Special basic science review

Journal of Pediatric Surgery VOL 35, NO 7

JULY 2000

SPECIAL BASIC SCIENCE REVIEW

Pathogenesis of Hirschsprung’s Disease By Giuseppe Martucciello, Isabella Ceccherini, Margherita Lerone, and Vincenzo Jasonni Genoa, Italy

Hirschsprung’s disease is an inherited disorder showing incomplete penetrance and variable expressivity. Genetic mapping and mutation screening of candidate genes, together with the study of several natural and knockout animal models, clearly have shown the involvement of several different genes in the pathogenesis of Hirschsprung’s disease. Among these genes, the RET proto-oncogene accounts for the highest proportion of both familial and sporadic cases, with a wide range of mutations scattered along its entire coding region. The low detection rate of RET mutations in Hirschsprung patients also led to different hypotheses, such as the existence of additional Hirschsprung genes. Different animal and human genetic studies have identified 6 Hirschsprung genes: RET proto-oncogene (RET), endothelin 3 (EDN3), endothelin B receptor gene (EDNRB), glial-cell-line–

derived neurotrophic factor (GDNF ), endothelin converting enzyme (ECE1), gene encoding the Sry-related transcription factor SOX10 (SOX10). Microenvironmental factors also can play a role in the pathogenesis of aganglionosis. The developmental process of the crest-derived progenitor cells is sensitive to the level of different molecules. The expression deficit of different factors (GDNF, NTN) in the hindgut, in the absence of genetic mutations, could determine a missed activation of the receptor system, causing enteric neuroblast migration arrest. J Pediatr Surg 35:1017-1025. Copyright r 2000 by W.B. Saunders Company.

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early, it has been called one of the primary organs in the development of the vertebrate embryo. But it has only a temporary existence, as it cells are soon dispersed throughout the body, differentiating into many tissues.’’2 Although first identified over a century ago, the importance of the neural crest in vertebral development has gained more appreciation over the last 50 years and wide acceptance over the last 20 years.3 Of all described human congenital malformations, about one third involves structures related to the neural crest. The neural crest contributes to a vast amount of structures throughout the body.

IRSCHSPRUNG’S DISEASE (HSCR) is a developmental disorder of the enteric nervous system (ENS) characterized by absence of ganglion cells in the myenteric and submucosal plexuses along a variable portion of the distal intestine. The most widely accepted etiopathogenetic hypothesis is based on a defect of craniocaudal migration of neuroblasts originating from the neural crest (NC) that, under normal circumstances, reach the small intestine in the seventh week of gestation and the rectum in the 12th week.1 The clinical forms with variable extension of the aganglionic segment could be interpreted as interruptions of the migration process in different gestational periods. The earlier is the migration arrest, the longer is the distal aganglionic intestinal portion. THE NEURAL CREST AND THE NEUROCRISTOPATHIES

The ganglion cells of the ENS derive from the NC, which is a very peculiar structure. ‘‘As it originates so Journal of Pediatric Surgery, Vol 35, No 7 (July), 2000: pp 1017-1025

INDEX WORDS: Hirschsprung’s disease, genetic mapping, gene mutation.

From the Departments of Pediatric Surgery and Molecular Genetics, G. Gaslini Children’s Hospital, University of Genoa, Italy. Address reprint requests to Giuseppe Martucciello, MD, Department of Pediatric Surgery, Giannina Gaslini Children’s Hospital, University of Genoa, Largo G. Gaslini, 5 16147 Genoa, Italy. Copyright r 2000 by W.B. Saunders Company 0022-3468/00/3507-0001$03.00/0 doi:10.1053/js.2000.7763 1017

