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Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly often accompanied by other structural anomalies and/or neurobehavioral manifestations. Rare de novo protein-coding variants and copy-number variations contribute to CDH in the population. However, most individuals with CDH remain genetically undiagnosed. Here, we perform integrated de novo and common-variant analyses using 1,469 CDH individuals, including 1,064 child-parent trios and 6,133 ancestry-matched, unaffected controls for the genome-wide association study. We identify candidate CDH variants in 15 genes, including eight novel genes, through deleterious de novo variants. We further identify two genomic loci contributing to CDH risk through common variants with similar effect sizes among Europeans and Latinx. Both loci are in putative transcriptional regulatory regions of developmental patterning genes. Estimated heritability in common variants is ∼19%. Strikingly, there is no significant difference in estimated polygenic risk scores between isolated and complex CDH or between individuals harboring deleterious de novo variants and individuals without these variants. The data support a polygenic model as part of the CDH genetic architecture., Competing Interests: Declaration of interests W.K.C. is on the Board of Directors of Prime Medicine. The authors declare no additional competing interests., (Copyright © 2024 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

MED27 is a subunit of the Mediator multiprotein complex, which is involved in transcriptional regulation. Biallelic MED27 variants have recently been suggested to be responsible for an autosomal recessive neurodevelopmental disorder with spasticity, cataracts and cerebellar hypoplasia. We further delineate the clinical phenotype of MED27-related disease by characterizing the clinical and radiological features of 57 affected individuals from 30 unrelated families with biallelic MED27 variants. Using exome sequencing and extensive international genetic data sharing, 39 unpublished affected individuals from 18 independent families with biallelic missense variants in MED27 have been identified (29 females, mean age at last follow-up 17 ± 12.4 years, range 0.1-45). Follow-up and hitherto unreported clinical features were obtained from the published 12 families. Brain MRI scans from 34 cases were reviewed. MED27-related disease manifests as a broad phenotypic continuum ranging from developmental and epileptic-dyskinetic encephalopathy to variable neurodevelopmental disorder with movement abnormalities. It is characterized by mild to profound global developmental delay/intellectual disability (100%), bilateral cataracts (89%), infantile hypotonia (74%), microcephaly (62%), gait ataxia (63%), dystonia (61%), variably combined with epilepsy (50%), limb spasticity (51%), facial dysmorphism (38%) and death before reaching adulthood (16%). Brain MRI revealed cerebellar atrophy (100%), white matter volume loss (76.4%), pontine hypoplasia (47.2%) and basal ganglia atrophy with signal alterations (44.4%). Previously unreported 39 affected individuals had seven homozygous pathogenic missense MED27 variants, five of which were recurrent. An emerging genotype-phenotype correlation was observed. This study provides a comprehensive clinical-radiological description of MED27-related disease, establishes genotype-phenotype and clinical-radiological correlations and suggests a differential diagnosis with syndromes of cerebello-lental neurodegeneration and other subtypes of 'neuro-MEDopathies'., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Congenital diaphragmatic hernia (CDH) is a relatively common and genetically heterogeneous structural birth defect associated with high mortality and morbidity. We describe eight unrelated families with an X-linked condition characterized by diaphragm defects, variable anterior body-wall anomalies, and/or facial dysmorphism. Using linkage analysis and exome or genome sequencing, we found that missense variants in plastin 3 (PLS3), a gene encoding an actin bundling protein, co-segregate with disease in all families. Loss-of-function variants in PLS3 have been previously associated with X-linked osteoporosis (MIM: 300910), so we used in silico protein modeling and a mouse model to address these seemingly disparate clinical phenotypes. The missense variants in individuals with CDH are located within the actin-binding domains of the protein but are not predicted to affect protein structure, whereas the variants in individuals with osteoporosis are predicted to result in loss of function. A mouse knockin model of a variant identified in one of the CDH-affected families, c.1497G>C (p.Trp499Cys), shows partial perinatal lethality and recapitulates the key findings of the human phenotype, including diaphragm and abdominal-wall defects. Both the mouse model and one adult human male with a CDH-associated PLS3 variant were observed to have increased rather than decreased bone mineral density. Together, these clinical and functional data in humans and mice reveal that specific missense variants affecting the actin-binding domains of PLS3 might have a gain-of-function effect and cause a Mendelian congenital disorder., Competing Interests: Declaration of interests M.L. is currently an employee of, and holds equity in, Illumina Inc. J.R.L. has stock ownership in 23andMe and is a paid consultant for Genome International., (Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

