Discovery and functional investigation of BMP4 as a new causative gene for human congenital heart disease

Abstract

Objective: Aggregating evidence highlights the strong genetic basis underpinning congenital heart disease (CHD). Here BMP4 was chosen as a prime candidate gene causative of human CHD predominantly because BMP4 was amply expressed in the embryonic hearts and knockout of Bmp4 in mice led to embryonic demise mainly from multiple cardiovascular developmental malformations. The aim of this retrospective investigation was to discover a novel BMP4 mutation underlying human CHD and explore its functional impact.

Methods: A sequencing examination of BMP4 was implemented in 212 index patients suffering from CHD and 236 unrelated non-CHD individuals as well as the family members available from the proband carrying a discovered BMP4 mutation. The impacts of the discovered CHD-causing mutation on the expression of NKX2-5 and TBX20 induced by BMP4 were measured by employing a dual-luciferase analysis system.

Results: A new heterozygous BMP4 mutation, NM_001202.6:c.318T>G;p.(Tyr106*), was found in a female proband affected with familial CHD. Genetic research of the mutation carrier's relatives unveiled that the truncating mutation was in co-segregation with CHD in the pedigree. The nonsense mutation was absent from 236 unrelated non-CHD control persons. Quantitative biologic measurement revealed that Tyr106*-mutant BMP4 failed to induce the expression of NKX2-5 and TBX20, two genes whose expression is lost in CHD.

Conclusion: The current findings indicate BMP4 as a new gene predisposing to human CHD, allowing for improved prenatal genetic counseling along with personalized treatment of CHD patients.

Keywords: BMP4; Congenital heart disease; molecular genetics; reporter gene assay; signal transduction.

Figure 1

A new BMP4 mutation causing familial congenital cardiovascular malformations. A. Sequence chromatogram traces revealing the heterozygous BMP4 mutation in the CHD-affected proband (Mutant) along with corresponding homozygous control in an unaffected family member of the proband (Wild type). A vertical arrow points to the nucleotide position where the heterozygous BMP4 mutation (c.318T>G) occurs. B. Schematic diagrams delineating the structural domains of human BMP4 proteins. The Tyr106*-mutant BMP4 protein (Mutant) was predicted to lose 303 amino acids at the carboxyl terminus (COOH). TGFβ: transforming growth factor-beta; NH2: amino terminus. C. Pedigree manifesting autosomal-dominant inheritance of ventricular septal defect and double-outlet right ventricle. An oblique arrow points to the index patient. A family member’s genotype is marked with “+” or “-”, of which “+” signifies a member harboring the heterozygous BMP4 mutation, while “-” signifies a member with no BMP4 mutation.

Figure 2

No induction of NKX2-5 expression by Tyr106*-mutant BMP4. In maintained HeLa cells, dual-luciferase measurement of the expression of the NKX2-5 promoter-driven firefly luciferase (NKX2-5-luc) in the presence of wild-type human BMP4-pcDNA^TM3.1(+) vector (BMP4) or Tyr106*-mutant human BMP10-pcDNA^TM3.1(+) vector (Tyr106*), separately or in combination, showed that Tyr106* failed to induce the expression of NKX2-5. For every expression vector, functional experiments were performed three times in triplicate. Here, “a” means P < 0.001, and “b” means P < 0.005, in comparison to BMP4 (600 ng).

Figure 3

Inability of Tyr106*-mutant BMP4 to induce TBX20 expression. Dual-reporter analysis of the expression of the TBX20 promoter-driven firefly luciferase (TBX20-luc) in grown HeLa cells in the presence of wild-type human BMP4-pcDNA^TM3.1(+) vector (BMP4) or Tyr106*-mutant human BMP10-pcDNA^TM3.1(+) vector (Tyr106*), singly or in both, revealed that Tyr106* failed to induce the expression of TBX20. For each expression vector, three independent biological measurements were conducted in triplicate. Here, “c” signifies P < 0.001, and “d” indicates P < 0.005, when compared with BMP4 (600 ng).