Dominant-Negative ALK2 Allele Associates With Congenital Heart Defects

Abstract

Background-Serious congenital heart defects occur as a result of improper atrioventricular septum (AVS) development during embryogenesis. Despite extensive knowledge of the genetic control of AVS development, few genetic lesions have been identified that are responsible for AVS-associated congenital heart defects. Methods and Results-We sequenced 32 genes known to be important in AVS development in patients with AVS defects and identified 11 novel coding single-nucleotide polymorphisms that are predicted to impair protein function. We focused on variants identified in the bone morphogenetic protein receptor, ALK2, and subjected 2 identified variants to functional analysis. The coding single-nucleotide polymorphisms R307L and L343P are heterozygous missense substitutions and were each identified in single individuals. The L343P allele had impaired functional activity as measured by in vitro kinase and bone morphogenetic protein-specific transcriptional response assays and dominant-interfering activity in vivo. In vivo analysis of zebrafish embryos injected with ALK2 L343P RNA revealed improper atrioventricular canal formation. Conclusion-These data identify the dominant-negative allele ALK2 L343P in a patient with AVS defects. (Circulation.2009;119:3062-3069.)Key Words: ALK2 protein, human Ⅲ genes Ⅲ heart defects, congenital Ⅲ screening Ⅲ signal transduction T he primitive heart tube of vertebrates consists of an inner layer of endothelial cells, the endocardium, and an outer muscular layer of myocardial cells. After formation of the heart tube, endothelial cells delaminate from the endocardium and migrate into an extracellular matrix, called cardiac jelly, which resides between the endocardium and myocardium. These invading endothelial cells undergo an endothelium-tomesenchyme transition (for detailed review, see elsewhere 1 ) and give rise to swellings known as endocardial cushions (ECs). ECs contribute to the valves and septa of the heart, and disruptions in their formation result in valvular and septal defects. 2 A number of signaling pathways, including vascular endothelial growth factor signaling, Notch, Wnt/␤-catenin, bone morphogenetic protein (BMP)/transforming growth factor-␤ signaling have been implicated in atrioventricular septum (AVS) development either in vitro or in vivo. 1 Clinical Perspective on p 3069This extensive knowledge of the genetic control of AVS development has yet to be translated into a broader clinical knowledge of the genetic determinants of congenital heart defects (CHDs). This is due largely to the complex pathogenesis of CHD and the scarcity of large families with multiple affected individuals suitable for conventional genetic analyses. More recently, candidate screening approaches have been used to circumvent this limitation. Such approaches, when coupled with kindred linkage and/or detailed functional analyses, can identify novel causative mutations in genes previously suspected to function in AVS development. [3][4][5] In an effort to identify genetic lesions that may cause C...

ALK2 mutation in a patient with Down's syndrome and a congenital heart defect

“…Next, endogenous ALK2 expression was knocked down by an RNAi construct as previously described. 14 Transfection with the ALK2 p.His286Asp variant demonstrated significantly lower levels of luciferase activity in the uninduced state (Po0.05), but a difference from wt ALK2 was not observed upon stimulation with BMP6 (Figure 2b). These results indicate that the ALK2 p.His286Asp receptor is capable of propagating a signal when stimulated with a BMP ligand, but this activity is impaired when the ALK2 p.His286Asp variant is in the presence of the wt ALK2 form of the receptor.…”

“…14 In the current study, we report on a patient with DS and a primum-type atrial septal defect who harbors three cSNPs in genes associated with endocardial cushion development. The cSNPs result in missense substitutions in the type I BMP receptor genes, ALK2 and ALK3, resulting in the genetic variants p.His286Asp and p.Glu414Lys, respectively, and in the epidermal growth factor receptor family gene, ERRB3, resulting in the p.Thr1169Ile variant.…”

“…One of these variants identified has a dominant-interfering effect on BMP signaling and is associated with primum-type atrial septal defect (ASD) type I. 14 Here we report an additional ALK2 variant that was identified in a patient with trisomy 21 and a primum-type ASD (ASD type I). In addition to the ALK2 variant, two other variations were detected in another type I BMP receptor gene, ALK3, and in the epidermal growth factor receptor family, ErbB3.…”

“…19 This included an injection with vector only or in combination with a short hairpin RNA, the latter to knockdown endogenous bovine ALK2 but not vector-based human ALK2, as described previously. 14 …”

The genetics of cardiovascular disease: new insights from emerging approaches

“…In this way, mutations of the ALK2 receptor identified in patients with AV septal defects were tested by injecting zebrafish with mRNA engineered to contain the putative disease-causing mutation. This induced AV canal defects, proving the causative nature of the mutation [90]. Other recent examples of reverse genetics where a putative human disease modulating gene has been validated using zebrafish include:…”