Dysregulation of CITED2 in abnormal lung development in the nitrofen rat model.

PURPOSE: CITED2 both modulates lung, heart and diaphragm development. The role of CITED2 in the pathogenesis of congenital diaphragmatic hernia (CDH) is unknown. We aimed to study CITED2 during abnormal lung development in the nitrofen model. METHODS: Timed-pregnant rats were given nitrofen on embryonic day (E) 9 to induce CDH. Fetal lungs were harvested on E15, 18 and 21. We performed RT-qPCR, RNAscope™ in situ hybridization and immunofluorescence staining for CITED2. RESULTS: We observed no difference in RT-qPCR (control: 1.09 ± 0.22 and nitrofen: 0.95 ± 0.18, p = 0.64) and in situ hybridization (1.03 ± 0.03; 1.04 ± 0.03, p = 0.97) for CITED2 expression in E15 nitrofen and control pups. At E18, CITED2 expression was reduced in in situ hybridization of nitrofen lungs (1.47 ± 0.05; 1.14 ± 0.07, p = 0.0006), but not altered in RT-qPCR (1.04 ± 0.16; 0.81 ± 0.13, p = 0.33). In E21 nitrofen lungs, CITED2 RNA expression was increased in RT-qPCR (1.04 ± 0.11; 1.52 ± 0.17, p = 0.03) and in situ hybridization (1.08 ± 0.07, 1.29 ± 0.04, p = 0.02). CITED2 protein abundance was higher in immunofluorescence staining of E21 nitrofen lungs (2.96 × 109 ± 0.13 × 109; 4.82 × 109 ± 0.25 × 109, p < 0.0001). CONCLUSION: Our data suggest that dysregulation of CITED2 contributes to abnormal lung development of CDH, as demonstrated by the distinct spatial-temporal distribution in nitrofen-induced lungs.

Characterization of the congenital diaphragmatic hernia model in C57BL/6J fetal mice: a step toward lineage tracing experiments.

PURPOSE: Lineage tracing is key to study the fate of individual cells and their progeny especially in developmental biology. To conduct these studies, we aimed to establish a reproducible model of CDH in the most commonly used genetic background strain that is C57BL/6J mice. METHODS: CDH was induced in C57BL/6J dams by maternal administration of nitrofen + bisdiamine at E8.5. Fetuses from olive oil-gavaged mothers served as controls. Lungs from CDH and control fetuses were compared for (1) growth via radial airspace count (RAC), mean linear intercept (MLI) and gene expression for Fgf10, Nrp1, and Ctnnb1; (2) maturation (Pdpn, Spc, Ager, Abca3, Eln, Acta2, Pdgfra) via gene and protein expression; (3) vascularization via gene and protein expression (CD31, Vegfa, Vegfr1/2, Epas1, Enos). STATISTICS: unpaired t-test or Mann-Whitney test. RESULTS: Nitrofen + bisdiamine administration resulted in 36% left-sided CDH (31% mortality). CDH fetuses had hypoplastic lungs and impaired growth (lower RAC, higher MLI, lower Fgf10, Nrp1, Ctnnb1), maturation (decreased Pdpn, Ager, Eln gene expression), and vascularization (decreased Cd31, Vegfr1/2; Epas1 and Enos). Lower protein expression was confirmed for PDPN, ELN and CD31. CONCLUSION: Modeling CDH in C57BL/6J mouse fetuses is effective in reproducing the classical CDH hallmarks. This model will be critical for lineage tracing experiments.

Embryology and anatomy of congenital diaphragmatic hernia.

Prenatal and postnatal treatment modalities for congenital diaphragmatic hernia (CDH) continue to improve, however patients still face high rates of morbidity and mortality caused by severe underlying persistent pulmonary hypertension and pulmonary hypoplasia. Though the majority of CDH cases are idiopathic, it is believed that CDH is a polygenic developmental defect caused by interactions between candidate genes, as well as environmental and epigenetic factors. However, the origin and pathogenesis of these developmental insults are poorly understood. Further, connections between disrupted lung development and the failure of diaphragmatic closure during embryogenesis have not been fully elucidated. Though several animal models have been useful in identifying candidate genes and disrupted signalling pathways, more studies are required to understand the pathogenesis and to develop effective preventative care. In this article, we summarize the most recent litterature on disrupted embryological lung and diaphragmatic development associated with CDH.

