Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).

Causes for miscarriages and congenital malformations can be genetic, environmental, or a combination of both. Genetic variants, hypoxia, malnutrition, or other factors individually may not affect embryo development, however, they may do so collectively. Biallelic loss-of-function variants in HAAO or KYNU, two genes of the nicotinamide adenine dinucleotide (NAD) synthesis pathway, are causative of congenital malformation and miscarriage in humans and mice. The variants affect normal embryonic development by disrupting the synthesis of NAD, a key factor in multiple biological processes, from its dietary precursor tryptophan, resulting in NAD deficiency. This study demonstrates that congenital malformations caused by NAD deficiency can occur independent of genetic disruption of NAD biosynthesis. C57BL/6J wild-type mice had offspring exhibiting similar malformations when their supply of the NAD precursors tryptophan and vitamin B3 in the diet was restricted during pregnancy. When the dietary undersupply was combined with a maternal heterozygous variant in Haao, which alone does not cause NAD deficiency or malformations, the incidence of embryo loss and malformations was significantly higher, suggesting a gene–environment interaction. Maternal and embryonic NAD levels were deficient. Mild hypoxia as an additional factor exacerbated the embryo outcome. Our data show that NAD deficiency as a cause of embryo loss and congenital malformation is not restricted to the rare cases of biallelic mutations in NAD synthesis pathway genes. Instead, monoallelic genetic variants and environmental factors can result in similar outcomes. The results expand our understanding of the causes of congenital malformations and the importance of sufficient NAD precursor consumption during pregnancy.

Congenital heart disease (CHD) is an enigma. It is the most common human birth defect and yet, even with the application of modern genetic and genomic technologies, only a minority of cases can be explained genetically. This is because environmental stressors also cause CHD. Here we propose a plausible non-genetic mechanism for induction of CHD by environmental stressors. We show that exposure of mouse embryos to short-term gestational hypoxia induces the most common types of heart defect. This is mediated by the rapid induction of the unfolded protein response (UPR), which profoundly reduces FGF signaling in cardiac progenitor cells of the second heart field. Thus, UPR activation during human pregnancy might be a common cause of CHD. Our findings have far-reaching consequences because the UPR is activated by a myriad of environmental or pathophysiological conditions. Ultimately, our discovery could lead to preventative strategies to reduce the incidence of human CHD.

CITED2 is a transcriptional co-factor with important roles in many organs of the developing mammalian embryo. Complete deletion of this gene causes severe malformation of the placenta, and results in significantly reduced embryonic growth and death from E14.5. The placenta is a complex organ originating from cells derived from three lineages: the maternal decidua, the trophectoderm, and the extra-embryonic mesoderm. Cited2 is expressed in many of these cell types, but its exact role in the formation of the placenta is unknown. Here we use a conditional deletion approach to remove Cited2 from overlapping subsets of trophectoderm and extra-embryonic mesoderm. We find that Cited2 in sinusoidal trophoblast giant cells and syncytiotrophoblasts is likely to have a non-cell autonomous role in patterning of the pericytes associated with the embryonic capillaries. This function is likely to be mediated by PDGF signaling. Furthermore, we also identify that loss of Cited2 in syncytiotrophoblasts results in the subcellular mislocalization of one of the major lactate transporters in the placenta, SLC16A3 (MCT4). We hypothesize that the embryonic growth retardation observed in Cited2 null embryos is due in part to a disorganized embryonic capillary network, and in part due to abnormalities of the nutrient transport functions of the feto-maternal interface.

Segmentation defects of the vertebrae (SDV) are caused by aberrant somite formation during embryogenesis and result in irregular formation of the vertebrae and ribs. The Notch signal transduction pathway plays a critical role in somite formation and patterning in model vertebrates. In humans, mutations in several genes involved in the Notch pathway are associated with SDV, with both autosomal recessive (MESP2, DLL3, LFNG, HES7) and autosomal dominant (TBX6) inheritance. However, many individuals with SDV do not carry mutations in these genes. Using whole-exome capture and massive parallel sequencing, we identified compound heterozygous mutations in RIPPLY2 in two brothers with multiple regional SDV, with appropriate familial segregation. One novel mutation (c.A238T:p.Arg80*) introduces a premature stop codon. In transiently transfected C2C12 mouse myoblasts, the RIPPLY2 mutant protein demonstrated impaired transcriptional repression activity compared with wild-type RIPPLY2 despite similar levels of expression. The other mutation (c.240-4T>G), with minor allele frequency <0.002, lies in the highly conserved splice site consensus sequence 5' to the terminal exon. Ripply2 has a well-established role in somitogenesis and vertebral column formation, interacting at both gene and protein levels with SDV-associated Mesp2 and Tbx6. We conclude that compound heterozygous mutations in RIPPLY2 are associated with SDV, a new gene for this condition.

Congenital heart disease (CHD) is the most common birth defect and brings with it significant mortality and morbidity. The application of exome and genome sequencing has greatly improved the rate of genetic diagnosis for CHD but the cause in the majority of cases remains uncertain. It is clear that genetics, as well as environmental influences, play roles in the aetiology of CHD. Here we address both these aspects of causation with respect to the Notch signalling pathway. In our CHD cohort, variants in core Notch pathway genes account for 20% of those that cause disease, a rate that did not increase with the inclusion of genes of the broader Notch pathway and its regulators. This is reinforced by case-control burden analysis where variants in Notch pathway genes are enriched in CHD patients. This enrichment is due to variation in NOTCH1. Functional analysis of some novel missense NOTCH1 and DLL4 variants in cultured cells demonstrate reduced signalling activity, allowing variant reclassification. Although loss-of-function variants in DLL4 are known to cause Adams-Oliver syndrome, this is the first report of a hypomorphic DLL4 allele as a cause of isolated CHD. Finally, we demonstrate a gene-environment interaction in mouse embryos between Notch1 heterozygosity and low oxygen- or anti-arrhythmic drug-induced gestational hypoxia, resulting in an increased incidence of heart defects. This implies that exposure to environmental insults such as hypoxia could explain variable expressivity and penetrance of observed CHD in families carrying Notch pathway variants.

No abstract

Mammalian embryos develop in a low oxygen environment. The transcription factor hypoxia inducible factor 1a (HIF1α) is a key element in the cellular response to hypoxia. Complete deletion of Hif1α from the mouse conceptus causes extensive placental, vascular and heart defects, resulting in embryonic lethality. However the precise role of Hif1α in each of these organ systems remains unknown. To further investigate, we conditionally-deleted Hif1α from mesoderm, vasculature and heart individually. Surprisingly, deletion from these tissues did not recapitulate the same severe heart phenotype or embryonic lethality. Placental insufficiency, such as occurs in the complete Hif1α null, results in elevated cellular hypoxia in mouse embryos. We hypothesized that subjecting the Hif1α conditional null embryos to increased hypoxic stress might exacerbate the effects of tissue-specific Hif1α deletion. We tested this hypothesis using a model system mimicking placental insufficiency. We found that the majority of embryos lacking Hif1α in the heart died when exposed to non-physiological hypoxia. This was a heart-specific phenomenon, as HIF1α protein accumulated predominantly in the myocardium of hypoxia-stressed embryos. Our study demonstrates the vulnerability of the heart to lowered oxygen levels, and that under such conditions of non-physiological hypoxia the embryo absolutely requires Hif1α to continue normal development. Importantly, these findings extend our understanding of the roles of Hif1α in cardiovascular development.