Functional analysis of cell lines derived from SMAD3-related Loeys-Dietz Syndrome patients provides insights into genotype-phenotype relations
Authors: de Wagenaar Nathalie P. , van den Bersselaar Lisa M. , Odijk Hanny J.H.M. , Stefens Sanne J.M. , Reinhardt Dieter P. , Roos-Hesselink Jolien W. , Kanaar Roland , Verhagen Judith M.A. , Brüggenwirth Hennie T. , van de Laar Ingrid M.B.H. , van der Pluijm Ingrid , Essers Jeroen 
Published: December 2023

AbstractIntroductionPathogenic (P) and likely pathogenic (LP) variants in theSMAD3gene cause Loeys-Dietz syndrome type 3 (LDS3), also known as aneurysms-osteoarthritis syndrome (AOS). The phenotype of LDS3 is highly variable and characterized by arterial aneurysms, dissections and tortuosity throughout the vascular system combined with skeletal, cutaneous and facial features.ObjectivesInvestigate the impact of P/LPSMAD3variants through conducting functional tests on patient-derived fibroblasts and vascular smooth muscle cells (VSMCs).The resulting knowledge will optimize interpretation ofSMAD3variants.Material and methodsWe conducted a retrospective analysis on clinical data from individuals with a P/LPSMAD3variant and utilized patient-derived VSMCs to investigate the functional impacts of dominant negative (DN) and haploinsufficient (HI) variants in SMAD3. Additionally, to broaden our cell model accessibility, we performed similar functional analyses on patient-derived fibroblasts carrying SMAD3 variants, differentiating them into myofibroblasts with the same variants. This enabled us to study the functional effects of DN and HI variants inSMAD3across both patient-derived myofibroblasts and VSMCs.ResultsIndividuals with dominant negative (DN) variants in the MH2 protein interaction 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 the onset of the first major event was notably younger in individuals with DN variants in MH2, 35.0 years [IQR 29.0-47.0], compared to 46.0 years [IQR 40.0-54.0] in those with HI variants (p=0.065). In functional assays, fibroblasts carrying DNSMAD3variants displayed reduced differentiation potential, contrasting with increased differentiation potential observed in fibroblasts with HISMAD3variants. Additionally, HISMAD3variant VSMCs showed elevated SMA expression, while exhibiting altered expression of alternative MYH11 isoforms. Conversely, DNSMAD3variant myofibroblasts demonstrated reduced extracellular matrix (ECM) formation compared to control cell lines. These findings collectively indicate distinct functional consequences between DN and HI variants inSMAD3across fibroblasts and VSMCs, potentially contributing to the observed differences in disease manifestation and age of onset of major events.ConclusionDistinguishing between P/LP HI and DNSMAD3variants can be achieved by assessing differentiation potential, and evaluating SMA and MYH11 expression. Notably, myofibroblast differentiation seems to be a suitable alternative in vitro test system in comparison to VSMCs. Moreover, there is a notable trend of aortic events occurring at younger age in individuals with a DNSMAD3variant in the MH2 domain, distinguishing them from those with a DN variant in the MH1 domain or a HI variant.