Abstract
Whole transcriptome RNA-sequencing was performed to quantify RNA expression changes in whole blood samples collected from steady state sickle cell anemia (SCA) and control subjects. Pediatric SCA and control subjects were recruited from Atlanta (GA)—based hospital(s) systems and consented for RNA sequencing. RNA sequencing was performed on an Ion Torrent S5 sequencer, using the Ion Total RNA-seq v2 protocol. Data were aligned to the hg19 reference genome and analyzed in the Partek Genomics studio package (v7.0). 223 genes were differentially expressed between SCA and controls (± 1.5 fold change FDR p < 0.001) and 441 genes show differential transcript expression (± 1.5 fold FDR p < 0.001). Differentially expressed RNA are enriched for hemoglobin associated genes and ubiquitin-proteasome pathway genes. Further analysis shows higher gamma globin gene expression in SCA (33-fold HBG1 and 49-fold HBG2, both FDR p < 0.05), which did not correlate with hemoglobin F protein levels. eQTL analysis identified SNPs in novel non-coding RNA RYR2 gene as having a potential regulatory role in HBG1 and HBG2 expression levels. Gene expression correlation identified JHDM1D-AS1(KDM7A-DT), a non-coding RNA associated with angiogenesis, enhanced GATA1 and decreased JAK-STAT signaling to correlate with HBG1 and HBG2 mRNA levels. These data suggest novel regulatory mechanisms for fetal hemoglobin regulation, which may offer innovative therapeutic approaches for SCA.
Highlights
Sickle cell anemia (SCA) is a genetically inherited blood disorder
In order to further study the pathologic mechanisms of sickle cell anemia (SCA), and identify potential therapeutic targets, we investigated peripheral blood whole transcriptome profiling in pediatric patients with SCA
One participant was withdrawn from the study because on subsequent chart review it was determined they were being medicated with hydroxyurea (HU) therapy at the time of blood sample collection
Summary
Homozygous hemoglobin SS is characterized by a single point mutation (T to A) of the beta hemoglobin gene (Hbb), resulting in a non-conservative protein mutation at the sixth codon (Glu to Val) (Pauling et al, 1949; Ingram, 1958). This mutation results in polymerization of the hemoglobin molecules under low oxygen conditions, and the classic sickling morphology of red blood cells (Scriver and Waugh, 1930). Using these circulating cells as sentinels, one can diagnose various brain insults using RNA expression patterns in peripheral blood, including stroke
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