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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Ann Med. Author manuscript; available in PMC 2008 January 4.
Published in final edited form as:
PMCID: PMC2174621

Molecular genetics of congenital diaphragmatic defects


Congenital diaphragmatic hernia (CDH) is a severe birth defect that is accompanied by malformations of the lung, heart, testis, and other organs. Patients with CDH may have any combination of these extradiaphragmatic defects, suggesting that CDH is often a manifestation of a global embryopathy. This review highlights recent advances in human and mouse genetics that have led to the identification of genes involved in CDH. These include genes for transcription factors, molecules involved in cell migration, and extracellular matrix components. The expression patterns of these genes in the developing embryo suggest that mesenchymal cell function is compromised in the diaphragm and other affected organs in patients with CDH. We discuss potential mechanisms underlying the seemingly random combination of diaphragmatic, pulmonary, cardiovascular, and gonadal defects in these patients.

Keywords: birth defects, diaphragmatic eventration, embryonic development, hernia, diaphragmatic, heart defects, congenital, sex reversal, gonadal

1. Introduction

Congenital diaphragmatic hernia (CDH) is a severe developmental abnormality that affects 1 per 3000 live births (1,2). The hallmark of the disorder is a defect in the muscular or tendinous portion of the diaphragm that allows abdominal viscera to protrude into the thoracic cavity. CDH is often accompanied by malformations of the lungs, heart, and/or other organs (3). The incidence of these extradiaphragmatic birth defects ranges from 35 percent to 60 percent in newborns with CDH and may be even greater in affected stillborns (1-4). Despite advances in surgical and medical management, the neonatal mortality rate of CDH remains significant, owing in part to concomitant birth defects (3).

Most cases of CDH are presumed to reflect a combination of genetic predisposition and environmental factors, but the etiopathogenesis of this disorder is poorly understood (4-6). This review focuses on recent advances in human and mouse genetics that have led to the identification of genes involved in CDH. For a discussion of potential environmental triggers of CDH, such as vitamin A deficiency or teratogens, the reader is referred to other review articles (7,8).

2. CDH as a global embryopathy

In both humans and animal models, mutation of a single gene can cause primary development defects in not only the diaphragm but also the lung, heart, testis, and other organs (Table 1) (9-11). In humans, CDH has been described as a part of known genetic syndromes (4). Thus, CDH and associated birth defects are manifestations of a global embryopathy reflecting disruption of fundamental cellular processes during organogenesis. For practical reasons, surgeons and clinical geneticists generally categorize individual cases of CDH as “isolated” or “complex.” The term “isolated”, however, tends to understate the pervasive nature of the embryopathy, because cases of “isolated” CDH are often accompanied by primary defects in lung development.

Table 1
Birth defects associated with CDH

A. Diaphragmatic defects

The primordial diaphragm forms through amalgamation of four embryonic structures: the septum transversum, esophageal mesentery, pleuroperitoneal folds (PPF), and body wall (Fig 1A) (12). Each of these four tissues is derived from mesoderm, as are the mesenchymal progenitors of several other organs, including the heart, lungs, and liver [e.g., mesenchymal cells in the septum transversum contribute to not only the central tendon of the diaphragm but also liver sinusoidal cells and certain cardiac cell types (Fig 1B)]. Fusion of the PPF with adjacent structures leads to closure of the pleuroperitoneal canals at week 5−8 in humans [corresponding to embryonic day (E)12.5-E13.5 in mice]. Subsequently the PPF is invaded by muscle precursors derived from the lateral dermomyotome of cervical somites.

Figure 1
A) Embryonic structures contributing to the primordial diaphragm. The diaphragm forms through amalgamation of the septum transversum, foregut mesentery, pleuroperitoneal folds, and body wall. Abbreviations: IVC (inferior vena cava), PPF (pleuroperitoneal ...

Several anatomical defects fall under the rubric of CDH (3). Bochdalek hernias, involving the posterolateral region of the diaphragm, account for the majority of cases (Fig 1C). This type of CDH is thought to reflect failure of fusion of the PPF with adjacent structures. Less common forms of CDH include the Morgagni hernia, which affects the retrosternal diaphragm, and the central-type hernias, involving the central tendon. Other malformations grouped with CDH include diaphragmatic agenesis and eventration (the abnormal elevation of the diaphragm due to paralysis or aberrant muscularization). Most cases of CDH, however, are presumed to result from malformation or dysfunction of the amuscular mesenchymal component of the primordial diaphragm rather than a primary defect in myogenesis (13).

