Stroke is a devastating complication of sickle cell anemia (SCA) that will occur in ~11% of all patients without intervention. In pediatric patients with SCA, most of these strokes are ischemic and strongly linked with arterial vasculopathy. There is a strong heritable component that affects development of stroke in SCA patients. However, our understanding of the genes involved is very limited. Our group reported an unbiased genome wide association study of stroke in SCA using whole exome sequencing. We identified and validated two particular missense mutations in the GOLGB1 (Y1212C) and ENPP1 (K173Q) genes as being protective against stroke in SCA. Our study goal is to define the functional role of GOLGB1 and ENPP1 as modulators of vascular endothelial injury and ischemic stroke risk. We have previously observed that: a) GOLGB1 levels affect Golgi structure in monocytes and endothelial cells; b) The GOLGB1 Y1212C variant is associated with more dispersed Golgi in monocytes isolated from SCA patients; and c) ENPP1 enzymatic activity is higher in SCA patients with the protective K allele of the ENPP1 K173Q variant. From this preliminary data, we hypothesize that 1) GOLGB1 affects Golgi structure, which in turn regulates secretory output of pro-thrombotic factors such as von Willebrand factor (vWF) that can affect stroke in SCA; and 2) that ENPP1 affects risk of thrombosis either through endothelial cell signaling or by hydrolysis of the pro-inflammatory ATP inorganic pyrophosphate (PPi). We plan to test these hypotheses by analyzing the function of GOLGB1 and ENPP1 in samples from patients with SCA. We will isolate primary monocyte and endothelial cells from a large cohort of SCA patients receiving care at Texas Children?s Hospital.We will test our central hypotheses by pursuing three aims:
Aim 1 : Examine the role of GOLGB1 in the structure and function of Golgi complexes in sickle cell anemia - We will use high resolution imaging to determine the role of GOLGB1 in maintaining Golgi structure and protein trafficking in monocyte and endothelial cells from SCA patients;
Aim 2 : Determine the effect of ENPP1 activity in sickle cell anemia - We will measure plasma and endothelial activity of the ENPP1 enzyme.We will also test whether ENPP1 modulates vascular dysfunction in patients with SCA;
Aim 3 : Determine impact of GOLGB1 and ENPP1 on endothelial activation in a microfluidic arterial model - We will combine thrombotic protein assays and RNA expression profiling to identify endothelial activation pathways affected by our candidate genes. Our proposed studies will characterize the functional impact of our two genes and their genetic variants in SCA patient samples. This will benefit our understanding of the pathophysiological pathways of stroke and potentially provide targets for drug therapy to prevent or treat stroke in SCA patients.
These exploratory studies are relevant to public health for two main reasons. First, they may provide the first functional evidence for two genes that may modulate risk of stroke in patients with sickle cell anemia. Secondly, identifying genes involved in stroke development should provide a better understanding of the molecular pathways involved, and possibly drive the development of new therapies.
