Fryns syndrome without diaphragmatic hernia. Report on a new case and review of the literature

Fryns syndrome is an autosomal recessive multiple congenital anomaly syndrome characterized by coarse facies, diaphragmatic hernia, distal limb hypoplasia and malformations of the cardiovascular, gastrointestinal, genitourinary and central nervous systems. Diaphragmatic hernia is a leading diagnostic feature in Fryns syndrome, recorded in more than 80% of cases. We report a newborn with clinical features of Fryns syndrome except the diaphragmatic hernia. Cases of Fryns syndrome without diaphragmatic hernia are reviewed. Even in the absence of diaphragmatic hernia, pulmonary anomalies are described in Fryns syndrome, especially pulmonary hypoplasia. Fetal mice, exposed to nitrofen, have a high incidence of congenital diaphragmatic hernia and other malformations similar to that seen in Fryns syndrome. Nitrofen might target molecular mechanisms similar to those involved in Fryns syndrome.     

Congenital diaphragmatic hernia, kidney agenesis and cardiac defects associated with Slit3-deficiency in mice

Slit3 along with Slit1 and Slit2 comprise the Slit family of proteins. The latter two proteins are known to be involved in axon guidance and cell migration during animal development. However, little is know about the functions of Slit3. We created a Slit3-deficient mouse model from an OmniBank ES cell line with a Slit3 allele trapped by insertional mutagenesis to analyze the in vivo functions of this protein. In this model, congenital diaphragmatic hernia is the most obvious phenotype. Herniation was found to be caused by a defective central tendon (CT) of the diaphragm that remained fused with the liver. Electron microscopic analyses of the defective CT revealed disorganized collagen fibrils that failed to form tight collagen bundles. The hearts of Slit3-deficient mice have an enlarged right ventricle. In addition, 20% of homozygous mice also showed a range of kidney defects that include unilateral or bilateral agenesis of the kidney and ureter, or varying degrees of renal hypoplasia. Thus, we concluded that Slit3 is involved in the development of multiple organ systems that include the diaphragm and the kidney. Slit3-deficient mice represent a genetic animal model for physiological and pathological studies of congenital diaphragmatic hernia.     

Linking animal models to human congenital diaphragmatic hernia

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a major life-threatening malformation, occurring in approximately 1 in 3,000 live births. Over the years, different animal models have been used to gain insight into the etiology of this complex congenital anomaly and to develop treatment strategies. However, to date the pathogenic mechanism is still not understood, and treatment remains difficult because of the associated pulmonary hypoplasia and pulmonary hypertension. METHODS: In this review, data available from several animal models will be discussed. The retinoic acid signaling pathway (RA pathway, retinoid pathway) will be addressed as a developmental pathway that is potentially disrupted in the pathogenesis of CDH. Furthermore, genetic factors involved in diaphragm and lung development will be discussed. CONCLUSIONS: With this review article, we aim to provide a concise overview of the current most important experimental genetic data available in the field of CDH.     

An infant with diaphragmatic hernia, anophthalmia and cardiac defect: evaluation by magnetic resonance imaging autopsy

We present an infant with diaphragmatic hernia, anophthalmia and cardiac defect evaluated by magnetic resonance imaging (MRI) autopsy. This female infant was born at 39th weeks by vaginal delivery and presented with diaphragmatic hernia, anophthalmia, cardiac defect and died due to respiratory problems at 28th hours of life. MRI autopsy showed internal organ abnormalities including congenital hernia of the left diaphragm, secondary hypoplasia of the left lung, atrial and ventricular septal defect, dilatation of calices of the kidneys, bilateral anophthalmia, hypoplasia of the optic nerves, hyperintensity of pituitary gland possibly due to bleeding and a cyst of the septum pellucidum. This article shows that MRI autopsy is a valuable method for the evaluation of cases with congenital anomalies if autopsy is not possible.     

Congenital diaphragmatic hernia: A retinoid‐signaling pathway disruption during lung development?

Congenital diaphragmatic hernia (CDH) usually occurs sporadically. The prognosis remains poor, with a 50% perinatal mortality rate. Most deaths result from hypoxemia due to lung hypoplasia and abnormal development of pulmonary vasculature that results in persistent pulmonary hypertension. Our current understanding of the pathogenesis of CDH is based on an assumption linking herniation of abdominal viscera into the thorax with compression of the developing lung. Pulmonary hypoplasia, however, can also result from reduced distension of the developing lung secondary to impaired fetal breathing movements. Moreover, a nitrofen-induced CDH model shows that lung hypoplasia precedes the diaphragmatic defect, leading to a "dual-hit hypothesis." Recent data reveal the role of a retinoid-signaling pathway disruption in the pathogenesis of CDH. We describe the clinical and epidemiological aspects of human CDH, the metabolic and molecular aspects of the retinoid-signaling pathway, and the implications of retinoids in the development of the diaphragm and the lung. Finally, we highlight the existing links between CDH and disruption of the retinoid-signaling pathway, which may suggest an eventual use of retinoids in the treatment of CDH.     

