使此类研究提供更多信息;（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。

项目成果

Genome-wide Association Study of Fetal Haemoglobin in Nigerian Patients with Sickle Cell Disease

尼日利亚镰状细胞病患者胎儿血红蛋白的全基因组关联研究

• 发表时间: 2023
• 作者: Ojewunmi O

Fetal-haemoglobin enhancing genotype at BCL11A reduces HbA(2) levels in patients with sickle cell anaemia.

• DOI: 10.1002/jha2.186
• 发表时间: 2021-08
• 期刊: EJHaem

COPILOT: a Containerised wOrkflow for Processing ILlumina genOtyping daTa

COPILOT：用于处理 ILlumina 基因分型数据的容器化工作流程

• DOI: 10.1101/2021.07.26.453753
• 发表时间: 2021
• 作者: Patel H