Differential expression of globin gene switching using single cell sequencing

Abstract

The aim of this project, was to probe and understand better the mechanism that surrounds the gamma to beta globin gene switch that leads to foetal and adult haemoglobin production respectively. Induction of gamma-globin reduces symptoms in beta thalassaemia and sickle cell disease patients but there is a clear need for drugs with a better efficacy, tolerability and patient response. At present, treatment involves lifelong monthly blood transfusions and iron chelation therapy, both of which impinge significantly on the quality of life and may lead to death prematurely. Beta thalassaemia alone costs the European Union 1.64 billion Euros per year in patient treatment. The globin gene switch was studied by looking at peripheral blood mononuclear cells obtained from peripheral blood of 13 thalassaemic patients which were divided into 4 different groups depending on their genotype subtype and 5 healthy adults (controls) at single cell level. Once single cell sequencing was carried out, next generation sequencing was performed and the data obtained was downloaded and further analysed on peripheral blood mononuclear cells and CD34+ cells to determine the genes which were responsible for globin gene switch perturbation, control and regulation. Quality control metrics, high dimensional single cell data, cluster composition and dot plots per sample, per group and per condition were obtained and helped to identify the highly expressed genes in the peripheral blood mononuclear cells and CD34+ cells. Such genes included Kruppel-Like Factor 10 and LIM Domain Only 2 among others, which showed a fold change of -2 or less in the β-thalassaemia patients versus in the controls. Uncovering the molecular basis of gamma-globin gene control might also serve as a model for other clinically significant single gene disorders. This study offered for the first time ever in Malta, a look at single cell level of a highly and well defined patient population in Malta and can be useful for targets required for therapeutic targeting in the treatment of β-haemoglobinopathies.