| | Molecular Basis of Hirschsprung’s Disease and Other Congenital Enteric NeuropathiesThe enteric nervous system (ENS) is a complex neural network that controls many essential functions in the gastrointestinal tract, including the regulation of intestinal peristalsis. Neural crest cells migrate, proliferate, and differentiate within the intestinal wall to give rise to the neurons and glial cells of the ENS. Failure of this process to occur normally during embryogenesis can lead to severe disorders of intestinal motility, the most common of which is Hirschsprung’s disease. Several proteins have been identified as being essential for normal ENS development, including members of the Ret and endothelin-3 signaling pathways. The analysis of transgenic mice harboring mutations in these critical genes has greatly enhanced our understanding of the molecular regulation of ENS development and of the etiology of intestinal aganglionosis. Building on this fundamental research, much has been learned recently about the genetics underlying the complex inheritance pattern of Hirschsprung’s disease. Continued progress in elucidating the molecular basis of normal and abnormal ENS development will greatly enhance our understanding of congenital enteric neuropathies and improve our ability to diagnose and treat children affected by these disabling conditions.
The enteric nervous system (ENS) comprises a complex neural network whose primary role is to regulate smooth muscle contractility in the gut to promote the propagation of nutrients along the gastrointestinal (GI) tract.1 The ENS is the largest component of the autonomic nervous system, containing more neurotransmitters and neuronal subtypes than any other part of the peripheral nervous system.2 It is estimated that at least 40 neuronal subtypes exist in the ENS.3 Among its most interesting features is that the ENS is able to regulate bowel function entirely in the absence of central nervous system input, a fact that, together with its complexity, has led to the ENS being termed “the second brain.”2 As one would expect from a system this complex, developmental anomalies occur. When the ENS is absent, as it is in Hirschsprung’s disease4 (HSCR), normal motility cannot occur and intestinal pseudoobstruction develops. Abnormal development of the ENS is also the underlying cause of other forms of congenital intestinal dysmotility, including intestinal neuronal dysplasia5 (IND) and various types of intestinal pseudoobstruction.6
Congenital neuromuscular disorders of the intestine are common and debilitating. Unfortunately, our current level of understanding of these conditions is limited by our rudimentary understanding of the ENS. If we are to understand the pathophysiology of neurointestinal disease in children and ultimately improve our ability to diagnose and treat these conditions, then we must first understand their etiology at the molecular and cellular levels. This review will summarize what is currently known about the development of the ENS, the principal molecular pathways essential for normal ENS formation, and the genetic basis of Hirschsprung’s disease and other congenital enteric neuropathies. Only when we have a thorough understanding of the underlying molecular and developmental etiology of these conditions will we be able to accurately classify patients based on their specific molecular and morphological pathology, develop precise clinical–pathological correlation, and ultimately lay the groundwork for improved treatment of these conditions.
Origin of the Enteric Nervous System  Most of what is known regarding development of the ENS is based on extensive experimental work done primarily in avian and rodent embryos. Neural crest cells (NCC) arise from the dorsal aspect of the neural tube. From there they migrate throughout the embryo to form a variety of structures, including the peripheral nervous system, pigment cells, bones of the head and face, and the cardiac outflow tract.7, 8 The cells that form the ENS leave the vagal region of the neural crest, migrate to the foregut, and then proceed in a proximal-to-distal wave of migration to colonize the entire gastrointestinal tract.9, 10 In the human embryo, this migration begins around week 4 of gestation, culminating with the arrival of NCC in the terminal colon by week 7.11 A smaller contribution of cells also arises from the sacral neural crest. These cells begin at the distal end of the gut and migrate in a proximal direction to contribute neurons and glial cells mostly to the large intestine.9, 10, 12, 13 During their migration along the gut, NCC undergo extensive cell proliferation to give rise to the large numbers of cells required to populate the entire gut. As they complete their journey, the cells cluster to form aggregates of cell bodies. These clusters, called ganglia, comprise ganglion