Microarray Analysis of Peripheral Blood Monocytes in Sickle Cell Anemia Patients with Cerebrovascular Stenosis.
Authors: Driscoll M. Catherine , Adenika Remi , Nagel Ronald L. , Hoffman Eric P. 
Published: October 2019

Abstract
Cerebrovascular disease (CVD) of both large and small cerebral vessels occurs in one-third of patients with sickle cell anemia (SCA) by age 20 years. Large vessel stenosis (LVS) occurs in ~10% of patients and is associated with overt stroke and/or (+) transcranial Doppler (TCD). Epistatic genetic modifiers are postulated to account for the significant phenotypic heterogeneity of SCA and likely contribute to CVD. Potential genetic modifers of CVD may be found in pathways for inflammation, adhesion, stress/immune reactions, angiogenesis, thrombosis, and oxidant generation. Peripheral blood monocytes are activated in SCA and contribute to inflammation and endothelial activation. The goal of this study is to determine if there is a difference in gene expression profiles of monocytes in patients with SCA and LVS, compared to SCA controls without CVD, and to normal African American (AA) controls. The study group consisted of 1) SCA patients with LVS [n = 6, (moyamoya = 3, no moyamoya =3)], 2) SCA patients without CVD and nornal TCD (n = 3), and 3) normal AA controls (n = 3). The 6 SCA-LVS patients were on chronic transfusion programs to maintain Hb S <30%. Monocytes were isolated by negative selection (RosetteSep Antibody) and total RNA extracted, converted to cRNA and hybridized to Affymetrix U133A GeneChip. Gene profiles were analyzed using dChip version 1.3 and gene lists were created for genes with a 2-fold or greater increase or decrease in expression with a Welch t-test p-values of 0.05 or less. Results: 1) SCA controls without CVD compared to AA controls revealed 50 differentially expressed genes (p<0.05), 16 with 2-fold or greater change. Differential gene expression was observed in pathways for angiogenesis (PD-ECGF, 5.1 fold, p = 0.025), cell signaling (lymphocyte antigen complex 6, 10.4 fold, p = 0.032, STAT1, 5.1 fold, p = 0.039), and inflammation (MARCO, 3.1 fold, p = 0.042). 2) SCA patients with LVS compared to SCA controls revealed 47 differentially expressed genes (p = <0.05) but only 6 genes with 2-fold or greater change. These included angiogenesis (PD-ECFG, -3.59 fold, p = 0.02), cell signaling (lymphocyte antigen complex 6, −4.61 fold, p = 0.02), adhesion (junction plakoglobin, −7.73 fold, p = 0.03), inflammation (LTA4H, 2.57 fold, p =0.009). These data suggest that patients with SCA and LVS overexpress leukotriene A4 hydrolase (LTA4H), an enzyme that catalyzes the final step in the leukotriene B4 (LTB4) pathway. LTB4 is a chemoattractant which triggers adhesion and aggregation of leukocytes to the endothelium. Five-lipoxygenase-activating protein (FLAP), a leukotriene pathway protein has recently been identified as a candidate gene for stroke and AMI risk in an Icelandic population. Also of interest, the SCA-LVS patients showed decreased expression of PD-ECFG and lymphocyte antigen complex 6, both genes being significantly overexpressed in SCA controls. Since the SCA-LVS patients are on chronic transfusion, underexpression could be due to the ameliorating effects of transfusion therapy. Validation studies of these findings are currently underway.