Congenital diaphragmatic hernia and early lethality in PIGL-related disorder.

We report on three male siblings who presented prenatally with a nearly identical combination of congenital anomalies and who died shortly after preterm birth. The first baby was a singleton pregnancy, and the other two babies were dichorionic diamniotic twins. Key features included: left-sided congenital diaphragmatic hernia, inferior vermian dysgenesis/hypoplasia, prenasal edema, cleft palate, micropenis/ambiguous genitalia (in 2 of 3 babies), bilateral renal pelvic dilatation (in twins, first baby showed slightly enlarged kidneys) and polyhydramnios (in 2 of 3). Whole genome sequencing performed on DNA from all three babies revealed homozygous missense PIGL gene variants: c.438C>A, p.(Phe146Leu). Both parents were heterozygous carriers of the variant. The reporting clinical laboratory classified the change as a variant of uncertain significance (VUS), and concluded "A genetic diagnosis of autosomal recessive CHIME syndrome is possible". The PIGL gene has been reported to cause two different autosomal recessive conditions: CHIME syndrome and Mabry syndrome. CHIME (Zunich neuroectodermal syndrome) is characterized by ocular Colobomas, Heart defects, Ichthyosiform dermatosis, Mental retardation (intellectual disability), and Ear anomalies, including conductive hearing loss. Mabry [aka hyperphosphatasia mental retardation syndrome (HPMRS)] is characterized by severe developmental delay, moderate to severe intellectual disability, distinctive facial features, brachytelephalangy, increased serum levels of alkaline phosphatase (ALP), and recurrent seizures. Neonatal demise and lack of postmortem examination precluded assessment of some key features (including seizures, developmental delay, ALP levels, colobomas and deafness), but overlapping features observed included cleft palate, brain anomalies, genitourinary abnormalities and prenasal edema. Notably, diaphragmatic hernia is not a common feature of either condition, but is a cardinal feature of Fryns syndrome. The genetic etiology of Fryns syndrome has not been definitively established, although, much like CHIME and Mabry syndrome, can be caused by variants in glycosylphosphatidylinositol (GPI) anchor pathway genes. Our findings suggest further overlap between inherited GPI deficiencies, and possible expansion of the clinical phenotype of PIGL-related disorders to include prenatal presentations with congenital diaphragmatic hernia. Although reported as a VUS, we present phenotypic and familial segregation evidence that supports likely pathogenicity of the c.438C>A variant.

Tenascin C is dysregulated in hypoplastic lungs of miR-200b-/- mice.

PURPOSE: We previously demonstrated that absence of miR-200b results in abnormal lung development in congenital diaphragmatic hernia due to imbalance between epithelial and mesenchymal cells. Tenascin C is a highly conserved extracellular matrix protein involved in epithelial to mesenchymal transition, tissue regeneration and lung development. Considering the involvement of Tenascin C and miR-200b and their potential interaction, we aimed to study Tenascin C during lung development in the absence of miR-200b. METHODS: We collected lungs of miR-200b-/- mice (male, 8 weeks). We performed Western blot (WB) analysis (N = 6) and immunofluorescence (N = 5) for Tenascin C and alpha smooth muscle actin and RT-qPCR for Tenascin C gene expression (N = 4). RESULTS: Using WB analysis, we observed a decreased total protein abundance of Tenascin C in miR-200b-/- lungs (miR-200b+/+: 3.8 × 107 ± 1 × 107; miR-200b-/-: 1.9 × 107 ± 5 × 106; p = 0.002). Immunofluorescence confirmed decreased total Tenascin C in miR-200b-/- lungs. Tenascin C was significantly decreased in the mesenchyme but relatively increased in the airways of mutant lungs. Total lung RNA expression of Tenascin C was higher in miR-200b-/- lungs. CONCLUSION: We report dysregulation of Tenascin C in lungs of miR-200b-/- mice. This suggests that absence of miR-200b results in abnormal Tenascin C abundance contributing to the lung hypoplasia observed in miR-200b-/- mice.

