De novo variants in FRYL are associated with developmental delay, intellectual disability, and dysmorphic features.

FRY-like transcription coactivator (FRYL) belongs to a Furry protein family that is evolutionarily conserved from yeast to humans. The functions of FRYL in mammals are largely unknown, and variants in FRYL have not previously been associated with a Mendelian disease. Here, we report fourteen individuals with heterozygous variants in FRYL who present with developmental delay, intellectual disability, dysmorphic features, and other congenital anomalies in multiple systems. The variants are confirmed de novo in all individuals except one. Human genetic data suggest that FRYL is intolerant to loss of function (LoF). We find that the fly FRYL ortholog, furry (fry), is expressed in multiple tissues, including the central nervous system where it is present in neurons but not in glia. Homozygous fry LoF mutation is lethal at various developmental stages, and loss of fry in mutant clones causes defects in wings and compound eyes. We next modeled four out of the five missense variants found in affected individuals using fry knockin alleles. One variant behaves as a severe LoF variant, whereas two others behave as partial LoF variants. One variant does not cause any observable defect in flies, and the corresponding human variant is not confirmed to be de novo, suggesting that this is a variant of uncertain significance. In summary, our findings support that fry is required for proper development in flies and that the LoF variants in FRYL cause a dominant disorder with developmental and neurological symptoms due to haploinsufficiency.

The H2Bub1-deposition complex is required for human and mouse cardiogenesis.

De novo variants affecting monoubiquitylation of histone H2B (H2Bub1) are enriched in human congenital heart disease. H2Bub1 is required in stem cell differentiation, cilia function, post-natal cardiomyocyte maturation and transcriptional elongation. However, how H2Bub1 affects cardiogenesis is unknown. We show that the H2Bub1-deposition complex (RNF20-RNF40-UBE2B) is required for mouse cardiogenesis and for differentiation of human iPSCs into cardiomyocytes. Mice with cardiac-specific Rnf20 deletion are embryonic lethal and have abnormal myocardium. We then analyzed H2Bub1 marks during differentiation of human iPSCs into cardiomyocytes. H2Bub1 is erased from most genes at the transition from cardiac mesoderm to cardiac progenitor cells but is preserved on a subset of long cardiac-specific genes. When H2Bub1 is reduced in iPSC-derived cardiomyocytes, long cardiac-specific genes have fewer full-length transcripts. This correlates with H2Bub1 accumulation near the center of these genes. H2Bub1 accumulation near the center of tissue-specific genes was also observed in embryonic fibroblasts and fetal osteoblasts. In summary, we show that normal H2Bub1 distribution is required for cardiogenesis and cardiomyocyte differentiation, and suggest that H2Bub1 regulates tissue-specific gene expression by increasing the amount of full-length transcripts.

Pleiotropic role of TRAF7 in skull-base meningiomas and congenital heart disease.

While somatic variants of TRAF7 (Tumor necrosis factor receptor-associated factor 7) underlie anterior skull-base meningiomas, here we report the inherited mutations of TRAF7 that cause congenital heart defects. We show that TRAF7 mutants operate in a dominant manner, inhibiting protein function via heterodimerization with wild-type protein. Further, the shared genetics of the two disparate pathologies can be traced to the common origin of forebrain meninges and cardiac outflow tract from the TRAF7-expressing neural crest. Somatic and inherited mutations disrupt TRAF7-IFT57 interactions leading to cilia degradation. TRAF7-mutant meningioma primary cultures lack cilia, and TRAF7 knockdown causes cardiac, craniofacial, and ciliary defects in Xenopus and zebrafish, suggesting a mechanistic convergence for TRAF7-driven meningiomas and developmental heart defects.

Neither cardiac mitochondrial DNA variation nor copy number contribute to congenital heart disease risk.