Defective pulmonary innervation and autonomic imbalance in congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is associated with significant mortality due to lung hypoplasia and pulmonary hypertension. The role of embryonic pulmonary innervation in normal lung development and lung maldevelopment in CDH has not been defined. We hypothesize that developmental defects of intrapulmonary innervation, in particular autonomic innervation, occur in CDH. This abnormal embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH. To define patterns of pulmonary innervation in CDH, human CDH and control lung autopsy specimens were stained with the pan-neural marker S-100. To further characterize patterns of overall and autonomic pulmonary innervation during lung development in CDH, the murine nitrofen model of CDH was utilized. Immunostaining for protein gene product 9.5 (a pan-neuronal marker), tyrosine hydroxylase (a sympathetic marker), vesicular acetylcholine transporter (a parasympathetic marker), or VIP (a parasympathetic marker) was performed on lung whole mounts and analyzed via confocal microscopy and three-dimensional reconstruction. Peribronchial and perivascular neuronal staining pattern is less complex in human CDH than control lung. In mice, protein gene product 9.5 staining reveals less complex neuronal branching and decreased neural tissue in nitrofen-treated lungs from embryonic day 12.5 to 16.5 compared with controls. Furthermore, nitrofen-treated embryonic lungs exhibited altered autonomic innervation, with a relative increase in sympathetic nerve staining and a decrease in parasympathetic nerve staining compared with controls. These results suggest a primary defect in pulmonary neural developmental in CDH, resulting in less complex neural innervation and autonomic imbalance. Defective embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH.     

Computed tomography in the diagnosis of pathological states of the lumbar diaphragm. 1: general data. Developmental defects and traumatic disorders]

CT was employed for investigation of 94 patients with pathological changes of the diaphragm. Congenital defects and unilateral aplasia of the diaphragm were observed in 5 of them. A new symptom of a pathological line of the diaphragm was recognized, characteristic for this type of patients. Teratodermoid formations with a typical CT picture were found in 3 patients. A tumor growth source was undetectable by CT. The results of investigation of 5 patients with traumatic diaphragmatic hernia have shown no particular advantages of CT over traditional radiation methods. In one case, a traumatic diaphragmatic cyst was correctly diagnosed by CT. CT was shown to be a method of choice in the diagnosis of congenital and traumatic diaphragmatic lesions.     

Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development

The teratogen nitrofen produces a congenital diaphragmatic hernia (CDH) and pulmonary hypoplasia in rodent fetuses that closely parallel observations made in humans. We hypothesized that these changes may be due to primary pulmonary hypoplasia and not herniation of the abdominal contents. Timed-pregnant rats were given nitrofen on day 9, and fetuses were harvested on days 13 through 21. Initial evagination of lung buds on gestational day 11 was not delayed in nitrofen-treated fetuses. On gestational day 13, however, there was a significant decrease in the number of terminal end buds in the lungs of nitrofen-exposed fetuses vs. controls. Thymidine-labeled lung epithelial and mesenchymal cells were significantly decreased in nitrofen-treated lungs. Lungs from nitrofen-treated fetuses exhibited wide septae with disorganized, compacted tissue, particularly around the air spaces. Expression of surfactant protein B and C mRNAs was significantly decreased in the nitrofen litters. In situ hybridization of fetal lung tissue at all gestational ages showed no difference in the expression of vascular endothelial growth factor, Flk-1, or Flt-1 mRNAs. Because closure of the diaphragm is completed on gestational day 16 in the rat, our results suggest that lung hypoplasia in this model of CDH is due at least in part to a primary effect of nitrofen on the developing lung.     

Gene expression in the developing diaphragm: significance for congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a frequently occurring birth defect and a source of potentially fatal neonatal respiratory distress. Recently, through the application of detailed karyotyping methods, several CDH-critical regions within the human genome have been identified. These regions typically contain several genes. Here we focused on genes from 15q26, the best-characterized CDH-critical region, as well as FOG2 and GATA4, genes singled out from CDH-critical regions at 8q22-8q23 and 8p23.1, respectively. We tested the hypothesis that these putative CDH-related genes are expressed within the developing diaphragm at the time of the hypothesized initial defect. Our results show that 15q26 contains a cluster of genes that are expressed in the developing rodent diaphragm, consistent with an association between deletions in this region and CDH. We then examined the protein expression pattern of positively identified genes within the developing diaphragm. Two major themes emerged. First, those factors strongly associated with CDH are expressed only in the nonmuscular, mesenchymal component of the diaphragm, supporting the hypothesis that CDH has its origins in a mesenchymal defect. Second, these factors are all coexpressed in the same cells. This suggests that cases of CDH with unique genetic etiology may lead to a common defect in these cells and supports the hypothesis that these factors may be members of a common pathway. This study is the first to provide a detailed examination of how genes associated with CDH are expressed in the developing diaphragm and provides an important foundation for understanding how the deletion of specific genes may contribute to abnormal diaphragm formation.     