cells, referring to the neuronal cell bodies, and enteroglial cells. The ganglia form two concentric rings on either side of the circular smooth muscle of the bowel wall, an inner submucosal (Meissner’s) plexus and an outer myenteric (Auerbach’s) plexus (Fig. 1). Once they reach their target location, the cells undergo differentiation into neurons and glia to form the mature ENS. Each of these critical events—migration, proliferation, and differentiation—relies on specific molecular signals expressed by migrating neural crest cells and by the intestinal microenvironment. A multitude of questions arises when one considers the complexity of forming an ENS. For instance, what drives the continued migration of neural crest cells along the gut? Why do some cells stop migrating at a particular location while the rest continue along as if destined to some distal target? What drives sacral crest-derived cells to migrate in the opposite direction to vagal crest-derived cells? What regulates the patterning of two perfectly concentric rings of ganglia within the gut wall? A recent explosion of interest in the ENS has brought answers to many of these questions, although much remains unknown. Molecular Regulation of Enteric Nervous System Development Endothelin-3 Pathway Genetic mutations in mice have revealed most of what is known about the molecular signals that control ENS formation (Table 1).14 Initial progress on understanding the molecular basis of ENS development began with the discovery of two naturally occurring mouse mutations. These mouse strains were referred to as “lethal spotted”15 and “piebald lethal”16 because of their abnormal pigmentation, a reflection of disordered development of melanocytes, another cell type derived from the neural crest. Both mutant strains exhibit congenital megacolon due to distal intestinal aganglionosis that is very reminiscent of human HSCR. Later experiments revealed that the “lethal spotted” mouse has a mutation in the gene encoding endothelin-3 (ET3),17 while “piebald lethal” has a mutation in the receptor to ET3, called endothelin receptor B (EDNRB).16 | | |  | Gene | Gene Product | Homozygous Phenotype⁎ | Heterozygous Phenotype⁎ | Refs. | |
|---|
| Ret | Receptor tyrosine kinase (co-receptor for GDNF) | AG of small and large intestine | Normal | 32, 35 | |
| GDNF | Glial-cell line derived neurotrophic factor (ligand for Ret) | AG of small and large intestine | Hypoganglionosis | 31, 32, 33, 40 | |
| GFRα1 | GDNF family receptor alpha-1 (co-receptor for GDNF) | AG of small and large intestine | Normal | 33, 34 | |
| ET3 | Endothelin-3 | Distal colon AG and pigmentation defects | Normal | 15, 17 | |
| EDNRB | Endothelin-B receptor | Distal colon AG and pigmentation defects | Hyperganglionosis in submucosal plexus (IND-like) | 16, 28 | |
| ECE1 | Endothelin converting enzyme-1 | Distal colon AG and pigmentation defects | Normal | 25 | |
| Sox10 | Transcription factor | Embryonic lethal | Distal colon AG and pigmentation defects | 44, 45, 46, 86 | |
| Phox2b | Transcription factor | Total intestinal AG | | 72 | |
| Mash1 | Transcription factor | AG of esophagus | | 87 | | | | |
|
⁎
Phenotypes vary in these models. The most common phenotype seen is described. |
A critical component of normal ENS development is the communication between crest-derived cells and their intestinal environment. ET3 is a 21-amino-acid peptide expressed in the gut wall, while its receptor, EDNRB, is present on the NCC. Absence of either gene in mice leads to aganglionosis limited to the distal colon.16, 17 While this suggests an essential role for ET3 signaling in colorectal ENS formation, its mechanism of action remains unclear. The most commonly held hypothesis suggests that the role of ET3 is to maintain a pool of NCC precursors by inhibiting their differentiation into neurons18, 19, 20, 88 and promoting their continued proliferation18, 20, 21, 22, 88 loss of ET3 activity would, therefore, both decrease the number of neural crest cells available and allow the precursor cells to become neurons prematurely. Whereas undifferentiated precursor NCC are capable of migrating, once those cells terminally differentiate into neurons they are postmitotic and unable to migrate any farther, explaining the absence of ganglia in the distal intestines of ET3 and EDNRB mutant animals.20, 23 ET3-EDNRB signaling also has an indirect effect on NCC through its influence on differentiation of mesenchymal cells in the gut wall. EDNRB is expressed by the intestinal smooth muscle, where it promotes smooth muscle cell differentiation. This in turn leads to a loss of laminin-1 expression within the gut wall.20 Laminin-1 normally promotes neuronal development.24 The loss of ET3-EDNRB activity would thereby lead to increased laminin-1 and an environment promoting neuronal differentiation. Additional evidence for the importance