Analysis of the foetal to adult globin gene switch mechanism by single cell RNA and TCR sequencing

Abstract

High levels of g-globin chains, pairing up with α-globin chains to produce foetal haemoglobin (HbF), greatly improves the symptoms associated with β -haemoglobinopathies. Multiple studies have been fuelled by this interesting phenomenon so as to identify new avenues for the treatment of this inherited blood disorder. A number of complications that are associated with thalassaemia, that include infections, iron overload and organ damage also obligate the need for better therapeutic approaches with the aim of reducing such co-morbidities. In this project, a state-of-the-art technology has been utilised for the first time on Maltese thalassaemic patients with the aim of addressing parts of the paradox surrounding globin gene switching. Peripheral blood mononuclear cells (PBMCs) were collected from 13 adult patients and 5 healthy controls. The PBMCs were further processed on a microchip, and were individually labelled. mRNA as well as specific targets of T-cell receptors (TCRs) were captured and reverse transcribed into cDNA, which was later amplified via Polymerase Chain Reaction. The final product was a Next Generation Sequencing Library that was sequenced at an -omic level with the appropriate tools. Cells with similar gene expression were clustered and differentially expressed genes (DEGs) for every cluster were identified so as to deduce the cell type corresponding to that cluster. Advanced bioinformatic analysis between the individual patient samples themselves, as well as between the homozygotes and compound heterozygotes as compared to the controls was carried out. Promising genes which could potentially play a role in the pathology of thalassaemia were also uncovered. Amongst these genes, most notably YY1 as well as KLF3, were both downregulated in the patient group (fold change < -2.0 and a p-value <0.05). Through TCR sequencing, it was also identified that thalassaemic patients exhibit lower diversity of T cell clonotypes, that could be one reason for increased infectious episodes encountered in these patients. Future drug development targeting directly the biomarkers identified throughout this study, as well as methods to possibly increase diversity of T cell clones in these patients could pave the way to ameliorate the status of such patients and improve their quality of life.