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Randomized Controlled Trial
. 2014 Oct;28(10):1740-51.
doi: 10.1210/me.2014-1147. Epub 2014 Aug 22.

Aromatase Inhibitor-Associated Bone Fractures: A Case-Cohort GWAS and Functional Genomics

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Free PMC article
Randomized Controlled Trial

Aromatase Inhibitor-Associated Bone Fractures: A Case-Cohort GWAS and Functional Genomics

Mohan Liu et al. Mol Endocrinol. .
Free PMC article

Abstract

Bone fractures are a major consequence of osteoporosis. There is a direct relationship between serum estrogen concentrations and osteoporosis risk. Aromatase inhibitors (AIs) greatly decrease serum estrogen levels in postmenopausal women, and increased incidence of fractures is a side effect of AI therapy. We performed a discovery case-cohort genome-wide association study (GWAS) using samples from 1071 patients, 231 cases and 840 controls, enrolled in the MA.27 breast cancer AI trial to identify genetic factors involved in AI-related fractures, followed by functional genomic validation. Association analyses identified 20 GWAS single nucleotide polymorphism (SNP) signals with P < 5E-06. After removal of signals in gene deserts and those composed entirely of imputed SNPs, we applied a functional validation "decision cascade" that resulted in validation of the CTSZ-SLMO2-ATP5E, TRAM2-TMEM14A, and MAP4K4 genes. These genes all displayed estradiol (E2)-dependent induction in human fetal osteoblasts transfected with estrogen receptor-α, and their knockdown altered the expression of known osteoporosis-related genes. These same genes also displayed SNP-dependent variation in E2 induction that paralleled the SNP-dependent induction of known osteoporosis genes, such as osteoprotegerin. In summary, our case-cohort GWAS identified SNPs in or near CTSZ-SLMO2-ATP5E, TRAM2-TMEM14A, and MAP4K4 that were associated with risk for bone fracture in estrogen receptor-positive breast cancer patients treated with AIs. These genes displayed E2-dependent induction, their knockdown altered the expression of genes related to osteoporosis, and they displayed SNP genotype-dependent variation in E2 induction. These observations may lead to the identification of novel mechanisms associated with fracture risk in postmenopausal women treated with AIs.

Trial registration: ClinicalTrials.gov NCT00283608.

Figures

Figure 1.
Figure 1.
A, GWAS Manhattan plot GWAS performed with P values adjusted for age, previous fracture, bisphosphonate use, and the first 3 eigenvectors. The 3 SNP signals that underwent functional genomic validation are highlighted. B, LocusZoom plot of the chromosome 20 region surrounding the THIL-TUBB1-CTSZ-SLMO2-ATP5E genes showing a plot of −log10 (P values) for observed (circles) and imputed SNPs (diamonds). C, LocusZoom plot of the chromosome 6 region surrounding the TRAM2-TMEM14A genes. D, LocusZoom plot of the chromosome 2 region surrounding the MAP4K4 gene.
Figure 2.
Figure 2.
Validation decision cascade used to test GWAS signals. The association analysis shown graphically in the Manhattan plot in Figure 1A resulted in the identification of 20 SNP signals with P < 5E-06. The figure depicts graphically the process for SNP signal and associated gene functional validation.
Figure 3.
Figure 3.
E2 induction time course for THIL, CTSZ, SLMO2, ATP5E, TRAM2, TEME14A, and MAP4K4 mRNA expression in hFOB-ERα cells exposed to 0.1 nmol/L E2 for differing periods of time. The values shown represent means of duplicate assays.
Figure 4.
Figure 4.
Effect of knockdown of SLMO2 (A) and TRAM2 (B) on the relative mRNA expression of genes involved in the osteoporosis genes primer panel in hFOB-ERα cells. Red lines represent basal control levels of expression. Data for the RANK-RANKL-OPG genes are highlighted with red rectangles.
Figure 5.
Figure 5.
LCL SNP-dependent variation of candidate gene expression in response to increasing concentrations of E2 for 3 homozygous W/W (blue lines) and 3 homozygous V/V (pink lines) LCLs. A, Relative CTSZ, SLMO2, and ATP5E mRNA expression (upper panels) and relative mRNA expression for OPG and THIL (lower panels). B, SNP-dependent variation in TMEM14A-TRAM2 expression in response to increasing concentrations of E2 for 3 homozygous W/W (blue lines) and 3 homozygous V/V (pink lines) LCLs (upper panels) and relative mRNA expression level for OPG (lower left panel). C, SNP-dependent variation in MAP4K4 expression in response to increasing concentrations of E2 for 3 homozygous W/W (blue lines) and 3 homozygous V/V (pink lines) AA LCLs (upper panel) and relative mRNA expression level for OPG (lower panel). *, P < .05 and **, P < .01 when compared with the alternative genotype at the same concentration of E2.

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