Clinical exome sequencing data reveal high diagnostic yields for congenital diaphragmatic hernia plus (CDH+) and new phenotypic expansions involving CDH.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a life-threatening birth defect that often co-occurs with non-hernia-related anomalies (CDH+). While copy number variant (CNV) analysis is often employed as a diagnostic test for CDH+, clinical exome sequencing (ES) has not been universally adopted. METHODS: We analysed a clinical database of ~12 000 test results to determine the diagnostic yields of ES in CDH+ and to identify new phenotypic expansions. RESULTS: Among the 76 cases with an indication of CDH+, a molecular diagnosis was made in 28 cases for a diagnostic yield of 37% (28/76). A provisional diagnosis was made in seven other cases (9%; 7/76). Four individuals had a diagnosis of Kabuki syndrome caused by frameshift variants in KMT2D. Putatively deleterious variants in ALG12 and EP300 were each found in two individuals, supporting their role in CDH development. We also identified individuals with de novo pathogenic variants in FOXP1 and SMARCA4, and compound heterozygous pathogenic variants in BRCA2. The role of these genes in CDH development is supported by the expression of their mouse homologs in the developing diaphragm, their high CDH-specific pathogenicity scores generated using a previously validated algorithm for genome-scale knowledge synthesis and previously published case reports. CONCLUSION: We conclude that ES should be ordered in cases of CDH+ when a specific diagnosis is not suspected and CNV analyses are negative. Our results also provide evidence in favour of phenotypic expansions involving CDH for genes associated with ALG12-congenital disorder of glycosylation, Rubinstein-Taybi syndrome, Fanconi anaemia, Coffin-Siris syndrome and FOXP1-related disorders.

Exome sequencing of fetuses with congenital diaphragmatic hernia supports a causal role for NR2F2, PTPN11, and WT1 variants.

BACKGROUND: To identify genes associated with congenital diaphragmatic hernia (CDH) to help understand the etiology and inform prognosis. METHODS: We performed exome sequencing on fetuses with CDH and their parents to identify rare genetic variants likely to mediate risk. We reviewed prenatal characteristics and neonatal outcomes. RESULTS: Data were generated for 22 parent-offspring trios. Six Likely Damaging (LD) variants were identified in five families (23 %). Three LD variants were in genes that contain variants in other CDH cohorts (NR2F2, PTPN11, WT1), while three were in genes that do not (CTR9, HDAC6, TP53). Integrating these data bolsters the evidence of association of NR2F2, PTPN11, and WT1 with CDH in humans. Of the five fetuses with a genetic diagnosis, one was terminated, two underwent perinatal demise, while two survived until repair. CONCLUSIONS: Exome sequencing expands the diagnostic yield of genetic testing in CDH. Correlating CDH patients' exomes with clinical outcomes may enable personalized counseling and therapies.

Genetic Diagnostic Strategies and Counseling for Families Affected by Congenital Diaphragmatic Hernia.

Congenital diaphragmatic hernia (CDH) is a relatively common and severe birth defect with variable clinical outcome and associated malformations in up to 60% of patients. Mortality and morbidity remain high despite advances in pre-, intra-, and postnatal management. We review the current literature and give an overview about the genetics of CDH to provide guidelines for clinicians with respect to genetic diagnostics and counseling for families. Until recently, the common practice was (molecular) karyotyping or chromosome microarray if the CDH diagnosis is made prenatally with a 10% diagnostic yield. Undiagnosed patients can be reflexed to trio exome/genome sequencing with an additional diagnostic yield of 10 to 20%. Even with a genetic diagnosis, there can be a range of clinical outcomes. All families with a child with CDH with or without additional malformations should be offered genetic counseling and testing in a family-based trio approach.

