Gavin Chapman

The spectrum of congenital malformations in VACTERL association varies among patients and can be differentially diagnosed with CHARGE syndrome, Fanconi anaemia, and others (reviewed in Solomon 2011). Despite overlapping clinical findings, the genetic causes of these diseases are distinct. In this context, unbiased whole genome sequencing can assist in differential diagnoses, as well as identify new gene-disease associations. In this report, we demonstrate that whole genome sequencing of a proband with suspected VACTERL association revealed two gene variants in the ribosomal genes RPL18 (NM_000979.4 c.397 G > C p.(Gly133Arg)) and RPS6 (NM_001010.3: c.370 C > G p.(Leu124Val)). Mutations in ribosomal genes are associated with Diamond-Blackfan anaemia, a condition that shares phenotypic similarities with Fanconi anaemia. Our modelling and functional assessment of the identified variants strongly indicate pathogenicity of the RPL18:p.(G133R) variant as it is novel, displays reduced expression and stability and abnormal intracellular distribution, and interferes with protein synthesis in cultured cells. The RPS6:p.(L124V) variant reduced protein expression and altered cytoplasmic distribution but does not interfere with protein synthesis in cultured cells. Overall, our study indicates the significant advantage of using unbiased whole genome sequencing for the examination of patients with complex congenital malformations.

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.

Nicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway — KYNU, HAAO, and NADSYN1 — have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain–specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1^+/– mothers, any B₃ vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1^–/– mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.

SUMMARY: Pluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across multiple developmental lineage trajectories. In this study, we engineered barcoded iPSCs to generate an atlas of multilineage differentiation from pluripotency, encompassing a time-course of WNT-induced differentiation perturbed using modulators of WNT, BMP, and VEGF signalling. Computational mapping of in vitro cell types to in vivo developmental lineages revealed a diversity of iPSC-derived cell types comprising mesendoderm lineage cell types including lateral plate and paraxial mesoderm, neural crest, and primitive gut. Coupling this atlas of in vitro differentiation with Summary data-based Mendelian Randomisation analysis of human complex traits, we identify the WNT-inhibitor protein TMEM88 as a putative regulator of mesendodermal cell types governing development of diverse cardiovascular and anthropometric traits. Using genetic loss of function models, we show that TMEM88 is required for differentiation of diverse endoderm and mesoderm cell lineages in vitro and that TMEM88 knockout in vivo results in a significant dysregulation of arterial blood pressure. This study provides an atlas of multilineage iPSC differentiation coupled with new molecular, computational, and statistical genetic tools to dissect genetic determinants of mammalian developmental physiology.

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme involved in over 400 cellular reactions. During embryogenesis, mammals synthesize NAD de novo from dietary l‐tryptophan via the kynurenine pathway. Biallelic, inactivating variants in three genes encoding enzymes of this biosynthesis pathway (KYNU, HAAO, and NADSYN1) disrupt NAD synthesis and have been identified in patients with multiple malformations of the heart, kidney, vertebrae, and limbs; these patients have Congenital NAD Deficiency Disorder HAAO and four families with biallelic variants in KYNU. These patients present similarly with multiple malformations of the heart, kidney, vertebrae, and limbs, of variable severity. We show that each variant identified in these patients results in loss‐of‐function, revealed by a significant reduction in NAD levels via yeast genetic complementation assays. For the first time, missense mutations are identified as a cause of malformation and shown to disrupt enzyme function. These missense and frameshift variants cause moderate to severe NAD deficiency in yeast, analogous to insufficient synthesized NAD in patients. We hereby expand the genotypic and corresponding phenotypic spectrum of Congenital NAD Deficiency Disorder.

The genetic causes of multiple congenital anomalies are incompletely understood. Here, we report novel heterozygous predicted loss-of-function (LoF) and predicted damaging missense variants in the WW domain binding protein 11 (WBP11) gene in seven unrelated families with a variety of overlapping congenital malformations, including cardiac, vertebral, tracheo-esophageal, renal and limb defects. WBP11 encodes a component of the spliceosome with the ability to activate pre-messenger RNA splicing. We generated a Wbp11 null allele in mouse using CRISPR-Cas9 targeting. Wbp11 homozygous null embryos die prior to E8.5, indicating that Wbp11 is essential for development. Fewer Wbp11 heterozygous null mice are found than expected due to embryonic and postnatal death. Importantly, Wbp11 heterozygous null mice are small and exhibit defects in axial skeleton, kidneys and esophagus, similar to the affected individuals, supporting the role of WBP11 haploinsufficiency in the development of congenital malformations in humans. LoF WBP11 variants should be considered as a possible cause of VACTERL association as well as isolated Klippel-Feil syndrome, renal agenesis or esophageal atresia.

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.