of ET3 signaling comes from mice deficient in endothelin converting enzyme-1 (ECE-1), the protease that cleaves ET3 from its larger precursor form. These mice exhibit features typical of ET3 and EDNRB mutant mice, including melanocyte abnormalities and intestinal aganglionosis.25 Aside from its role in NCC proliferation and differentiation, the ET3-EDNRB signaling pathway has other effects on the ENS that were recently identified. One intriguing effect is that ET3 inhibits the chemoattraction of NCC to glial cell line-derived neurotrophic factor (GDNF).22, 26, 88 As discussed in further detail below, GDNF is chemoattractive to migrating NCC,27 promoting their continued migration along the GI tract. However, GDNF is strongly expressed at the junction of the small and large intestine in mouse27 and chick88 embryos, leaving one to wonder how NCC are able to migrate past this strong GDNF-expressing zone. Interestingly, ET3 is also strongly expressed at that location.28, 88 By inhibiting the chemoattraction to GDNF, the presence of ET3 may allow NCC to migrate past the GDNF-expressing region and into the large intestine. In summary, ET3-EDNRB signaling influences crest cell proliferation, differentiation, and migration to promote complete colonization of the entire intestinal tract. Consequently, in the absence of this signaling pathway, the distal intestine remains devoid of ganglion cells. Mice harboring null mutations in genes of the ET3 pathway exhibit a contracted distal colon with markedly distended intestine proximal to the transition zone. The extent of aganglionosis varies among animals, ranging from the most distal 2 to 3 mm of rectum to the entire colon, although rectosigmoid transition zones are most commonly found.16, 17 Unlike these homozygous mutants, mice harboring heterozygous mutations for EDNRB exhibit ENS findings similar to IND, with very large and more numerous ganglia in the submucosal plexus.29 This observation demonstrates that different levels of gene expression can produce a spectrum of ENS abnormalities and suggests that HSCR and IND may be phenotypic variants of a similar underlying enteric neuropathy. Ret Pathway The most important genetic pathway in the pathogenesis of HSCR is the GDNF-Ret signaling pathway. GNDF is a neurotrophic factor expressed in the intestinal wall. It binds to a receptor complex, present on enteric NCC, which consists of the Ret receptor and GDNF family receptor-α1 (GFRα1). Null mutations of any of these three genes in mice result in the absence of enteric ganglia throughout the small and large intestine.30, 31, 32, 33, 34, 35, 36 The role of GDNF signaling supports the survival and proliferation of neuronal precursors in the gut.19, 22, 37, 38 By so doing, GDNF ensures that sufficient precursor cells are available to populate the entire intestine. If GDNF or its receptors are absent, then only enough precursor cells are present to populate the esophagus and stomach. GDNF signaling also stimulates the differentiation of migrating and proliferating neural crest cells into nonmigrating and nonproliferating neurons. This action may seem contradictory to its role in ensuring an adequate pool of precursor cells. However, the role of GDNF appears to change over time. Early during intestinal development, GDNF promotes the survival and proliferation of precursor NCC, while later it supports the development of neurons.19, 38 The early action of GDNF thereby ensures the production of a large pool of cells. Once that pool has been created and migration is well underway, the cells can then differentiate as they reach their target. To ensure that differentiation occurs at the proper time and location in the bowel wall, this pro-differentiation action of GDNF is modulated by ET3, which inhibits neuronal differentiation.19 This molecular interaction creates a critical balance that regulates the development of terminally differentiated neurons in the gut while maintaining an adequate pool of precursor cells to continue colonizing the bowel. In addition to its effects on NCC survival, proliferation, and differentiation, GDNF is also a potent chemoattractive factor for enteric NCC, promoting the migration of those cells along the length of the gut.27 The expression of GDNF is highly regulated in a spatial and temporal manner such that it is most highly expressed in the intestinal mesenchyme just ahead of the migrating wavefront of NCC, serving to attract those cells toward it.39 As mentioned above, ET3 modulates this action of GDNF, inhibiting its ability to attract NCC, a role that seems particularly important as NCC reach the colon.22, 26, 88 Complete absence of GDNF-Ret signaling in mice leads to total intestinal aganglionosis from the stomach to the rectum.30, 31, 32 Animals heterozygous for a GDNF null mutation, where one allele of the normal GDNF gene is still