Identifying the Genetic Causes of Congenital Anomalies

Structural organ anomalies (malformations) are common and cause significant mortality and morbidity worldwide. The aim of this study was to identify monogenic causes of malformations to understand their pathogenesis, enhance clinical care and ultimately ease the burden of disease. We used family-based exome (ES) or genome sequencing (GS), cohort-wide analyses, collaborative clinical characterisation, and laboratory validation to identify causative variants in well-known and novel genes. We first studied a cohort of 44 patients with vertebral defects. Via ES, we found the genetic cause in 9/44 individuals from 8/43 families. Two of these families had spondylocostal dysostosis due to pathogenic variants in DLL3 and TBX6. The other six families had multiple congenital anomalies (MCA), with disruptive variants in HAAO, KYNU, SMAD4, STAG2, TBX6, and WBP11. As WBP11, HAAO, and KYNU were not associated with MCA, our laboratory studied their function. We found heterozygous WBP11 variants in two families from our cohort and five additional families via GeneMatcher. Our laboratory produced a Wbp11 mouse model. Our clinical and laboratory findings showed that WBP11 haploinsufficiency in humans and mice leads to MCA with incomplete penetrance and variable expressivity. We next studied patients with cardiac defects. Via GS and GeneMatcher, we identified two additional patients with biallelic HAAO and KYNU variants respectively, who both have hypoplastic left heart and other MCA. HAAO and KYNU encode enzymes in the synthesis of nicotinamide adenine dinucleotide (NAD). Biochemical and animal studies showed that pathogenic biallelic variants in HAAO and KYNU cause NAD deficiency in humans and mice, revealing a novel mechanism that results in MCA. In Haao and Kynu mice, niacin supplementation alleviated NAD deficiency and prevented MCA. Hence, our discovery has revealed a potential preventor of malformations due to genetic and environmental causes of NAD deficiency. Similarly, combining GS with functional assays led us to confirm the pathogenicity of a variant in a gene known to cause MCA, NOTCH1. This highlights the need to establish efficient pipelines to functionally characterise the increasing number of variants identified as ES and GS become more widely used in investigating malformations. We found genetic causes of malformations via family sequencing and functional confirmation, improving the care of families in our study and beyond

NAD deficiency, congenital malformations, and niacin supplementation

BACKGROUND: Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. METHODS: We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 system. RESULTS: Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. CONCLUSIONS: 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

Relationship between child rearing practices and selected adolescent behavior

This study is aimed to determine the relationship between certain child-rearing practices of mothers and some patterns of adolescent behavior. The subjects chosen were 210 students of De La Salle University who are in the Dean\u27s List. Structured questionnaires for mothers and students were used in data gathering and correlations between measures for the different categories were computed. Significant correlations were obtained between the achievement-oriented behavior of their children between the mother\u27s child rearing practices emphasizing sharing and cooperation and the children\u27s sociability behavior and, between the mother\u27s permissive - rearing practices and the self-reliant behavior of their children. Thus, as concluded, the mother is still but not as much significant a factor in personality development in the adolescent years than in the earlier years. the adolescents may have been exposed to and influenced by other factors such as their personal interactions in school and the community

NAD deficiency, congenital malformations, and niacin supplementation

Background Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients..

Myhre syndrome is caused by dominant-negative dysregulation of SMAD4 and other co-factors

Myhre syndrome is a connective tissue disorder characterized by congenital cardiovascular, craniofacial, respiratory, skeletal, and cutaneous anomalies as well as intellectual disability and progressive fibrosis. It is caused by germline variants in the transcriptional co-regulator SMAD4 that localize at two positions within the SMAD4 protein, I500 and R496, with I500 V/T/M variants more commonly identified in individuals with Myhre syndrome. Here we assess the functional impact of SMAD4-I500V variant, identified in two previously unpublished individuals with Myhre syndrome, and provide novel insights into the molecular mechanism of SMAD4-I500V dysfunction. We show that SMAD4-I500V can dimerize, but its transcriptional activity is severely compromised. Our data show that SMAD4-I500V acts dominant-negatively on SMAD4 and on receptor-regulated SMADs, affecting transcription of target genes. Furthermore, SMAD4-I500V impacts the transcription and function of crucial developmental transcription regulator, NKX2-5. Overall, our data reveal a dominant-negative model of disease for SMAD4-I500V where the function of SMAD4 encoded on the remaining allele, and of co-factors, are perturbed by the continued heterodimerization of the variant, leading to dysregulation of TGF and BMP signaling. Our findings not only provide novel insights into the mechanism of Myhre syndrome pathogenesis but also extend the current knowledge of how pathogenic variants in SMAD proteins cause disease

Functional genomics and gene-environment interaction highlight the complexity of congenital heart disease caused by Notch pathway variants

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

Heterozygous loss of WBP11 function causes multiple congenital defects in humans and mice

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