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NC cells from all axial levels appear morphologically similar; however, they follow migratory pathways that depend largely on their rostro-caudal level of origin. On completion of migration, they differentiate into diverse cell types, including neurons and glia of the sensory, sympathetic, and parasympathetic ganglia, neuroendocrine cells, adrenal medulla, pigmented cells, and facial cartilage (Fig 1).4 Diseases arising from NC are particularly diverse in clinical presentation, including endocrinologic, cutaneous, neurological, digestive, or other syndromes.5 For this constellation of conditions, in 1974 Bolande6 suggested the name neurocristopathies. In his historic publication, the author chose to divide neural crest diseases into 2 basic forms. The first includes the simple neurocristopathies, which are characterized by a single pathological process, generally unifocal and localized. The second is represented by neurocristopathy syndromes and complex neurocristopathies that correspond to multifocal and varied associations of simple neurocristopathies6 (Table 1). Taking into account the embryological origin of enteric ganglion cells from NC lines, HSCR therefore was classified as a simple neurocristopathy. The vagal neural crest generally is considered to be the source of both neurons and supportive cells of the enteric nervous system along the digestive tract.7 Apart from this, the vagal neural crest also originates cardiac ganglia8 and is involved in the development of cardiac outflow tract,9 thymic stromal cells,10 and the parathyroids.11 Using ablation experiments, Besson et al12 showed that

MARTUCCIELLO ET AL

Table 1. The Neurocristopathies According to Bolande Classification6 (1981) Simple neurocristopathies Dysgenetic Hirschsprung’s disease Albinism Mandibulofacial dysostosis Otocephaly Neoplastic Neuroblastoma Pheochromocitoma Medullary thyroid carcinoma (MTC only) Noncromaffin paraganglioma Carcinoid tumors Complex neurocristopathies Neurofibromatosis (von Recklinghausen disease) Multiple endocrine neoplsia (MEN) type 1 MEN2A MEN2B Neurocutaneous melanosis Familial neuroblastoma with Hirschsprung’s disease

the size and the location of the ablations influenced both the incidence and the type of cardiac defects. Using similar ablation experiments Peters-van der Sanden et al13,14 studied regional differences within the vagal neural crest with regard to the formation of the enteric nervous system. They were able to show that ablation of the total vagal neural crest resulted in total aganglionosis from anus through midgut, the foregut (esophagus, stomach, duodenum) being normally ganglionic. Ablation of the vagal crest adjacent to somites 3 to 5 resulted in aganglionosis of the hindgut only. Ablations of the vagal neural crest not including this segment had no effect on the formation of the enteric nervous system. These portions of the NC could use multipotent cells, able to form any of multiple NC derivatives (including enteric neuroblasts), or subgroups of unipotent cell populations that are able to originate the enteric neuroblast as a single NC derivative. Alternatively, the NC can be a mixture of multipotent and unipotent cells.4 In any case, to be able to contribute to ENS development, NC cells have to leave the neural tube, migrate to the gut, enter it at some point, and start their cranio-caudal migration along the hindgut.15 These events are regulated by molecular and cellular mechanisms that are not completely understood. THE MAJOR GENE FOR HSCR

Fig 1. Differentiation of neural crest cells in mammals. The elements of the neural crest are Retⴙ before migration. The neural crest progenitors differentiate into sympatho-adrenal progenitors that are NFⴙ, Retⴚ and THⴙ. The enteric neuroblasts that migrate along the primitive intestine become again Retⴙ.

The first step toward the understanding of the molecular basis of HSCR as well as of the nature of its genetic transmission was the observation of a young female patient with total colonic aganglionosis (TCA), carrying a de novo interstitial deletion of chromosome 10: 46,XX,del 10q11.21-q21.2.16 Following the hypothesis that a major gene responsible for HSCR had to be present in the DNA portion