present, display severe intestinal hypoganglionosis,40 again demonstrating, as in the case of ET3 mutants, that gene dosage plays a role in determining the severity of the phenotype. Another neurotrophic factor closely related to GDNF, neurturin, can also activate the Ret receptor. Mouse mutations in this gene result in hypoganglionosis specifically affecting the myenteric plexus.41, 42 Similar to these mouse models, patients with HSCR often have a region of hypoganglionosis in the transition zone and sometimes exhibit IND proximal to the aganglionic segment.43 This clinical observation, together with the available mouse models, suggests that these enteric neuropathies may share common underlying developmental and molecular abnormalities. Sox10 Dominant megacolon (Dom) refers to a mouse strain that arose from a spontaneous mutation and exhibits colonic aganglionosis and pigmentation defects.44 The responsible mutation was found to be in the Sox10 gene, a transcription factor expressed in the neural crest and in migrating ENS precursors.45, 46 Why mutations in Sox10 lead to colonic aganglionosis is not entirely understood. However, expression of EDNRB is disrupted in the ENS of Dom mice.46 Recent analysis of the EDNRB gene revealed an enhancer element of the gene that is activated as NCC reach the colon. Mutations in this regulatory region of EDNRB lead to varying degrees of colonic aganglionosis. Interestingly, that enhancer element has several binding sites for Sox10, positioning this transcriptional factor as a regulator of EDNRB expression and possibly explaining its mechanism of action.47 Genetic Mutations in Human HSCR HSCR occurs in approximately 1 in 5000 live births. The condition is characterized by the absence of ganglion cells along a variable length of the distal intestine. In about 80% of cases, the aganglionic segment is limited to the rectosigmoid, a condition referred to as short-segment HSCR (S-HSCR). In the remaining 20% the aganglionosis extends further proximally and is referred to as long-segment HSCR (L-HSCR).48 HSCR is clearly a hereditary disease. However, the pattern of inheritance is not a simple Mendelian one. One of the interesting hallmarks of this complex genetic disorder is that the recurrence risk within a family ranges from 1 to 33% and that risk depends on gender and the length of aganglionosis.48, 49 For example, the risk for HSCR is 1% for a female sibling of an affected male with short-segment disease, while it is 33% for a male sibling of a female patient with long-segment disease.50 Genetic analyses have identified the involvement of multiple genes (Table 2). If a mutation in any one of these genes were sufficient to account for the disease, we would expect the same phenotype among members of a single family. However, this appears not to be the case for HSCR. Even in large affected families any particular mutation has low penetrance and the resulting phenotype is highly variable. As will be discussed below, this complex pattern of inheritance appears to be due to the segregation of multiple genetic mutations. The phenotype observed is thus the result of the cumulative effects of mutations in more than one gene. Consequently, the reduced penetrance of a given mutation and the high variability of disease expression (ie, the presence and length of aganglionosis) are not random events, but rather depend on an individual’s genetic background. Since multiple factors influence the occurrence and phenotypic expression of HSCR, genes associated with HSCR are best considered “susceptibility genes,” since a given mutation simply increases the odds of having the disease while not being predictive of the abnormality.49 | | | | Gene | Gene Product | Clinical Phenotype | % of Cases⁎ | Refs. | |
|---|
| Ret | Receptor tyrosine kinase (co-receptor for GDNF) | ns-HSCR | 50% familial15–35% sporadic | 48, 50, 57, 58, 59 | |
| GDNF | Glial-cell line derived neurotrophic factor (ligand for Ret) | ns-HSCR | Rare | 1, 62 | |
| Neurturin | Ligand for Ret | ns-HSCR | 1 case | 63 | |
| ET3 | Endothelin-3 | SW and ns-HSCR | <5% | 57 | |
| EDNRB | Endothelin-B receptor | SW and ns-HSCR | 5% | 48, 49, 50, 64, 65, 66 | |
| ECE1 | Endothelin converting enzyme-1 | Syndrome of craniofacial, cardiac, and autonomic defects | Rare | 78 | |
| Sox10 | Transcription factor | SW | Rare | 69 | |
| Phox2b | Transcription factor | Haddad syndrome | Rare | 70 | |
| Sip1 | Transcription factor | Mowat–Wilson syndrome | Rare | 73, 74, 75 | |
| 9q31 | Genetic locus | L-HSCR | | 54 | |
| 3p21 | Genetic locus | S-HSCR | | 55 | |
| 19q12 | Genetic locus | S-HSCR | | 55 | | | | |
|
⁎
Percent of HSCR cases with identifiable gene mutation. |
