A metabolic signature for NADSYN1-dependent congenital NAD deficiency disorder.

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.

Polygenic Risk in Families With Spontaneous Coronary Artery Dissection.

Importance: Spontaneous coronary artery dissection (SCAD) is a poorly understood cause of acute coronary syndrome that predominantly affects women. Evidence to date suggests a complex genetic architecture, while a family history is reported for a minority of cases. Objective: To determine the contribution of rare and common genetic variants to SCAD risk in familial cases, the latter via the comparison of a polygenic risk score (PRS) with those with sporadic SCAD and healthy controls. Design, Setting, and Participants: This genetic association study analyzed families with SCAD, individuals with sporadic SCAD, and healthy controls. Genotyping was undertaken for all participants. Participants were recruited between 2017 and 2021. A PRS for SCAD was calculated for all participants. The presence of rare variants in genes associated with connective tissue disorders (CTD) was also assessed. Individuals with SCAD were recruited via social media or from a single medical center. A previously published control database of older healthy individuals was used. Data were analyzed from January 2022 to October 2023. Exposures: PRS for SCAD comprised of 7 single-nucleotide variants. Main Outcomes and Measures: Disease status (familial SCAD, sporadic SCAD, or healthy control) associated with PRS. Results: A total of 13 families with SCAD (27 affected and 12 unaffected individuals), 173 individuals with sporadic SCAD, and 1127 healthy controls were included. A total of 188 individuals with SCAD (94.0%) were female, including 25 of 27 with familial SCAD and 163 of 173 with sporadic SCAD; of 12 unaffected individuals from families with SCAD, 6 (50%) were female; and of 1127 healthy controls, 672 (59.6%) were female. Compared with healthy controls, the odds of being an affected family member or having sporadic SCAD was significantly associated with a SCAD PRS (where the odds ratio [OR] represents an increase in odds per 1-SD increase in PRS) (affected family member: OR, 2.14; 95% CI, 1.78-2.50; adjusted P = 1.96 × 10-4; sporadic SCAD: OR, 1.63; 95% CI, 1.37-1.89; adjusted P = 5.69 × 10-4). This association was not seen for unaffected family members (OR, 1.03; 95% CI, 0.46-1.61; adjusted P = .91) compared with controls. Further, those with familial SCAD were overrepresented in the top quintile of the control PRS distribution (OR, 3.70; 95% CI, 2.93-4.47; adjusted P = .001); those with sporadic SCAD showed a similar pattern (OR, 2.51; 95% CI, 1.98-3.04; adjusted P = .001). Affected individuals within a family did not share any rare deleterious variants in CTD-associated genes. Conclusions and Relevance: Extreme aggregation of common genetic risk appears to play a significant role in familial clustering of SCAD as well as in sporadic case predisposition, although further study is required.

Myeloid-CITED2 Deficiency Exacerbates Diet-Induced Obesity and Pro-Inflammatory Macrophage Response.

Macrophages are the principal component of the innate immune system that are found in all tissues and play an essential role in development, homeostasis, tissue repair, and immunity. Clinical and experimental studies have shown that transcriptionally dynamic pro-inflammatory macrophages are involved in the pathogenesis of diet-induced obesity and insulin resistance. However, cell-intrinsic mechanisms must exist that bridle uncontrolled pro-inflammatory macrophage activation in metabolic organs and disease pathogenesis. In this study, we show that CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2) is an essential negative regulator of pro-inflammatory macrophage activation and inflammatory disease pathogenesis. Our in vivo studies show that myeloid-CITED2 deficiency significantly elevates high-fat diet (HFD)-induced expansion of adipose tissue volume, obesity, glucose intolerance, and insulin resistance. Moreover, myeloid-CITED2 deficiency also substantially augments HFD-induced adipose tissue inflammation and adverse remodeling of adipocytes. Our integrated transcriptomics and gene set enrichment analyses show that CITED2 deficiency curtails BCL6 signaling and broadly elevates BCL6-repressive gene target expression in macrophages. Using complementary gain- and loss-of-function studies, we found that CITED2 deficiency attenuates, and CITED2 overexpression elevates, inducible BCL6 expression in macrophages. At the molecular level, our analyses show that CITED2 promotes BCL6 expression by restraining STAT5 activation in macrophages. Interestingly, siRNA-mediated knockdown of STAT5 fully reversed elevated pro-inflammatory gene target expression in CITED2-deficient macrophages. Overall, our findings highlight that CITED2 restrains inflammation by promoting BCL6 expression in macrophages, and limits diet-induced obesity and insulin resistance.

