Human Congenital Diaphragmatic Hernia Research Articles

Fetal hypoplastic lungs have multilineage inflammation that is reversed by amniotic fluid stem cell extracellular vesicle treatment.

Antenatal administration of extracellular vesicles from amniotic fluid stem cells (AFSC-EVs) reverses features of pulmonary hypoplasia in models of congenital diaphragmatic hernia (CDH). However, it remains unknown which lung cellular compartments and biological pathways are affected by AFSC-EV therapy. Herein, we conducted single-nucleus RNA sequencing (snRNA-seq) on rat fetal CDH lungs treated with vehicle or AFSC-EVs. We identified that intra-amniotically injected AFSC-EVs reach the fetal lung in rats with CDH, where they promote lung branching morphogenesis and epithelial cell differentiation. Moreover, snRNA-seq revealed that rat fetal CDH lungs have a multilineage inflammatory signature with macrophage enrichment, which is reversed by AFSC-EV treatment. Macrophage enrichment in CDH fetal rat lungs was confirmed by immunofluorescence, flow cytometry, and inhibition studies with GW2580. Moreover, we validated macrophage enrichment in human fetal CDH lung autopsy samples. Together, this study advances knowledge on the pathogenesis of pulmonary hypoplasia and further evidence on the value of an EV-based therapy for CDH fetuses.

Comparative Transcriptome Analysis of Human and Mouse Canalicular Lungs in Fetal Diaphragmatic Hernia

BackgroundThe nitrofen model of congenital diaphragmatic hernia (CDH) is widely used in translational research. However, the molecular pathways associated with pulmonary hypoplasia in this model compared to the human CDH phenotype have not been well described. The aim of this study was to investigate differentially expressed genes (DEG) and signaling pathways in early stage fetal lungs in mouse and human CDH. MethodsCDH lung tissue was obtained from human fetuses (21-23 weeks gestation) and nitrofen mouse pups (E15.5). NovaSeq Flowcell RNA-seq was performed to evaluate differentially expressed transcriptional and molecular pathways (DEGs) in fetal mice with CDH, compared with age-matched normal mouse lungs and human CDH samples. ResultsThere were thirteen overlapping DEGs in human and mouse CDH lung samples compared to controls. These genes were involved in extracellular matrix, myogenesis, cilia, and immune modulation pathways. Human CDH was associated with an upregulation of collagen formation and extracellular matrix reorganization whereas mouse CDH was associated with an increase in muscular contraction. The most common cell types upregulated in human and mouse CDH samples were ciliated airway cells. ConclusionsThis study highlights the unique gene transcriptional patterns in early fetal mouse and human lungs with CDH. These data have implications when determining the translational potential of novel therapies in CDH using nitrofen-based animal models. Level of EvidenceLevel IV Study TypeBasic science/case series

Decreased β-catenin Protein in Lungs From Human Congenital Diaphragmatic Hernia Archival Pathology Specimens: A Case-control Study

BackgroundLung hypoplasia contributes to congenital diaphragmatic hernia (CDH) associated morbidity and mortality. Changes in lung wingless-type MMTV integration site family member (Wnt)-signalling and its downstream effector beta-catenin (CTNNB1), which acts as a transcription coactivator, exist in animal CDH models but are not well characterized in humans. We aim to identify changes to Wnt-signalling gene expression in human CDH lungs and hypothesize that pathway expression will be lower than controls. MethodsWe identified 51 CDH cases and 10 non-CDH controls with archival formalin-fixed paraffin-embedded (FFPE) autopsy lung tissue from 2012 to 2022. 11 liveborn CDH cases and an additional two anterior diaphragmatic hernias were excluded from the study, leaving 38 CDH cases. Messenger ribonucleic acid (mRNA) expression of Wnt-signalling effectors WNT2B and CTNNB1 was determined for 19 CDH cases and 9 controls. A subset of CDH cases and controls lung sections were immunostained for β-catenin. Clinical variables were obtained from autopsy reports. ResultsMedian gestational age was 21 weeks. 81% (n = 31) of hernias were left-sided. 47% (n = 18) were posterolateral. Liver position was up in 81% (n = 31) of cases. Defect size was Type C or D in 58% (n = 22) of cases based on autopsy photos, and indeterminable in 42% (n = 16) of cases. WNT2B and CTNNB1 mRNA expression did not differ between CDH and non-CDH lungs. CDH lungs had fewer interstitial cells expressing β-catenin protein than non-CDH lungs (13.2% vs 42.4%; p = 0.006). ConclusionThere appear to be differences in the abundance and/or localization of β-catenin proteins between CDH and non-CDH lungs. Level of EvidenceLevel III. Type of StudyCase-Control Study.

