UNIVERSITY OF MALTA LIFE SCIENCE RESEARCH SEMINARS Web: http://www.um.edu.mt/events/scisem/ Email: [email protected] Abstract form Title: How does the absolute MCHbF in adult F-cells correlate with genomic variants in HPFH? Presenter: Jeanesse Scerri Contact address: C1, Sir Leslie Rundle Str. Mtarfa Tel: 79459473 Fax: Email: [email protected] Presentation date: 26th May, 2014 Abstract Around birth, a shift from γ- to β-globin gene expression causes a switch from foetal haemoglobin (HbF) to adult haemoglobin (HbA). Residual amounts of HbF are synthesized throughout life and localized in erythrocytes termed F-cells. Increased HbF levels are known to ameliorate symptoms of β-haemoglobinopathies. Krüppel-like factor 1 (KLF1) plays a central role in the developmental globin gene switch; carriers of KLF1 mutations have hereditary persistence of foetal haemoglobin (HPFH) and show variably elevated (10-40%) HbF. This variation may be due to differential expression of other modifier genes. The KLF1 interactome can be further defined by identifying potential molecular targets and observing their expression at the cellular level in comparison with HbF expression and distribution in F-cells. While analysing next generation sequencing (NGS) data of a Maltese family with HPFH, calculated cMCHbF provided an independent classification from HbF levels, according to which genomic variants were observed. The mean corpuscular HbF (MCHbF) is normally estimated by dividing the amount of HbF, determined by HPLC, by the number of F-cells, obtained by flow cytometry. Since HbF may be unequally distributed among F-cells, an imaging cytometry protocol enables quantification of HbF per F-cell by fluorescent emission measurements. A new intracellular anti-HbF antibody-labelling technique was thus optimized for use with the Nikon Eclipse Ti inverted fluorescence microscope. This technique resulted in images of superior quality from which the mean HbF could be determined for each F-cell observed. The technique was applied on blood from six family members, three of which had HPFH. The data obtained will be used to study the association of KLF1 and other genes, obtained through NGS data analyses, with quantitated qMCHbF in normal and HPFH individuals, to identify new potential therapeutic targets for β-haemoglobinopathies.