HSCR PATHOGENESIS

encompassing the deletion, human-hamster somatic cell hybrids retaining the deleted (Hy185-O) and the nondeleted (Hy179-Q) chromosome 10 were produced from lymphocytes of the TCA patient using an immunomagnetic positive selection method.17 The availability of these 2 somatic cell hybrids allowed the mapping of a series of chromosome 10–specific polymorphic markers, either inside or outside the deletion. Using 7 polymorphic DNA markers (microsatellites) mapped within the deletion, a genetic linkage analysis was performed in 15 HSCR families. The results of this study confirmed that in all these families a gene responsible for HSCR was located on chromosome 10. At the same time, another study, performed by analyzing 5 independent HSCR pedigrees, confirmed genetic linkage to the proximal portion of 10q.18,19 The refinement of the genetic and physical maps of the proximal portion of the long arm of chromosome 10 took advantage of the following facts: 1. One or more genes responsible for familial medullary thyroid carcinoma (FMTC), multiple endocrine neoplasia type 2A (MEN2A), and type 2B (MEN2B) had been previously mapped to the same chromosomal region, and, consequently, a number of new highly polymorphic markers were developed.20,21 2. Two additional interstitial deletions of chromosome 10q associated with HSCR disease were observed.22,23 The characterization of the smallest region of overlap (SRO) among the 3 deleted chromosomes 10 carried by the corresponding cell hybrids obtained in the meantime by immunomagnetic selection, allowed to narrow the candidate HSCR region to an interval of less than 250 Kb.22 The RET proto-oncogene was the only cloned gene known to be located in this interval.24

Fig 2. RET proto-oncogene mutations identified in the gene structure by different investigators. 䊊, Romeo et al,68 1994; 䊉, Yin et al,37 1994; 䊊䊊, Seri et al,36 1997; 䊉䊉, Borst et al,69 1995; 䊐, Edery et al,32 1994; 䊏, Attie´ et al,31 1995; 䉫, Mulligan et al,38 1994; 䉬, Angrist et al,29 1995; 䉰, Pelet et al,34 1994. (Modified with permission from (Original figure Martucciello.45) rSpringer-Verlag.)

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To test the RET proto-oncogene as a candidate gene for HSCR, a strategy was devised to detect point mutations of this gene coding sequence in HSCR patients. In particular, starting from the published cDNA sequence25,26 and using a polymerase chain reaction (PCR)-based approach, the exon-intron structure of this gene was reconstructed.27 The intronic sequences flanking the 58 and 38 of each of its first 20 exons (known at the time) were used to design primers and to amplify each exon, which was then subjected to single strand conformational polymorphism (SSCP) analysis.28 Missense and nonsense mutations and a few base-pair deletions or insertions of the RET proto-oncogene have been identified since then in HSCR patients by different groups29-37 although in different proportions in familial and sporadic cases and in the long- and short-segment forms of the disease. In particular, 64 mutations have been reported among the 281 HSCR patients analyzed so far (22.8%).29,36 These data are summarized in Fig 2. About 74% of point mutations in the series studied at Gaslini Children’s Hospital were identified in longsegment patients, accounting only for 25% of our cases. Therefore, a breakdown of the HSCR patients populations studied in the different laboratories (Table 2) may yield a possible explanation of the different rates of mutation detection reported.29,31,37 The high proportion of patients with long-segment HSCR (44 of 80) studied by Attie´ et al31 may account for the apparently higher efficiency in detecting mutations, because 76% (25 of 33) were found in patients with this form of the disease. HIRSCHSPRUNG’S DISEASE AND MEN2

It is well established that receptor tyrosine kinases play important roles in normal differentiation and growth of

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MARTUCCIELLO ET AL Table 2. RET Proto-Oncogene Mutations in Hirschsprung’s (HSCR) Patients Gaslini Children’s Hospital, Genoa Romeo et al (1994)68 Yin et al (1994)37 Seri et al (1995)36 Patients Studied (%)

Long-segment HSCR Short-segment HSCR Undetermined Total

Mutations Identified (%)