B. Pulmonary defects

Lung abnormalities, such as hypoplasia, retarded acinar development, emphysematous changes, or excessive muscularization of peripheral pulmonary arteries, are evident in most patients with CDH (14-16). Traditionally it was assumed that these lung defects were solely the result of external compression by herniating abdominal viscera, but it is now apparent that the pulmonary manifestations in CDH often reflect intrinsic defects in lung development. In experimental models, pulmonary hypoplasia precedes the appearance of diaphragmatic hernias, implying that there are primary disturbances in the lung developmental program (17,18). Postmortem analysis of the lungs of infants with unilateral CDH reveals reduced numbers of bronchi, alveoli, and arteries in both the ipsilateral and contralateral lungs (19). Finally, genes implicated in development of the primordial diaphragm are often co-expressed in mesenchymal cells of the embryonic lung (Fig 2A,B). Consequently, mutations impairing diaphragm development may have independent effects on lung morphogenesis, a process that requires coordinated interactions between endoderm-derived airway epithelial cells and mesoderm-derived mesenchymal cells (20).

Figure 2
Genes implicated in diaphragm morphogenesis are expressed concomitantly in mesenchymal cells of the lungs and heart

C. Cardiovascular defects

A wide variety of cardiac anomalies have been associated with CDH, including atrial septal defects (ASD), ventricular septal defects (VSD), atrioventricular canal (AV canal), tetralogy of Fallot, univentricular anatomy, truncus arteriosus, and other malformations (Fig 3A-C) (16). Some of the defects are plausibly related to a defect in the field of mesenchymal cells that contribute to the heart and diaphragm. For example, the proepicardium, which arises from the septum transversum, contributes cells to the atrioventricular cushion (21), thus suggesting a common developmental basis for endocardial cushion defects and CDH. Abnormalities of the great vessels have also been linked to CDH; these include stenosis of the aorta or pulmonary arteries, aortic aneurysm, aortic tortuosity, and anomalous pulmonary venous return (Fig 3A, ,4A4A).

Figure 3
A) Partial anomalous pulmonary venous return in a patient with CDH and a complete, unbalanced AV canal defect. In this angiogram contrast was injected into the pulmonary circulation via a Blalock-Taussig shunt. Levophase shows blood returning from the ...
Figure 4
Examples of extradiaphragmatic defects associated with CDH in humans and mice with known gene defects

D. Other birth defects

Like the diaphragm and heart, the liver is derived in part from septum transversum mesenchyme, and hepatic lesions, such as hemangioendotheliomas and hamartomas, have been observed in patients with CDH (22,23). Interestingly, CDH has also been linked to cryptorchidism and 46XY pseudohermaphroditism (24-27), which are presumed to reflect intrinsic defects in testicular somatic cell differentiation, a process that involves mesenchymal cell migration from adjoining mesonephros (28). CDH may also be accompanied by developmental eye defects (e.g., microphthamia) (29), spinal anomalies (30), or limb-reduction defects (31).

3. Gene mutations that impair mesenchymal cell function

In an effort to explain the association of diaphragmatic and extradiaphragmatic defects, the mesenchymal hit hypothesis has been proposed (17,32-34). This hypothesis posits that: 1) similar signaling pathways are involved in the differentiation of mesenchymal cells in all of the affected organs; and 2) the function of mesenchymal cells in the affected organs is disrupted by genetic or environmental triggers. Consequently, a major goal of CDH research is to characterize genes and signaling pathways critical for early mesenchymal cell function during morphogenesis of the diaphragm, lungs, cardiovascular system, and testes.

Genes involved in CDH have been identified through the analysis of recurrent chromosomal abnormalities in patients with CDH and the characterization of mutant mice. The genes identified so far encode proteins that fall into two broad groups: A) transcriptional regulators, and B) factors involved in cell migration, mesodermal patterning, or components of the extracellular matrix (ECM; Table 2).

