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Functional analysis of cell lines derived from SMAD3-related Loeys-Dietz syndrome patients provides insights into
Nathalie P de Wagenaar1,2, Lisa M van den Bersselaar3, Hanny J H M Odijk1
1Department of Molecular Genetics, Oncode Institute, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD, Rotterdam, The Netherlands.
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.
Insights
Pathogenic SMAD3 variants cause Loeys-Dietz syndrome type 3. Functional tests reveal differences in patient cells, distinguishing between dominant-negative and haploinsufficient variants for better interpretation.
Area of Science:
- Genetics and Molecular Biology
- Cardiovascular Research
- Cell Biology
Background:
- Pathogenic (P)/likely pathogenic (LP) SMAD3 variants are associated with Loeys-Dietz syndrome type 3 (LDS3).
- LDS3 is characterized by vascular abnormalities including aneurysms, dissections, and tortuosity, alongside osteoarthritis.
- Understanding the functional impact of different SMAD3 variant types is crucial for accurate clinical interpretation.
Purpose of the Study:
- To investigate the functional impact of P/LP SMAD3 variants using patient-derived cells.
- To differentiate between dominant-negative (DN) and haploinsufficient (HI) SMAD3 variants through functional assays.
- To enhance the interpretation of SMAD3 variants in the context of LDS3.
Main Methods:
- Retrospective analysis of clinical data from individuals with P/LP SMAD3 variants.
- Functional analyses using patient-derived vascular smooth muscle cells (VSMCs) and fibroblasts.
- In vitro differentiation of fibroblasts into myofibroblasts for cellular assessment.
Main Results:
- Dominant-negative (DN) SMAD3 variants in the MH2 domain were associated with more major vascular events at a younger age compared to haploinsufficient (HI) variants.
- DN SMAD3 variant fibroblasts showed reduced differentiation potential, while HI variants exhibited increased potential.
- HI SMAD3 variant VSMCs displayed elevated SMA expression and altered MYH11 isoform expression; DN SMAD3 variant myofibroblasts showed reduced extracellular matrix formation.
Conclusions:
- Functional assessment of differentiation potential, SMA, and MYH11 expression can distinguish between P/LP HI and DN SMAD3 variants.
- Observed cellular differences in fibroblasts and VSMCs correlate with distinct disease manifestations in LDS3 patients.
- Myofibroblast differentiation serves as a viable in vitro model for studying SMAD3 variant effects, offering an alternative to VSMCs.
