Loss-of-function mutations in growth differentiation factor-1 (GDF1) are associated with congenital heart defects in humans

Abstract

Congenital heart defects (CHDs) are among the most common birth defects in humans (incidence 8-10 per 1,000 live births). Although their etiology is often poorly understood, most are considered to arise from multifactorial influences, including environmental and genetic components, as well as from less common syndromic forms. We hypothesized that disturbances in left-right patterning could contribute to the pathogenesis of selected cardiac defects by interfering with the extrinsic cues leading to the proper looping and vessel remodeling of the normally asymmetrically developed heart and vessels. Here, we show that heterozygous loss-of-function mutations in the human GDF1 gene contribute to cardiac defects ranging from tetralogy of Fallot to transposition of the great arteries and that decreased TGF- beta signaling provides a framework for understanding their pathogenesis. These findings implicate perturbations of the TGF- beta signaling pathway in the causation of a major subclass of human CHDs.

Figure 1.

Structural analysis of GDF1 mutations. A, Alignment of human GDF1 (hGDF1) and structurally related members of the TGF-β family, including human BMP2 (hBMP2), human BMP7 (hBMP7), zebrafish DVR (zDVR), and murine Gdf1 (mGDF1). Amino acid residues showing absolute identity are shown with white letters against a blue background; those positions with conservative substitutions are shown against a yellow background. The positions of the five mutations considered in this study are indicated by arrowheads. The amino acid numbering is based on that of the mature processed ligand. The β-sheet elements of the winglike finger projections F1 and F2 are indicated by blackened arrows above the alignment, whereas the α-helical core is denoted by the cylinder above the alignment. B, Ribbon diagram of human GDF1, showing the position of the mutations clustered in the knuckle region of the monomer. (Note that the G9S variant lies within the poorly conserved N-terminal region of the protein where the crystal structure is unknown, precluding accurate modeling of this mutation.)

Figure 2.

GDF1 activity assay by morphological phenotype. Twenty picograms of mRNA synthesized from humanized wild-type (WT) and mutant GDF1 constructs were injected at the one-cell stage. Embryos were allowed to develop for 27 h and were assayed for severity of developmental defects. Representative samples from injected clutches are shown. WT humanized GDF1 exhibited the severest developmental defects indicative of strongest activity. Mutant GDF1 constructs exhibited varying degrees of decreased activity compared with WT GDF1. Uninjected WT embryos are included for comparison. Twenty picograms of LacZ mRNA was injected as control and did not exhibit any phenotype (data not shown). mGDF1 = murine GDF1.

Figure 3.

GDF1 activity assay by gsc induction. As in figure 2, injected embryos were fixed and stained for gsc. gsc is a higher-threshold target of GDF1; thus, higher amounts of mRNA (200 pg) were injected per embryo. mGDF1 = murine GDF1.

Figure 4.

GDF1 activity assay by induction of ntl expression. Twenty picograms of mRNA synthesized from humanized wild-type (WT) and mutant GDF1 constructs were injected at the one-cell stage. Embryos were allowed to develop for 5 h and were fixed and stained for ntl, a downstream target of GDF1. Representative animal samples from injected clutches are shown. Mutant GDF1 constructs exhibited varying degrees of decreased activity compared with WT GDF1, as assessed by the extent of ectopic ntl induction. Stained samples from uninjected WT embryos are included for comparison. mGDF1 = murine GDF1.

Figure 5.

Classification and quantification of morphological phenotypes. Embryos aged 27 h injected with GDF1 constructs were sorted into classes I–IV (from wild type [WT] to those with the severest developmental defects) and were quantified. All mutant constructs exhibited varying degrees of decreased activity compared with WT humanized GDF1. mGDF1 = murine GDF1. Red font indicates the predominant class.