Nicotinamide Adenine Dinucleotide Deficiency and Its Impact on Mammalian Development.

Significance: Nicotinamide adenine dinucleotide (NAD) is an important molecule synthesized from tryptophan or vitamin B3 and involved in numerous cellular reactions. NAD deficiency during pregnancy causes congenital NAD deficiency disorder (CNDD) characterized by multiple congenital malformations and/or miscarriage. Studies in genetically engineered mice replicating mutations found in human patient cases show that CNDD can be prevented by dietary supplements. Recent Advances: A growing number of patient reports show that biallelic loss-of-function of genes involved in NAD de novo synthesis (KYNU, HAAO, NADSYN1) cause CNDD. Other factors that limit the availability of NAD precursors, for example, limited dietary precursor supply or absorption, can cause or contribute to NAD deficiency and result in CNDD in mice. Molecular flux experiments allow quantitative understanding of NAD precursor concentrations in the circulation and their usage by different cells. Studies of NAD-consuming enzymes and contributors to NAD homeostasis help better understand how perturbed NAD levels are implicated in various diseases and adverse pregnancy outcomes. Critical Issues: NAD deficiency is one of the many known causes of adverse pregnancy outcomes, but its prevalence in the human population and among pregnant women is unknown. Since NAD is involved in hundreds of diverse cellular reactions, determining how NAD deficiency disrupts embryogenesis is an important challenge. Future Directions: Furthering our understanding of the molecular fluxes between the maternal and embryonic circulation during pregnancy, the NAD-dependent pathways active in the developing embryo, and the molecular mechanisms by which NAD deficiency causes adverse pregnancy outcomes will provide direction for future prevention strategies. Antioxid. Redox Signal. 39, 1108-1132.

Quantitative trait and transcriptome analysis of genetic complexity underpinning cardiac interatrial septation in mice using an advanced intercross line.

Unlike single-gene mutations leading to Mendelian conditions, common human diseases are likely to be emergent phenomena arising from multilayer, multiscale, and highly interconnected interactions. Atrial and ventricular septal defects are the most common forms of cardiac congenital anomalies in humans. Atrial septal defects (ASD) show an open communication between the left and right atria postnatally, potentially resulting in serious hemodynamic consequences if untreated. A milder form of atrial septal defect, patent foramen ovale (PFO), exists in about one-quarter of the human population, strongly associated with ischaemic stroke and migraine. The anatomic liabilities and genetic and molecular basis of atrial septal defects remain unclear. Here, we advance our previous analysis of atrial septal variation through quantitative trait locus (QTL) mapping of an advanced intercross line (AIL) established between the inbred QSi5 and 129T2/SvEms mouse strains, that show extremes of septal phenotypes. Analysis resolved 37 unique septal QTL with high overlap between QTL for distinct septal traits and PFO as a binary trait. Whole genome sequencing of parental strains and filtering identified predicted functional variants, including in known human congenital heart disease genes. Transcriptome analysis of developing septa revealed downregulation of networks involving ribosome, nucleosome, mitochondrial, and extracellular matrix biosynthesis in the 129T2/SvEms strain, potentially reflecting an essential role for growth and cellular maturation in septal development. Analysis of variant architecture across different gene features, including enhancers and promoters, provided evidence for the involvement of non-coding as well as protein-coding variants. Our study provides the first high-resolution picture of genetic complexity and network liability underlying common congenital heart disease, with relevance to human ASD and PFO.

Australian Genomics: Outcomes of a 5-year national program to accelerate the integration of genomics in healthcare.

Australian Genomics is a national collaborative partnership of more than 100 organizations piloting a whole-of-system approach to integrating genomics into healthcare, based on federation principles. In the first five years of operation, Australian Genomics has evaluated the outcomes of genomic testing in more than 5,200 individuals across 19 rare disease and cancer flagship studies. Comprehensive analyses of the health economic, policy, ethical, legal, implementation and workforce implications of incorporating genomics in the Australian context have informed evidence-based change in policy and practice, resulting in national government funding and equity of access for a range of genomic tests. Simultaneously, Australian Genomics has built national skills, infrastructure, policy, and data resources to enable effective data sharing to drive discovery research and support improvements in clinical genomic delivery.

ConanVarvar: a versatile tool for the detection of large syndromic copy number variation from whole-genome sequencing data.