The well-established manifestation of mitochondrial mutations in functional cardiac disease (e.g., mitochondrial cardiomyopathy) prompted the hypothesis that mitochondrial DNA (mtDNA) sequence and/or copy number (mtDNAcn) variation contribute to cardiac defects in congenital heart disease (CHD). MtDNAcns were calculated and rare, non-synonymous mtDNA mutations were identified in 1,837 CHD-affected proband-parent trios, 116 CHD-affected singletons, and 114 paired cardiovascular tissue/blood samples. The variant allele fraction (VAF) of heteroplasmic variants in mitochondrial RNA from 257 CHD cardiovascular tissue samples was also calculated. On average, mtDNA from blood had 0.14 rare variants and 52.9 mtDNA copies per nuclear genome per proband. No variation with parental age at proband birth or CHD-affected proband age was seen. mtDNAcns in valve/vessel tissue (320 ± 70) were lower than in atrial tissue (1,080 ± 320, p = 6.8E-21), which were lower than in ventricle tissue (1,340 ± 280, p = 1.4E-4). The frequency of rare variants in CHD-affected individual DNA was indistinguishable from the frequency in an unaffected cohort, and proband mtDNAcns did not vary from those of CHD cohort parents. In both the CHD and the comparison cohorts, mtDNAcns were significantly correlated between mother-child, father-child, and mother-father. mtDNAcns among people with European (mean = 52.0), African (53.0), and Asian haplogroups (53.5) were calculated and were significantly different for European and Asian haplogroups (p = 2.6E-3). Variant heteroplasmic fraction (HF) in blood correlated well with paired cardiovascular tissue HF (r = 0.975) and RNA VAF (r = 0.953), which suggests blood HF is a reasonable proxy for HF in heart tissue. We conclude that mtDNA mutations and mtDNAcns are unlikely to contribute significantly to CHD risk.

Network assisted analysis of de novo variants using protein-protein interaction information identified 46 candidate genes for congenital heart disease.

De novo variants (DNVs) with deleterious effects have proved informative in identifying risk genes for early-onset diseases such as congenital heart disease (CHD). A number of statistical methods have been proposed for family-based studies or case/control studies to identify risk genes by screening genes with more DNVs than expected by chance in Whole Exome Sequencing (WES) studies. However, the statistical power is still limited for cohorts with thousands of subjects. Under the hypothesis that connected genes in protein-protein interaction (PPI) networks are more likely to share similar disease association status, we developed a Markov Random Field model that can leverage information from publicly available PPI databases to increase power in identifying risk genes. We identified 46 candidate genes with at least 1 DNV in the CHD study cohort, including 18 known human CHD genes and 35 highly expressed genes in mouse developing heart. Our results may shed new insight on the shared protein functionality among risk genes for CHD.

Single-cell reconstruction and mutation enrichment analysis identifies dysregulated cardiomyocyte and endothelial cells in congenital heart disease.

Congenital heart disease (CHD) is one of the most prevalent neonatal congenital anomalies. To catalog the putative candidate CHD risk genes, we collected 16,349 variants [single-nucleotide variants (SNVs) and Indels] impacting 8,308 genes in 3,166 CHD cases for a comprehensive meta-analysis. Using American College of Medical Genetics (ACMG) guidelines, we excluded the 0.1% of benign/likely benign variants and the resulting dataset consisted of 83% predicted loss of function variants and 17% missense variants. Seventeen percent were de novo variants. A stepwise analysis identified 90 variant-enriched CHD genes, of which six (GPATCH1, NYNRIN, TCLD2, CEP95, MAP3K19, and TTC36) were novel candidate CHD genes. Single-cell transcriptome cluster reconstruction analysis on six CHD tissues and four controls revealed upregulation of the top 10 frequently mutated genes primarily in cardiomyocytes. NOTCH1 (highest number of variants) and MYH6 (highest number of recurrent variants) expression was elevated in endocardial cells and cardiomyocytes, respectively, and 60% of these gene variants were associated with tetralogy of Fallot and coarctation of the aorta, respectively. Pseudobulk analysis using the single-cell transcriptome revealed significant (P < 0.05) upregulation of both NOTCH1 (endocardial cells) and MYH6 (cardiomyocytes) in the control heart data. We observed nine different subpopulations of CHD heart cardiomyocytes of which only four were observed in the control heart. This is the first comprehensive meta-analysis combining genomics and CHD single-cell transcriptomics, identifying the most frequently mutated CHD genes, and demonstrating CHD gene heterogeneity, suggesting that multiple genes contribute to the phenotypic heterogeneity of CHD. Cardiomyocytes and endocardial cells are identified as major CHD-related cell types.NEW & NOTEWORTHY Congential heart disease (CHD) is one of the most prevalent neonatal congenital anomalies. We present a comprehensive analysis combining genomics and CHD single-cell transcriptome. Our study identifies 90 potential candidate CHD risk genes of which 6 are novel. The risk genes have heterogenous expression suggestive of multiple genes contributing to the phenotypic heterogeneity of CHD. Cardiomyocytes and endocardial cells are identified as major CHD-related cell types.