Rare and de novo variants in 827 congenital diaphragmatic hernia probands implicate LONP1 as candidate risk gene.

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.

Transamniotic Stem Cell Therapy for Experimental Congenital Diaphragmatic Hernia: Structural, Transcriptional, and Cell Kinetics Analyses in the Nitrofen Model.

PURPOSE: We examined select pulmonary effects and donor cell kinetics after transamniotic stem cell therapy (TRASCET) in a model of congenital diaphragmatic hernia (CDH). METHODS: Pregnant dams (n = 58) received nitrofen on gestational day 9.5 (E9) to induce fetal CDH. Fetuses (n = 681) were divided into 4 groups: untreated (n = 99) and 3 groups receiving volume-matched intra-amniotic injections on E17 of either saline (n = 142), luciferase-labeled amniotic fluid-derived mesenchymal stem cells (afMSCs; n = 299), or acellular recombinant luciferase (n = 141). Pulmonary morphometry, quantitative gene expression of pulmonary vascular tone mediators, or screening for labeled afMSCs were performed at term (E22). Statistical comparisons were by Mann-Whitney U-test, nested ANOVA, and Wald test. RESULTS: TRASCET led to significant downregulation of endothelial nitric oxide synthase and endothelin receptor-A expressions compared to both untreated and saline groups (both p < 0.001). TRASCET also led to a significant decrease in arteriole wall thickness compared to the untreated group (p < 0.001) but not the saline group (p = 0.180). Donor afMSCs were identified in the bone marrow and umbilical cord (p = 0.035 and 0.015, respectively, vs. plain luciferase controls). CONCLUSIONS: The effects of TRASCET in experimental CDH appear to be centered on the pulmonary vasculature and to derive from circulating donor cells.

Resolving the heterogeneity of diaphragmatic mesenchyme: a novel mouse model of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a relatively common developmental defect with considerable mortality and morbidity. Formation of the diaphragm is a complex process that involves several cell types, each with different developmental origins. Owing to this complexity, the aetiology of CDH is not well understood. The pleuroperitoneal folds (PPFs) and the posthepatic mesenchymal plate (PHMP) are transient structures that are essential during diaphragm development. Using several mouse models, including lineage tracing, we demonstrate the heterogeneous nature of the cells that make up the PPFs. The conditional deletion of Wilms tumor 1 homolog (Wt1) in the non-muscle mesenchyme of the PPFs results in CDH. We show that the fusion of the PPFs and the PHMP to form a continuous band of tissue involves movements of cells from both sources. The PPFs of mutant mice fail to fuse with the PHMP and exhibit increased RALDH2 (also known as ALDH1A2) expression. However, no changes in the expression of genes (including Snai1, Snai2, Cdh1 and Vim) implicated in epithelial-to-mesenchymal transition are observed. Additionally, the mutant PPFs lack migrating myoblasts and muscle connective tissue fibroblasts (TCF4+/GATA4+), suggesting possible interactions between these cell types. Our study demonstrates the importance of the non-muscle mesenchyme in development of the diaphragm.

Pulmonary hypertension is an important co-morbidity in developmental lung diseases of infancy: Bronchopulmonary dysplasia and congenital diaphragmatic hernia.

Bronchopulmonary dysplasia (BPD) following preterm birth and congenital diaphragmatic hernia (CDH) are both forms of developmental lung disease that may result in persistent pulmonary and pulmonary vascular morbidity in childhood. The pulmonary vascular disease (PVD) which accompanies BPD and CDH is due to developmental abnormalities and ongoing perinatal insults. This may be accompanied by evidence of elevated right heart pressures and pulmonary vascular resistance, leading to diagnosis of pulmonary hypertension (PH). The development of PH in these conditions is associated with increased morbidity and mortality in the vulnerable BPD and CDH populations. We present a review of PVD pathogenesis and evaluation in BPD and CDH and discuss management of related sequelae of PH co-morbidity for affected infants.