Table 2
Genes implicated in CDH in humans or mice

A. Transcription factors

Retrospective surveys suggest that germline mutations in transcription factor genes account for only a small percentage of cases of CDH in humans (35,36); nevertheless, these cases highlight the pivotal role of transcriptional regulation in the pathogenesis of CDH. In mice, homozygous mutations in certain transcription factor genes elicit CDH and other anomalies that result in embryonic lethality. In humans, transcription factor haploinsufficiency is an established cause of CDH, especially in cases with concomitant extradiaphragmatic birth defects. The identified transcription factors directly or indirectly regulate expression of genes critical for mesenchymal cell function in the diaphragmatic substratum as well as the lungs, heart, and testis. Deficiency of these transcription factors leads to impaired structural integrity, increased apoptosis, and anomalous cell sorting in the tissues.

1. WT1

The product of the Wilms tumor suppressor gene, WT1, is a zinc finger transcription factor expressed in the amuscular diaphragm, pleural/abdominal mesothelial cells, epicardium, testicular somatic cells, and in the developing kidney (37-39). Heterozygous loss-of-function mutations in WT1 cause a series of overlapping clinical syndromes [e.g., WAGR syndrome (Wilms tumor-Aniridia-Genitourinary anomalies-Mental Retardation), Denys-Drash syndrome, Frasier syndrome, and Meacham syndrome]. Clinical features of these syndromes include genitourinary (GU) abnormalities (e.g., male pseudohermaphroditism), diaphragmatic defects, and cardiac malformations (40-42). Mice homozygous for Wt1 deletion mutations die between E13 and E15. In addition to major GU malformations, Wt1–/– mice exhibit Bochdalek-type diaphragmatic hernias and cardiac anomalies (abnormally developed epicardium with perturbations of the coronary vessels) (37-39,43).

2. FOG-2

Friend of GATA-2 (FOG-2) is another zinc finger transcription factor that is expressed in embryonic diaphragm, lung mesenchyme, epicardium, myocardium, and testicular somatic cells [see Fig 2A and (44,45)]. The human FOG2 (ZFPM2) gene resides on chromosome 8q23, and rearrangements of 8q22−23 have been observed in patients with CDH (46-48). Patients who are heterozygous for FOG2 loss-of-function point mutations are predisposed to diaphragm anomalies (eventration), pulmonary hypoplasia, and/or cardiac malformations (36,49). Fog2–/– mice, generated through either targeted or chemical mutagenesis, recapitulate this spectrum of anomalies [eventration, hypoplasia of the right middle and accessory lobes of the lung, cardiac malformations (including aberrant coronary vessels), and male-to-female sex reversal (36,50,51)].

3. GATA-4

This zinc finger transcription factor, which interacts with FOG-2 during embryonic development, has also been implicated in CDH and associated anomalies. The human GATA4 gene is found on chromosome 8p23.1, and microdeletion of this region is a recurring chromosomal abnormality in patients with CDH (52-56). Like FOG-2 and WT-1, GATA-4 is expressed in mesenchymal cells of the developing diaphragm, lungs, and heart [see Fig 2B and (57)] and in testicular somatic cells (45). Gata4–/– mice die by E9 from defects in ventral morphogenesis and heart tube fusion, precluding studies of diaphragm development in these mice (58,59). However, C57Bl/6 mice heterozygous for a Gata4 deletion mutation are predisposed to midline diaphragmatic hernias, dilated distal airways, thickened pulmonary mesenchyme and valvuloseptal cardiac malformations [see Fig 4B-D and (57)]. Such defects are not seen in Gata4 heterozygotes maintained on mixed genetic backgrounds (58,59), suggesting that strain-specific modifier genes impact penetrance. Confirming the importance of GATA4-FOG2 interactions to normal organogenesis, mice homozygous for a Gata4 missense mutation that disrupts GATA4-FOG2 interactions develop pulmonary hypoplasia, cardiac malformations, and male-to-female sex reversal (51,60,61). Cardiac malformations have been reported in patients heterozygous for GATA4 loss-of-function mutations (62-64); whether point mutations or small deletions in the GATA4 gene can cause CDH or primary pulmonary defects in humans is unknown.


Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is a nuclear orphan receptor expressed during embryonic development in a variety of tissues, including mesodermal derivatives in the diaphragm, lungs, and heart (65). In CDH patients a minimally deleted region has been identified on human chromosome 15q26.1−26.2 (65-68). COUP-TFII (NR2F2) is one of the four known genes residing within this critical region, suggesting that COUP-TFII deficiency contributes to the formation of CDH in individuals with 15q deletions. Supporting this premise, Cre-lox mediated mutagenesis of the Coup-TFII gene in the foregut mesenchyme and PPF of mice has been shown to cause Bochdalek-type CDH (65). Coup-TFII null mice die by E10.5 with cardiac malformations and impaired angiogenesis (69), while Coup-TFII+/– mice and have impaired gonadal somatic cell function (70). Interestingly, COUP-TFII has been shown to physically interact with FOG-2 (71), implying that these two factors may cooperate in diaphragm morphogenesis.

5. bHLH factors

Basic helix-loop-helix (bHLH) transcription factors have been implicated in diaphragm development in mice. One of these transcription factors, capsulin (POD1, epicardin), is expressed in branchial muscle, the primordial diaphragm, and in mesenchymal cells of the heart, kidney, lung, gut, spleen, and testis (72). In myoblasts and other progenitor cells, capsulin appears to function as a negative regulator of differentiation by acting as a transcriptional repressor (73). Mice homozygous for a capsulin null mutation die shortly after birth due to pulmonary and cardiac defects; these animals also exhibit asplenia, male-to-female sex reversal, and renal anomalies (74,75). Interestingly, mice homozygous deficient for both capsulin and the related bHLH transcription factor, MyoR, lack facial musculature and have CDH (76). Mice homozygous deficient for another bHLH factor, MyoD, exhibit abnormal muscularization of the diaphragm on a dystrophin-deficient (mdx) background (77). No mutation in Capsulin, MyoR, or MyoD has been reported in a patient with CDH.

6. Retinoic acid receptors

Disruptions in the retinoid signaling pathway during embryogenesis may contribute to the pathogenesis of CDH (7,8). Retinoic acid receptor (RAR)-α,-β2 double mutant mice exhibit CDH and heart and lung abnormalities, a constellation of defects also seen in vitamin A deficient animals (78). Similarly, teratogens that disrupt retinoid acid production, such as nitrofen, cause CDH and primary lung defects in rodents (7,8). Mutations in STRA6, the membrane receptor that mediates cellular uptake of vitamin A (79), cause a spectrum of anomalies including CDH, congenital heart malformations, lung hypoplasia, and anophthalmia (80).

B. Factors involved in cell-cell signaling, mesodermal patterning, or ECM biosynthesis

Proper cell sorting and guidance is critical for normal organ development. Loss-of-function mutations in genes involved in cell migration and mesodermal patterning have been shown to cause diaphragmatic and extradiaphragmatic defects in humans and animal models. These genes encode growth/guidance factors, their cognate receptors, and components of the ECM, such as elastic fibers, which serve structural functions and bind growth/differentiation factors.

1. HGF signaling pathway

Hepatocyte growth factor (HGF) is produced in the PPF, lung mesenchyme, and other mesenchymal cell types. Its receptor tyrosine kinase, c-Met, is expressed in myoblast precursors and in certain epithelial cells. In Hgf–/– or cMet–/– mouse embryos the diaphragmatic substratum forms normally, but its muscularization is impaired due to a defect in myoblast migration (81,82). Similarly, mouse embryos deficient in Grab1, a docking protein that binds to phosphorylated c-Met and facilitates intracellular signaling, lack diaphragmatic musculature (83). HGF-signaling has also been linked to testicular differentiation in the mouse (84). In Fog2–/– mice, there is impaired expression of HGF mRNA in the PPF, suggesting that Hgf may be one of the target genes for this transcription factor in the developing diaphragm (36). It is unclear whether mutations in the genes encoding HGF, c-Met, and Grab1 cause CDH or testicular defects in humans.

