Dysregulated smooth muscle cell proliferation and gene expression underlie <i>ACTA2</i> variant-associated aortopathy

Pathogenic variants affecting alpha-2 smooth muscle actin (<i>ACTA2</i>) 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 <i>ACTA2</i>-associated TAA remain poorly defined, particularly for variants of uncertain significance. In this study, we investigated the transcriptional and cellular effects of a novel <i>ACTA2</i> 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, <i>ACTA2</i>-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 <i>FOSB</i>, <i>FOS</i>, <i>JUN</i>, and <i>DEPP1</i>. 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 <i>ACTA2</i>-p.Met49Thr specimen. Taken together, these findings implicate a novel pathogenic <i>ACTA2</i> variant that drives transcriptional reprogramming and proliferative VSMC remodeling, supporting that <i>ACTA2</i>-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.<b>NEW & NOTEWORTHY</b> This study uncovers how a novel <i>ACTA2</i> 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 <i>ACTA2</i>-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.