Haploinsufficient variants in SMAD5 are associated with isolated congenital heart disease

Abstract

Mothers against decapentaplegic homolog 5 (SMAD5) is a transcriptional regulator that functions within the TGF-β signaling cascade. Evidence from animal studies show that it is crucial for dorsoventral patterning, left-right asymmetry, cardiac looping, and other embryonic processes. However, its role in human development has not been explored, and the contribution of SMAD5 variants to congenital disease is unknown. Here, we report SMAD5 variants identified in six unrelated families with seven individuals presenting with congenital heart disease (CHD). Isolated congenital heart defects are observed in six individuals who carry de novo or inherited missense, nonsense, frameshift, or copy-number variants in SMAD5. A multi-organ phenotype is observed in one individual with a de novo SMAD5 variant that alters an amino acid crucial for SMAD5 multimerization. Septal defects, identified in four individuals, are the most common cardiac lesion in our cohort, with hypoplastic left heart also observed in two individuals. In silico assessment of SMAD5 missense variants predicts disrupted binding to co-factors, and in vitro functional assessment shows changes in SMAD5 gene and protein expression, as well as impaired activation of a BMP4-responsive promoter by the variants. Our findings suggest haploinsufficiency as the underlying molecular mechanism in five of the six families, resulting in isolated CHD, with a SMAD5 dominant-negative variant identified in one family leading to multiple congenital defects. Here, we provide evidence that SMAD5 variants lead to CHD and offer a basis for future exploration of SMAD5 variants in both CHD and post-natal disease.

Keywords: BMP signaling; SMAD5 signaling; TGF-β signaling; cardiac septal defects; congenital heart disease; hypoplastic left heart.

Graphical abstract

Figure 1

Pedigrees of families with SMAD5 variants (A) Genotypes of sequenced individuals are displayed below each family member. +/+ indicates absence of SMAD5 variant. Affected individuals are indicated by filled shapes. Black arrows indicate the proband of each family (F). Individuals who carry a variant without a CHD diagnosis are indicated by a black circle within a white shape. Gray-filled shapes indicate individuals with non-descriptive CHD phenotypes. The Sanger sequencing chromograph for F2 can be found in Figure S2. (B) Schematic of the chromosomal deletion observed in affected individual in F3 (hg37). Numbers are assigned to protein-coding genes deleted within the region (1: SLC25A48; 2: IL9; 3: FBXL21; 4: LECT2; 5: TGFβ1; 6: SMAD5; 7: TRPC7; 8: SPOCK1). Arrows indicate directionality of the gene. (C) Missense and protein-truncating variants observed in affected individuals in this cohort are positioned on a schematic of SMAD5 protein sequence (ENST00000545279.6, NM_005903.7, NP_005894.3). L3 loop (F416–T432) is shown within the MH2 domain. (D) Missense variant constraint across the SMAD5 sequence is derived from gnomAD (version 4.1.0). 0.0 is intolerant to missense variation, and 1.0+ is tolerant to missense variation. (E) Sequence conservation between selected vertebrates of residues affected by the missense variants in our cohort. (F) Sequence conservation between all human SMAD proteins of residues affected by the missense variants in our cohort. Residue impacted by the variant in MH1 domain (p.V78F) is highlighted in pink, and the residues affected by the variants in MH2 domain (p.N361D and p.T430I) are highlighted in blue. Sequence alignments were created using the Uniprot Align tool. ASD, atrial septal defect; AVSD, atrioventricular septal defect; HLH, hypoplastic left heart; MH1, Mad homolog 1; MH2, Mad homolog 2; ToF, tetralogy of Fallot; VSD, ventricular septal defect.

Figure 2

Structural and functional analysis of the SMAD5 missense variants (A–C) Impact of missense variants p.T430I (A), p.V78F (B), and p.N361D (C) on SMAD5 homotrimer (predicted), SMAD5-DNA (predicted), and SMAD5-cofactor (predicted) complexes. (A) p.T430 (teal) predominantly interacts with neighboring monomers within the homotrimer, and p.T430I slightly shifts the residue side chain away from the binding partner to reduce affinity. (B) p.V78F (blue) stabilizes the conformation of SMAD5 away from DNA binding, reducing the possibility of steric hindrance and enabling higher affinity to DNA. (C) p.N361D (raspberry) reduces the polar bonds directing its orientation away from co-factor, MAN1, binding, leading to a higher affinity to MAN1. The models were generated using PDB structures 6TBZ, 5ZOK, and AlphaFold2, and analyses were performed using PyMOL (version 2.5.4). (D) Expression of SMAD5 mRNA in cultured HEK293T cells transiently transfected with wild-type (WT) SMAD5 or variant plasmids. n = 4; ∗p < 0.05; one-way ANOVA with Tukey’s post hoc test. (E) Expression of SMAD5 protein in cultured HEK293T cells transiently co-transfected with FLAG-SMAD5 variant plasmids and GFP. Cells were lysed and assessed for levels of FLAG-SMAD5 variants by ELISA. FLAG-SMAD5 levels were normalized to GFP. n = 8–12; ∗∗p < 0.01; ∗∗∗∗p < 0.0001; one-way ANOVA with Tukey’s post hoc test. (F) Protein stability of SMAD5 variants in cultured HEK293T cells co-transfected with FLAG-SMAD5 variants and GFP. Cells were treated with cycloheximide or vehicle for 8 h, lysed, and assessed for levels of FLAG-SMAD5 variants by ELISA. FLAG-SMAD5 levels were normalized to GFP. Protein levels of FLAG-SMAD5 variants are presented relative to their levels in vehicle-treated cell lysates. n = 8–12; ∗∗∗∗p < 0.0001; one-way ANOVA with Tukey’s post hoc test. (G–I) Transcriptional activation ability of the SMAD5 variants p.T430I, p.V78F, and p.N361D was tested on a BMP-activated (Xvent2-luc) promoter (columns a–d) in HEK293T cells. Impact of the variants on WT-SMAD5 activation of the Xvent2-luc promoter was also assessed (columns e–h), where cells were transfected with 2× WT-SMAD5 or co-transfected with WT-SMAD5 and variant. Fold change was calculated by normalizing variant activity over vector-only activity. n = 4–5; ∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; one-way ANOVA with Tukey’s post hoc test. All data are presented as mean ± SD.