EZH1, a polycomb repressive complex-2 component, is involved in a myriad of cellular processes. EZH1 represses transcription of downstream target genes through histone 3 lysine27 (H3K27) trimethylation (H3K27me3). Genetic variants in histone modifiers have been associated with developmental disorders, while EZH1 has not yet been linked to any human disease. However, the paralog EZH2 is associated with Weaver syndrome. Here we report a previously undiagnosed individual with a novel neurodevelopmental phenotype identified to have a de novo missense variant in EZH1 through exome sequencing. The individual presented in infancy with neurodevelopmental delay and hypotonia and was later noted to have proximal muscle weakness. The variant, p.A678G, is in the SET domain, known for its methyltransferase activity, and an analogous somatic or germline mutation in EZH2 has been reported in patients with B-cell lymphoma or Weaver syndrome, respectively. Human EZH1/2 are homologous to fly Enhancer of zeste (E(z)), an essential gene in Drosophila, and the affected residue (p.A678 in humans, p.A691 in flies) is conserved. To further study this variant, we obtained null alleles and generated transgenic flies expressing wildtype [E(z)WT] and the variant [E(z)A691G]. When expressed ubiquitously the variant rescues null-lethality similar to the wildtype. Overexpression of E(z)WT induces homeotic patterning defects but notably the E(z)A691G variant leads to dramatically stronger morphological phenotypes. We also note a dramatic loss of H3K27me2 and a corresponding increase in H3K27me3 in flies expressing E(z)A691G, suggesting this acts as a gain-of-function allele. In conclusion, here we present a novel EZH1 de novo variant associated with a neurodevelopmental disorder. Furthermore, we found that this variant has a functional impact in Drosophila., Competing Interests: Conflicts of interest The author(s) declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phosphoinositides (PIs) are membrane phospholipids produced through the local activity of PI kinases and phosphatases that selectively add or remove phosphate groups from the inositol head group. PIs control membrane composition and play key roles in many cellular processes including actin dynamics, endosomal trafficking, autophagy, and nuclear functions. Mutations in phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] phosphatases cause a broad spectrum of neurodevelopmental disorders such as Lowe and Joubert syndromes and congenital muscular dystrophy with cataracts and intellectual disability, which are thus associated with increased levels of PI(4,5)P2. Here, we describe a neurodevelopmental disorder associated with an increase in the production of PI(4,5)P2 and with PI-signaling dysfunction. We identified three de novo heterozygous missense variants in PIP5K1C, which encodes an isoform of the phosphatidylinositol 4-phosphate 5-kinase (PIP5KIγ), in nine unrelated children exhibiting intellectual disability, developmental delay, acquired microcephaly, seizures, visual abnormalities, and dysmorphic features. We provide evidence that the PIP5K1C variants result in an increase of the endosomal PI(4,5)P2 pool, giving rise to ectopic recruitment of filamentous actin at early endosomes (EEs) that in turn causes dysfunction in EE trafficking. In addition, we generated an in vivo zebrafish model that recapitulates the disorder we describe with developmental defects affecting the forebrain, including the eyes, as well as craniofacial abnormalities, further demonstrating the pathogenic effect of the PIP5K1C variants., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Rationale: Congenital diaphragmatic hernia (CDH) is characterized by incomplete closure of the diaphragm and lung hypoplasia. The pathophysiology of lung defects in CDH is poorly understood. Objectives: To establish a translational model of human airway epithelium in CDH for pathogenic investigation and therapeutic testing. Methods: We developed a robust methodology of epithelial progenitor derivation from tracheal aspirates of newborns. Basal stem cells (BSCs) from patients with CDH and preterm and term non-CDH control subjects were derived and analyzed by bulk RNA sequencing, assay for transposase accessible chromatin with sequencing, and air-liquid interface differentiation. Lung sections from fetal human CDH samples and the nitrofen rat model of CDH were subjected to histological assessment of epithelial defects. Therapeutics to restore epithelial differentiation were evaluated in human epithelial cell culture and the nitrofen rat model of CDH. Measurements and Main Results: Transcriptomic and epigenetic profiling of CDH and control BSCs reveals a proinflammatory signature that is manifested by hyperactive nuclear factor kappa B and independent of severity and hernia size. In addition, CDH BSCs exhibit defective epithelial differentiation in vitro that recapitulates epithelial phenotypes found in fetal human CDH lung samples and fetal tracheas of the nitrofen rat model of CDH. Furthermore, blockade of nuclear factor kappa B hyperactivity normalizes epithelial differentiation phenotypes of human CDH BSCs in vitro and in nitrofen rat tracheas in vivo . Conclusions: Our findings have identified an underlying proinflammatory signature and BSC differentiation defects as a potential therapeutic target for airway epithelial defects in CDH.

FOXP1 encodes a transcription factor involved in tissue regulation and cell-type-specific functions. Haploinsufficiency of FOXP1 is associated with a neurodevelopmental disorder: autosomal dominant mental retardation with language impairment with or without autistic features. More recently, heterozygous FOXP1 variants have also been shown to cause a variety of structural birth defects including central nervous system (CNS) anomalies, congenital heart defects, congenital anomalies of the kidney and urinary tract, cryptorchidism, and hypospadias. In this report, we present a previously unpublished case of an individual with congenital diaphragmatic hernia (CDH) who carries an approximately 3.8 Mb deletion. Based on this deletion, and deletions previously reported in two other individuals with CDH, we define a CDH critical region on chromosome 3p13 that includes FOXP1 and four other protein-coding genes. We also provide detailed clinical descriptions of two previously reported individuals with CDH who carry de novo, pathogenic variants in FOXP1 that are predicted to trigger nonsense-mediated mRNA decay. A subset of individuals with putatively deleterious FOXP4 variants has also been shown to develop CDH. Since FOXP proteins function as homo- or heterodimers and the homologs of FOXP1 and FOXP4 are expressed at the same time points in the embryonic mouse diaphragm, they may function together as a dimer, or in parallel as homodimers, to regulate gene expression during diaphragm development. Not all individuals with heterozygous, loss-of-function changes in FOXP1 develop CDH. Hence, we conclude that FOXP1 acts as a susceptibility factor that contributes to the development of CDH in conjunction with other genetic, epigenetic, environmental, and/or stochastic factors., Competing Interests: Conflict of Interest None declared., (Thieme. All rights reserved.)

Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet associated with recognized Mendelian disorders. Using exome sequencing, we identified a rare homozygous splice variant (c.87+3A>G) in ATP5PO, the complex V subunit which encodes the oligomycin sensitivity conferring protein, in three individuals from two unrelated families, with clinical suspicion of a mitochondrial disorder. These individuals had a similar, severe infantile and often lethal multi-systemic disorder that included hypotonia, developmental delay, hypertrophic cardiomyopathy, progressive epileptic encephalopathy, progressive cerebral atrophy, and white matter abnormalities on brain MRI consistent with Leigh syndrome. cDNA studies showed a predominant shortened transcript with skipping of exon 2 and low levels of the normal full-length transcript. Fibroblasts from the affected individuals demonstrated decreased ATP5PO protein, defective assembly of complex V with markedly reduced amounts of peripheral stalk proteins, and complex V hydrolytic activity. Further, expression of human ATP5PO cDNA without exon 2 (hATP5PO-∆ex2) in yeast cells deleted for yATP5 (ATP5PO homolog) was unable to rescue growth on media which requires oxidative phosphorylation when compared to the wild type construct (hATP5PO-WT), indicating that exon 2 deletion leads to a non-functional protein. Collectively, our findings support the pathogenicity of the ATP5PO c.87+3A>G variant, which significantly reduces but does not eliminate complex V activity. These data along with the recent report of an affected individual with ATP5PO variants, add to the evidence that rare biallelic variants in ATP5PO result in defective complex V assembly, function and are associated with Leigh syndrome., (© 2022 SSIEM.)