BACKGROUND: A wide range of tools are available for the detection of copy number variants (CNVs) from whole-genome sequencing (WGS) data. However, none of them focus on clinically-relevant CNVs, such as those that are associated with known genetic syndromes. Such variants are often large in size, typically 1-5 Mb, but currently available CNV callers have been developed and benchmarked for the discovery of smaller variants. Thus, the ability of these programs to detect tens of real syndromic CNVs remains largely unknown. RESULTS: Here we present ConanVarvar, a tool which implements a complete workflow for the targeted analysis of large germline CNVs from WGS data. ConanVarvar comes with an intuitive R Shiny graphical user interface and annotates identified variants with information about 56 associated syndromic conditions. We benchmarked ConanVarvar and four other programs on a dataset containing real and simulated syndromic CNVs larger than 1 Mb. In comparison to other tools, ConanVarvar reports 10-30 times less false-positive variants without compromising sensitivity and is quicker to run, especially on large batches of samples. CONCLUSIONS: ConanVarvar is a useful instrument for primary analysis in disease sequencing studies, where large CNVs could be the cause of disease.

The International Society of Differentiation: Past, present, and future.

The International Society of Differentiation was born from the First International Conference on Cell Differentiation conceived by D.V. and held in Nice, France in 1971. The conference also resulted in the creation of the journal of the Society named Differentiation. The Society advocates for the field of differentiation through the journal Differentiation, organizing and supporting international scientific conferences, honoring scientific achievements, and supporting trainees.

Maternal heterozygosity of Slc6a19 causes metabolic perturbation and congenital NAD deficiency disorder in mice.

Nicotinamide adenine dinucleotide (NAD) is a key metabolite synthesised from vitamin B3 or tryptophan. Disruption of genes encoding NAD synthesis enzymes reduces NAD levels and causes congenital NAD deficiency disorder (CNDD), characterised by multiple congenital malformations. SLC6A19 (encoding B0AT1, a neutral amino acid transporter), represents the main transporter for free tryptophan in the intestine and kidney. Here, we tested whether Slc6a19 heterozygosity in mice limits the tryptophan available for NAD synthesis during pregnancy and causes adverse pregnancy outcomes. Pregnant Slc6a19+/- mice were fed diets depleted of vitamin B3, so that tryptophan was the source of NAD during gestation. This perturbed the NAD metabolome in pregnant Slc6a19+/- females, resulting in reduced NAD levels and increased rates of embryo loss. Surviving embryos were small and exhibited specific combinations of CNDD-associated malformations. Our results show that genes not directly involved in NAD synthesis can affect NAD metabolism and cause CNDD. They also suggest that human female carriers of a SLC6A19 loss-of-function allele might be susceptible to adverse pregnancy outcomes unless sufficient NAD precursor amounts are available during gestation. This article has an associated First Person interview with the first author of the paper.

Using novel data linkage of congenital heart disease biobank data with administrative health data to identify cardiovascular outcomes to inform genomic analysis.

Introduction: Contemporary care of congenital heart disease (CHD) is largely standardised, however there is heterogeneity in post-surgical outcomes that may be explained by genetic variation. Data linkage between a CHD biobank and routinely collected administrative datasets is a novel method to identify outcomes to explore the impact of genetic variation. Objective: Use data linkage to identify and validate patient outcomes following surgical treatment for CHD. Methods: Data linkage between clinical and biobank data of children born from 2001-2014 that had a procedure for CHD in New South Wales, Australia, with hospital discharge data, education and death data. The children were grouped according to CHD lesion type and age at first cardiac surgery. Children in each 'lesion/age at surgery group' were classified into 'favourable' and 'unfavourable' cardiovascular outcome groups based on variables identified in linked administrative data including; total time in intensive care, total length of stay in hospital, and mechanical ventilation time up to 5 years following the date of the first cardiac surgery. A blind medical record audit of 200 randomly chosen children from 'favourable' and 'unfavourable' outcome groups was performed to validate the outcome groups. Results: Of the 1872 children in the dataset that linked to hospital or death data, 483 were identified with a 'favourable' cardiovascular outcome and 484 were identified as having a 'unfavourable' cardiovascular outcome. The medical record audit found concordant outcome groups for 182/192 records (95%) compared to the outcome groups categorized using the linked data. Conclusions: The linkage of a curated biobank dataset with routinely collected administrative data is a reliable method to identify outcomes to facilitate a large-scale study to examine genetic variance. These genetic hallmarks could be used to identify patients who are at risk of unfavourable cardiovascular outcomes, to inform strategies for prevention and changes in clinical care.

Viewing teratogens through the lens of nicotinamide adenine dinucleotide (NAD+).

