Understanding regulation of zeta-globin transcription as the first step towards embryonic globin induction in patients with severe alpha-thalassemia

It is estimated by the World Health Organization that 250,000 individuals with severe hemoglobinopathies are born each year. A significant proportion of these suffer from @alpha;-thalassemia, which is one of the most common human monogenic disorders known with a carrier rate of &GT;1% among all tropical and subtropical populations that have been studied. A common cause of alpha-thalassemia is an intra chromosomal deletion, termed the Southeast Asian (SEA) deletion, which removes both adult alpha-globin genes leaving the embryonically expressed zeta-globin gene intact. The SEA deletion is very common in some areas of the world, including Northern Thailand, where it is present at an allele frequency of approximately 15%. Individuals homozygous for the SEA deletion die of severe anaemia and tissue hypoxia in the third trimester of pregnancy, a condition termed Hb Bart's Hydrops Fetalis Syndrome (BHFS).
Although BHFS has hitherto been considered a universally fatal disorder, an increasing number of patients have survived because of prenatal and immediate postnatal blood transfusion. The first aim of this work is to fully document the natural history and clinical outcomes of long-term survivors with the BHFS to gain insight into whether this disease should now be considered manageable and to assess whether the burden of treatment is too great. To achieve this, I have initiated a BHFS survivor registry and recruited 60 cases. Analysis suggests that as many as 82% of the BHFS survivors have favorable long-term neurodevelopmental outcomes. However, 50% of the patients suffer from severe growth retardation and 14% have inoperable limb defects. In addition, the majority (83%) have the burden of life-long transfusion dependence. Previous work suggests that zeta-globin can functionally substitute for alpha globin in adulthood, therefore there is a need for improved understanding of the regulation of the embryonic zeta-globin gene to allow development of targeted therapeutic approaches for embryonic hemoglobin induction to ameliorate BHFS.
The second aim of this work is to investigate the cis- and trans-regulation of the zeta-globin gene with the ultimate aim of preventing silencing of this gene or reactivating its expression in definitive hematopoiesis. I have investigated the zeta-globin cis-regulatory network during murine primitive erythropoiesis using a DNaseI hypersensitivity assay coupled with an approach (termed Capture-C) to determine the cis-acting regulatory landscape of the zeta-globin gene. Interestingly, the data show that all five previously characterized DNaseI Hypersensitive Sites (DHSs) 5' of the α-globin cluster are present in primitive erythroid cells and that no extra sites are present at this developmental stage. These DHSs interact with both the zeta-globin and alpha-globin gene promoters in primitive erythroid cells as determined by Capture-C. I have also identified differential contributions of the individual alpha-globin cis-acting elements on alpha- and zeta-globin expression during primitive erythropoiesis using mouse lines harboring specific cis-element deletions.
To identify novel trans-acting regulators of zeta-globin I have compared the transcriptomes of primitive and definitive erythroblasts using high-throughput sequencing and gene expression arrays. I have prioritized differentially expressed genes, using ontology analysis, for future functional testing using a Cas9/CRISPR library screen. In the last part of this work, I have integrated information on the cis- and trans-regulation of zeta-globin in an attempt to gain insight into an unusual BHFS survivor, who remarkably survived the first year of life with minimal transfusion, most likely because of persistent expression of zeta-globin.

Advisor: Higgs, D
Babbs, C