30 (24.80) 17 (73.90) 59 (48.80) 4 (17.40) 32 (26.40) 2 (8.70) 121 (100) 23 (100) Rate of mutation, 19%

various types of cells. Binding of growth factors to the receptors results in receptor dimerization and subsequent activation of their intrinsic tyrosine kinases. However, point mutations and rearrangements of these genes may lead to constitutive activation of the corresponding receptors responsible for tumor development. The RET proto-oncogene encodes a receptor tyrosine kinase and was first identified as a chimeric oncogene activated by a rearrangement that occurred during a classical NIH 3T3 transfection assay.24 Nearly 10 years after RET protooncogene fortuitous discovery, germline mutations of the RET proto-oncogene were identified in patients with either inherited cancer syndrome, multiple endocrine neoplasia type 2, (MEN2) and HSCR. These mutations, which cause either activation or loss of RET function, represent the best documented example of an allelic series causing phenotypic diversity. MEN2A mutations result in nonconservative substitutions for 6 cysteines in the RET extracellular domain.24 However, in a few HSCR patients without any clinical symptoms of MEN2A or sporadic medullary thyroid carcinoma (MTC), missense mutations in the cysteine residues 609 and 620 have been identified (Fig 2).38,39 In addition, several pedigrees have been reported in which HSCR cosegregates with either MEN2A or FMTC (familial MTC), and in all of them (with a single exception) a documented germline mutation in cysteine 609 or 620 follows the segregation of the disease (Table 3). The appearance of the HSCR phenotype within such pedigrees cannot be reconciled easily with the gain of function that is associated with the dominant oncogenic effect of MEN2A mutations. Although an extensive study of the gastrointestinal phenotype of MEN2B has never been carried out, the existing clinical observations supported the conclusion of a great variability of bowel involvement in MEN2B. According to Anderson et al,40 Lucaya et al,41 Grun and Eberle42 and Verdy et al,43 there are distinctive radiological signs in the barium enema of MEN2B patients, namely (1) an abnormal haustral pattern in the bariumfilled colon, (2) thick mucosal folds in postevacuation film, (3) colonic diverticula, and (4) megacolon associated with a narrowed distal segment.

Necker-Enfants Malades, Paris Attie´ et al (1995)31 Patients Studied (%)

Case Wester Reserve University, Cleveland Angrist et al (1995)29

Mutations Identified (%)

44 (55.00) 25 (75.75) 25 (31.25) 8 (24.25) 11 (13.75) ⫺ (⫺) 80 (100) 33 (100) Rate of mutation, 41%

Patients Studied (%)

Mutations Identified (%)

29 (36.25) 2 (25.00) 16 (20.00) 16 (37.50) 35 (43.75) 35 (37.50) 80 (100) 8 (100) Rate of mutation, 10%

This last radiological finding suggests the hypothesis that short aganglionosis and proximal intestinal ganglioneuromatosis could be present in different parts of MEN2B bowel as already proposed.43 An accurate enzymo-histochemical study on a larger number of cases will clarify how patients with MEN2B with documented exon 16 mutations should be classified on the basis of their intestinal phenotype. Recently Romeo et al44 also described the first association between HSCR and MEN2B in a 3-year-old baby girl (Table 3). Enzymo-histochemical diagnosis of HSCR was obtained on the basis of acetylcholinesterase on rectal suction biopsy results. The patient was analyzed for the whole RET gene, and a typical exon 16 Met918Thr mutation was detected. After 2 years the MEN2B phenoTable 3. Activating Mutations (MEN2A, MTC, MEN2B Mutations) in Hirschsprung’s Disease HSCH-Associated Phenotype

Study

Mulligan et al,38 1994 Mulligan et al,38 1994 Mulligan et al,38 1994 Mulligan et al,38 1994 Cretien et al,70 1994 Borst et al,69 1995 Borst et al,69 1995 Caron et al,71 1996 Blank et al,72 1996 Seri et al,36 1997, and Romeo et al,44 1998 Romeo et al,44 1998 Angrist et al,29 1994 Mulligan et al,38 1994 Angrist et al,29 1994

Ret Exon Mutation

MEN2A MEN2A MEN2A FMTC MEN2A MEN2A MEN2A MEN2A MEN2A FMTC

Cys618 Arg Cys620 Arg Cys620 Arg Cys620 Arg Cys620 Arg Cys620 Ser Cys620 Ser Cys618 Arg Cys620 Arg Cys620 Arg Cys620 Ser Cys620 Arg Cys609 Trp Cys609 Trp