BACKGROUND: Nicotinamide adenine dinucleotide (NAD+) depletion is associated with numerous diseases in humans. Recently it was revealed that genetic blockage of the NAD+ synthesis pathway in humans causes birth defects in multiple organ systems and miscarriage. Additionally, mice with NAD+ deficiency created through dietary restriction of tryptophan and vitamin B3 were shown to have congenital anomalies affecting virtually every organ system along with miscarriage. Perturbations in NAD+/NADH affect mechanisms of teratogenesis presented by Wilson and others, including genetic alterations, altered energy sources, and lack of precursors and substrates needed for biosynthesis. METHODS: Medical literature was evaluated to demonstrate how perturbations in NAD+/NADH affect mechanisms of teratogenesis. In addition, literature describing several different teratogens of various types (infectious, physical, maternal health factors, drugs) was reviewed showing the impact of these teratogens on NAD+ and NAD+/NADH ratios. RESULT: Many teratogens affect NAD+ by altering its metabolism, decreasing its intracellular availability, or decreasing its production, which in turn is a plausible mechanism for the creation of birth defects. CONCLUSION: Looking at teratogens through the lens of their impact on NAD+ could provide valuable insight into the mechanism by which some teratogens cause birth defects and miscarriage.

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.

Insights into the genetic architecture underlying complex, critical congenital heart disease.

Congenital heart disease (CHD) has a multifactorial aetiology, raising the possibility of an underlying genetic burden, predisposing to disease but also variable expression, including variation in disease severity, and incomplete penetrance. Using whole genome sequencing (WGS), the findings of this study, indicate that complex, critical CHD is distinct from other types of disease due to increased genetic burden in common variation, specifically among established CHD genes. Additionally, these findings highlight associations with regulatory genes and environmental "stressors" in the final presentation of disease.

An image analysis protocol using CellProfiler for automated quantification of post-ischemic cardiac parameters.

Quantitative assessment of post-ischemic cardiac remodeling is often hampered by tissue complexity and structural heterogeneity of the scar. Automated quantification of microscopy images offers an unbiased approach to reduce inter-observer variability. Here, we present a CellProfiler-based analytical pipeline for the high-throughput analysis of confocal images to quantify post-ischemic cardiac parameters. We describe image preprocessing and the quantification of capillary rarefaction, immune cell infiltration, cell death, and proliferating fibroblasts. This protocol can be adapted to other tissue types. For complete details on the use and execution of this profile, please refer to Janbandhu et al. (2021).

Exploring the Genetic Architecture of Spontaneous Coronary Artery Dissection Using Whole-Genome Sequencing.

BACKGROUND: Spontaneous coronary artery dissection (SCAD) is a cause of acute coronary syndrome that predominantly affects women. Its pathophysiology remains unclear but connective tissue disorders (CTD) and other vasculopathies have been observed in many SCAD patients. A genetic component for SCAD is increasingly appreciated, although few genes have been robustly implicated. We sought to clarify the genetic cause of SCAD using targeted and genome-wide methods in a cohort of sporadic cases to identify both common and rare disease-associated variants. METHODS: A cohort of 91 unrelated sporadic SCAD cases was investigated for rare, deleterious variants in genes associated with either SCAD or CTD, while new candidate genes were sought using rare variant collapsing analysis and identification of novel loss-of-function variants in genes intolerant to such variation. Finally, 2 SCAD polygenic risk scores were applied to assess the contribution of common variants. RESULTS: We identified 10 cases with at least one rare, likely disease-causing variant in CTD-associated genes, although only one had a CTD phenotype. No genes were significantly associated with SCAD from genome-wide collapsing analysis, however, enrichment for TGF (transforming growth factor)-ß signaling pathway genes was found with analysis of 24 genes harboring novel loss-of-function variants. Both polygenic risk scores demonstrated that sporadic SCAD cases have a significantly elevated genetic SCAD risk compared with controls. CONCLUSIONS: SCAD shares some genetic overlap with CTD, even in the absence of any major CTD phenotype. Consistent with a complex genetic architecture, SCAD patients also have a higher burden of common variants than controls.

Benchmarking the Effectiveness and Accuracy of Multiple Mitochondrial DNA Variant Callers: Practical Implications for Clinical Application.