POGZ -related disorders (also known as White-Sutton syndrome) encompass a wide range of neurocognitive abnormalities and other accompanying anomalies. Disease severity varies widely among POGZ patients and studies investigating genotype-phenotype association are scarce. Therefore, our aim was to collect data on previously unreported POGZ patients and perform a large-scale phenotype-genotype comparison from published data. Overall, 117 POGZ patients' genotype and phenotype data were included in the analysis, including 12 novel patients. A severity scoring system was developed for the comparison. Mild and severe phenotypes were compared with the types and location of the variants and the predicted presence or absence of nonsense-mediated RNA decay (NMD). Missense variants were more often associated with mild phenotypes ( p = 0.0421) and truncating variants predicted to escape NMD presented with more severe phenotypes ( p < 0.0001). Within this group, variants in the prolin-rich region of the POGZ protein were associated with the most severe phenotypes ( p = 0.0004). Our study suggests that gain-of-function or dominant negative effect through escaping NMD and the location of the variants in the prolin-rich domain of the protein may play an important role in the severity of manifestations of POGZ -associated neurodevelopmental disorders.

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly that is often accompanied by other anomalies. Although the role of genetics in the pathogenesis of CDH has been established, only a small number of disease-associated genes have been identified. To further investigate the genetics of CDH, we analyzed de novo coding variants in 827 proband-parent trios and confirmed an overall significant enrichment of damaging de novo variants, especially in constrained genes. We identified LONP1 (lon peptidase 1, mitochondrial) and ALYREF (Aly/REF export factor) as candidate CDH-associated genes on the basis of de novo variants at a false discovery rate below 0.05. We also performed ultra-rare variant association analyses in 748 affected individuals and 11,220 ancestry-matched population control individuals and identified LONP1 as a risk gene contributing to CDH through both de novo and ultra-rare inherited largely heterozygous variants clustered in the core of the domains and segregating with CDH in affected familial individuals. Approximately 3% of our CDH cohort who are heterozygous with ultra-rare predicted damaging variants in LONP1 have a range of clinical phenotypes, including other anomalies in some individuals and higher mortality and requirement for extracorporeal membrane oxygenation. Mice with lung epithelium-specific deletion of Lonp1 die immediately after birth, most likely because of the observed severe reduction of lung growth, a known contributor to the high mortality in humans. Our findings of both de novo and inherited rare variants in the same gene may have implications in the design and analysis for other genetic studies of congenital anomalies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Early infantile epileptic encephalopathy-44 (EIEE44, MIM: 617132) is a previously described condition resulting from biallelic variants in UBA5 , a gene involved in a ubiquitin-like post-translational modification system called UFMylation. Here we report five children from four families with biallelic pathogenic variants in UBA5 All five children presented with global developmental delay, epilepsy, axial hypotonia, appendicular hypertonia, and a movement disorder, including dystonia in four. Affected individuals in all four families have compound heterozygous pathogenic variants in UBA5 All have the recurrent mild c.1111G > A (p.Ala371Thr) variant in trans with a second UBA5 variant. One patient has the previously described c.562C > T (p. Arg188*) variant, two other unrelated patients have a novel missense variant, c.907T > C (p.Cys303Arg), and the two siblings have a novel missense variant, c.761T > C (p.Leu254Pro). Functional analyses demonstrate that both the p.Cys303Arg variant and the p.Leu254Pro variants result in a significant decrease in protein function. We also review the phenotypes and genotypes of all 15 previously reported families with biallelic UBA5 variants, of which two families have presented with distinct phenotypes, and we describe evidence for some limited genotype-phenotype correlation. The overlap of motor and developmental phenotypes noted in our cohort and literature review adds to the increasing understanding of genetic syndromes with movement disorders-epilepsy., (© 2021 Briere et al.; Published by Cold Spring Harbor Laboratory Press.)

The Mediator multiprotein complex functions as a regulator of RNA polymerase II-catalyzed gene transcription. In this study, exome sequencing detected biallelic putative disease-causing variants in MED27, encoding Mediator complex subunit 27, in 16 patients from 11 families with a novel neurodevelopmental syndrome. Patient phenotypes are highly homogeneous, including global developmental delay, intellectual disability, axial hypotonia with distal spasticity, dystonic movements, and cerebellar hypoplasia. Seizures and cataracts were noted in severely affected individuals. Identification of multiple patients with biallelic MED27 variants supports the critical role of MED27 in normal human neural development, particularly for the cerebellum. ANN NEUROL 2021;89:828-833., (© 2021 American Neurological Association.)

