FOXO3 Regulates Fetal Hemoglobin Levels in Sickle Cell Anemia. V. Sheehan1, Y. Zhang1, J. Crosby2,3, R. Ware5, E. Boerwinkle3,4 1) Pediatrics, Baylor College of Medicine, Houston, TX; 2) The University of Texas Graduate School of Biomedical Sciences at Houston; Department of Biostatistics, Bioinformatics, and Systems Biology, University of Texas, Houston, TX; 3) Human Genetics Center, University of Texas, Houston, TX; 4) Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX; 5) Division of Hematology, Cincinnati Childrens Hospital Medical Center, Cincinnati, OH.
Although they ostensibly have a monogenetic disease, individuals with sickle cell anemia (SCA) exhibit wide variability in their degree of clinical severity. One of the most powerful and reproducible predictors of disease severity is the level of endogenous fetal hemoglobin (HbF), composed of two -globin and two -globin chains. We have used whole exome sequencing (WES) to find new genetic variants associated with baseline HbF in SCA, using 171 pediatric SCA genomes from participants in two clinical trials, HUSTLE (NCT NCT00305175) and SWiTCH (NCT 00122980). WES allows identification of both common and rare exonic variants; in order to capture the association between the phenotype variants with a minor allele frequency (MAF) below 1%, burden tests maximize power by grouping low frequency variants together by gene. Burden analysis (T1), found seven unique non-synonymous variations in a Forkhead box O transcription factor, FOXO3, to be significantly associated with lower HbF (p=5.6x10-4, -value ln HbF -0.66). All variants produced the same effect, a lowering of HbF. HbF values were normalized using natural log transformation to permit analysis, and adjusted for age, BCL11A, and XmnI variant status. Each individual was heterozygous for a variant. In order to verify the association between FOXO3 and endogenous HbF levels, we performed functional studies in K562 cells, an erythroid leukemia cell line that expresses -globin. We knocked down FOXO3 in K562 cells using silencing RNA (siRNA). RT-qPCR and Western blot analysis detected a substantial decrease in -globin expression with FOXO3 knockdown. This knockdown of FOXO3 recapitulated the patient phenotype of lower HbF levels with the inheritance of a FOXO3 mutation. We then examined the effect of overexpression of FOXO3 on -globin expression. K562 cells were transfected with expression plasmids encoding wild-type FOXO3 (FOXO3-WT) or a plasmid containing a constitutively active FOXO3 with three serine residues altered, so it could not be phosphorylated and sent to the cytoplasm where it is inactive (FOXO3-TM). A significant increase in -globin expression was observed in K562 cells transfected with FOXO3-WT. FOXO3-TM overexpressing cell lines had an even greater increase in -globin expression compared to FOXO3-WT. Our results indicate that FOXO3 participates in regulating -globin gene expression in K562 cells, and corroborate the association found through WES of sickle cell patient samples.
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