2. Slit2 and Robo1

An example of a ligand-receptor pair involved in cell migration is Slit3 and its receptor, Robo1. Slit proteins are secreted molecules that serve to repel axons and direct mesodermal cell migration. Slit3 is expressed in the primordial diaphragm and other tissues, and a majority of Slit3–/– mice develop central-type diaphragmatic hernias, cardiac defects, and renal anomalies (85,86). Robo1 encodes a surface transmembrane protein found in developing brain, lung, heart, liver, muscle, and kidney (87). Robo1 transcripts are alternatively spliced in a tissue-specific fashion (87). Most Robo1–/– mice die at birth of respiratory failure, and histological examination of newborn lung reveals abnormal mesenchymal cellularity (88). A subset of the homozygotes also exhibit CDH. Recent studies suggest that heparan sulfate is an integral component of the Slit-Robo signaling complex (89). Interestingly, mutations in a heparan sulfate proteoglycan, glypican-3, cause CDH in humans (see below). To date, neither a SLIT3 nor ROBO1 mutation has been detected in a patient with CDH. However, duplication of the distal region of chromosome 11q, which includes the ROBO3 and ROBO4 genes, is associated with CDH in humans (90).

3. Ephrin-B1

Eph receptor tyrosine kinases and their ligands, ephrins, play a critical role in cell sorting during a number of developmental processes, including diaphragm morphogenesis (91). Loss-of-function mutations in the ephrin-B1 (EFNB1) gene cause craniofrontonasal syndrome (CFNS), an X-linked dominant disorder in which the phenotype is paradoxically more severe in females than in males (92,93). In addition to CDH, female patients with CFNS exhibit craniosynostosis, frontonasal dysplasia, and limb anomalies (94). Such birth defects are also seen in female Efnb1+/– mice. Due to X-inactivation, EFNB1+/– females are mosaic for cells that express the mutant allele. The presence of pools of cells that are either Ephrin-B1-positive or Ephrin-B-negative is thought to disrupt normal tissue boundary formation and impair sorting of invading receptor-positive cells (95,96).

4. Glypican-3

CDH is one of the manifestations of Simpson-Golabi-Behmel syndrome (SGBS), which, like CFNS, is an X-linked disorder that exhibits semidominant inheritance. Along with CDH, patients with SGBS exhibit macrosomia, abnormal facial features, skeletal abnormalities, cardiac malformations, and a predisposition to Wilms tumor and other embryonal tumors of childhood (97,98). SGBS is caused by loss-of-function mutations in the glypican-3 (GPC3) gene, which encodes a heparan sulfate proteoglycan that is bound to the cell surface through a glycosyl-phosphatidylinositol anchor. Glypican-3 binds and thereby regulates the activity of a wide array of growth factors (e.g., fibroblast growth factors, bone morphogeneic proteins), growth factor antagonists (e.g., Noggin), and guidance molecules (e.g., Slit-Robo complex) that play a critical role in morphogenesis (89,99,100).

5. Components of the Notch signaling pathway

Diaphragmatic hernias also occur in patients with spondylocostal dysostosis (SCD), a syndrome marked by aberrant mesodermal patterning, caused by defects in the Notch signaling pathway (101-104). Notch signaling mediates short range interactions involved in cell fate decision and boundary formation. Notch proteins are cell surface receptors that function as membrane-tethered transcription factors. In response to ligand binding, Notch receptors undergo proteolytic processing, allowing the transcription factor domain to translocate to the nucleus. SCD is caused by recessive mutations in the genes for Delta like-3 (a Notch ligand), Lunatic Fringe (a glycosyltransferase that modifies and thereby regulates Notch receptors), and MESP2 (a downstream transcription factor) (105-107). Mutations in another gene implicated in the Notch signaling, NIPBL, cause Cornelia de Lange syndrome, a dominantly inherited developmental disorder characterized by CDH, growth and mental retardation, facial anomalies, limb defects, cardiac malformations, and GU abnormalities (108).

6. Components of the ECM

Impaired formation of the ECM, caused by disruptions in either collagen or elastic fibers, can lead to developmental defects in a wide range of organs, including the diaphragm, lungs, heart, and great vessels.

a) Elastin

Mutations in the human elastin (ELN) gene cause supravalvular aortic stenosis (109) or autosomal dominant cutis laxa (110), a syndrome characterized by systemic connective-tissue abnormalities, including excess inelastic skin, vascular tortuosity, aortic aneurysm, joint laxity, CDH, and emphysematous lungs. Similarly, mice harboring homozygous mutations in the Eln gene develop lethal obstructive arterial disease, distal airway dilatation, and other emphysematous changes (111,112).

