Functional analysis of cell lines derived from SMAD3-related Loeys-Dietz syndrome patients provides insights into genotype-phenotype relation

Nathalie P de Wagenaar; Lisa M van den Bersselaar; Hanny J H M Odijk; Sanne J M Stefens; Dieter P Reinhardt; Jolien W Roos-Hesselink; Roland Kanaar; Judith M A Verhagen; Hennie T Brüggenwirth; Ingrid M B H van de Laar; Ingrid van der Pluijm; Jeroen Essers

doi:10.1093/hmg/ddae044

Functional analysis of cell lines derived from SMAD3-related Loeys-Dietz syndrome patients provides insights into genotype-phenotype relation

Hum Mol Genet. 2024 Mar 27:ddae044. doi: 10.1093/hmg/ddae044. Online ahead of print.

Authors

Nathalie P de Wagenaar^{1

2}, Lisa M van den Bersselaar³, Hanny J H M Odijk¹, Sanne J M Stefens¹, Dieter P Reinhardt^{4

5}, Jolien W Roos-Hesselink², Roland Kanaar¹, Judith M A Verhagen³, Hennie T Brüggenwirth³, Ingrid M B H van de Laar³, Ingrid van der Pluijm^{1

6}, Jeroen Essers^{1

6

7}

Affiliations

¹ Department of Molecular Genetics, Oncode Institute, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD, Rotterdam, The Netherlands.
² Department of Cardiology and European Reference Network for Rare Multisystemic Vascular Disease (VASCERN), HTAD Rare Disease Working Group, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD, Rotterdam, The Netherlands.
³ Department of Clinical Genetics and European Reference Network for Rare Multisystemic Vascular Disease (VASCERN), HTAD Rare Disease Working Group, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD, Rotterdam, The Netherlands.
⁴ Faculty of Medicine and Health Sciences, McGill University, 3640 University Street, Montreal, QC H3A 0C7, Canada.
⁵ Faculty of Dental Medicine and Oral Health Sciences, McGill University, 3640 University Street, Montreal, QC H3A 0C7, Canada.
⁶ Department of Vascular Surgery, Cardiovascular Institute, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD, Rotterdam, The Netherlands.
⁷ Department of Radiotherapy, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD, Rotterdam, The Netherlands.

PMID: 38538566
DOI: 10.1093/hmg/ddae044

Abstract

Rationale: Pathogenic (P)/likely pathogenic (LP) SMAD3 variants cause Loeys-Dietz syndrome type 3 (LDS3), which is characterized by arterial aneurysms, dissections and tortuosity throughout the vascular system combined with osteoarthritis.

Objectives: Investigate the impact of P/LP SMAD3 variants with functional tests on patient-derived fibroblasts and vascular smooth muscle cells (VSMCs), to optimize interpretation of SMAD3 variants.

Methods: A retrospective analysis on clinical data from individuals with a P/LP SMAD3 variant and functional analyses on SMAD3 patient-derived VSMCs and SMAD3 patient-derived fibroblasts, differentiated into myofibroblasts.

Results: Individuals with dominant negative (DN) SMAD3 variant in the MH2 domain exhibited more major events (66.7% vs. 44.0%, P = 0.054), occurring at a younger age compared to those with haploinsufficient (HI) variants. The age at first major event was 35.0 years [IQR 29.0-47.0] in individuals with DN variants in MH2, compared to 46.0 years [IQR 40.0-54.0] in those with HI variants (P = 0.065). Fibroblasts carrying DN SMAD3 variants displayed reduced differentiation potential, contrasting with increased differentiation potential in HI SMAD3 variant fibroblasts. HI SMAD3 variant VSMCs showed elevated SMA expression and altered expression of alternative MYH11 isoforms. DN SMAD3 variant myofibroblasts demonstrated reduced extracellular matrix formation compared to control cell lines.

Conclusion: Distinguishing between P/LP HI and DN SMAD3 variants can be achieved by assessing differentiation potential, and SMA and MYH11 expression. The differences between DN and HI SMAD3 variant fibroblasts and VSMCs potentially contribute to the differences in disease manifestation. Notably, myofibroblast differentiation seems a suitable alternative in vitro test system compared to VSMCs.

Keywords: Loeys-Dietz syndrome; SMAD3; aneurysmsosteoarthritis syndrome; functional assay.

Grants and funding

Erasmus MC Mrace