MORC2 encodes an ATPase that plays a role in chromatin remodeling, DNA repair, and transcriptional regulation. Heterozygous variants in MORC2 have been reported in individuals with autosomal-dominant Charcot-Marie-Tooth disease type 2Z and spinal muscular atrophy, and the onset of symptoms ranges from infancy to the second decade of life. Here, we present a cohort of 20 individuals referred for exome sequencing who harbor pathogenic variants in the ATPase module of MORC2. Individuals presented with a similar phenotype consisting of developmental delay, intellectual disability, growth retardation, microcephaly, and variable craniofacial dysmorphism. Weakness, hyporeflexia, and electrophysiologic abnormalities suggestive of neuropathy were frequently observed but were not the predominant feature. Five of 18 individuals for whom brain imaging was available had lesions reminiscent of those observed in Leigh syndrome, and five of six individuals who had dilated eye exams had retinal pigmentary abnormalities. Functional assays revealed that these MORC2 variants result in hyperactivation of epigenetic silencing by the HUSH complex, supporting their pathogenicity. The described set of morphological, growth, developmental, and neurological findings and medical concerns expands the spectrum of genetic disorders resulting from pathogenic variants in MORC2., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The translocase of outer mitochondrial membrane (TOMM) complex is the entry gate for virtually all mitochondrial proteins and is essential to build the mitochondrial proteome. TOMM70 is a receptor that assists mainly in mitochondrial protein import. Here, we report two individuals with de novo variants in the C-terminal region of TOMM70. While both individuals exhibited shared symptoms including hypotonia, hyper-reflexia, ataxia, dystonia and significant white matter abnormalities, there were differences between the two individuals, most prominently the age of symptom onset. Both individuals were undiagnosed despite extensive genetics workups. Individual 1 was found to have a p.Thr607Ile variant while Individual 2 was found to have a p.Ile554Phe variant in TOMM70. To functionally assess both TOMM70 variants, we replaced the Drosophila Tom70 coding region with a Kozak-mini-GAL4 transgene using CRISPR-Cas9. Homozygous mutant animals die as pupae, but lethality is rescued by the mini-GAL4-driven expression of human UAS-TOMM70 cDNA. Both modeled variants lead to significantly less rescue indicating that they are loss-of-function alleles. Similarly, RNAi-mediated knockdown of Tom70 in the developing eye causes roughening and synaptic transmission defect, common findings in neurodegenerative and mitochondrial disorders. These phenotypes were rescued by the reference, but not the variants, of TOMM70. Altogether, our data indicate that de novo loss-of-function variants in TOMM70 result in variable white matter disease and neurological phenotypes in affected individuals., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The frequent occurrence of congenital heart defects (CHDs) in chromosome abnormality syndromes is well-known, and among aneuploidy syndromes, distinctive patterns have been delineated. We update the type and frequency of CHDs in the aneuploidy syndromes involving trisomy 13, 18, 21, and 22, and in several sex chromosome abnormalities (Turner syndrome, trisomy X, Klinefelter syndrome, 47,XYY, and 48,XXYY). We also discuss the impact of noninvasive prenatal screening (mainly, cell-free DNA analysis), critical CHD screening, and the growth of parental advocacy on their surgical management and natural history. We encourage clinicians to view the cardiac diagnosis as a "phenotype" which supplements the external dysmorphology examination. When detected prenatally, severe CHDs may influence decision-making, and postnatally, they are often the major determinants of survival. This review should be useful to geneticists, cardiologists, neonatologists, perinatal specialists, other pediatric specialists, and general pediatricians. As patients survive (and thrive) into adulthood, internists and related adult specialists will also need to be informed about their natural history and management., (© 2019 Wiley Periodicals, Inc.)

Objective: To characterize the phenotypic spectrum associated with GNAO1 variants and establish genotype-protein structure-phenotype relationships., Methods: We evaluated the phenotypes of 14 patients with GNAO1 variants, analyzed their variants for potential pathogenicity, and mapped them, along with those in the literature, on a three-dimensional structural protein model., Results: The 14 patients in our cohort, including one sibling pair, had 13 distinct, heterozygous GNAO1 variants classified as pathogenic or likely pathogenic. We attributed the same variant in two siblings to parental mosaicism. Patients initially presented with seizures beginning in the first 3 months of life (8/14), developmental delay (4/14), hypotonia (1/14), or movement disorder (1/14). All patients had hypotonia and developmental delay ranging from mild to severe. Nine had epilepsy, and nine had movement disorders, including dystonia, ataxia, chorea, and dyskinesia. The 13 GNAO1 variants in our patients are predicted to result in amino acid substitutions or deletions in the GNAO1 guanosine triphosphate (GTP)-binding region, analogous to those in previous publications. Patients with variants affecting amino acids 207-221 had only movement disorder and hypotonia. Patients with variants affecting the C-terminal region had the mildest phenotypes., Significance: GNAO1 encephalopathy most frequently presents with seizures beginning in the first 3 months of life. Concurrent movement disorders are also a prominent feature in the spectrum of GNAO1 encephalopathy. All variants affected the GTP-binding domain of GNAO1, highlighting the importance of this region for G-protein signaling and neurodevelopment., (Wiley Periodicals, Inc. © 2019 International League Against Epilepsy.)

Congenital diaphragmatic hernia (CDH) is a severe birth defect that is often accompanied by other congenital anomalies. Previous exome sequencing studies for CDH have supported a role of de novo damaging variants but did not identify any recurrently mutated genes. To investigate further the genetics of CDH, we analyzed de novo coding variants in 362 proband-parent trios including 271 new trios reported in this study. We identified four unrelated individuals with damaging de novo variants in MYRF (P = 5.3x10(-8)), including one likely gene-disrupting (LGD) and three deleterious missense (D-mis) variants. Eight additional individuals with de novo LGD or missense variants were identified from our other genetic studies or from the literature. Common phenotypes of MYRF de novo variant carriers include CDH, congenital heart disease and genitourinary abnormalities, suggesting that it represents a novel syndrome. MYRF is a membrane associated transcriptional factor highly expressed in developing diaphragm and is depleted of LGD variants in the general population. All de novo missense variants aggregated in two functional protein domains. Analyzing the transcriptome of patient-derived diaphragm fibroblast cells suggest that disease associated variants abolish the transcription factor activity. Furthermore, we showed that the remaining genes with damaging variants in CDH significantly overlap with genes implicated in other developmental disorders. Gene expression patterns and patient phenotypes support pleiotropic effects of damaging variants in these genes on CDH and other developmental disorders. Finally, functional enrichment analysis implicates the disruption of regulation of gene expression, kinase activities, intra-cellular signaling, and cytoskeleton organization as pathogenic mechanisms in CDH., Competing Interests: The authors have declared that no competing interests exist.