Mechanisms of Congenital Heart Disease Caused by NAA15 Haploinsufficiency.

[Figure: see text].

SMC5 Plays Independent Roles in Congenital Heart Disease and Neurodevelopmental Disability.

Up to 50% of patients with severe congenital heart disease (CHD) develop life-altering neurodevelopmental disability (NDD). It has been presumed that NDD arises in CHD cases because of hypoxia before, during, or after cardiac surgery. Recent studies detected an enrichment in de novo mutations in CHD and NDD, as well as significant overlap between CHD and NDD candidate genes. However, there is limited evidence demonstrating that genes causing CHD can produce NDD independent of hypoxia. A patient with hypoplastic left heart syndrome and gross motor delay presented with a de novo mutation in SMC5. Modeling mutation of smc5 in Xenopus tropicalis embryos resulted in reduced heart size, decreased brain length, and disrupted pax6 patterning. To evaluate the cardiac development, we induced the conditional knockout (cKO) of Smc5 in mouse cardiomyocytes, which led to the depletion of mature cardiomyocytes and abnormal contractility. To test a role for Smc5 specifically in the brain, we induced cKO in the mouse central nervous system, which resulted in decreased brain volume, and diminished connectivity between areas related to motor function but did not affect vascular or brain ventricular volume. We propose that genetic factors, rather than hypoxia alone, can contribute when NDD and CHD cases occur concurrently.

Histone H2B monoubiquitination regulates heart development via epigenetic control of cilia motility.

Genomic analyses of patients with congenital heart disease (CHD) have identified significant contribution from mutations affecting cilia genes and chromatin remodeling genes; however, the mechanism(s) connecting chromatin remodeling to CHD is unknown. Histone H2B monoubiquitination (H2Bub1) is catalyzed by the RNF20 complex consisting of RNF20, RNF40, and UBE2B. Here, we show significant enrichment of loss-of-function mutations affecting H2Bub1 in CHD patients (enrichment 6.01, P = 1.67 × 10-03), some of whom had abnormal laterality associated with ciliary dysfunction. In Xenopus, knockdown of rnf20 and rnf40 results in abnormal heart looping, defective development of left-right (LR) asymmetry, and impaired cilia motility. Rnf20, Rnf40, and Ube2b affect LR patterning and cilia synergistically. Examination of global H2Bub1 level in Xenopus embryos shows that H2Bub1 is developmentally regulated and requires Rnf20. To examine gene-specific H2Bub1, we performed ChIP-seq of mouse ciliated and nonciliated tissues and showed tissue-specific H2Bub1 marks significantly enriched at cilia genes including the transcription factor Rfx3 Rnf20 knockdown results in decreased levels of rfx3 mRNA in Xenopus, and exogenous rfx3 can rescue the Rnf20 depletion phenotype. These data suggest that Rnf20 functions at the Rfx3 locus regulating cilia motility and cardiac situs and identify H2Bub1 as an upstream transcriptional regulator controlling tissue-specific expression of cilia genes. Our findings mechanistically link the two functional gene ontologies that have been implicated in human CHD: chromatin remodeling and cilia function.