Epithelial cell-adhesion protein cadherin 26 is dysregulated in congenital diaphragmatic hernia and congenital pulmonary airway malformation.

BACKGROUND: Congenital diaphragmatic hernia (CDH) and congenital pulmonary airway malformation (CPAM) are two inborn pathologies of the lung of unknown origin. Alterations of gene expression in airway epithelial cells are involved in the pathobiology of both diseases. We previously found decreased expression of the epithelial cell adhesion protein cadherin 26 (CDH26) in hypoplastic mice lungs. Here, our objective was to describe the expression and localization of CDH26 in hypoplastic CDH lungs and hyperproliferative CPAM tissues. METHODS: After ethical approval, we used human lung tissues from CDH and CPAM cases and age-matched control samples from a previously established biobank. Furthermore, lungs from the nitrofen rat model of CDH were included in the study. We performed immunohistochemistry and western blot analysis with antibodies against CDH26 to examine protein localization and abundance. Statistical analysis was performed using Mann-Whitney U test with significance set at p < 0.05. RESULTS: We observed an overexpression of CDH26 within the epithelium of cystic CPAM lesions compared to normal airways within the same lung and compared to control lungs. Western blot demonstrated a downregulation of CDH26 in the nitrofen rat model of CDH compared to healthy controls. Immunohistochemistry could not show consistent differences between CDH and control in human and rat lungs. In the studied human lung samples, CDH26 was localized to the apical part of the airway epithelial cells. CONCLUSION: CDH26 is differentially expressed in human CPAM lung tissues and may be downregulated in nitrofen-induced hypoplastic rat lungs compared to control lungs. Disruption of CDH26 associated pathways in lung development may be involved in the pathogenesis of lung hypoplasia or cystic lung disease.

A Case Report of Donnai-Barrow Syndrome.

BACKGROUND: A genetic disorder should be considered when an infant presents with multiple congenital anomalies. Because of the acute presentation of an infant with multiple life-threatening defects, a genetic diagnosis of a rare disorder took weeks to delineate. CLINICAL FINDINGS: This case describes a late preterm infant who presented at birth with congenital diaphragmatic hernia, tetralogy of Fallot, cleft lip, low-set ears, and hypertelorism. PRIMARY DIAGNOSIS: Donnai-Barrow syndrome was the final diagnosis confirmed by a defect observed on the LRP2 (2q31.1) gene using sequence analysis. This is a rare disorder that presents with a variety of phenotypic features in infants. INTERVENTIONS: Initial neonatal resuscitation in the delivery room included intubation, positive pressure ventilation, and oxygen supplementation. Extracorporeal membrane oxygenation therapy was initiated from day of life 3 to 15. Initial surgery included correction of the congenital diaphragmatic hernia, and further surgical procedures included tracheostomy, gastrostomy tube, circumcision, ventricular septal defect repair, and cleft lip repair. Physical, occupational, and speech therapies were also initiated. OUTCOMES: The infant was transported to a pediatric rehabilitation facility at 6 months of life for further management of his chronic lung disease requiring tracheostomy with ventilator dependence. PRACTICE RECOMMENDATIONS: Early recognition and diagnosis of genetic syndromes can improve family education and guide treatment interventions. An underlying syndrome should be suspected when an infant presents with multiple congenital defects. Infants with Donnai-Barrow syndrome should have thorough cardiac, neurologic, ophthalmologic, audiologic, and renal examinations due to the gene mutation effects on those systems.

miR-200 family expression during normal and abnormal lung development due to congenital diaphragmatic hernia at the later embryonic stage in the nitrofen rat model.