Gonadal agenesis in a 46,XY female with multiple malformations and positive testing for the sex-determining region of the Y chromosome.

A full-term 46,XY female newborn presented with respiratory failure due to a right-sided diaphragmatic hernia. During surgical repair, exploration revealed isolated dextrocardia and hypoplasia of the right lung. Neither gonads nor wolffian or müllerian structures could be palpated. Cardiac catheterization demonstrated defects of the ventricular septum, hypoplasia of the right pulmonary artery, persistence of the left vena cava superior and a patent ductus arteriosus. Anthropometric data were normal at birth, but fell below the 3rd percentile during follow-up. Body proportions displayed a predominance of the upper compared to the lower segment. Endocrine studies indicated no defect of steroid biosynthesis and no functional gonadal tissue. Using genetic analyses of various loci within the testis-determining region of the Y chromosome, a mutation could not be detected. The patient died from pneumonia at the age of 19 months. Postmortem examination confirmed the diagnosis of gonadal agenesis.     

Pathogenesis of nitrofen-induced congenital diaphragmatic hernia in fetal rats

Allan, Douglas W., and John J. Greer. Pathogenesis ofnitrofen-induced congenital diaphragmatic hernia in fetal rats. J. Appl. Physiol. 83(2): 338-347, 1997.Congenital diaphragmatic hernia (CDH) is a developmental anomalycharacterized by the malformation of the diaphragm and impaired lungdevelopment. In the present study, we tested several hypothesesregarding the pathogenesis of CDH, including those suggesting that theprimary defect is due to abnormal 1)lung development, 2) phrenic nerveformation, 3) developmentalprocesses underlying diaphragmatic myotube formation, 4) pleuroperitoneal canal closure,or 5) formation of the primordial diaphragm within the pleuroperitoneal fold. The2,4-dichloro-phenyl-p-nitrophenyl ether (nitrofen)-induced CDH rat model was used for thisstudy. The following parameters were compared between normal andherniated fetal rats at various stages of development:1) weight, protein, and DNA contentof lungs; 2) phrenic nerve diameter,axonal number, and motoneuron distribution;3) formation of the phrenic nerve intramuscular branching pattern and diaphragmatic myotube formation; and 4) formation of the precursor ofthe diaphragmatic musculature, the pleuroperitoneal fold. Wedemonstrated that previously proposed theories regarding the primaryrole of the lung, phrenic nerve, myotube formation, and the closure ofpleuroperitoneal canal in the pathogenesis of CDH are incorrect.Rather, the primary defect associated with CDH, at least in thenitrofen rat model, occurs at the earliest stage of diaphragmdevelopment, the formation of the pleuroperitoneal fold.

     

MiR-449a Affects Epithelial Proliferation during the Pseudoglandular and Canalicular Phases of Avian and Mammal Lung Development

Congenital diaphragmatic hernia is associated with pulmonary hypoplasia and respiratory distress, which result in high mortality and morbidity. Although several transgenic mouse models of lung hypoplasia exist, the role of miRNAs in this phenotype is incompletely characterized. In this study, we assessed microRNA expression levels during the pseudoglandular to canalicular phase transition of normal human fetal lung development. At this critical time, when the distal respiratory portion of the airways begins to form, microarray analysis showed that the most significantly differentially expressed miRNA was miR-449a. Prediction algorithms determined that N-myc is a target of miR-449a and identified the likely miR-449a:N-myc binding sites, confirmed by luciferase assays and targeted mutagenesis. Functional ex vivo knock-down in organ cultures of murine embryonic lungs, as well as in ovo overexpression in avian embryonic lungs, suggested a role for miR-449a in distal epithelial proliferation. Finally, miR-449a expression was found to be abnormal in rare pulmonary specimens of human fetuses with Congenital Diaphragmatic Hernia in the pseudoglandular or canalicular phase. This study confirms the conserved role of miR-449a for proper pulmonary organogenesis, supporting the delicate balance between expansion of progenitor cells and their terminal differentiation, and proposes the potential involvement of this miRNA in human pulmonary hypoplasia.     

