Timing of NAD Deficiency During Organogenesis Dictates Defect Type and Penetrance.

Major congenital malformations are common, and most cases have no known etiology because of complex interactions between genetic and environmental factors and variable phenotypic outcomes. Congenital NAD Deficiency Disorder (CNDD), a cause of multiple congenital malformations and embryo loss, exemplifies this variability in phenotypic presentation, even between siblings with the same underlying genetic variants. Mouse models show that CNDD is caused by embryonic nicotinamide adenine dinucleotide (NAD) deficiency because of the embryos' genetic inability to synthesize NAD and/or insufficient maternal provision of NAD precursors to embryos. But it is unknown when during pregnancy embryos become susceptible to developing malformations and what drives the malformation variability. Here, we induced CNDD in wild-type mice via the maternal diet and longitudinally tracked affected and unaffected embryos in utero. We compared 3-day interval measurements of the maternal blood NAD metabolome with embryo phenotype using Fast Spin Echo Magnetic Resonance Imaging, mass spectrometry, and micro-computed tomography. Malformations varied between litters, but they correlated with different embryo growth dynamics. Mice with lower maternal NAD Salvage Pathway metabolite levels and minimal levels of derived excretion metabolites from embryonic day 6.5 onward had smaller embryos with more malformations. This showed that altered embryo growth and reduced maternal NAD precursor availability during organogenesis resulted in CNDD. Variability in the timing of maternal metabolic perturbation corresponded to variability in organ and tissue defect types between litters. As embryo phenotypes are directly linked to maternal NAD precursor availability prior to and during organogenesis, this suggests NAD-derived metabolites are potential biomarkers predicting CNDD.