INTRODUCTION: Congenital diaphragmatic hernia (CDH) is a life-threatening disease associated with pulmonary hypoplasia. CDH occurs approximately 1 in every 2000-3000 live births, and the pathophysiology is unknown. MicroRNAs are short, non-coding RNAs that control gene expression through post-transcriptional regulation. Based on our previous work, we hypothesized that the miR-200 family is differentially expressed in normal and abnormal lung development. We aimed to examine the expression of the miR-200 family during normal and hypoplastic lung development due to CDH. METHODS: We performed reverse transcriptase polymerase chain reaction (RT-qPCR) and fluorescent in situ hybridization (FISH) to study the expression levels and distribution of the miR-200 family members on embryonic day 21 (E21) rat control and nitrofen-induced hypoplastic CDH lungs. RESULTS: RT-qPCR showed up-regulation of miR-200a in hypoplastic CDH lungs. FISH showed contrasting expression patterns for miR- 200a, miR-200c, and miR-429 between control and hypoplastic CDH lungs, while we could not detect miR-141 in control and hypoplastic CDH lungs. CONCLUSION: We demonstrate a specific expression pattern of miR-200 family members in hypoplastic CDH lungs different from control lungs. This study suggests that disruption of miR-200 family expression plays a role in the pathogenesis of pulmonary hypoplasia associated with CDH.

Transgenic animal models of congenital diaphragmatic hernia: a comprehensive overview of candidate genes and signaling pathways.

Congenital diaphragmatic hernia (CDH) is a relatively common and life-threatening birth defect, characterized by incomplete formation of the diaphragm. Because CDH herniation occurs at the same time as preacinar airway branching, normal lung development becomes severely disrupted, resulting almost invariably in pulmonary hypoplasia. Despite various research efforts over the past decades, the pathogenesis of CDH and associated lung hypoplasia remains poorly understood. With the advent of molecular techniques, transgenic animal models of CDH have generated a large number of candidate genes, thus providing a novel basis for future research and treatment. This review article offers a comprehensive overview of genes and signaling pathways implicated in CDH etiology, whilst also discussing strengths and limitations of transgenic animal models in relation to the human condition.

Extracranial midline defects in a patient with craniofrontonasal syndrome with a novel EFNB1 mutation.

We report a female patient with craniofrontonasal syndrome (CFNS) who in addition showed other cranial and extracranial midline defects including partial corpus callosum agenesis, ocular melanocytosis, pigmentary glaucoma, duplex collecting system, uterus didelphys, and septate vagina. She was found to have a novel pathogenic variant in exon 5 of EFNB1, c.646G>T (p.Glu216*) predicted to cause premature protein truncation. From our review, we found at least 39 published CFNS patients with extracranial midline defects, comprising congenital diaphragmatic hernia, congenital heart defects, umbilical hernia, hypospadias, and less frequently, sacrococcygeal teratomas, and internal genital anomalies in females. These findings support that the EFNB1 mutations have systemic consequences disrupting morphogenetic events at the extracranial midline. Though these are not rigorously included as midline defects, we found at least 10 CFNS patients with congenital anomalies of the kidney and urinary tract, all females. Additionally, uterus didelphys and ocular melanocytosis observed in our patient are proposed also as a previously unreported EFNB1-related midline defects. In addition, this case may be useful for considering the intentional search for genitourinary anomalies in future patients with CFNS, which will be helpful to define their frequency in this entity.

Basic and translational science advances in congenital diaphragmatic hernia.

Congenital Diaphragmatic Hernia (CDH) is a birth defect that is characterized by lung hypoplasia, pulmonary hypertension and a diaphragmatic defect that allows herniation of abdominal organs into the thoracic cavity. Although widely unknown to the public, it occurs as frequently as cystic fibrosis (1:2500). There is no monogenetic cause, but different animal models revealed various biological processes and epigenetic factors involved in the pathogenesis. However, the pathobiology of CDH is not sufficiently understood and its mortality still ranges between 30 and 50%. Future collaborative initiatives are required to improve our basic knowledge and advance novel strategies to (prenatally) treat the abnormal lung development. This review focusses on the genetic, epigenetic and protein background and the latest advances in basic and translational aspects of CDH research.