To understand the complex genetics of HSCR, patients can be divided into syndromic and nonsyndromic cases. The latter group includes both sporadic and familial forms. The majority of HSCR patients have the sporadic form of the disease, which tends to be S-HSCR. Nonsyndromic familial cases account for 10 to 15% of cases and are usually L-HSCR.51 Syndromic HSCR comprises a minority of cases and describes patients with aganglionosis associated with multiple extra-intestinal abnormalities. These patients include those with Down syndrome, which make up 2 to 8% of all HSCR,49 and several other syndromes that are discussed below. The first mutations identified in HSCR patients were in the Ret gene52, 53 and this is clearly the dominant gene in HSCR.54, 55, 56 Since that time, several large multigenerational families have been studied, most of whom are inbred Mennonite kindreds harboring a high risk for the disease. All reported families with HSCR, except one, are linked to the Ret locus.54 In 50% of these families a specific mutation in the Ret coding sequence can be identified,48 whereas in sporadic S-HSCR Ret mutations are identified in less than 35%,50, 57, 58, 59 leaving the genetic cause of the much more common S-HSCR poorly understood. Gabriel and coworkers55 tackled this issue by scanning the genomes of 49 families with S-HSCR. They found three susceptibility loci that cosegregated in affected individuals. These loci were found to be both necessary and sufficient to account for the risk of HSCR in these families. One locus encodes the Ret gene (located at 10q11) while the other two chromosomal loci, 3p21 and 19q12, involve unidentified genes that appear to function by modifying the expression of Ret. This important study gives strong evidence for multigenic inheritance in HSCR. It also emphasizes the importance of noncoding Ret mutations in S-HSCR, which may contribute to the lower penetrance in S-HSCR as compared with L-HSCR. These findings help to explain why the inheritance pattern of S-HSCR is non-Mendelian.55, 60 In a search for other susceptibility loci, Bolk and coworkers54 studied 12 HSCR families, most with L-HSCR, and identified a susceptibility allele at 9q31. As expected, these families had a very high rate of linkage to the Ret locus, with 50% having an identifiable mutation. Interestingly, the association of HSCR with the 9q31 locus occurred specifically in those families that, despite the linkage, lacked an identifiable Ret mutation.54 One could hypothesize that these noncoding mutations likely decrease, but do not eliminate, Ret function. As in the case of S-HSCR, such hypomorphic Ret mutations may account for the incomplete genetic penetrance and variable phenotypic expression observed. The 9q31 allele seems to work together with these “weak” Ret mutations to modify penetrance and increase the risk of HSCR in these families. Based on this new susceptibility allele, these investigators reanalyzed their data on S-HSCR55 (discussed above) and found no significant association of disease with the 9q31 locus in those patients. However, when they specifically looked at S-HSCR families with “mild” Ret alleles (ie, noncoding mutations), an association with 9q31 was identified, although the effect of this Ret modifier was much weaker than in familial L-HSCR.55 Several known genes have been found to harbor mutations in patients with nonsyndromic HSCR. Mutations in GDNF, the ligand for Ret, have been identified in rare instances. These GDNF mutations, however, may not be sufficient to induce HSCR since most patients also have Ret mutations.61, 62 Similarly, a mutation has been identified in neurturin, another Ret ligand with strong homology to GDNF. This family also had a Ret mutation.63 GNDF and neurturin are further examples of susceptibility genes since the disease only occurs in the presence of additional mutations. Heterozygous EDNRB mutations have been found in about 5% of patients with nonsyndromic HSCR,48, 49, 50, 64, 65, 66 while mutations in the ligand, ET3, are much less common.57 Studying multiple Mennonite families, Carrasquillo and coworkers67 found a significant association between the transmissions of two susceptibility loci to affected patients. One locus is a noncoding sequence variant in the Ret gene and the other is a coding mutation in the EDNRB gene. Joint transmission of these two alleles in affected individuals suggests a genetic interaction between Ret and EDNRB in the etiology of HSCR. Interestingly, a functional interaction between these genes has been demonstrated in mice, where the combination of both mutations leads to dramatically more severe aganglionosis than either mutation alone.67, 68 Several conclusions can be drawn from these studies. First, Ret mutations, either coding or noncoding, are central to all cases of nonsyndromic HSCR. The single reported family with no linkage to Ret54 is likely due to the failure to detect segregation of an allele when both parents are homozygous for a common noncoding sequence variant.56 Second, strong