Congenital diaphragmatic hernia (CDH), characterized by malformation of the diaphragm and hypoplasia of the lungs, is one of the most common and severe birth defects, and is associated with high morbidity and mortality rates. There is growing evidence demonstrating that genetic factors contribute to CDH, although the pathogenesis remains largely elusive. Single-nucleotide polymorphisms have been studied in recent whole-exome sequencing efforts, but larger copy number variants (CNVs) have not yet been studied on a large scale in a case control study. To capture CNVs within CDH candidate regions, we developed and tested a targeted array comparative genomic hybridization platform to identify CNVs within 140 regions in 196 patients and 987 healthy controls, and identified six significant CNVs that were either unique to patients or enriched in patients compared with controls. These CDH-associated CNVs reveal high-priority candidate genes including HLX , LHX1 , and HNF1B We also discuss CNVs that are present in only one patient in the cohort but have additional evidence of pathogenicity, including extremely rare large and/or de novo CNVs. The candidate genes within these predicted disease-causing CNVs form functional networks with other known CDH genes and play putative roles in DNA binding/transcription regulation and embryonic development. These data substantiate the importance of CNVs in the etiology of CDH, identify CDH candidate genes and pathways, and highlight the importance of ongoing analysis of CNVs in the study of CDH and other structural birth defects., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

N-alpha-acetylation is a common co-translational protein modification that is essential for normal cell function in humans. We previously identified the genetic basis of an X-linked infantile lethal Mendelian disorder involving a c.109T>C (p.Ser37Pro) missense variant in NAA10, which encodes the catalytic subunit of the N-terminal acetyltransferase A (NatA) complex. The auxiliary subunit of the NatA complex, NAA15, is the dimeric binding partner for NAA10. Through a genotype-first approach with whole-exome or genome sequencing (WES/WGS) and targeted sequencing analysis, we identified and phenotypically characterized 38 individuals from 33 unrelated families with 25 different de novo or inherited, dominantly acting likely gene disrupting (LGD) variants in NAA15. Clinical features of affected individuals with LGD variants in NAA15 include variable levels of intellectual disability, delayed speech and motor milestones, and autism spectrum disorder. Additionally, mild craniofacial dysmorphology, congenital cardiac anomalies, and seizures are present in some subjects. RNA analysis in cell lines from two individuals showed degradation of the transcripts with LGD variants, probably as a result of nonsense-mediated decay. Functional assays in yeast confirmed a deleterious effect for two of the LGD variants in NAA15. Further supporting a mechanism of haploinsufficiency, individuals with copy-number variant (CNV) deletions involving NAA15 and surrounding genes can present with mild intellectual disability, mild dysmorphic features, motor delays, and decreased growth. We propose that defects in NatA-mediated N-terminal acetylation (NTA) lead to variable levels of neurodevelopmental disorders in humans, supporting the importance of the NatA complex in normal human development., (Copyright © 2018 American Society of Human Genetics. All rights reserved.)

Congenital Diaphragmatic Hernia (CDH) is a common and often lethal birth defect characterized by diaphragmatic structural defects and pulmonary hypoplasia. CDH is isolated in 60% of newborns, but may also be part of a complex phenotype with additional anomalies. We performed whole exome sequencing (WES) on 87 individuals with isolated or complex CDH and on their unaffected parents, to assess the contribution of de novo mutations in the etiology of diaphragmatic and pulmonary defects and to identify new candidate genes. A combined analysis with 39 additional trios with complex CDH, previously published, revealed a significant genome-wide burden of de novo variants compared to background mutation rate and 900 control trios. We identified an increased burden of likely gene-disrupting (LGD, i.e. nonsense, frameshift, and canonical splice site) and predicted deleterious missense (D-mis) variants in complex and isolated CDH patients. Overall, an excess of predicted damaging de novo LGD and D-mis variants relative to the expected frequency contributed to 21% of complex cases and 12% of isolated CDH cases. The burden of de novo variants was higher in genes expressed in the developing mouse diaphragm and heart. Some overlap with genes responsible for congenital heart defects and neurodevelopmental disorders was observed in CDH patients within our cohorts. We propose that de novo variants contribute significantly to the development of CDH.

Congenital diaphragmatic hernia (CDH) is one of the most common and lethal congenital anomalies, and significant evidence is available in support of a genetic contribution to its etiology, including single-gene knockout mice associated with diaphragmatic defects, rare monogenetic disorders in humans, familial aggregation, and association of CDH with chromosomal abnormalities. Structural lung defects in the form of lung hypoplasia are almost invariably seen in patients with CDH and frequently in animal models of this condition. Better understanding of the mechanisms of pulmonary defects in CDH has the potential for creating targeted therapies, particularly in postnatal stages, when therapeutics can have maximum clinical impact on the surviving cohorts. Successful treatment of CDH is dependent on the integration of human genomic and genetic data with developmental expression profiling, mouse knockouts, and gene network and pathway modeling, which have generated a large number of candidate genes and pathways for follow-up studies. In particular, defective alveolarization appears to be a common and potentially actionable phenotype in both patients and animal models., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

COUP-TFII (NR2F2) is mapped to the 15q26 deletion hotspot associated with the common and highly morbid congenital diaphragmatic hernia (CDH). Conditional homozygous deletions of COUP-TFII in mice result in diaphragmatic defects analogous to the human Bochdalek-type hernia phenotype. Despite evidence from animal models however, mutations in the coding sequence of COUP-TFII have not been reported in patients, prompting the speculation that additional coding or non-coding sequences in the 15q26 locus are necessary for diaphragmatic hernias to develop. In this report, we describe a case of a patient with a heterozygous de novo COUP-TFII frameshift mutation, presenting with CDH and an atrial septal defect. The p.Pro33AlafsTer77 mutation specifically disrupts protein isoform 1 which contains the DNA binding domain. In addition, we review other COUP-TFII sequence variations and deletions that have been described in cases of CDH. We conclude that COUP-TFII mutations can cause diaphragmatic hernias, and should be included in the differential diagnosis of CDH patients, particularly those with comorbid congenital heart defects. © 2016 Wiley Periodicals, Inc., Competing Interests: The authors declare that there are no conflicts of interest., (© 2016 Wiley Periodicals, Inc.)

