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

Rationale: Pathogenic (P)/ likely pathogenic (LP) SMAD3 variants cause Loeys-Dietz syndrome type 3 (LDS3), also known as aneurysms-osteoarthritis syndrome (AOS). LDS3 is characterized by a highly variable phenotype of arterial aneurysms, dissections and tortuosity throughout the vascular system combined with osteoarthritis. Objectives: Investigate the impact of P/LP SMAD3 variants through conducting functional tests on patient-derived fibroblasts and vascular smooth muscle cells (VSMCs).This knowledge will optimize interpretation of SMAD3 variants. Methods and Results: We conducted a retrospective analysis on clinical data from individuals with a P/LP SMAD3 variant and utilized patient-derived VSMCs for functional analyses. Additionally, we performed similar functional analyses on SMAD3 patient-derived fibroblasts, differentiated into myofibroblasts. Individuals with dominant negative (DN) variants in the MH2 domain of SMAD3 exhibited a higher frequency of major events (66.7% vs. 44.0%, p=0.054), occurring at a younger age compared to those with haploinsufficient (HI) variants. Moreover, the age at time of the 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). In functional assays, fibroblasts carrying DN SMAD3 variants displayed reduced differentiation potential, contrasting with increased differentiation potential observed in fibroblasts with HI SMAD3 variants. Additionally, HI SMAD3 variant VSMCs showed elevated SMA expression and altered expression of alternative MYH11 isoforms. Conversely, DN SMAD3 variant myofibroblasts demonstrated reduced extracellular matrix (ECM) formation compared to control cell lines. Conclusion: Distinguishing between P/LP HI and DN SMAD3 variants can be achieved by assessing differentiation potential, and evaluating SMA and MYH11 expression. The distinct functional consequences between DN and HI variants in SMAD3 fibroblasts and VSMCs potentially contribute to the observed differences in disease manifestation and age of onset of major events. Notably, myofibroblast differentiation seems to be a suitable alternative in vitro test system compared to VSMCs. ### Competing Interest Statement The authors have declared no competing interest.