Dysregulated smooth muscle cell proliferation and gene expression underlie ACTA2 variant-associated aortopathy

Pathogenic variants affecting alpha-2 smooth muscle actin (ACTA2) account for approximately 20% of nonsyndromic familial thoracic aortic aneurysms (TAA) and confer a high risk of dissection; however, the cell type-specific transcriptional mechanisms underlying ACTA2-associated TAA remain poorly defined, particularly for variants of uncertain significance. In this study, we investigated the transcriptional and cellular effects of a novel ACTA2 p.Met49Thr mutation identified in a young male in his early twenties, who developed a dissected ascending aortic aneurysm without traditional risk factors. Using clinically archived formalin-fixed paraffin-embedded (FFPE) aortic tissue, we isolated intact nuclei and performed single-nucleus RNA sequencing (snRNA-Seq) to generate 17,938 transcriptomes. Relative to a nongenetic hypertensive TAA control, ACTA2-p.Met49Thr aortic tissue displayed marked expansion of vascular smooth muscle cells (VSMCs) (70.6% vs. 39.7%), accompanied by upregulation of proliferation-associated transcripts including FOSB, FOS, JUN, and DEPP1. Lineage tracing via trajectory analysis revealed transcriptional progression from quiescent to pro-proliferative VSMC states, enriching for human loci associated with aortic strain and diameter. Histological evaluation corroborated these findings, demonstrating medial hypercellularity, elastic fiber fragmentation, and adventitial fibrosis enriched within the ACTA2-p.Met49Thr specimen. Taken together, these findings implicate a novel pathogenic ACTA2 variant that drives transcriptional reprogramming and proliferative VSMC remodeling, supporting that ACTA2-associated aortopathy progression occurs via functional cell state transitions. In addition, this work demonstrates the feasibility of FFPE-compatible snRNA-Seq for clinical variant annotation.NEW & NOTEWORTHY This study uncovers how a novel ACTA2 variant (p.Met49Thr) likely drives thoracic aortic aneurysm through a pro-proliferative shift in vascular smooth muscle cell states. Using FFPE-compatible single-nucleus RNA-sequencing (snRNA-seq), this study links transcriptomic profiling of the human aorta to vascular traits, highlighting a pathologic cell state transition in ACTA2-mutant aortopathy. More broadly, it also serves as a proof-of-concept to leverage snRNA-seq technology using archived human tissue for clinical phenotype-to-variant characterization.