Hofstra et al,39 1998

FMTC HSCH only (sporadic) HSCH only (familial) HSCH only (familial) Three HSCH only (1) HSCR (2) MEN2A (3) MTC

Romeo et al,44 1998

MEN2B

Romeo et al,44 1998

FMTC

Romeo et al,44 1998

FMTC

Data from Romeo et al.44

Cys609 (1 case) and Cys620 Arg (2 cases) Met 918 Thr (ATG:ACG) Cys620 Ser (TGC:TCC) Cys620 Arg (TGC:CGC)

HSCR PATHOGENESIS

type also was characterized by MTC, so the patient underwent thyroidectomy. A possible explanation for the coexistence of MEN2 and HSCR (or congenital megacolon) phenotypes in the same patients might be based on the activation of an apoptotic program in the embryonic enteric ganglion cells as a response to the inappropriate mitogenic signal transduced by a mutant activated Ret tyrosine kinase.44 Alternatively, the presence of additional RET mutations, the influence of allelic variants of RET, of modifier genes or of environmental factors might account for these observations. Following the hypothesis of possible additional RET mutations, we screened all the exons of RET in genomic DNA from patients with such unusual associations. Neither additional RET mutations nor rare DNA variants could be detected in any of the patients. The hypothesis of further constitutional RET mutations in HSCR patients is supported neither by these findings nor by those of previous reports. Future investigations aimed at studying the molecular basis of the association between MEN2 and HSCR also should take into account the variability of gastrointestinal manifestations in patients affected by MEN2B. Another important clinical implication is that these new genetic data suggest that a subgroup of HSCR patients could be at risk for MEN2 or at least for MTC. In view of the wide age range at onset of these tumors, this risk cannot be discounted. To quantify this risk, it must be considered that (1) the rate of RET mutations in the Hirschsprung population can be estimated around 20% of cases and (2) the mutation involving the cysteine residues on exon 10, known to predispose to the development of MEN2A or FMTC, is present in at least 5% of all HSCR mutated patients.45,46

Fig 3. Activation of Ret receptor by GDNF and NTN. (A and B) GDNF ligand binding to GDNFR-␣ forms an activated complex with Ret receptor. (C and D), NTN binding to NTNR-␣ activates Ret receptor.

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GDNF DEFICIT IN HIRSCHSPRUNG’S DISEASE

The first identified Ret receptor ligand was the glial cell line–derived neurotrophic factor (GDNF). The GDNF mature protein consists of 134 amino acids with a molecular mass of 32 to 42 kD in dimer form.47 GDNF is proposed to act as a ligand for a multisubunit receptor in which the glycosylphosphatidylinositol-linked protein GDNFR-␣ provides the ligand binding, and RET provides the signalling component (Fig 3). Functional assays have shown that, in the absence of either GDNF or GDNFR-␣, Ret signalling is reduced or absent.48,49 Because the number of reported mutations in RET did not account for all the HSCR cases studied, GDNF appeared to be an ideal candidate for mutation analysis studies. The GDNF gene comprises 2 known exons: exon 1 (151 bp) and exon 2 (485 bp) and is therefore a small target for potential mutagenesis. At the Gaslini Children’s Hospital of Genoa, a GDNF mutation analysis was performed in 98 cases of intestinal dysganglionosis, 43 of which had different forms of aganglionosis. Patient screening identified 2 heterozygous mutations in exon 2. The first, R143R, was a transversion G-A at nucleotide 492, identified in a case of total colonic aganglionosis; the second, I211M, was a missense heterozygous mutation caused by a transversion C-G at nucleotide 630 in a classic form of HSCR (unpublished data). Our genetic results confirmed the infrequency of GDNF mutations in association with HSCR. In our series, mutations were detected in 4.6% of all HSCR cases studied, in agreement with mutation rates reported by other investigators ranging from 0.9% to 5.5%.50,51 We also investigated a possible expression deficit of

