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

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.

Our project aims to dissect quantitative trait loci (QTL) for the variable persistence of fetal haemoglobin (HbF) in patients with sickle cell disease (SCD). The HbF trait is clinically important: its variable expression contributes significantly to differences in disease severity.
Our research strategy is three-pronged: starting with genetic fine-mapping in four ethnically-diverse patient populations and a total of >3,000 patients, we will move on to functional studies in primary erythroid cells derived from individuals carrying specific variant haplotypes and will finally focus our observations by investigating human erythroid cells lines where individual genetic variants have been introduced through genome editing.
Our main goals are (1) to identify novel molecular mechanisms by which common genetic variants at the HbF QTLs BCL11A and HBS1L-MYB affect pathways for the regulation of HbF; (2) to identify at least two novel causal SNPs driving the set of subloci occupying these QTLs and to weave those and robust sentinel SNPs into a universally-applicable polygenic score for the prediction of fetal haemoglobin and, ultimately, disease severity. (3) Our final goal is to help build research capacity in Africa: our collaborators in Tanzania and Nigeria will not only provide patients cohorts, but will also send trainees (one of them a post doc) to take charge of the genome-wide SNP data created for their patients, building a sustainable resource for future research.
The post doc will investigate functional effects of key variants at 2 subloci by testing for changes in transcription factor binding (EMSA, ChIP), chromosome conformation (4C-seq) and gene expression, including HbF production. Novel pathways identified by our work are intended to ultimately lead to the development of affordable treatments of this devastating condition that each year causes thousands of childhood deaths in Sub-Saharan Africa.

Sickle cell disease is one of the commonest genetic diseases with over 300,000 annual births worldwide, of which about 70% occur in sub-Saharan Africa, most of the affected children will die before the age of 5. In the UK and other Western countries, the disease is present mostly through the African diaspora and shows significant clinical diversity. The main known factor that can alleviate the disease is the persistence of fetal haemoglobin (HbF) that leads to varying HbF levels being present beyond the first year of life and throughout adulthood.
HbF induction by pharmacological agents or by gene editing approaches is currently a major therapeutic target. The most important impact of our work will be that it identifies naturally-occurring molecular mechanisms, caused by common genetic variation, that lead to raised HbF in some patients and thus to milder disease. It will be desirable to replicate such genetic effects through therapy. An important outcome would be the development of affordable drugs that will be available to the large number of patients living in low-and-middle-income countries (LMIC). Thus, an immediate beneficiary of our work will be the pharmaceutical industry, where regulatory factors identified by us can become targets in drug screening or for therapeutic gene editing.

It has long been a dream of geneticists to explain human variation in health and disease through underlying genetic variability. Success so far has been restricted mostly to rare and severe conditions where single gene defects have a clear impact. In contrast, relatively small fractions of common biological variability and common disease risk have been explained by genetic variants. We aim to explain 30-40% of HbF variability in sickle cell patients through common genetic variants and to build a polygenic score that can be calculated to predict HbF levels and, to a certain degree, disease severity. Beneficiaries of such a system will be researchers and clinicians conducting clinical or drug trials, in Africa, the UK, and elsewhere, where this score will help to adjust for genetic background variability that obscures test outcomes.

Our work will also help to understand fundamental erythroid biology and globin gene regulation as well as the effects of human common genetic variation occupying super-enhancer structures. The erythroid system has long been at the forefront of regenerative medicine and stem cell therapy. Our work will help to explain how red blood cell progenitors grow and equip themselves with haemoglobin, feeding into efforts in haematopoietic stem cell transplantation, gene therapy and in-vitro erythropoiesis.

We hope that our collaborators in Tanzania and Nigeria will gain significant benefit from our joint work. We strive to contribute to the scientific capacity for sickle cell research and to the growing international reputation of the groups at Muhimbili University in Dar es Salaam, Tanzania, and at the Universities of Lagos and Abuja in Nigeria. We will provide training and teaching material for our partners and will create genome-wide genetic datasets for the Nigerian patient research cohorts, which will support researchers' independence as partners in international collaborations.