A coding variant in RARG confers susceptibility to anthracycline-induced cardiotoxicity in childhood cancer

Abstract

Anthracyclines are used in over 50% of childhood cancer treatment protocols, but their clinical usefulness is limited by anthracycline-induced cardiotoxicity (ACT) manifesting as asymptomatic cardiac dysfunction and congestive heart failure in up to 57% and 16% of patients, respectively. Candidate gene studies have reported genetic associations with ACT, but these studies have in general lacked robust patient numbers, independent replication or functional validation. Thus, the individual variability in ACT susceptibility remains largely unexplained. We performed a genome-wide association study in 280 patients of European ancestry treated for childhood cancer, with independent replication in similarly treated cohorts of 96 European and 80 non-European patients. We identified a nonsynonymous variant (rs2229774, p.Ser427Leu) in RARG highly associated with ACT (P = 5.9 × 10(-8), odds ratio (95% confidence interval) = 4.7 (2.7-8.3)). This variant alters RARG function, leading to derepression of the key ACT genetic determinant Top2b, and provides new insight into the pathophysiology of this severe adverse drug reaction.

Figure 1. A pharmacogenetic association with susceptibility to anthracycline-induced cardiotoxicity is situated within RARG

a) Association results are shown for genotyped (circles) and imputed (squares) SNPs along with recombination rates for a 122 kb region of chromosome 12q13.13. Each point represents the nominal P-value (left y-axis) for the stage 1 cohort. P-values are from logistic regression analysis using an additive model, adjusted for age, cumulative dose, tumor type (ALL, Ewing’s sarcoma and rhabdomyosarcoma) and cardiac radiation therapy. SNPs are colored according to their pairwise correlation (r²) with rs2229774 (purple circle) using the 1000 Genomes CEU reference population. Overlaid are the recombination rates (right y-axis) for estimating putative recombination hotspots also based upon the 1000 Genomes CEU population. b) The linkage disequilibrium (D′) based upon the 1000 Genomes CEU population depicted for this region similarly demonstrates the associated haplotype is localized to RARG. Details of D′ color coding are provided in Online Methods.

Figure 2. Functional characterization of RARG^S427L reveals impaired transcriptional regulation

a) Transcriptional activation of luciferase coupled to an optimized retinoic acid response element (RARE) by transiently transfected RARG wild type (WT) or RARG^S427L in HEK293T cells. Averages of RARE-activation from aggregate data (n = 48; three independent experiments of sixteen replicates) are presented. Inset, Immunoblot of 20μg HEK293T lysate generated 48 hours post-transfection with empty vector (negative), or the indicated construct using anti-DDK 4C5 (top panel) and anti-GAPDH (bottom panel) antibodies. Untagged wild type RARG has an estimated molecular weight of 50.4 kDa. Molecular sizes are indicated on the left. b) Relative Top2b expression in untransfected or RARG-transfected H9c2 cells in the presence or absence of ATRA. Average values of relative Top2b expression from aggregate data (n = 12; four independent experiments of three replicates) are presented. (c) Repression of Top2b expression in RARG WT- or RARG^S427L-transfected H9c2 cells compared to untransfected cells. Averages of normalized Top2b repression from aggregate data (n = 12; two independent experiments of six replicates) are presented. ** and *** denote P < 0.005 and P < 0.0001, respectively, using t-test (a and c) or one-way ANOVA with Tukey post-test analyses (b). Error bars; s.e.m.