Modeling SMAD2 Mutations in Induced Pluripotent Stem Cells Provides Insights Into Cardiovascular Disease Pathogenesis.

BACKGROUND: SMAD2 is a coregulator that binds a variety of transcription factors in human development. Heterozygous SMAD2 loss-of-function and missense variants are identified in patients with congenital heart disease (CHD) or arterial aneurysms. Mechanisms that cause distinct cardiovascular phenotypes remain unknown. We aimed to define transcriptional and epigenetic effects of SMAD2 variants and their role in CHD. We also assessed the function of SMAD2 missense variants of uncertain significance. METHODS AND RESULTS: Rare SMAD2 variants (minor allele frequency ≤10(-5)) were identified in exome sequencing of 11 336 participants with CHD. We constructed isogenic induced pluripotent stem cells with heterozygous or homozygous loss-of-function and missense SMAD2 variants identified in CHD probands. Wild-type and mutant induced pluripotent stem cells were analyzed using bulk RNA sequencing, chromatin accessibility (Assay for Transposase-Accessible Chromatin With Sequencing), and integrated with published SMAD2/3 chromatin immunoprecipitation data. Cardiomyocyte differentiation and contractility were evaluated. Thirty participants with CHD had heterozygous loss-of-function or missense SMAD2 variants. SMAD2 haploinsufficiency altered chromatin accessibility at promoters and dysregulated expression of 385 SMAD regulated genes, including 10 CHD-associated genes. Motifs enriched in differential Assay for Transposase-Accessible Chromatin peaks predicted that SMAD2 haploinsufficiency disrupts interactions with transcription factors NANOG (homeobox protein NANOG), ETS, TEAD3/4 (transcriptional enhanced associate domain 3/4), CREB1 (cAMP response element binding protein 1), and AP1 (activator protein 1). Compared with SMAD2-haploinsufficient cells, induced pluripotent stem cells with R114C or W274C variants exhibited distinct and shared chromatin accessibility and transcription factor binding changes. CONCLUSIONS: SMAD2 haploinsufficiency disrupts transcription factor binding and chromatin interactions critical for cardiovascular development. Differences between the molecular consequences of loss-of-function and missense variants likely contribute to phenotypic heterogeneity. These findings indicate opportunities for molecular analyses to improve reclassification of SMAD2 variants of uncertain clinical significance.