The role of FLVCR1 isoforms on inter-erythrocytic distribution of human foetal haemoglobin

Abstract

Introduction: We sought to expand the genetic repertoire underlying the developmental switching of gto bglobin genes with haematological and molecular exploration among two families from Malta with the KLF1 p. K288X truncation. The data revealed competitive inter- play between KLF1 and FLVCR1 isoforms on BCL11A that acted on the inter-erythrocytic distribution of Hb F among F-Erythrocytes, the MC-HbF of adults. Methods: HbF concentration, expressed as the percentage of total haemoglobin was determined with the Bio-Rad VARIANTTM Haemoglobin Testing System (Bio-Rad Laboratories, California, USA). F-Erythrocytes were quantified by flow cytometry on a BD FACSCaliburTM cytometer (Becton Dickinson Biosciences, California, USA) The cMC-Hb.F was confirmed by semi- automated quantitative imaging immuno-cytometry (qMC-Hb.F) that gave a mean value of 6.2 pg ranging from 3.0 to 9.8 pg in the KLF1± heterozygotes. Human erythroid progenitor cells (HEPs) were cultured from samples of ten family members and allowed to differentiate for two days. Cells were classified morphologically by microscopy and counted with an electronic cell counter (CASY-1, Schärfe System). Lentiviral transfection of buffy coat HEPs was conducted using clones obtained from The RNAi Consortium (Sigma-Aldrich, St.Louis) Results: The two families from Malta segregated a unique truncation mutation of the KLF1 locus such that the protein product was inactivated and expressed HbF over a broad range of 230 - 2480 mg/dL in peripheral blood. Non-linearity between the HbF (mg/dL) and the F-Erythrocyte numbers (N.1012/dL) suggested independent gene control of the inter-erythrocytic distribution of the HbF calculated as the Mean Corpuscular HbF or cMC- HbF (0 – 9.2 pg) The cMC-HbF correlated well with direct immune-cytometric quantification (qMC-HbF) Bio-Informatic studies with exome sequences from both families revealed a strong connection between the cMC- HbF and a new mutation in FLVCR1 (p.F473L; SIFT -5.37; Polyphen 0.029). FLVCR1 is a known Haeme transporter producing two isoforms. FLVCR1a is plasma membrane bound and FLVCR1b is bound to the mitochondrial membrane. In vitro transcriptomics and knockdowns showed that the FLVCR1 mutation decreased the output of BCL11A independent of KLF1 and it shifted the relative amounts of the two isoforms with increasing cMC-HbF. Conclusion: The interplay between the two FLVCR1 iso-forms could regulate intra-cellular concentration of Haeme and the total rate of translation with differential effects on specific messengers depending on the strength of the translation initiation complex. These data widen the understanding of globin gene switching and the possibilities of therapeutic intervention to increase Hb F levels in human haemoglobinopathies through alternative pathways.