Molecular insights using spatial transcriptomics of the distal lung in congenital diaphragmatic hernia.

Abnormal pulmonary vascular development and function in congenital diaphragmatic hernia (CDH) is a significant factor leading to pulmonary hypertension. The lung is a very heterogenous organ and has marked cellular diversity that is differentially responsive to injury and therapeutic agents. Spatial transcriptomics provides the unmatched capability of discerning the differences in the transcriptional signature of these distinct cell subpopulations in the lung with regional specificity. We hypothesized that the distal lung parenchyma (selected as a region of interest) would show a distinct transcriptomic profile in the CDH lung compared with control (normal lung). We subjected lung sections obtained from male and female CDH and control neonates to spatial transcriptomics using the Nanostring GeoMx platform. Spatial transcriptomic analysis of the human CDH and control lung revealed key differences in the gene expression signature. Increased expression of alveolar epithelial-related genes (SFTPA1 and SFTPC) and angiogenesis-related genes (EPAS1 and FHL1) was seen in control lungs compared with CDH lungs. Response to vitamin A was enriched in the control lungs as opposed to abnormality of the coagulation cascade and TNF-alpha signaling via NF-kappa B in the CDH lung parenchyma. In male patients with CDH, higher expression of COL1A1 (ECM remodeling) and CD163 was seen. Increased type 2 alveolar epithelial cells (AT-2) and arterial and lung capillary endothelial cells were seen in control lung samples compared with CDH lung samples. To the best of our knowledge, this is the first use of spatial transcriptomics in patients with CDH that identifies the contribution of different lung cellular subpopulations in CDH pathophysiology and highlights sex-specific differences.NEW & NOTEWORTHY This is the first use of spatial transcriptomics in patients with congenital diaphragmatic hernia (CDH) that identifies the contribution of different lung cellular subpopulations in CDH pathophysiology and highlights sex-specific differences.

Intact cord resuscitation in newborns with congenital diaphragmatic hernia: insights from a lamb model.

Congenital diaphragmatic hernia (CDH) is a rare condition characterized by pulmonary hypoplasia, vascular dystrophy, and pulmonary hypertension at birth. Validation of the lamb model as an accurate representation of human CDH is essential to translating research findings into clinical practice and understanding disease mechanisms. This article emphasizes the importance of validating the lamb model to study CDH pathogenesis and develop innovative therapeutics. At 78 days of gestation, the fetal lamb's left forelimb was exposed through a midline laparotomy and hysterotomy, and a supra diaphragmatic thoracotomy was performed to allow the digestive organs to ascend into the thoracic cavity. At 138 ± 3 days of gestation, lambs were delivered via a cesarean section; then, with umbilical cord intact during 1 hour, the lambs were mechanically ventilated with gentle ventilation in a pressure-controlled mode for 2 h. CDH lambs exhibited a lower left lung-to-body weight ratio of 5.3 (2.03), p < 0.05, and right lung-to-body weight ratio of 8.2 (3.1), p < 0.05. They reached lower Vt/kg (tidal volume per kg) during the course of the resuscitation period with 1.2 (0.7) ml/kg at 10 min and 3 (1.65) ml/kg at 60 min (p < 0.05). Compliance of the respiratory system was lower in CDH lambs with 0.5 (0.3) ml/cmH2O at 60 min (p < 0.05) and 0.9 (0.26) ml/cmH2O at 120 min (p < 0.05). Differences between pre- and postductal SpO2 were higher with 15.1% (21.4%) at 20 min and 6.7% (14.5%) at 80 min (p < 0.05). CDH lambs had lower differences between inspired and expired oxygen fractions with 4.55% (6.84%) at 20 min and 6.72% (8.57%) at 60 min (p < 0.05). CDH lamb had lower left ventricle [2.73 (0.5) g/kg, p < 0.05] and lower right ventricle [0.69 (0.8), p < 0.05] to left ventricle ratio. CDH lambs had significantly lower tidal volume than control lambs due to lower compliance of the respiratory system and higher airway resistance. These respiratory changes are characteristic of CDH infants and are associated with higher mortality rates. CDH lambs also exhibited pulmonary hypertension, pulmonary hypoplasia, and left ventricle hypoplasia, consistent with observations in human newborns. To conclude, our lamb model successfully provides a reliable representation of CDH and can be used to study its pathophysiology and potential interventions.