evidence exists for the multigenic inheritance of HSCR. Multiple genes may be necessary to modulate the expression of the Ret protein. While a severe Ret mutation (one in the coding sequence) may be sufficient to cause HSCR, weaker (ie, noncoding) mutations require the effects of those additional gene mutations to produce the disease.54, 55, 56 This would account for the low penetrance of hypomorphic Ret alleles, since in the absence of additional mutations the phenotype will be normal. The identity of the genes at 3p21, 19q12, and 9q31 is currently unknown. They are likely to encode genes that modulate Ret expression or that are critical to a particular aspect of ENS development. An important piece of this genetic puzzle was recently filled by Emison and coworkers56 who wanted to understand the nature of the mutation in the >50% of HSCR patients with Ret-linkage but no identifiable mutation. They studied 126 HSCR patients and their parents, focusing on families with Ret linkage, but no coding sequence mutation. The authors identified a common noncoding sequence variant in an enhancer-like element in the first intron of the Ret gene. This sequence polymorphism is highly associated with HSCR, has low penetrance, and can account for the failure to find Ret coding mutations in nearly all cases. Moreover, the mutation decreases enhancer function in vitro, yielding functional support for its relevance. These results further support multigenic inheritance in HSCR and demonstrate the importance of common noncoding sequence variants in the inheritance of complex genetic diseases. Syndromic HSCR Shah–Waardenburg’s syndrome or Waardenburg’s syndrome type 4 represents the association of pigmentation abnormalities, sensorineural deafness, and HSCR. Homozygous mutations in the EDNRB or ET3 genes have been identified in patients with this syndrome,48, 49 yielding a phenotype reminiscent of mice harboring these mutations.15, 16 Other individuals with Shah–Waardenburg’s syndrome have been found to have mutations in the transcription factor Sox10.69 Unlike EDNRB/ET3, Sox10 mutations are generally inherited in an autosomal-dominant pattern and exhibit high penetrance.48 The association of pigmentary defects with aganglionosis in this syndrome is similar to the Dom mouse mutant, which also harbors a Sox10 mutation.45, 46 Congenital central hypoventilation syndrome, or Ondine’s curse, is a rare and serious disorder where involuntary ventilatory drive is lost, especially during sleep. The condition is associated with other abnormalities of the autonomic nervous system, including HSCR and neuroblastoma.70 HSCR is present in about 15 to 20% of cases, a combination referred to as Haddad syndrome.71 The causative gene for Ondine’s curse is the transcription factor, Phox2b,70 a protein expressed by enteric neurons. Mice lacking Phox2b exhibit near-total aganglionosis, except for the presence of ganglion cells in the esophagus,72 a phenotype very similar to that of Ret-null mice.30, 31, 32 Interestingly, Phox2b is necessary for the expression of Ret, likely accounting for this phenotype and possibly explaining the association of HSCR and Ondine’s curse.72 HSCR has been found in patients with mental retardation, microcephaly, and dysmorphic facial features, an association referred to as Mowat–Wilson syndrome.48 Mutations have been identified in these patients in Smad interacting protein-1 (Sip1), a transcriptional repressor involved in signaling by the TGF-β family of proteins.73, 74, 75 The precise role of Sip1 in ENS development is unknown. However, among the TGF-β family of signaling molecules, bone morphogenetic proteins (BMPs) play an important role in the ENS, where they regulate migration, ganglion formation, and neuronal development.76, 77 Sip1 may function by regulating BMP activity or another Smad-dependent pathway yet to be identified. The fourth major syndromic form of HSCR involves the endothelin pathway. Endothelins are 21-amino-acid proteins generated by the proteolytic cleavage of preproendothelins. In the case of ET3, the responsible protease is endothelin-converting enzyme-1 (ECE-1). Homozygous mutations in this gene in the mouse lead to cardiac and craniofacial abnormalities as well as colonic aganglionosis.25 A mutation in this gene was identified in a patient with HSCR, cardiac and craniofacial abnormalities, and autonomic dysfunction.78 Molecular Basis of Other Neurointestinal Diseases Chronic intestinal pseudoobstruction in children can be due to abnormalities of the muscles or nerves of the gut. Neuropathic causes include abnormalities of neuronal or glial cell morphology or density, as well as rare disorders of neuronal cytology, such as ganglion cell inclusion bodies.3 Recent characterization of several rodent models has provided information regarding the molecular basis of some of these enteric