Early development of the primordial mammalian diaphragm and cellular mechanisms of nitrofen‐induced congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a frequently occurring cause of neonatal respiratory distress and is associated with high mortality and long‐term morbidity. Evidence from animal models suggests that CDH has its origins in the malformation of the pleuroperitoneal fold (PPF), a key structure in embryonic diaphragm formation. The aims of this study were to characterize the embryogenesis of the PPF in rats and humans, and to determine the potential mechanism that leads to abnormal PPF development in the nitrofen model of CDH. Analysis of rat embryos, and archived human embryo sections, allowed the timeframe of PPF formation to be determined for both species, thus delineating a critical period of diaphragm development in relation to CDH. Experiments on nitrofen‐exposed NIH 3T3 cells in vitro led us to hypothesize that nitrofen might cause diaphragmatic hernia in vivo by two possible mechanisms: through decreased cell proliferation or by inducing apoptosis. Data from nitrofen‐exposed rat embryos indicates that the primary mechanism of nitrofen teratogenesis in the PPF is through decreased cell proliferation. This study provides novel insight into the embryogenesis of the PPF in rats and humans, and it indicates that impaired cell proliferation might contribute to abnormal diaphragm development in the nitrofen model of CDH. Birth Defects Research (Part A) 2010. © 2009 Wiley‐Liss, Inc.     

Structure of the primordial diaphragm and defects associated with nitrofen-induced CDH.

The goals of this study were to further our understanding of diaphragm embryogenesis and the pathogenesis of congenital diaphragmatic hernia (CDH). Past work suggests that the pleuroperitoneal fold (PPF) is the primary source of diaphragmatic musculature. Furthermore, defects associated with an animal model of CDH can be traced back to the formation of the PPF. This study was designed to elucidate the anatomic structure of the PPF and to determine which regions of the PPF malform in the well-established nitrofen model of CDH. This was achieved by producing three-dimensional renderings constructed from serial transverse sections of control and nitrofen-exposed rats at embryonic day 13.5. Renderings of left- and right-sided defects demonstrated that the malformations were always limited to the dorsolateral portions of the caudal regions of the PPF. These data provide an explanation of why the holes in diaphragmatic musculature associated with CDH are characteristically located in dorsolateral regions. Moreover, these data provide further evidence against the widely stated hypothesis that a failure of pleuroperitoneal canal closure underlies the pathogenesis of nitrofen-induced CDH.     

Diaphragm defects occur in a CDH hernia model independently of myogenesis and lung formation

Congenital diaphragmatic hernia (CDH) is a significant clinical problem in which a portion of the diaphragmatic musculature fails to form, resulting in a hole in the diaphragm. Here we use animal models of CDH to test two hypotheses regarding the pathogenesis. First, the origin of the defect results from the malformation of the amuscular mesenchymal component of the primordial diaphragm rather than with the process of myogenesis. Second, the defect in the primordial diaphragmatic tissue is not secondary to defects in the developing lung. In c-met(-/-) mouse embryos, in which diaphragm muscle fibers do not form because of a defect in muscle precursor migration, the amuscular substratum forms fully. We show that a defect characteristic of CDH can be induced in the amuscular membrane. In Fgf10(-/-) mouse embryos that have lung agenesis we show that the primordial diaphragm does not depend on signals from lung tissue for proper development and that diaphragmatic malformation is a primary defect in CDH. These data suggest that the pathogenesis of CDH involves mechanisms fundamentally different from previously proposed hypotheses.     

Pulmonary vascular development goes awry in congenital lung abnormalities

Pulmonary vascular diseases of the newborn comprise a wide range of pathological conditions with developmental abnormalities in the pulmonary vasculature. Clinically, pulmonary arterial hypertension (PH) is characterized by persistent increased resistance of the vasculature and abnormal vascular response. The classification of PH is primarily based on clinical parameters instead of morphology and distinguishes five groups of PH. Congenital lung anomalies, such as alveolar capillary dysplasia (ACD) and PH associated with congenital diaphragmatic hernia (CDH), but also bronchopulmonary dysplasia (BPD), are classified in group three. Clearly, tight and correct regulation of pulmonary vascular development is crucial for normal lung development. Human and animal model systems have increased our knowledge and make it possible to identify and characterize affected pathways and study pivotal genes. Understanding of the normal development of the pulmonary vasculature will give new insights in the origin of the spectrum of rare diseases, such as CDH, ACD, and BPD, which render a significant clinical problem in neonatal intensive care units around the world. In this review, we describe normal pulmonary vascular development, and focus on four diseases of the newborn in which abnormal pulmonary vascular development play a critical role in morbidity and mortality. In the future perspective, we indicate the lines of research that seem to be very promising for elucidating the molecular pathways involved in the origin of congenital pulmonary vascular disease. Birth Defects Research (Part C) 102:343–358, 2014. © 2014 Wiley Periodicals, Inc.