Genetic Basis for Congenital Heart Disease: Revisited: A Scientific Statement From the American Heart Association.

This review provides an updated summary of the state of our knowledge of the genetic contributions to the pathogenesis of congenital heart disease. Since 2007, when the initial American Heart Association scientific statement on the genetic basis of congenital heart disease was published, new genomic techniques have become widely available that have dramatically changed our understanding of the causes of congenital heart disease and, clinically, have allowed more accurate definition of the pathogeneses of congenital heart disease in patients of all ages and even prenatally. Information is presented on new molecular testing techniques and their application to congenital heart disease, both isolated and associated with other congenital anomalies or syndromes. Recent advances in the understanding of copy number variants, syndromes, RASopathies, and heterotaxy/ciliopathies are provided. Insights into new research with congenital heart disease models, including genetically manipulated animals such as mice, chicks, and zebrafish, as well as human induced pluripotent stem cell-based approaches are provided to allow an understanding of how future research breakthroughs for congenital heart disease are likely to happen. It is anticipated that this review will provide a large range of health care-related personnel, including pediatric cardiologists, pediatricians, adult cardiologists, thoracic surgeons, obstetricians, geneticists, genetic counselors, and other related clinicians, timely information on the genetic aspects of congenital heart disease. The objective is to provide a comprehensive basis for interdisciplinary care for those with congenital heart disease.

Genetics and Genomics of Congenital Heart Disease.

Congenital heart disease is the most common birth defect, and because of major advances in medical and surgical management, there are now more adults living with congenital heart disease (CHD) than children. Until recently, the cause of the majority of CHD was unknown. Advances in genomic technologies have discovered the genetic causes of a significant fraction of CHD, while at the same time pointing to remarkable complexity in CHD genetics. This review will focus on the evidence for genetic causes underlying CHD and discuss data supporting both monogenic and complex genetic mechanisms underlying CHD. The discoveries from CHD genetic studies draw attention to biological pathways that simultaneously open the door to a better understanding of cardiac development and affect clinical care of patients with CHD. Finally, we address clinical genetic evaluation of patients and families affected by CHD.

The heterotaxy gene GALNT11 glycosylates Notch to orchestrate cilia type and laterality.

Heterotaxy is a disorder of left-right body patterning, or laterality, that is associated with major congenital heart disease. The aetiology and mechanisms underlying most cases of human heterotaxy are poorly understood. In vertebrates, laterality is initiated at the embryonic left-right organizer, where motile cilia generate leftward flow that is detected by immotile sensory cilia, which transduce flow into downstream asymmetric signals. The mechanism that specifies these two cilia types remains unknown. Here we show that the N-acetylgalactosamine-type O-glycosylation enzyme GALNT11 is crucial to such determination. We previously identified GALNT11 as a candidate disease gene in a patient with heterotaxy, and now demonstrate, in Xenopus tropicalis, that galnt11 activates Notch signalling. GALNT11 O-glycosylates human NOTCH1 peptides in vitro, thereby supporting a mechanism of Notch activation either by increasing ADAM17-mediated ectodomain shedding of the Notch receptor or by modification of specific EGF repeats. We further developed a quantitative live imaging technique for Xenopus left-right organizer cilia and show that Galnt11-mediated Notch1 signalling modulates the spatial distribution and ratio of motile and immotile cilia at the left-right organizer. galnt11 or notch1 depletion increases the ratio of motile cilia at the expense of immotile cilia and produces a laterality defect reminiscent of loss of the ciliary sensor Pkd2. By contrast, Notch overexpression decreases this ratio, mimicking the ciliopathy primary ciliary dyskinesia. Together our data demonstrate that Galnt11 modifies Notch, establishing an essential balance between motile and immotile cilia at the left-right organizer to determine laterality, and reveal a novel mechanism for human heterotaxy.

