Identification of ALK2 variant in DS. (a) Schematic representation of the ALK2 protein and structural domains: signal peptide (SP), ligand-binding domain (LBD) , transmembrane domain (TM), GS domain (GS) and kinase domain (KD). Arrows indicate approximate locations of p.His286Asp in ALK2. (b) Sequencing chromatogram with alignment below (aligned by ClustalW) showing the ALK2 p.His286Asp variant in genomic DNA from the proband with DS and a primum-type atrial septal defect. The protein alignment shows strong conservation between species of the ALK2 p.His286Asp residue. (c) TGFβRI (pdb entry 1PY5) as a model for ALK2, showing H268 in red, ADP in blue and the β-sheet in yellow. (d) Family pedigree of proband (III:2). Only the father, without DS, was in possession of the ALK2 variant and did not have a congenital heart defect by echocardiography. Carriers of variants are depicted with a number: 1(ALK2 p.His286Asp), 2 (ALK3 p.Glu415Lys) or 3 (ErbB3 p.Thr1169Ile), and individuals with or without CHDs are shaded black or white, respectively. Gray coloration indicates the genotype or phenotype was unable to be determined. Deceased individuals are annotated by a diagonal strike-through. (e) Echocardiogram of proband carrying the p.His286Asp variant (III:2) before surgery, demonstrating a large primum-type atrial septal defect (arrow) and overriding AV valve.

Representative luciferase assay on bovine aortic endothelial (BAE) cells transiently transfected with a BMP-responsive element fused to luciferase, renilla (to control the transfection) and ALK2 constructs. ALK2 activity was measured as relative luciferase units (RLU), without (white) and with (black) induction by BMP6 protein. (a) Significantly higher RLU was observed for ALK2 constitutively active (CA) without induction, whereas significantly lower RLU was observed for ALK2 dominant negative (DN) with and without BMP6 induction. For the p.His286Asp variant, both without and with induction with BMP6 significantly lower RLU was observed. (b) Luciferase assay on BAE cells as described above, including transfection of the bovine ALK2 RNAi construct to knockdown endogenous bovine ALK2 but not vector-based human ALK2. A similar pattern for ALK2 CA, ALK2 DN and wt ALK2 was observed as with injection of vector only. However, transfection with the p.His286Asp variant demonstrated significantly lower RLU without BMP6 induction, whereas induction with BMP6 did not demonstrate a significant difference between wt ALK2 and the p.His286Asp variant. RLU were measured as luciferase activity/renilla activity and expressed relative to unstimulated wt ALK2 activity. RLU are depicted as mean±SEM, where n=5 (3 replicates per experiment). Statistical significance, compared with wt ALK2, was determined by t-test, where ^*P<0.05.

Rescue experiments in laf mutants. Rescue experiments in the zebrafish alk2/8 mutant, lost-a-fin (laf). (a) Injection of wt ALK2 or p.His286Asp RNA at the single-cell stage in laf/Alk2 mutants. Wt ALK2 RNA rescues phenotype in 65% of mutant embryos, while injection of p.His286Asp rescues the phenotype in 51% of mutant embryos. This is significantly less effective compared with wt RNA injection. Statistical significance was determined by Student's t-test, where ^*P<0.05. (b) laf embryo identifiable by its C1 dorsalization phenotype, with cardiac edema and absent ventral fin. (c) Rescued laf zebrafish sibling after injection with ALK2 p.His286Asp demonstrating wt phenotype. (d) wt zebrafish.