Zinc finger protein, FOG2 family member 2 (ZFPM2) (previously named FOG2) gene defects result in the highly morbid congenital diaphragmatic hernia (CDH) in humans and animal models. In a cohort of 275 CDH patient exomes, we estimated the prevalence of damaging ZFPM2 mutations to be almost 5%. Genetic analysis of a multigenerational family identified a heritable intragenic ZFPM2 deletion with an estimated penetrance of 37.5%, which has important implications for genetic counseling. Similarly, a low penetrance ZFPM2 frameshift mutation was observed in a second multiplex family. Isolated CDH was the predominant phenotype observed in our ZFPM2 mutation patients. Findings from the patients described herein indicate that ZFPM2 point mutations or deletions are a recurring cause of CDH., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Congenital diaphragmatic hernia (CDH) is a common and severe birth defect. Despite its clinical significance, the genetic and developmental pathways underlying this disorder are incompletely understood. In this study, we report a catalog of variants detected by a whole exome sequencing study on 275 individuals with CDH. Predicted pathogenic variants in genes previously identified in either humans or mice with diaphragm defects are enriched in our CDH cohort compared with 120 size-matched random gene sets. This enrichment was absent in control populations. Variants in these critical genes can be found in up to 30.9% of individuals with CDH. In addition, we filtered variants by using genes derived from regions of recurrent copy number variations in CDH, expression profiles of the developing diaphragm, protein interaction networks expanded from the known CDH-causing genes, and prioritized genes with ultrarare and highly disruptive variants, in 11.3% of CDH patients. These strategies have identified several high priority genes and developmental pathways that likely contribute to the CDH phenotype. These data are valuable for comparison of candidate genes generated from whole exome sequencing of other CDH cohorts or multiplex kindreds and provide ideal candidates for further functional studies. Furthermore, we propose that these genes and pathways will enhance our understanding of the heterogeneous molecular etiology of CDH.

Background: Notch signaling in vascular smooth muscle precursors is required for smooth muscle differentiation. Jagged1 expression on endothelium activates Notch in vascular smooth muscle precursors including those of neural crest origin to initiate the formation of a smooth muscle layer in a maturing blood vessel., Methods and Results: Here, we show that Jagged1 is a direct Notch target in smooth muscle, resulting in a positive feedback loop and lateral induction that propagates a wave of smooth muscle differentiation during aortic arch artery development. In vivo, we show that Notch inhibition in cardiac neural crest impairs Jagged1 messenger RNA expression and results in deficient smooth muscle differentiation and resultant aortic arch artery defects. Ex vivo, Jagged1 ligand activates Notch in neural crest explants and results in activation of Jagged1 messenger RNA, a response that is blocked by Notch inhibition. We examine 15 evolutionary conserved regions within the Jagged1 genomic locus and identify a single Notch response element within the second intron. This element contains a functional Rbp-J binding site demonstrated by luciferase reporter and chromatin immunoprecipitation assays and is sufficient to recapitulate aortic arch artery expression of Jagged1 in transgenic mice. Loss of Jagged1 in neural crest impairs vascular smooth muscle differentiation and results in aortic arch artery defects., Conclusions: Taken together, these results provide a mechanism for lateral induction that allows for a multilayered smooth muscle wall to form around a nascent arterial endothelial tube and identify Jagged1 as a direct Notch target., (© 2011 American Heart Association, Inc.)

Notch signaling is vital for proper cardiovascular development and function in both humans and animal models. Indeed, mutations in either JAGGED or NOTCH cause congenital heart disease in humans and NOTCH mutations are associated with adult valvular disease. Notch typically functions to mediate developmental interactions between adjacent tissues. Here we show that either absence of the Notch ligand Jagged1 or inhibition of Notch signaling in second heart field tissues results in murine aortic arch artery and cardiac anomalies. In mid-gestation, these mutants displayed decreased Fgf8 and Bmp4 expression. Notch inhibition within the second heart field affected the development of neighboring tissues. For example, faulty migration of cardiac neural crest cells and defective endothelial-mesenchymal transition within the outflow tract endocardial cushions were observed. Furthermore, exogenous Fgf8 was sufficient to rescue the defect in endothelial-mesenchymal transition in explant assays of endocardial cushions following Notch inhibition within second heart field derivatives. These data support a model that relates second heart field, neural crest, and endocardial cushion development and suggests that perturbed Notch-Jagged signaling within second heart field progenitors accounts for some forms of congenital and adult cardiac disease.

The Notch ligand Jagged1 (Jag1) is essential for vascular remodeling and has been linked to congenital heart disease in humans, but its precise role in various cell types of the cardiovascular system has not been extensively investigated. We show that endothelial-specific deletion of Jag1 results in embryonic lethality and cardiovascular defects, recapitulating the Jag1 null phenotype. These embryos show striking deficits in vascular smooth muscle, whereas endothelial Notch activation and arterial-venous differentiation appear normal. Endothelial Jag1 mutant embryos are phenotypically distinct from embryos in which Notch signaling is inhibited in endothelium. Together, these results imply that the primary role of endothelial Jag1 is to potentiate the development of neighboring vascular smooth muscle.

Notch receptors and their cognate ligands transduce crucial signals between cells in various tissues, and have been conserved across millions of years of evolution. Mutations in Notch signalling components result in congenital heart defects in humans and mice, demonstrating an essential role for Notch in cardiovascular development. The results of recent experiments implicate this signalling pathway in many stages of heart development, and provide mechanistic insight into the vital functions of Notch in the aetiology of several common forms of paediatric and adult cardiac disease.