Overactivated Epithelial NF-κB Disrupts Lung Development in Congenital Diaphragmatic Hernia.

Abnormal lung development is the main cause of morbidity and mortality in neonates with congenital diaphragmatic hernia (CDH), a common birth defect (1:2500) of largely unknown pathobiology. Recent studies discovered that inflammatory processes, and specifically NF-κB associated pathways are enriched in human and experimental CDH. However, the molecular signaling of NF-κB in abnormal CDH lung development and its potential as a therapeutic target requires further investigation. Using sections and hypoplastic lung explant cultures from the nitrofen rat model of CDH and human fetal CDH lungs, we demonstrate that NF-κB and its downstream transcriptional targets are hyperactive during abnormal lung formation in CDH. NF-κB activity was especially elevated in the airway epithelium of nitrofen and human CDH lungs at different developmental stages. Fetal rat lung explants had impaired pseudoglandular airway branching after exposure to nitrofen, together with increased phosphorylation and transcriptional activity of NF-κB. Dexamethasone, the broad and clinically applicable anti-inflammatory NF-κB antagonist, rescued lung branching and normalized NF-κB signaling in hypoplastic lung explants. Moreover, specific NF-κB inhibition with curcumenol similarly rescued ex vivo lung hypoplasia and restored NF-κB signaling. Lastly, we showed that prenatal intraperitoneal dexamethasone administration to pregnant rat dams carrying fetuses with hypoplastic lungs, significantly improves lung branching and normalizes NF-κB in vivo. Our results indicate that NF-κB is aberrantly activated in human and nitrofen CDH lungs. Anti-inflammatory treatment with dexamethasone and/ or specific NF-κB inhibition should be investigated further as a therapeutic avenue to target lung hypoplasia in CDH.

Role of microRNAs in Congenital Diaphragmatic Hernia-Associated Pulmonary Hypertension.

Epigenetic regulators such as microRNAs (miRNAs) have a key role in modulating several gene expression pathways and have a role both in lung development and function. One of the main pathogenetic determinants in patients with congenital diaphragmatic hernia (CDH) is pulmonary hypertension (PH), which is directly related to smaller lung size and pulmonary microarchitecture alterations. The aim of this review is to highlight the importance of miRNAs in CDH-related PH and to summarize the results covering this topic in animal and human CDH studies. The focus on epigenetic modulators of CDH-PH offers the opportunity to develop innovative diagnostic tools and novel treatment modalities, and provides a great potential to increase researchers' understanding of the pathophysiology of CDH.

A Tracheal Aspirate-derived Airway Basal Cell Model Reveals a Proinflammatory Epithelial Defect in Congenital Diaphragmatic Hernia.

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 invitro 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 invitro and in nitrofen rat tracheas invivo. Conclusions: Our findings have identified an underlying proinflammatory signature and BSC differentiation defects as a potential therapeutic target for airway epithelial defects in CDH.

Proteomic Profiling of Hypoplastic Lungs Suggests an Underlying Inflammatory Response in the Pathogenesis of Abnormal Lung Development in Congenital Diaphragmatic Hernia.

The pathogenesis of lung hypoplasia in congenital diaphragmatic hernia (CDH), a common birth defect, is poorly understood. The diaphragmatic defect can be repaired surgically, but the abnormal lung development contributes to a high mortality in these patients. To understand the underlying pathobiology, we compared the proteomic profiles of fetal rat lungs at the alveolar stage (E21) that were either exposed to nitrofen in utero (CDH lungs, n=5) or exposed to vehicle only (non-CDH control lungs, n=5). Pathway analysis of proteomic datasets showed significant enrichment in inflammatory response proteins associated with cytokine signaling and Epstein Barr Virus in nitrofen CDH lungs. Among the 218 significantly altered proteins between CDH and non-CDH control lungs were Tenascin C, CREBBP, LYN, and STAT3. We showed that Tenascin C was decreased around the distal airway branches in nitrofen rat lungs and human CDH lungs, obtained from stillborn fetuses that did not receive pre- or postnatal treatment. In contrast, STAT3 was significantly increased in the airway epithelium of nitrofen lungs at E21. STAT3 inhibition after direct nitrofen exposure to fetal rat lung explants (E14.5) partially rescued the hypoplastic lung phenotype ex vivo by increasing peripheral lung budding. Moreover, we demonstrated that several STAT3-associated cytokines (IL-15, IL-9, andIL-2) are increased in fetal tracheal aspirates of CDH survivors compared with nonsurvivors after fetoscopic endoluminal tracheal occlusion. With our unbiased proteomics approach, we showed for the first time that downstream inflammatory processes are likely involved in the pathogenesis of abnormal lung development in CDH.

