Intermediate molecular phenotypes to identify genetic markers of anthracycline-induced cardiotoxicity risk

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  Figure 1.. Scheme of the general approach. A) This paper studies the different susceptibility to anthracycline cardiotoxicity (CDA) in a cohort of mice generated by backcrossing. The degree of CDA was quantified at the histopathological level using an Ariol slide scanner in the subepicardial and subendocardial zones of the heart. In addition, the levels in the myocardium of different molecules (intermediate phenotypes) associated with cardiotoxicity were quantified. Subsequently, genetic regions or quantitative trait loci (QTL) associated with CDA (cdaQTL) and other QTLs associated with intramyocardial levels of intermediate molecular phenotypes (ipQTL) were identified. B) We then evaluate whether some of the ipQTLs contribute significantly to the phenotypic variation (susceptibility) of CDA, despite not being directly linked to it. To do this, we evaluate using genetic models whether the phenotypic variation explained by the ipQTL together with the cdaQTL exceeded that explained by the cdaQTL alone. If so, the genetic determinant linked to the myocardial levels of this intermediate molecular phenotype contributes to CDA susceptibility. C) The genes responsible for ipQTLs are unknown;…

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  Figure 2.. Anthracycline-induced cardiotoxicity differs according to genetic background, therapy regime, and age. A) Cardiotoxicity due to anthracyclines (CDA) is evaluated in a cohort of mice generated by backcrossing. The mice are treated with chemotherapy, and cardiotoxicity is quantified at the histopathological level. B, C) Comparison of cardiotoxicity between mouse strains of homogeneous genetic background. After combined chemotherapy, FVB mice showed less global heart fibrosis (B) and less subendocardial fibrosis (C) than F1 mice. No differences exist between the parental strains after chemotherapy with doxorubicin alone (not shown). Note the distribution of heart fibrosis as a continuum in F1BX mice (A, B). D-F) In the genetically heterogeneous cohort of F1BX mice, the combined treatment is more cardiotoxic than doxorubicin alone in terms of the degree of global (D), subendocardial (E), and subepicardial (F) fibrosis. G-J) CDA was higher in the subendocardial zone than in the heart’s subepicardial location in F1BX1 mice, in fibrosis (G, H) and cardiomyocyte area (I, J). Mann–Whitney U test. K, L) CDA in the whole cohort of F1BX mice based on age and type of chemotherapy. Global…

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  Figure 3.. Intermediate molecular phenotypes are associated with cardiotoxicity due to anthracyclines (CDA). A) Quantification in the myocardium of F1BX mice of different intermediate molecular phenotypes after chemotherapy. B-I) Principal component analyses classify mice with high and low CDA susceptibility based on the levels of intermediate molecular phenotypes in the myocardium under different conditions: fibrosis after doxorubicin (B), cardiomyocyte area after the combined therapy (C), subendocardial zone fibrosis (combined therapy) (D), subepicardial zone fibrosis (combined therapy) (E), subendocardial zone fibrosis (doxorubicin) (F), subepicardial fibrosis zone (doxorubicin) (G), subendocardial cardiomyocyte area after combined therapy (H), subepicardial cardiomyocyte area (combined therapy) (I). Mice with high (brown) and low (blue) levels of fibrosis and cardiomyocyte area were differentiated by the median. This figure is related to Supplemental Table S3.

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  Figure 4.. Identification of quantitative trait loci associated with intermediate molecular phenotypes (ipQTLs) of CDA susceptibility. A) F1BX1 mice generated by backcrossing were genotyped using 1499 SNPs on an Illumina platform. Linkage analysis was performed to detect ipQTLs. B) The heatmap shows the global scenario of ipQTLs after doxorubicin or combined therapy. Each square represents a chromosome; its number is on the left. Each square’s intensity mark signifies the degree of association (LOD score) between the genetic markers and the phenotype according to the indicated scale. The marks’ location of each square occupies the relative area within each chromosome (centromere and telomere positions above and below, respectively). Only linkages with a LOD score > 1.5 (suggestive) are represented. R/qtl software was used to identify ipQTLs. The exact location of each ipQTL in each chromosome and the associated genetic markers are shown in Supplemental_Table_S5A.xls (doxorubicin therapy) and Supplemental_Table_S5B.xls (combined therapy).

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  Figure 5.. Quantitative trait loci linked to cardiotoxicity due to anthracyclines (cda-QTLs). A) The genotyping of the F1BX mouse cohort permitted to locate cdaQTLs directly linked to the CDA susceptibility measured at the histopathological level. B, C) The heatmaps show the global scenario of the cda-QTLs linked to cardiac fibrosis and cardiomyocyte area under different conditions (age and type of therapy): after chemotherapy with doxorubicin (B) or after combined treatment (C). For both panels, B and C: only QTLs with a LOD score > 1.5 were plotted along each chromosome. Analyses were carried out with R/qtl software. Each square represents one chromosome; its number is indicated on the left. According to the indicated scale, the intensity of marks within each square indicates the degree of association (LOD score) between the genetic markers and the phenotype. The mark’s location corresponds to its relative location within the chromosome (centromere and telomere positions above and below, respectively). The exact locations of each cda-QTL and its associated genetic markers are shown in Supplemental Table S5. D) The cda-QTL6 was associated with CDA under different conditions in old mice.…

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  Figure 6.. Percentage of global fibrosis explained by cda-QTL6 or cda-QTL13 and genetic models. A) Genetic models between ipQTLs and cdaQTL are generated to assess whether an ipQTL contributes to the phenotypic variation of CDA. If the variability of the model significantly exceeds that of the cdaQTL, the ipQTL would contribute to the phenotypic variation of CDA and susceptibility. B) Above, the diagram shows the percentage of global fibrosis explained by cda-QTL6 after treatment with doxorubicin (red). The circles on the right show the name of the cda-QTL6 under these conditions, difQTL1 (doxorubicin-induces fibrosis QTL1), which appears linked to global fibrosis in all mice, and odifQTL2 (old mice difQTL2), which seems to be associated with global fibrosis in all mice. Below is the percentage of global fibrosis explained by cda-QTL13 following treatment with doxorubicin (red) or combined therapy (yellow). The circles on the right show the other names of the cda-QTL13 for those conditions: ydifQTL2 (young mice doxorubicin-induced fibrosis QTL2), ycifQTL3 (young mice combined therapy-induced fibrosis QTL3), and cifQTL4 (combined therapy-induced fibrosis QTL3), the latter linked to global…

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  Figure 7.. Genetic models of CDA risk generated by bootstrapping 100 times and LASSO multivariate regression. A-C) CDA risk model for the cohort of breast cancer patients. The genetic model (A) and the ROC curves of the training set (B) and the testing set (C) are shown. D-E) Genetic risk model for the pediatric patient cohort. The genetic model (D) and the ROC curves for the training set (E) and the testing set (F) are shown. Cohorts were split at 80% for the training and 20% for the testing set. FPR, false positive rate.