The cardiac outflow tract develops as a result of a complex interplay among several cell types, including cardiac neural crest cells, endothelial cells, and cardiomyocytes. In both humans and mice, mutations in components of the Notch signaling pathway result in congenital heart disease characterized by cardiac outflow tract defects. However, the specific cell types in which Notch functions during cardiovascular development remain to be defined. In addition, in vitro studies have provided conflicting data regarding the ability of Notch to promote or inhibit smooth muscle differentiation, while the physiological role for Notch in smooth muscle formation during development remains unclear. In this study, we generated mice in which Notch signaling was specifically inactivated in derivatives of the neural crest. These mice exhibited cardiovascular anomalies, including aortic arch patterning defects, pulmonary artery stenosis, and ventricular septal defects. We show that Notch plays a critical, cell-autonomous role in the differentiation of cardiac neural crest precursors into smooth muscle cells both in vitro and in vivo, and we identify specific Notch targets in neural crest that are implicated in this process. These results provide a molecular and cellular framework for understanding the role of Notch signaling in the etiology of congenital heart disease.

Cardiac neural crest cells undergo extensive cell rearrangements during the formation of the aorticopulmonary septum in the outflow tract. However, the morphogenetic mechanisms involved in this fundamental process remain poorly understood. To determine the function of the Ca2+-dependent cell adhesion molecule, N-cadherin, in murine neural crest, we applied the Cre/loxP system and created mouse embryos genetically mosaic for N-cadherin. Specifically, deletion of N-cadherin in neural crest cells led to embryonic lethality with distinct cardiovascular defects. Neural crest cell migration and homing to the cardiac outflow tract niche were unaffected by loss of N-cadherin. However, N-cadherin-deficient neural crest cells were unable to undergo the normal morphogenetic changes associated with outflow tract remodeling, resulting in persistent truncus arteriosus in the majority of mutant embryos. Other mutant embryos initiated aorticopulmonary septum formation; however, the neural crest cells were unable to elongate and align properly along the midline and remained rounded with limited contact with their neighbors. Interestingly, rotation of the outflow tract was incomplete in these mutants suggesting that alignment of the channels is dependent on N-cadherin-generated cytoskeletal forces. A second cardiac phenotype was observed where loss of N-cadherin in the epicardium led to disruption of heterotypic cell interactions between the epicardium and myocardium resulting in a thinned ventricular myocardium. Thus, we conclude that in addition to its role in myocardial cell adhesion, N-cadherin is required for neural crest cell rearrangements critical for patterning of the cardiac outflow tract and in the maintenance of epicardial-myocardial cell interactions.

Pax3 is a transcription factor expressed in the dorsal neural tube and somite of the developing embryo. It plays critical roles in pre-migratory neural crest cells and in myogenic precursors of skeletal muscle. Pax3-deficient Splotch embryos display neural tube and neural crest defects and lack hypaxial muscles. We have created a new allele of Splotch by replacing the first coding exon with a gene encoding Cre recombinase. This functions as a null allele and no Pax3 protein is detected in homozygous embryos. Heterozygous Pax3(Cre/+) mice display a white belly spot, as do Splotch heterozygotes. Homozygous Pax3(Cre/Cre) embryos are embryonic lethal. We have used Pax3(Cre/+) mice to fate-map Pax3 derivatives in the developing mouse. As expected, neural crest and some somitic derivatives are identified. However, we also detect previously unappreciated derivatives of Pax3-expressing precursors in the colonic epithelium of the hindgut and within the urogenital system.

To address how heritable patterns of gene expression are acquired during the differentiation of Th1 and Th2 cells, we analyzed the nuclear position of lineage-restricted cytokine genes and their upstream regulators by 3-dimensional fluorescence in situ hybridization. During Th1 differentiation, GATA-3 and c-maf loci, which encode upstream regulators of Th2 cytokines, were progressively repositioned to centromeric heterochromatin as defined by a gamma-satellite repeat probe and/or the nuclear periphery, compartments that have been associated with transcriptional repression. A third transcription factor locus, T-bet, which controls Th1-specific programs, was subject to de novo CpG methylation in a Th2 cell clone. In contrast, we did not find repositioning of the cytokine gene loci IL-2, IL-3, IL-4 or IFN-gamma during T helper cell differentiation. Instead, IFN-gamma was constitutively associated with the nuclear periphery, even when primed for expression in Th1 cells. Our results suggest that Th1/Th2 lineage commitment and differentiation involve repositioning of the regulators of cytokine expression, rather than the cytokine genes themselves.

The ability of NK and T cells to produce IFN-gamma is critical for resistance to numerous intracellular pathogens but the kinetics of these responses differ. Consistent with this is a requirement for naive T cells to become activated and undergo proliferation-dependent epigenetic changes to the IFN-gamma locus that allow them to produce IFN-gamma. The data presented here reveal that unlike T cells, murine NK cells produce IFN-gamma under conditions of short-term cytokine stimulation, and these events are independent of proliferation and cell cycle progression. Furthermore, analysis of the IFN-gamma locus in NK cells reveals that this locus is constitutively demethylated. The finding that NK cells do not need to remodel the IFN-gamma locus to produce IFN-gamma, either because they do not exhibit epigenetic repression or they have undergone prior remodeling during development, provides a molecular basis for the innate and adaptive regulation of the production of this cytokine.

How a single cell gives rise to progeny with differing fates remains poorly understood. We examined cells lacking methyl CpG binding domain protein-2 (MBD2), a molecule that has been proposed to link DNA methylation to silent chromatin. Helper T cells from Mbd2(-/-) mice exhibit disordered differentiation. IL-4, the signature of a restricted set of progeny, is expressed ectopically in Mbd2(-/-) parent and daughter cells. Loss of MBD2-mediated silencing renders the normally essential activator, Gata-3, dispensable for IL-4 induction. Gata-3 and MBD2 act in competition, wherein each factor independently, and quantitatively, regulates the binary choice of whether heritable IL-4 expression is established. Gata-3 functions, in part, to displace MBD2 from methylated DNA. These results suggest that activating and silencing signals integrate to provide spatially and temporally restricted patterns of gene activity.