b) Collagen

Ehlers-Danlos syndrome (EDS) includes a group of connective tissue disorders characterized by abnormal collagen metabolism. Clinical manifestations of EDS include skin hyperextensibility, joint laxity, CDH, and other anomalies. There are several subtypes of EDS which differ in terms of the severity of skin and joint findings, involvement of other tissues, and mode of inheritance. CDH has been linked to several of these subtypes, including Type IV EDS, an autosomal dominant condition characterized by pulmonary arterial stenoses, systemic arterial tortuosity, and aneurysms (113,114). Type IV EDS is caused by mutations in COL3A1 (type III collagen), which is expressed during embryonic development in the skin, vessel walls, heart, and other organs (113,114).

c) Crosslinking enzymes

The crosslinking of collagens and elastin, which is critical for structural integrity of the ECM, is catalyzed by lysyl oxidase (Lox), an extracellular cuproenzyme. Lox–/– mice die perinatally of aortic aneurysms and other cardiovascular abnormalities. These animals also exhibit central-type diaphragmatic hernias (115-117). No human LOX mutations have been reported; however, mutation of the human ATP7A gene, which encodes a the α-subunit of a Cu2+-transporter required for biosynthesis of the Lox holoenzyme, causes Menkes disease, a lethal X-linked disorder marked by skin laxity, vascular tortuosity, aortic aneurysms, and CDH (118).

d) Fibulins

The fibulins are a family of six extracellular proteins that contain series of calcium-binding EGF-like modules and a distinctive C-terminal domain. These proteins serve to link elastic fibers to the surface of cells. Mutations in human Fibulin-4 (FBLN4) cause autosomal recessive cutis laxa with vascular tortuosity, aortic aneurysm, joint laxity, CDH, and emphysematous lungs (Fig 4A) (119). Fbln4–/– mice die as neonates and exhibit dilated distal airways and severe vascular defects, such as aortic tortuosity and aneurysm (120). Mutations in human Fibulin-5 (FBLN5) also cause recessive cutis laxa syndrome. These patients develop early emphysema and supravalvular aortic stenosis (121), and Fbln5–/– mice have a corresponding phenotype (122).

e) Fibrillin

Another ECM protein implicated in CDH is fibrillin, a major component of microfibrils in both elastic and nonelastic connective tissue. Mutations in the fibrillin-1 gene (FBN1) cause Marfan syndrome, an autosomal dominant condition characterized by aortic root dilatation, tall stature, kyphoscoliosis, ocular abnormalities, joint laxity, myxomatous mitral valve defects, and congenital diaphragmatic eventration (123,124). Congenital distal airway dilatation is seen in about 10 percent of patients with Marfan syndrome, and mice that make low amounts of fibrillin-1 develop aortic root abnormalities and have emphysema-like lung abnormalities at birth (125). In the lungs, aorta, and mitral valves of these mice there is evidence of excess accumulation of the active form of transforming growth factor-β (TGF-β). Fibrillin-1 deficiency appears to alter the matrix sequestration of the large latent form of TGF-β, and this renders the cytokine more prone to activation. Remarkably, the lung, aortic, and mitral valve defects in fibrillin-1 deficient mice can be prevented by administration of either a TGF-β neutralizing antibody or the angiotensin II type 1 receptor antagonist, losartan (126,127). This demonstrates that unraveling the molecular mechanism of a gene defect can lead to a pharmacological intervention that can, in a mouse model, prevent developmental anomalies associated with CDH, namely distal airway dilatation and valvular defects.

4. Mechanisms underlying the ostensibly random combination of diaphragmatic and extradiaphragmatic defects in patients with CDH

As noted above, CDH and related birth defects occur in random combinations, but the mechanistic basis for this phenotypic variability has remained an enigma. Recent studies suggest that bimodal gene expression, triggered by haploinsufficiency for transcription factors or signaling molecules, may underlie this phenomenon.

Transcriptional regulation is commonly viewed as a “rheostatic” process in which promoter activity increases proportionally in response to rising levels of a particular transcriptional activator (128-130). Under the rheostatic model, transcription factor haploinsufficiency leads to uniform reductions in target gene levels in all mesenchymal cells. An alternative to this model is the binary model of transcriptional activation (128-130). This model implies that genes exist in either an “on” or an “off” state and that transcription factors regulate the probability of a gene occupying either state. Accordingly, transcription factor haploinsufficiency impacts the bimodal distribution of cells that either express or do not express a target gene.