neuropathies, although most forms of severe dysmotility remain poorly understood. IND is an often discussed and very controversial entity, primarily because of disagreement regarding its existence and/or clinicopathologic criteria for its diagnosis.79 IND type B, which comprises about 98% of described cases, presents in infants or older children as severe constipation. It can occur alone or proximal to the aganglionic segment in a patient with HSCR.43 Diagnostic criteria vary but include hyperplasia of the submucosal plexus, the presence of “giant ganglia” (>7 ganglion cells per ganglion), and ectopic ganglia in the lamina propria.3, 80 Homozygous deletion of the Hox11L1 gene (also called Enx, Ncx, and Tlx2) in mice results in a phenotype of severe intestinal pseudoobstruction.81, 82 These animals develop abdominal distension with severe and lethal megacolon. Histologic analysis of the ENS shows significantly more numerous and larger ganglia than normal in the myenteric plexus. The authors suggest that these mice can serve as models of IND. Unfortunately, the morphology of the submucosal plexus, on which the diagnosis of IND is based, is not described in these mice. Hox11L1 is a transcription factor expressed in enteric neurons. Mutations of this gene have not been identified in humans. Nonetheless, understanding the mechanism by which Hox11L1 controls density of ganglia in the ENS, and how hyperganglionosis leads to pseudoobstruction, will yield valuable information for understanding these problems in humans. The heterozygous EDNRB-deficient rat may be a better model of IND since the colons of these animals demonstrate larger and denser ganglia in the submucosal plexus.29 However, while the histology is reminiscent of IND, the rats show no clinical evidence of functional intestinal obstruction and develop entirely normally. A search for mutations in the Ret, GDNF, EDNRB, and ET3 genes in 20 patients with IND failed to uncover any abnormalities in these HSCR-related genes in humans.57 Another animal model of intestinal pseudoobstruction is the fmc (familial megacecum and colon) rat, a spontaneous mutant line that exhibits severe abdominal distension, with massive dilation of the cecum and proximal colon, similar to HSCR.83 Intestinal motility is delayed, but the ENS and intestinal muscle layers are grossly normal. Mice overexpressing the Hoxa4 gene also display congenital megacolon.84 In these transgenic mice the terminal colon is hypoganglionic and has ganglia that look more like extraenteric peripheral nerves than enteric ganglia. Further characterization of these models may shed important light on the etiology of intestinal pseudoobstruction. Hypoganglionosis is rarely diagnosed in clinical practice, principally because the normal density of ganglia varies greatly and no standards have been established. Small series and case reports have been published attributing hypoganglionosis as the cause of colonic dysmotility.85 Several animal models exhibit hypoganglionosis, although no genetic cause has been identified in humans. As discussed above, mice heterozygous for mutations in the GDNF gene display hypoganglionosis.40 Similarly, heterozygous Sox10 mutant mice display a variety of phenotypes, ranging from distal colonic aganglionosis to severe hypoganglionosis.86 The coexistence of aganglionosis with proximal hypoganglionosis in the transition zone is well recognized clinically. The fact that mutations in the same gene can lead to both phenotypes, partly depending on gene dosage, lends a molecular basis to that clinical observation. Conclusions Tremendous progress has been made in understanding the development of the ENS, with the identification of many genes critical in this process. Based on that knowledge, primarily generated through the use of transgenic animal models, we have been able to identify many of the genetic causes of HSCR. This has proven to be a complex genetic disorder that has become a paradigm for understanding multigenic diseases. Despite our progress in the genetics of HSCR, virtually nothing is known regarding the molecular and genetic causes of the many other types of neurointestinal diseases. We can be confident that the coming years will bring continued growth in our understanding of the molecular basis of ENS development and the pathophysiology of congenital enteric neuropathies. That fundamental knowledge will be essential as we aim to improve our ability to diagnose and treat children with these chronic and disabling conditions. References 1.
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Department of Pediatric Surgery, Massachusetts General Hospital, Boston, MA; and Harvard Medical School, Boston, MA.  Address reprint requests to: Allan M. Goldstein, MD, Massachusetts General Hospital, Warren 1153, Boston, MA 02114.
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