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

BackgroundTo identify genes associated with congenital diaphragmatic hernia (CDH) to help understand the etiology and inform prognosis. MethodsWe 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. ResultsData 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. ConclusionsExome sequencing expands the diagnostic yield of genetic testing in CDH. Correlating CDH patients’ exomes with clinical outcomes may enable personalized counseling and therapies.

Isolated congenital diaphragm hernia associated with homozygous SLIT3 gene variant in dizygous twins

Congenital diaphragmatic hernia (CDH) is a serious life-threatening birth defect characterized by abnormal development in the muscular or tendinous portion of the diaphragm during embryogenesis. Despite its high incidence, the etiology of CDH hasn't been fully understood. Genetic factors are important in pathogenesis; however, few single genes have been definitively implicated in human CDH. SLIT1, SLIT2, and SLIT3 (slit guidance ligand) are three human homologs of the drosophila Slit gene. They interact with roundabout (Robo) homolog receptors to affect cell migration, adhesion, cell motility, and angiogenesis and play important roles in cell signaling pathways including the guidance of axons. In this report, we presented dizygous twin babies with CDH related to the SLIT3 gene variant. Previous studies showed that Slit3 null mice had congenital diaphragmatic hernias on or near the ventral midline portion of the central tendon. This is the first report of homozygous SLIT3 variant associated with CDH in humans.

EP127 - Evidence that FGFRL1 contributes to congenital diaphragmatic hernia development in humans

Fibroblast growth factor receptor-like 1 (FGFRL1) encodes a transmembrane protein that is related to fibroblast growth factor receptors but lacks an intercellular tyrosine kinase domain. in vitro studies suggest that FGFRL1 inhibits cell proliferation and promotes cell differentiation and cell adhesion. Mice that lack FGFRL1 die shortly after birth from respiratory distress and have abnormally thin diaphragms whose muscular hypoplasia allows the liver to protrude into the thoracic cavity. Haploinsufficiency of FGFRL1 has been hypothesized to contribute to the development of congenital diaphragmatic hernia (CDH) associated with Wolf-Hirschhorn syndrome. However, data from both humans and mice suggest that disruption of one copy of FGFRL1 alone is insufficient to cause diaphragm defects. Here we report a female fetus with CDH whose 4p16.3 deletion allows us to refine the Wolf-Hirschhorn syndrome CDH critical region to an approximately 1.9 Mb region that contains FGFRL1. We also report a male infant with isolated left-sided diaphragm agenesis who carried compound heterozygous missense variants in FGFRL1. These cases provide additional evidence that deleterious FGFRL1 variants may contribute to the development of CDH in humans.

Pbx1, Meis1, and Runx1 Expression Is Decreased in the Diaphragmatic and Pulmonary Mesenchyme of Rats with Nitrofen-Induced Congenital Diaphragmatic Hernia.

Congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia (PH) are thought to originate from mesenchymal defects in pleuroperitoneal folds (PPFs) and primordial lungs. Pre-B-cell leukemia homeobox 1 (Pbx1), its binding partner myeloid ecotropic integration site 1 (Meis1), and runt-related transcription factor 1 (Runx1) are expressed in diaphragmatic and lung mesenchyme, functioning as transcription cofactors that modulate mesenchymal cell proliferation. Furthermore, Pbx1 -/- mice develop diaphragmatic defects and PH similar to human CDH. We hypothesized that diaphragmatic and pulmonary Pbx1, Meis1, and Runx1 expression is decreased in the nitrofen-induced CDH model. Time-mated rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms (n = 72) and lungs (n = 48) were microdissected on D13, D15, and D18, and were divided into control and nitrofen-exposed specimens. Diaphragmatic and pulmonary gene expression levels of Pbx1, Meis1, and Runx1 were analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence-double-staining for Pbx1, Meis1, and Runx1 was combined with mesenchymal/myogenic markers Gata4 and myogenin to evaluate protein expression. Relative mRNA expression of Pbx1, Meis1, and Runx1 was significantly decreased in PPFs (D13), developing diaphragms/lungs (D15), and muscularized diaphragms/differentiated lungs (D18) of nitrofen-exposed fetuses compared with controls. Confocal-laser-scanning-microscopy revealed markedly diminished Pbx1, Meis1, and Runx1 immunofluorescence in diaphragmatic and pulmonary mesenchyme, associated with less proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15, and D18 compared with controls. Decreased Pbx1, Meis1, and Runx1 expression during diaphragmatic development and lung branching morphogenesis may reduce mesenchymal cell proliferation, causing malformed PPFs and disrupted airway branching, thus leading to diaphragmatic defects and PH in the nitrofen-induced CDH model.

Maldevelopment of intrapulmonary bronchial cartilage in congenital diaphragmatic hernia

Pulmonary hypoplasia is an important cause of morbidity and mortality in infants with congenital diaphragmatic hernia (CDH). This study aimed to verify our hypothesis that the abnormal development of bronchial cartilage as well as alveolar immaturity, might play a central role in hypoplasia of the lung in human CDH. We retrospectively analyzed autopsied lungs from 10 CDH cases and compared with nine age-matched controls to assess the bronchial cartilage and alveolar maturity using morphological techniques. Ki-67 and thyroid transcription factor-1 (TTF-1) expression in the alveoli significantly increased in bilateral lungs with CDH. The shortest distance from the bronchial cartilage to the pleura was significantly shorter in ipsilateral (left) lungs with CDH, showing a positive correlation with the radial alveolar count (RAC). Regarding the small bronchial cartilages less than 20 000 μm2 , the average cartilage area significantly decreased in left lungs with CDH, and tended to decrease in right lungs with CDH. In addition, cartilage around the bronchi less than 200 μm in diameter tended to be smaller in left lungs with CDH. In contrast, regarding the cartilage around the bronchi 200 to 400 μm in diameter, the ratio of the total cartilage area relative to the bronchial diameter tended to be higher in left lungs with CDH, although there was a large variation. These opposite directional cartilage abnormalities around the distal and more proximal bronchi support our hypothesis that abnormal development of bronchial cartilage might play an important role in the hypoplastic lung in CDH.

Deficiency of FRAS1-related extracellular matrix 1 (FREM1) causes congenital diaphragmatic hernia in humans and mice.

Deficiency of FRAS1-related extracellular matrix 1 (FREM1) causes congenital diaphragmatic hernia in humans and mice.

Inhibition of retinoic acid signaling induces aberrant pericyte coverage and differentiation resulting in vascular defects in congenital diaphragmatic hernia.

The mortality and morbidity of patients with congenital diaphragmatic hernia (CDH) is primarily caused by treatment-resistant, persistent pulmonary hypertension. Structural vascular changes, exemplified by extensive muscularization, are already present early in gestation, but the origin of these abnormalities is unknown. Understanding the origin of the vascular defects is important to improve treatment modalities. Here, we show that the distribution of pericytes is different and may thereby potentially initiate the vascular pathology in CDH. Transient inhibition of retinoic acid (RA) signaling early during pregnancy, the basis of the CDH mouse model, led to an increase in the number of pericytes, thereby affecting the angiogenic potential of pericytes in the fetuses. Pericytes of CDH lungs showed reduced proliferation and an increased ACTA2 expression, which indicates that these pericytes are more contractile than in control lung pericytes. This resulted in increased pericyte coverage of pulmonary vessels and reduced expansion of the capillary bed, the earliest pathological sign of the structural changes in CDH. Furthermore, the pericytes had reduced and altered collagen IV deposition in CDH, pointing to a loss of basal membrane integrity between pericytes and endothelial cells. Inhibition of RA signaling in vitro resulted in reduced migration of pericytes, reduced angiogenesis, and loss of collagen IV expression. Importantly, we confirmed our findings in lungs of human CDH patient samples. In summary, inhibition of RA signaling affects the lung pericyte population, leading to increased contractility, reduced pulmonary angiogenesis, and aberrant lung development, as observed in CDH.

