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

Area of Science:

• Genetics
• Molecular Biology
• Developmental Biology

Background:

• SMAD2 is a key regulator in human development, interacting with transcription factors.
• Heterozygous SMAD2 loss-of-function and missense variants are linked to congenital heart disease (CHD) and arterial aneurysms.
• The molecular mechanisms underlying distinct cardiovascular phenotypes caused by SMAD2 variants are not fully understood.

Purpose of the Study:

• To investigate the transcriptional and epigenetic impacts of SMAD2 variants in the context of CHD.
• To elucidate the functional consequences of SMAD2 missense variants of uncertain clinical significance.

Main Methods:

• Exome sequencing of 11,336 participants with CHD to identify rare SMAD2 variants.
• Generation of isogenic induced pluripotent stem cells (iPSCs) with heterozygous or homozygous SMAD2 variants.
• Analysis of iPSCs using bulk RNA sequencing, Assay for Transposase-Accessible Chromatin with Sequencing (ATAC-seq), and integration with SMAD2/3 ChIP-seq data.
• Evaluation of cardiomyocyte differentiation and contractility.

Main Results:

• Thirty CHD participants carried heterozygous SMAD2 variants.
• SMAD2 haploinsufficiency altered chromatin accessibility and dysregulated 385 SMAD-regulated genes, including 10 CHD-associated genes.
• SMAD2 haploinsufficiency was predicted to disrupt interactions with transcription factors like NANOG, ETS, TEAD3/4, CREB1, and AP1.
• Specific SMAD2 missense variants (R114C, W274C) induced distinct molecular changes compared to haploinsufficiency.

Conclusions:

• SMAD2 haploinsufficiency disrupts critical transcription factor binding and chromatin interactions during cardiovascular development.
• Discrepancies in molecular outcomes between loss-of-function and missense SMAD2 variants contribute to phenotypic diversity in CHD.
• These findings offer a basis for molecular analyses to improve the clinical classification of SMAD2 variants of uncertain significance.