De novo mutations in histone-modifying genes in congenital heart disease.

Congenital heart disease (CHD) is the most frequent birth defect, affecting 0.8% of live births. Many cases occur sporadically and impair reproductive fitness, suggesting a role for de novo mutations. Here we compare the incidence of de novo mutations in 362 severe CHD cases and 264 controls by analysing exome sequencing of parent-offspring trios. CHD cases show a significant excess of protein-altering de novo mutations in genes expressed in the developing heart, with an odds ratio of 7.5 for damaging (premature termination, frameshift, splice site) mutations. Similar odds ratios are seen across the main classes of severe CHD. We find a marked excess of de novo mutations in genes involved in the production, removal or reading of histone 3 lysine 4 (H3K4) methylation, or ubiquitination of H2BK120, which is required for H3K4 methylation. There are also two de novo mutations in SMAD2, which regulates H3K27 methylation in the embryonic left-right organizer. The combination of both activating (H3K4 methylation) and inactivating (H3K27 methylation) chromatin marks characterizes 'poised' promoters and enhancers, which regulate expression of key developmental genes. These findings implicate de novo point mutations in several hundreds of genes that collectively contribute to approximately 10% of severe CHD.

Robust identification of deletions in exome and genome sequence data based on clustering of Mendelian errors.

Multiple tools have been developed to identify copy number variants (CNVs) from whole exome (WES) and whole genome sequencing (WGS) data. Current tools such as XHMM for WES and CNVnator for WGS identify CNVs based on changes in read depth. For WGS, other methods to identify CNVs include utilizing discordant read pairs and split reads and genome-wide local assembly with tools such as Lumpy and SvABA, respectively. Here, we introduce a new method to identify deletion CNVs from WES and WGS trio data based on the clustering of Mendelian errors (MEs). Using our Mendelian Error Method (MEM), we identified 127 deletions (inherited and de novo) in 2,601 WES trios from the Pediatric Cardiac Genomics Consortium, with a validation rate of 88% by digital droplet PCR. MEM identified additional de novo deletions compared with XHMM, and a significant enrichment of 15q11.2 deletions compared with controls. In addition, MEM identified eight cases of uniparental disomy, sample switches, and DNA contamination. We applied MEM to WGS data from the Genome In A Bottle Ashkenazi trio and identified deletions with 97% specificity. MEM provides a robust, computationally inexpensive method for identifying deletions, and an orthogonal approach for verifying deletions called by other tools.

Robust identification of mosaic variants in congenital heart disease.

Mosaicism due to somatic mutations can cause multiple diseases including cancer, developmental and overgrowth syndromes, neurodevelopmental disorders, autoinflammatory diseases, and atrial fibrillation. With the increased use of next generation sequencing technology, multiple tools have been developed to identify low-frequency variants, specifically from matched tumor-normal tissues in cancer studies. To investigate whether mosaic variants are implicated in congenital heart disease (CHD), we developed a pipeline using the cancer somatic variant caller MuTect to identify mosaic variants in whole-exome sequencing (WES) data from a cohort of parent/affected child trios (n = 715) and a cohort of healthy individuals (n = 416). This is a novel application of the somatic variant caller designed for cancer to WES trio data. We identified two cases with mosaic KMT2D mutations that are likely pathogenic for CHD, but conclude that, overall, mosaicism detectable in peripheral blood or saliva does not account for a significant portion of CHD etiology.

The NIMA-like kinase Nek2 is a key switch balancing cilia biogenesis and resorption in the development of left-right asymmetry.

