A yeast based assay establishes the pathogenicity of novel missense ACTA2 variants associated with aortic aneurysms

Cristina Calderan; Ugo Sorrentino; Luca Persano; Eva Trevisson; Geppo Sartori; Leonardo Salviati; Maria Andrea Desbats

doi:10.1038/s41431-024-01591-1

A yeast based assay establishes the pathogenicity of novel missense ACTA2 variants associated with aortic aneurysms

Eur J Hum Genet. 2024 Mar 15. doi: 10.1038/s41431-024-01591-1. Online ahead of print.

Authors

Cristina Calderan^{1

2}, Ugo Sorrentino¹, Luca Persano³, Eva Trevisson¹, Geppo Sartori², Leonardo Salviati^{1

4}, Maria Andrea Desbats⁵

Affiliations

¹ Clinical Genetics Unit, Department of Women's and Children's Health, University of Padua, and Istituto di Ricerca Pediatrica (IRP) Città della Speranza, Padua, Italy.
² Department of Biomedical Sciences, University of Padua, Padua, Italy.
³ Pediatric Oncohematology Laboratory, Department of Women's and Children's Health, University of Padua, and Istituto di Ricerca Pediatrica (IRP) Città della Speranza, Padua, Italy.
⁴ CIR-Myo Myology Center, University of Padua, Padua, Italy.
⁵ Clinical Genetics Unit, Department of Women's and Children's Health, University of Padua, and Istituto di Ricerca Pediatrica (IRP) Città della Speranza, Padua, Italy. mariaandrea.desbats@unipd.it.

PMID: 38486025
DOI: 10.1038/s41431-024-01591-1

Abstract

The ACTA2 gene codes for alpha-smooth muscle actin, a critical component of the contractile apparatus of the vascular smooth muscle cells. Autosomal dominant variants in the ACTA2 gene have been associated to familial non-syndromic thoracic aortic aneurysm/dissection (TAAD). They are thought to act through a dominant-negative mechanism. These variants display incomplete penetrance and variable expressivity, complicating the validation of ACTA2 variants pathogenicity by family segregation studies. In this study, we developed a yeast based assay to test putative TAAD-associated ACTA2 variants. We identified five new heterozygous ACTA2 missense variants in TAAD patients through next generation sequencing. We decided to test their pathogenicity in Saccharomyces cerevisiae, since yeast actin is very similar to human alpha-smooth muscle actin, and the residues at which the TAAD-associated variants occur in ACTA2 are well conserved. A wild type yeast strain was transformed with a vector expressing the different mutant alleles, to model the heterozygous condition of patients. Then, we evaluated yeast growth by spot test and cytoskeletal and mitochondrial morphology by fluorescence microscopy. We found that mutant yeast strains displayed only mild growth defects but a significant increase in the percentage of cells with abnormal mitochondrial distribution and abnormal organization of the actin cytoskeleton compared to controls. All variants appeared to interfere with the activity of wild type actin in yeast, suggesting a dominant-negative pathogenic mechanism. Our results demonstrate the utility of using the yeast actin model system to validate the pathogenicity of TAAD-associated ACTA2 variants.