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

European Journal of Human Genetics (2024)Cite this article

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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.

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Fig. 1: Effect of thoracic aortic aneurysm and/or dissection-associated actin variants on yeast growth at 30 °C on solid media (Spot test assay).
Fig. 2: Differences in actin protein levels expressed from pYES2 vector in presence of different concentrations of galactose.
Fig. 3: Effect of thoracic aortic aneurysm and/or dissection-associated actin variants on actin-dependent yeast mitochondrial morphology.
Fig. 4: Effect of thoracic aortic aneurysm and/or dissection-associated actin variants on yeast cytoskeletal morphology.

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Data availability

The authors declare that all supporting data are available either within the article or upon request to the corresponding author. The variants identified in this work have been submitted to ClinVar by our group (c.218T > C, SCV004024262; c.648G > C, SCV004024222; c.655T > C, SCV004024263), or previously submitted by others (c.78C > A, VCV000580143.10; c.79G > A, VCV001329101.4; c.352C > T, VCV001053987.14, and c.772C > T, VCV000018278.16).

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Acknowledgements

This research was part of a doctoral dissertation by CC with the title “Development of yeast models for validating novel pathogenic mutations associated with human aortic aneurysm and primary Coenzyme Q10 deficiency” [40].

Funding

This work was supported by a grant from “IRP—Città della Speranza” to LS.

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Authors and 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

    Cristina Calderan, Ugo Sorrentino, Eva Trevisson, Leonardo Salviati & Maria Andrea Desbats

  2. Department of Biomedical Sciences, University of Padua, Padua, Italy

    Cristina Calderan & Geppo Sartori

  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

    Luca Persano

  4. CIR-Myo Myology Center, University of Padua, Padua, Italy

    Leonardo Salviati

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Contributions

CC, MAD and LS conceived the study concept; LS acquired funding; US, ET and LS collected patients’ samples and clinical data, and performed the molecular diagnosis; CC performed functional studies and microscopic analysis; MAD, LP, GS and LS provided their expertise for the experiments; CC, MAD, GS and LS wrote and edited the manuscript; all authors reviewed and commented on the final draft manuscript.

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Correspondence to Maria Andrea Desbats.

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Ethical approval was not needed in this study because we only used data from patients obtained in our diagnostic genetics unit. All genetic studies were performed with the informed consent of the patients which also accepted the use of results for research studies. All experiments were performed without employing biological material from the patients.

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Calderan, C., Sorrentino, U., Persano, L. et al. A yeast based assay establishes the pathogenicity of novel missense ACTA2 variants associated with aortic aneurysms. Eur J Hum Genet (2024). https://doi.org/10.1038/s41431-024-01591-1

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