Vertebrate left-right (LR) asymmetry originates at a transient left-right organizer (LRO), a ciliated structure where cilia play a crucial role in breaking symmetry. However, much remains unknown about the choreography of cilia biogenesis and resorption at this organ. We recently identified a mutation affecting NEK2, a member of the NIMA-like serine-threonine kinase family, in a patient with congenital heart disease associated with abnormal LR development. Here, we report how Nek2 acts through cilia to influence LR patterning. Both overexpression and knockdown of nek2 in Xenopus result in abnormal LR development and reduction of LRO cilia count and motility, phenotypes that are modified by interaction with the Hippo signaling pathway. nek2 knockdown leads to a centriole defect at the LRO, consistent with the known role of Nek2 in centriole separation. Nek2 overexpression results in premature ciliary resorption in cultured cells dependent on function of the tubulin deacetylase Hdac6. Finally, we provide evidence that the known interaction between Nek2 and Nup98, a nucleoporin that localizes to the ciliary base, is important for regulating cilium resorption. Together, these data show that Nek2 is a switch balancing ciliogenesis and resorption in the development of LR asymmetry.

The genetics of congenital heart disease understanding and improving long-term outcomes in congenital heart disease: a review for the general cardiologist and primary care physician.

PURPOSE OF REVIEW: This review has two purposes: to provide an updated review of the genetic causes of congenital heart disease (CHD) and the clinical implications of these genetic mutations, and to provide a clinical algorithm for clinicians considering a genetics evaluation of a CHD patient. RECENT FINDINGS: A large portion of congenital heart disease is thought to have a significant genetic contribution, and at this time a genetic cause can be identified in approximately 35% of patients. Through the advances made possible by next generation sequencing, many of the comorbidities that are frequently seen in patients with genetic congenital heart disease patients can be attributed to the genetic mutation that caused the congenital heart disease. These comorbidities are both cardiac and noncardiac and include: neurodevelopmental disability, pulmonary disease, heart failure, renal dysfunction, arrhythmia and an increased risk of malignancy. Identification of the genetic cause of congenital heart disease helps reduce patient morbidity and mortality by improving preventive and early intervention therapies to address these comorbidities. SUMMARY: Through an understanding of the clinical implications of the genetic underpinning of congenital heart disease, clinicians can provide care tailored to an individual patient and continue to improve the outcomes of congenital heart disease patients.

DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes.

Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-of-function DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed high-resolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-left-right dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.

Increased frequency of de novo copy number variants in congenital heart disease by integrative analysis of single nucleotide polymorphism array and exome sequence data.

RATIONALE: Congenital heart disease (CHD) is among the most common birth defects. Most cases are of unknown pathogenesis. OBJECTIVE: To determine the contribution of de novo copy number variants (CNVs) in the pathogenesis of sporadic CHD. METHODS AND RESULTS: We studied 538 CHD trios using genome-wide dense single nucleotide polymorphism arrays and whole exome sequencing. Results were experimentally validated using digital droplet polymerase chain reaction. We compared validated CNVs in CHD cases with CNVs in 1301 healthy control trios. The 2 complementary high-resolution technologies identified 63 validated de novo CNVs in 51 CHD cases. A significant increase in CNV burden was observed when comparing CHD trios with healthy trios, using either single nucleotide polymorphism array (P=7×10(-5); odds ratio, 4.6) or whole exome sequencing data (P=6×10(-4); odds ratio, 3.5) and remained after removing 16% of de novo CNV loci previously reported as pathogenic (P=0.02; odds ratio, 2.7). We observed recurrent de novo CNVs on 15q11.2 encompassing CYFIP1, NIPA1, and NIPA2 and single de novo CNVs encompassing DUSP1, JUN, JUP, MED15, MED9, PTPRE SREBF1, TOP2A, and ZEB2, genes that interact with established CHD proteins NKX2-5 and GATA4. Integrating de novo variants in whole exome sequencing and CNV data suggests that ETS1 is the pathogenic gene altered by 11q24.2-q25 deletions in Jacobsen syndrome and that CTBP2 is the pathogenic gene in 10q subtelomeric deletions. CONCLUSIONS: We demonstrate a significantly increased frequency of rare de novo CNVs in CHD patients compared with healthy controls and suggest several novel genetic loci for CHD.