Robust epistasis between the genes encoding a TGF effector and its regulatory microRNA governs modification of cardiovascular phenotypes in TGF vasculopathies. J. Calderon1, H. Dietz1,2 1) Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD; 2) Howard Hughes Medical Institute, Chevy Chase, MD.
The Loeys-Dietz Syndrome (LDS) spectrum disorders share a predisposition for widespread and aggressive vascular lesions (tortuosity and aneurysms) in association with primary heterozygous mutations in genes encoding critical TGF signaling effectors (TGFBR1, TGFBR2, SMAD3 and TGFB2). Many studies document that the C57BL/6J (B6) background exacerbates TGF deficiency phenotypes when compared to 129SvE (SV129), but the source of modification is unknown. While postnatal aneurysm progression is blunted upon crossing LDS mutations onto B6, LDS mice show near-complete penetrance of perinatal death due to truncus arteriosus (TA), a congenital heart defect that has been associated with loss of TGF signaling in the neural crest, once the B6 contribution becomes substantial. This discrete readout in LDS was utilized to map the relevant modifier alleles. Pure SV129 Tgfbr2G357W/+ mice were bred to F2 WT mice with extensive recombination between B6 and SV129 chromosomes and E17.5 fetuses were phenotyped for TA. GWA analysis revealed a single major B6 locus associated with TA on chr 9 (LOD=9); a minor linkage signal was apparent on chr X (LOD=2.75). Combinatorial analyses revealed that TA was dependent on B6 contribution at the chr 9 locus, but was thereafter dramatically influenced by B6 contribution at the chr X locus in a dosage-dependent manner. Tgfbr2 emerged as a promising candidate gene in the region of association on chr 9, both by virtue of its known function and the presence of strain-specific variation in its 3UTR that is predicted to alter mRNA conformation. In an unbiased search for miRNAs that target Tgfbr2, miR-106a emerged as a prominent candidate because it is located within the suggestive peak of association on chr X. A luciferase reporter allele harboring the B6 Tgfbr2 3'UTR showed dramatically reduced activity when compared to its 129 counterpart (p<10E-9) in transfection studies, with a disproportionately increased activity upon addition of a miR-106a antagonist. These results suggest greater sensitivity of the B6 version of Tgfbr2 to the repressive activity of miR-106a. We further showed that vascular smooth muscle cells from pure B6 animals express significantly less Tgfbr2 protein and exhibit blunted activation of intracellular signaling mediators in response to TGF, when compared to 129. These data reveal a complex architecture of modification that may prove relevant to other Mendelian disorders and inform therapeutic strategies for LDS.
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