Abstract
Various degree of anemia post chemotherapy was frequently observed among patients even with the same treatment protocol and similar intensity. In this study, we performed whole exon sequencing in 31 individuals having multiple RBC transfusions (MRTs) and 31 patients with no RBC transfusion (NRT), all from the SCMC-ALL 2005 cohort. For children with different age and mass, the RBC transfusion amount was normalized by body surface area, and only post-remission transfusions were calculated to minimize the effect of the disease itself. Patients who received more than 8 RBC units/m2 were defined as MRT. By a genome-wide association study (GWAS), 1708 single nucleotide polymorphisms (SNPs) of 281 genes passed the criterion of the primary sort: a call rate of >95% and P<0.01. Of interest, most of the SNPs were located in introns adjacent to exons, suggesting that these polymorphisms are relevant to chemotherapy-induced anemia by regulating gene expression. Thus, these genes should be highly expressed in erythroid. Sorted according to the Differentiation Map Portal (DMAP) database, 10 genes were at the top of the list TFDP2, CHPT1, LPIN2, TNS1, BSG, ANXA7, EPB42, RAP1GAP, ARHGEF12, and ABCC4 which expressed in erythroid 2 fold above the average. Indeed, some of these genes have well-known associations with RBC function, such as with cytotoxicity susceptibility to chemotherapy agents and with erythropoiesis. Of note, 3 of them are related to small GTPases, including ARHGEF12, a RhoA specific guanine exchange factor.
Among SNPs in ARHGEF12, the most significant association was with rs76693355 (P=3.469E-03, odds ratio 5.864). Because whole exome sequencing can miss most regulatory SNPs located in introns, we screened all SNPs with linkage disequilibrium 0.2<r2<1 related to rs76693355 in the 5-kb flanking regions of ARHGEF12 and found rs10892563 at a binding site of the erythroid-specific transcription factor GATA1. The minor allele of this variant disrupted this binding site, which was confirmed by dual luciferase assay in vitro and ARHGEF12 expression quantitation of sorted the CD71-positive erythroid cells from banked bone marrow samples of ALL patients (A). Targeted SNP genotyping of 452 ALL patients enrolled in the SCMC-2005 protocols showed that the genotype frequencies of rs10892563 were CC 7.52%, CT 41.37%, and TT 51.11%. The average normalized RBC transfusion was 4.533 units/m2 in patients with CC, and 2.353 and 2.335 in patients with CT and TT genotypes, respectively. All patients who were homozygous needed RBC transfusion to maintain hemoglobin above 65 g/L, whereas among those who were heterozygous or wild-type, the RBC transfusion frequencies were 61.497% and 70.996%. Considering MRTs frequency, both those who were homozygous and heterozygous had significantly higher than patients carrying the wild type (B). The above results suggested that the ARHGEF12 polymorphism rs10892563 affects chemotherapy-induced anemia susceptibility.
To study whether ARHGEF12 related chemotherapy-induced anemia susceptibility is due to ARHGEF12 regulated erythroid regeneration, conditional knockout mouse model was explored. At steady state, arhgef12 deletion selectively perturbed erythroid development in the stage where erythroid precursors and erythroblasts differentiate to late erythroblast (C,D). When challenged by hemolysis induced by Phenylhydrazine at dose of 40mg/kg, arhgef12null mice exhibited worsened anemia and delayed recovery (E). Whle increasing phenylhydrazine dose to 80mg/kg, arhgef12-null mice failed to generate enough mature RBCs to support life (F).
In conclusion, ARHGEF12 plays an important role in erythroid hematopoiesis, and its genetic polymorphism may also influence anemia susceptibility of individuals experience chemotherapy.
No relevant conflicts of interest to declare.
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