Mitochondrial DNA (mtDNA) mutations contribute to human disease across a range of severity, from rare, highly penetrant mutations causal for monogenic disorders to mutations with milder contributions to phenotypes. mtDNA variation can exist in all copies of mtDNA or in a percentage of mtDNA copies and can be detected with levels as low as 1%. The large number of copies of mtDNA and the possibility of multiple alternative alleles at the same DNA nucleotide position make the task of identifying allelic variation in mtDNA very challenging. In recent years, specialized variant calling algorithms have been developed that are tailored to identify mtDNA variation from whole-genome sequencing (WGS) data. However, very few studies have systematically evaluated and compared these methods for the detection of both homoplasmy and heteroplasmy. A publicly available synthetic gold standard dataset was used to assess four mtDNA variant callers (Mutserve, mitoCaller, MitoSeek, and MToolBox), and the commonly used Genome Analysis Toolkit "best practices" pipeline, which is included in most current WGS pipelines. We also used WGS data from 126 trios and calculated the percentage of maternally inherited variants as a metric of calling accuracy, especially for homoplasmic variants. We additionally compared multiple pathogenicity prediction resources for mtDNA variants. Although the accuracy of homoplasmic variant detection was high for the majority of the callers with high concordance across callers, we found a very low concordance rate between mtDNA variant callers for heteroplasmic variants ranging from 2.8% to 3.6%, for heteroplasmy thresholds of 5% and 1%. Overall, Mutserve showed the best performance using the synthetic benchmark dataset. The analysis of mtDNA pathogenicity resources also showed low concordance in prediction results. We have shown that while homoplasmic variant calling is consistent between callers, there remains a significant discrepancy in heteroplasmic variant calling. We found that resources like population frequency databases and pathogenicity predictors are now available for variant annotation but still need refinement and improvement. With its peculiarities, the mitochondria require special considerations, and we advocate that caution needs to be taken when analyzing mtDNA data from WGS data.

Quantitative 3D analysis and visualization of cardiac fibrosis by microcomputed tomography.

Following myocardial infarction, damaged myocardium is replaced with a fibrotic scar that preserves cardiac structural integrity. Scar area measured from sample 2D images of serial heart sections does not faithfully measure the extent of fibrosis due to structural heterogeneity caused by tissue dynamics. Here, we present an X-ray microcomputed tomography (micro-CT) workflow that generates accurate volumetric quantification of scar and surviving myocardium in infarcted mouse hearts. This workflow could be applied to other fibrotic organs or hearts from other species. For complete details on the use and execution of this protocol, please refer to Janbandhu et al. (2021).

Whole genome sequencing in transposition of the great arteries and associations with clinically relevant heart, brain and laterality genes.

BACKGROUND: The most common cyanotic congenital heart disease (CHD) requiring management as a neonate is transposition of great arteries (TGA). Clinically, up to 50% of TGA patients develop some form of neurodevelopmental disability (NDD), thought to have a significant genetic component. A "ciliopathy" and links with laterality disorders have been proposed. This first report of whole genome sequencing in TGA, sought to identify clinically relevant variants contributing to heart, brain and laterality defects. METHODS: Initial whole genome sequencing analyses on 100 TGA patients focussed on established disease genes related to CHD (n = 107), NDD (n = 659) and heterotaxy (n = 74). Single variant as well as copy number variant analyses were conducted. Variant pathogenicity was assessed using the American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. RESULTS: Fifty-five putatively damaging variants were identified in established disease genes associated with CHD, NDD and heterotaxy; however, no clinically relevant variants could be attributed to disease. Notably, case-control analyses identified significantly more predicted-damaging, silent and total variants in TGA cases than healthy controls in established CHD genes (P < .001), NDD genes (P < .001) as well as across the three gene panels (P < .001). CONCLUSION: We present compelling evidence that the majority of TGA is not caused by monogenic rare variants and is most likely oligogenic and/or polygenic in nature, highlighting the complex genetic architecture and multifactorial influences on this CHD sub-type and its long-term sequelae. Assessment of variant burden in key heart, brain and/or laterality genes may be required to unravel the genetic contributions to TGA and related disabilities.

A new era of genetic testing in congenital heart disease: A review.

Genetic and genomic testing in pediatric CHD is becoming increasingly routine, and can have important psychosocial, clinical and reproductive implications. In this paper we highlight important challenges and considerations when providing genetics consults and testing in pediatric CHD and illustrate the role of a dedicated CHD genetics clinic. Key lessons include that a) a genetic diagnosis can have clinical utility that justifies testing early in life, b) adequate genetic counselling is crucial to ensure families are supported, understand the range of possible results, and are prepared for new or unexpected health information, and c) further integration of the clinical genetics and cardiology workflows will be required to effectively manage the burgeoning information arising from genetic testing. Our experience demonstrates that a dedicated CHD genetics clinic is a valuable addition to a multidisciplinary team providing care to children with CHD.