Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease

Abstract

The vascular smooth muscle cell (SMC)-specific isoform of alpha-actin (ACTA2) is a major component of the contractile apparatus in SMCs located throughout the arterial system. Heterozygous ACTA2 mutations cause familial thoracic aortic aneurysms and dissections (TAAD), but only half of mutation carriers have aortic disease. Linkage analysis and association studies of individuals in 20 families with ACTA2 mutations indicate that mutation carriers can have a diversity of vascular diseases, including premature onset of coronary artery disease (CAD) and premature ischemic strokes (including Moyamoya disease [MMD]), as well as previously defined TAAD. Sequencing of DNA from patients with nonfamilial TAAD and from premature-onset CAD patients independently identified ACTA2 mutations in these patients and premature onset strokes in family members with ACTA2 mutations. Vascular pathology and analysis of explanted SMCs and myofibroblasts from patients harboring ACTA2 suggested that increased proliferation of SMCs contributed to occlusive diseases. These results indicate that heterozygous ACTA2 mutations predispose patients to a variety of diffuse and diverse vascular diseases, including TAAD, premature CAD, ischemic strokes, and MMD. These data demonstrate that diffuse vascular diseases resulting from either occluded or enlarged arteries can be caused by mutations in a single gene and have direct implications for clinical management and research on familial vascular diseases.

Figure 1

Vascular Diseases in Families with ACTA2 Mutations (A) Pedigrees of 12 families with ACTA2 mutations in which the mutation segregates with a number of vascular diseases, including TAAD, premature onset of CAD, and stroke. The disease and mutation status of individuals are as indicated in the figure legend. (B) Three families with ACTA2 R258 mutations with early age at onset of stroke and MMD. (C) Pedigrees of patients with ACTA2 mutations identified from Texgen cohort. Pedigrees of two patients with nonfamilial TAAD and de novo ACTA2 mutations and of one patient with nonfamilial TAAD, a sister with MMD, and a mother with premature CAD due to an ACTA2 mutation. Numbers and letters under each circle or square in the pedigree represent the following: the first line shows the individual ID number; the second line shows age at onset of the vascular disease(s), which is color coded based on the vascular disease (see the figure legend); and an asterisk next to a symbol indicates that individuals had MMD. Individuals in (C) are labeled with their sample ID number. Circles represent females, and squares represent males. A slash through a circle or square indicates a deceased person. A pound sign (#) indicates an individual who may have carried the ACTA2 mutation, on the basis of their location in the pedigree, but was not assumed to harbor a mutation in the genetic and clinical analysis of ACTA2-mutation carriers.

Figure 2

Genotype-Phenotype Correlation between Location of Mutation and Vascular Diseases (A) Location of all ACTA2 mutations and the specific phenotypes observed in patients heterozygous for those mutations. The mutations are listed in sequential order and align with the exon in which they are located. “No.” indicates the number of identified individuals who carry each specific mutation. The headings TAAD, Stroke, and CAD denote the number of individuals affected by each of these conditions. An asterisk indicates a mutation leading to the onset of ischemic strokes prior to the age of 20 years. (B) The ribbon diagram of SM α-actin, identifying the location of the different mutations at the protein-structure level. The recurrent ACTA2 mutation leading to ischemic strokes at a young age (p.R258C/H, shown in green) lies within subdomain 4, whereas mutations occurring in multiple individuals and leading primarily to premature CAD (p.R149C and p.R118Q, shown in red) all lie within the hydrophobic cleft.

Figure 3

ACTA2 Mutations Lead to Increased SMC and Myofibroblast Proliferation in Culture (A) Coronary artery from 28-year-old TAA441:IV:4, stained with Movat's stain and SM α-actin antibody. The coronary artery showed 70% narrowing of the vessel due to a fibrocellular atherosclerotic plaque (top two panels; magnification 40×; scale bar represents 1.0 mm). Higher magnification (200×; scale bar represents 200 μm) revealed that these cells contain SM α-actin. (B) The epicardial arteries from a 50-year-old man, TAA441:III:7, and a 53-year-old woman, TAA015:III:1. H&E, Movat, and α-actin immunostaining of the tissue showed thickened vessels within the myocardium (magnification 200×; scale bar represents 200 μm). (C) Cell-proliferation assays (BrdU incorporation) illustrate that SMCs explanted from patients heterozygous for an ACTA2 mutation (n = 2) proliferate more rapidly than matched control SMCs (n = 2). (D) Myofibroblasts (fibroblasts exposed to TGF-β1 for 72 hr) from patients heterozygous for ACTA2 mutations (n = 9) proliferate more rapidly than matched controls (n = 10). Data are expressed as means ± SEM, and p values are indicated. Immunoblotting for SM α-actin from the cell lysates confirms that TGF-β1 exposure increased cellular SM α-actin.