Pulmonary hypertension secondary to congenital diaphragmatic hernia: factors and pathways involved in pulmonary vascular remodeling.

Congenital diaphragmatic hernia (CDH) is a severe birth defect that is characterized by pulmonary hypoplasia and pulmonary hypertension (PHTN). PHTN secondary to CDH is a result of vascular remodeling, a structural alteration in the pulmonary vessel wall that occurs in the fetus. Factors involved in vascular remodeling have been reported in several studies, but their interactions remain unclear. To help understand PHTN pathophysiology and design novel preventative and treatment strategies, we have conducted a systematic review of the literature and comprehensively analyzed all factors and pathways involved in the pathogenesis of pulmonary vascular remodeling secondary to CDH in the nitrofen model. Moreover, we have linked the dysregulated factors with pathways involved in human CDH. Of the 358 full-text articles screened, 75 studies reported factors that play a critical role in vascular remodeling secondary to CDH. Overall, the impairment of epithelial homeostasis present in pulmonary hypoplasia results in altered signaling to endothelial cells, leading to endothelial dysfunction. This causes an impairment of the crosstalk between endothelial cells and pulmonary artery smooth muscle cells, resulting in increased smooth muscle cell proliferation, resistance to apoptosis, and vasoconstriction, which clinically translate into PHTN.

Neonatal cardiopulmonary transition in an ovine model of congenital diaphragmatic hernia

ObjectiveInfants with a congenital diaphragmatic hernia (CDH) are at high risk of developing pulmonary hypertension after birth, but little is known of their physiological transition at birth. We aimed to...

Genetic profile of isolated congenital diaphragmatic hernia revealed by targeted next-generation sequencing.

Congenital diaphragmatic hernia (CDH) is characterized by a defective closure of the diaphragm occurring as an isolated defect in 60% of cases. Lung size, liver herniation, and pulmonary circulation are major prognostic indices. Isolated CDH genetics is heterogeneous and poorly understood. Whether genetic lesions are also outcome determinants has never been explored. To identify isolated CDH genetic causes, to fine map the mutational burden, and to search for a correlation between the genotype and the disease severity and outcome. Targeted massively parallel sequencing of 143 human and mouse CDH causative and candidate genes in a cohort of 120 fetuses with isolated CDH and detailed outcome measures. Pathogenic and likely pathogenic variants were identified in 10% of the cohort. These variants affect both known CDH causative genes, namely, ZFPM2, GATA4, and NR2F2, and new genes, namely, TBX1, TBX5, GATA5, and PBX1. In addition, mutation burden analysis identified LBR, CTBP2, NSD1, MMP14, MYOD1, and EYA1 as candidate genes with enrichment in rare but predicted deleterious variants. No obvious correlation between the genotype and the phenotype or short-term outcome has been found. Targeted resequencing identifies a genetic cause in 10% of isolated CDH and identifies new candidate genes.

The role of FREM2 and FRAS1 in the development of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) has been reported twice in individuals with a clinical diagnosis of Fraser syndrome, a genetic disorder that can be caused by recessive mutations affecting FREM2 and FRAS1. In the extracellular matrix, FREM2 and FRAS1 form a self-stabilizing complex with FREM1, a protein whose deficiency causes sac CDH in humans and mice. By sequencing FREM2 and FRAS1 in a CDH cohort, and searching online databases, we identified five individuals who carried recessive or double heterozygous, putatively deleterious variants in these genes which may represent susceptibility alleles. Three of these alleles were significantly enriched in our CDH cohort compared with ethnically matched controls. We subsequently demonstrated that 8% of Frem2ne/ne and 1% of Fras1Q1263*/Q1263* mice develop the same type of anterior sac CDH seen in FREM1-deficient mice. We went on to show that development of sac hernias in FREM1-deficient mice is preceded by failure of anterior mesothelial fold progression resulting in the persistence of an amuscular, poorly vascularized anterior diaphragm that is abnormally adherent to the underlying liver. Herniation occurs in the perinatal period when the expanding liver protrudes through this amuscular region of the anterior diaphragm that is juxtaposed to areas of muscular diaphragm. Based on these data, we conclude that deficiency of FREM2, and possibly FRAS1, are associated with an increased risk of developing CDH and that loss of the FREM1/FREM2/FRAS1 complex, or its function, leads to anterior sac CDH development through its effects on mesothelial fold progression.