The accumulation of a separate population of “non-expressing” mesenchymal cells in sensitive areas of a particular organ could trigger a focal structural abnormality, such as a diaphragmatic hernia or valvuloseptal cardiac defect. Alternatively, the presence of expressing and non-expressing cells might impair morphogenesis by disrupting signaling molecules that influence cell migration. Indeed, the bimodal population of expressing and non-expressing cells in EFNB1+/– females (95) is reminiscent of the situation encountered under the binary model of transcription factor haploinsufficiency. Similarly, bimodal expression of an anti-apoptotic factor could be the direct pathogenic mechanism of CDH and related defects. Gata4 heterozygosity has been associated with a small but significant increase in the number of apoptotic cells in the stressed heart and in the developing diaphragm of the mouse (57,131). One can imagine that the increased incidence yield a higher probability of a cluster of cells in the diaphragm or heart that all die, leading to a structural defect.

Another potential cause of bimodal gene expression is somatic mutagenesis. Recently, investigators have detected somatic mutations in GATA4 and other “cardiac” transcription factors in the hearts of patients who died of congenital heart disease (132). Thus, somatic mutations that arise during cardiogenesis may be a novel molecular cause of congenital heart disease, and it is conceivable that somatic mutations in transcription factor genes contribute to the pathogenesis of CDH.

5. Future directions

Over the past decade basic research on CDH has been dominated by a teratogen-induced model of CDH (exposure of fetal rodents to nitrofen). Although the nitrofen model has improved our understanding of the early anatomical derangements associated with CDH and highlighted the importance of retinoic acid signaling in diaphragm morphogenesis (7,8), this model has provided limited insight into the genetic basis for CDH. We are entering a new era in CDH research; the characterization of chromosomal abnormalities in patients with CDH and the analysis of mutant mice have improved our understanding of the pathophysiology of this serious embryopathy.

One challenge for the future is to elucidate the hierarchy of the genes implicated in CDH [e.g., decreased expression of HGF mRNA in Fog2–/– mice (36)]. Another challenge is to determine how environmental triggers, such as teratogen exposure or vitamin A deficiency, interact with gene mutations to cause CDH. Based on comparisons of PPF abnormalities in nitrofen-treated rats, vitamin A deficient rats, and Wt1−/− mice, it has been proposed that teratogen-induced, dietary, and genetic models of congenital diaphragmatic hernia share a common mechanism of pathogenesis (43).

Once the factors and pathways involved in CDH and related anomalies are delineated, it may be possible to design rational therapeutic interventions. Recent experiments showing that developmental defects of the lung and heart in a mouse model of Marfan syndrome can be prevented with anti-TGF-β therapies offer hope that we can identify transplacental therapies to prevent (or lessen the degree of) developmental defects in the diaphragm and other organs.


We thank Dr. Debra Rita for providing the CT scan. This research was supported by NIH DK52574, Mallinckrodt Foundation, Finnish Pediatric Research Foundation, and the Juselius Foundation. PYJ is a Scholar of the Child Health Research Center of Excellence in Developmental Biology at Washington University School of Medicine (K12 HD001487).


atrial septal defect
atrioventricular canal
basic helix-loop-helix
congenital diaphragmatic hernia
craniofrontonasal syndrome
chicken ovalbumin upstream promoter-transcription factor II
embryonic day
extracellular matrix
Ehlers-Danlos syndrome
epidermal growth factor
friend of GATA
hepatocyte growth factor
lysyl oxidase
pleuroperitoneal folds
retinoic acid receptor
spondylocostal dysostosis
transforming growth factor-β
ventricular septal defect


Key messages

• Genetic triggers of CDH can disrupt mesenchymal cell function in not only the primordial diaphragm but also the developing lungs, heart, great vessels, gastrointestinal tract, and testes.

• CDH and associated birth defects can result from mutations in genes encoding transcription factors, molecules involved in cell migration, and extracellular matrix components.

• Bimodal gene expression may underlie the ostensibly random combination of diaphragmatic, pulmonary, cardiovascular, and gonadal defects in patients with CDH.

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