Fetal haemoglobin response to hydroxycarbamide treatment and sar1a promoter polymorphisms in sickle cell anaemia

Reactivation of fetal haemoglobin (HbF) expression in adult life could provide an effective therapeutic approach for patients with haemoglobinopathies and thalassaemias. Among HbF induction agents, hydroxycarbamide (HC) has been successfully used to treat sickle cell disease (SCD) and selected thalassaemia syndromes (Rodgers et al, 1990; Zeng et al, 1995; Fucharoen et al, 1996). Hydroxycarbamide is believed to exert its beneficial effects in SCD via a variety of mechanisms, including augmented HbF synthesis by erythroid regeneration or NO-related increases in soluble guanylate cyclase activity and cyclic guanidine monophosphate (cGMP) that stimulate HBG expression (Cokic et al, 2003). Additional mechanisms may also include myelosuppression with a reduction in circulating neutrophils, increased erythrocyte water content, modified erythrocyte endothelial cell interactions and altered vascular tone by increasing NO bioavailability (Okpala, 2005). Many patients treated with HC increase HbF levels from baseline, with wide variability in the magnitude of this response. The underlying reasons for this spectrum of HC responsiveness are poorly understood (Steinberg et al, 1997; Bakanay et al, 2005). Presently, a major research priority is the identification of genetic variants underlying both human diseases and differences in pharmacological responsiveness to drugs (Collins et al, 2003). In SCD, this paradigm is being applied to elucidate the pathogenesis of vaso-occlusive and vascular complications and to develop individualized SCD therapies (Steinberg, 2005). Genetic variants in drug-metabolizing enzymes, transporters or targets could potentially influence the efficacy or toxicity of drugs used in SCD. The HC-induced small guanosine triphosphate (GTP)-binding protein, secretion-associated and RAS-related (SAR) protein has recently been shown to play a pivotal role in HBG induction by causing cell apoptosis and G1/S-phase arrest by reduction of phosphatidylinositol 3 (PI3) kinase and extracellular protein-related kinase (ERK) phosphorylation with increased p21 and GATA-2 expression (Tang et al, 2005). SAR1A belongs to the small GTPase superfamily and encodes a GTP-binding protein SAR1a. This protein plays a key role in initializing transport from the endoplasmic reticulum (ER) to the Golgi apparatus. The localization of SAR1A in the endoplasmic reticulum and its association with HBG expression demonstrated in our recent study suggest that SAR1A also plays a special role in haemoglobin regulation (Tang et al, 2005). However, the precise pathway(s) through which SAR1A regulates HBG remain unknown. SAR1A may increase the transport of membrane-bound transcription factor precursors from the ER to the Golgi. The proteolytic cleavage of the precursor proteins in the Golgi activate cytosolic fragments that enter the nucleus and regulate erythroid-specific transcription factors, such as GATA, eventually modulating HBG expression. Moreover, previous studies have illuminated a pivotal role of the p38 mitogen-activated protein kinase (MAPK) pathway during GTP-mediated erythroid differentiation of K562 cells with the accumulation of HBG mRNA (Osti et al, 1997; Moosavi et al, 2007). Activators of the soluble guanylate cyclase (sGC) and protein kinase G (PKG) pathways have been implicated in the regulation of HBG expression in both erythroleukemic cells and in primary erythroblasts (Ikuta et al, 2001). Expression of HBG and the sGC alpha subunit are correlated, indicating that GTP-binding proteins may participate in HBG induction. Preliminary data from our study indicated that HC inducibility is transcriptionally regulated and localized to elements in the SAR1A promoter. Accordingly, we hypothesized that DNA sequence variation within the SAR1A promoter might explain differences in individual responses to HC therapy. To test this hypothesis, we identified the single nucleotide polymorphism (SNPs) in the SAR1A promoter by DNA sequencing and examined these variants in an association study of sickle cell anaemia patients treated with HC.