Abstract Body: Background: ACTA2 encodes smooth muscle α-actin, and pathogenic variants in ACTA2 at arginine 179 (R179) cause smooth muscle dysfunction syndrome with early onset aortic and cerebrovascular disease. ACTA2 R179 variants disrupt smooth muscle cell (SMC) differentiation from neural crest cells (NCCs). The regulatory shifts that derail NCC derived SMC differentiation remain incompletely defined.
Methods: Patient-derived (ACTA2 R179; n=3) and control-derived (n=4) induced pluripotent stem cell (iPSC) lines were differentiated through NCCs to SMCs and profiled by bulk RNA sequencing. We tested a stage×group interaction across stages and quantified a within-donor paired differentiation delta, Δ(SMC–NCC), for each line to reduce baseline differences between iPSC lines. Pathway and transcription factor (TF) activity footprints were inferred using PROGENy and DoRothEA/VIPER. Robustness was assessed by leave-one-line-out analyses and a diagnostic-based sensitivity refit excluding the control line with the largest Cook’s distance.
Results: SDC2 (Syndecan-2) was among the top stage-dependent signal in the stage×group interaction test (FDR≈0.018). In paired within-line NCC→SMC changes (Δ=SMC–NCC), ACTA2 R179 mutant lines showed a reduced NCC→SMC induction of SDC2 relative to controls (median Δ difference [mutant−control] ≈ −1.74; p≈0.057), with direction preserved in leave-one-line-out and diagnostic sensitivity refits. Footprint analyses identified established pathways, but also hypothesis-generating regulatory shifts alongside decreased SDC2, including decreased STAT2 and ZNF263 TF activity, increased PRDM14 TF activity, and altered signaling footprints in the mutant cells. In silico perturbation analyses further suggested that reduced SDC2 is associated with transcriptomic movement towards a de-differentiated SMC phenotype. SDC2 encodes a heparan sulfate proteoglycan that can act as a co-receptor modulating growth factor signaling in other differentiation contexts; its role in NCC-derived SMC differentiation remains uncharacterized. In-progress validation studies with Crispr knockout of SDC2 in control iPSC lines will confirm the identified signaling changes and differentiation-state changes are causally linked to changes in SDC2.
Conclusions: Stage dependent, within donor paired NCC→SMC transcriptomics identify SDC2 as a robust, reproducible signature of ACTA2 R179 smooth muscle dysfunction and prioritize SDC2 centered mechanisms for future functional testing.