Type 1 helper T (T(H)1) cells are essential for cellular immunity, but their ontogeny, maturation and durability remain poorly understood. By constructing a dominant-negative form of T-bet, we were able to determine the role played by this lineage-inducing trans-activator in the establishment and maintenance of heritable T(H)1 gene expression. Optimal induction of interferon-gamma (IFN-gamma) expression required genetic interaction between T-bet and its target, the homeoprotein Hlx. In fully mature T(H)1 cells, reiteration of IFN-gamma expression and stable chromatin remodeling became relatively independent of T-bet activity and coincided with demethylation of DNA. In contrast, some lineage attributes, such as expression of IL-12R beta 2 (interleukin 12 receptor beta 2), required ongoing T-bet activity in mature T(H)1 cells and their progeny. These findings suggest that heritable states of gene expression might be maintained by continued expression of the inducing factor or by a mechanism that confers a stable imprint of the induced state.

Naïve CD4(+) helper T (T(H)) cells respond to stimulation by terminally differentiating into two mature classes, T(H)1 cells, which express interferon gamma (IFN-gamma), and T(H)2 cells, which express interleukin 4 (IL-4). The transcriptional activators T-bet and Gata-3 mediate commitment to the T(H)1 and T(H)2 fates, respectively, including chromatin remodeling of signature genes. The cytokine IL-12 fosters growth of committed T(H)1 cells, while IL-4 fosters growth of committed T(H)2 cells. IL-12 and IL-4 also play critical roles in commitment by promoting transcriptional silencing of Gata-3 and T-bet, respectively. We now show that both T-bet and Gata-3 are induced in a cell cycle-independent manner in bipotent progenitor cells. In contrast, both lineage-restricted gene induction by the activator proteins and heritable silencing of the transcription of each activator, the hallmarks of terminal differentiation, are cell cycle dependent. We found that cells that cannot cycle remain uncommitted and bipotent in response to the most polarizing signals for maturation. These results provide mechanistic insight into a mammalian model of terminal differentiation by illustrating that cell cycle-coupled epigenetic effects, as originally described in yeast, may represent an evolutionarily conserved strategy for organizing signaling and cell fate.

Cytotoxic T lymphocyte antigen (CTLA)-4 plays an essential role in immunologic homeostasis. How this negative regulator of T cell activation executes its functions has remained controversial. We now provide evidence that CTLA-4 mediates a cell-intrinsic counterbalance to restrict the clonal expansion of proliferating CD4(+) T cells. The regulation of CTLA-4 expression and function ensures that, after approximately 3 cell divisions of expansion, most progeny will succumb to either proliferative arrest or death over the ensuing three cell divisions. The quantitative precision of the counterbalance hinges on the graded, time-independent induction of CTLA-4 expression during the first three cell divisions. In contrast to the limits imposed on unpolarized cells, T helper type 1 (Th1) and Th2 effector progeny may be rescued from proliferative arrest by interleukin (IL)-12 and IL-4 signaling, respectively, allowing appropriately stimulated progeny to proceed to the stage of tissue homing. These results suggest that the cell-autonomous regulation of CTLA-4 induction may be a central checkpoint of clonal expansion of CD4(+) T cells, allowing temporally and spatially restricted growth of progeny to be dictated by the nature of the threat posed to the host.

How cytokines control differentiation of helper T (TH) cells is controversial. We show that T-bet, without apparent assistance from interleukin 12 (IL-12)/STAT4, specifies TH1 effector fate by targeting chromatin remodeling to individual interferon-gamma (IFN-gamma) alleles and by inducing IL-12 receptor beta2 expression. Subsequently, it appears that IL-12/STAT4 serves two essential functions in the development of TH1 cells: as growth signal, inducing survival and cell division; and as trans-activator, prolonging IFN-gamma synthesis through a genetic interaction with the coactivator, CREB-binding protein. These results suggest that a cytokine does not simply induce TH fate choice but instead may act as an essential secondary stimulus that mediates selective survival of a lineage.

The heterogeneous nuclear ribonucleoproteins (hnRNP) associate with pre-mRNA in the nucleus and play an important role in RNA processing and splice site selection. In addition, hnRNP A proteins function in the export of mRNA to the cytoplasm. Although the hnRNP A proteins are predominantly nuclear, hnRNP A1 shuttles rapidly between the nucleus and the cytoplasm. HnRNP A2, whose cytoplasmic overexpression has been identified as an early biomarker of lung cancer, has been less well studied. Cytosolic hnRNP A2 overexpression has also been noted in brain tumors, in which it has been correlated with translational repression of Glucose Transporter-1 expression. We now examine the role of arginine methylation on the nucleocytoplasmic localization of hnRNP A2 in the HEK-293 and NIH-3T3 mammalian cell lines. Treatment of either cell line with the methyltransferase inhibitor adenosine dialdehyde dramatically shifts hnRNP A2 localization from the nuclear to the cytoplasmic compartment, as shown both by immunoblotting and by immunocytochemistry. In vitro radiolabeling with [(3)H]AdoMet of GST-tagged hnRNP A2 RGG mutants, using recombinant protein arginine methyltransferase (PRMT1), shows (i) that hnRNP A2 is a substrate for PRMT1 and (ii) that methylated residues are found only in the RGG domain. Deletion of the RGG domain (R191-G253) of hnRNP A2 results in a cytoplasmic localization phenotype, detected both by immunoblotting and by immunocytochemistry. These studies indicate that the RGG domain of hnRNP A2 contains sequences critical for cellular localization of the protein. The data suggest that hnRNP A2 may contain a novel nuclear localization sequence, regulated by arginine methylation, that lies in the R191-G253 region and may function independently of the M9 transportin-1-binding region in hnRNP A2., (Copyright 2000 Academic Press.)