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GDNF protein in the enteric nervous system (ENS) of Hirschsprung patient not mutated for the GDNF gene. Immunohistochemistry testing was performed in 30 HSCR and 10 control cases with GDNF D-20 (Santa Cruz Biotechnology) an affinity-purified rabbit polyclonal antibody raised against a peptide corresponding to aminoacids 186-205, mapping within the carboxy terminal domain of human glial cell line–derived neurotrophic factor.52 GDNF immunoreactivity was localized in the ganglia of myenteric and submucous plexuses. In the normal colon and in the ganglionic segment of HSCR, a strong granular red staining was obtained in the satellite elements and on the cellular membranes of the ganglion cells (Fig 4). In the ganglionic intestine, GDNF-positive nerve fibers were not observed. The small ganglia of the hypoganglionic segment presented a reduced GDNF immunoreactivity when compared with the proximal normoganglionic segment. In the distal aganglionic segment of HSCR, different enzymo and immunohistochemical markers (SDH, LDH, ANE, NSE) showed absence of ganglion cells. The muscular interstitium presented trunks of nerve fibers and persistence of some small cellular elements of glial origin that presented GFAP and S-100 protein immunoreactivity. GDNF immunoreactivity was absent in the aganglionic segment of HSCR (Fig 5). GDNF expression deficit in the distal aganglionic segment could be an important cofactor in HSCR pathogenesis. Absence of GDNF in the distal hindgut could determine a missed activation and phosphorylation of the Ret receptor in the absence of RET proto-oncogene mutations causing enteric neuroblast migration arrest and Hirschsprung’s disease. In addition to GDNF, RET has at least 3 other ligands

Fig 4. GDNF immunoreactivity in the ganglionic segment of Hirschsprung’s disease. GDNF is positive in the satellite elements and in the surface membrane of ganglion cells.

MARTUCCIELLO ET AL

Fig 5. GDNF immunoreactivity in the aganglionic segment of Hirschsprung’s disease. GDNF-negative nerve trunks and glial derivatives.

that are member of the GDNF family: neurturin (NTN), persephin (PSP), and artemin (ART). They have 7 conserved cysteine residues with similar spacing, making them distant members of the transforming growth factorbeta (TGF-␤) superfamily (Fig 3).53 OTHER HSCR GENES AND SYNDROMIC HSCR

The low detection rate of RET mutations in HSCR patients also led to different hypotheses, such as the existence of additional HSCR genes. The availability of animal models of HSCR has played a crucial role in identifying some of these genes (Table 4). Until today, different animal and human genetic studies have identified 6 HSCR genes: RET proto-oncogene, endothelin 3 (EDN3), endothelin B Receptor genes (EDNRB), GDNF, ECE1, and gene encoding the Sryrelated transcription factor SOX10 (SOX10). Although RET mutations account for about 20% of sporadic HSCR patients, the other genes seem to be involved in a minority of cases (1% to 5%). Moreover, some of these genes (EDN3, EDNRB, SOX10) control rare HSCR forms associated with other neural crest defects. It is the case of the EDN3-EDNRB signaling pathway. The endothelin-induced signalling pathway intrinsically is necessary to the development of those neural crest–derived cells committed to innervate the colon.54 Further evidence of this was provided by Baynash et al55 who showed that targeted disruption of the gene encoding one of the ligands for the endothelin-B receptor, endothelin-3 (EDN3), also leads to an autosomal recessive phenotype of white spotting and megacolon, and that EDN3 is mutated in the mouse phenotype lethal spotting,

HSCR PATHOGENESIS

which maps to mouse chromosome 2. Furthermore, the EDNRB gene is deleted partially in the autosomal recessive rat mutant lethal spotting, which also has the phenotype of white spotting and congenital aganglionosis.56 Endothelins ET-1, ET-2, ET-3 (coded by EDN1, EDN2, EDN3 genes, respectively) are small, 21-residue peptides that are derived from a 2-step proteolytic cleavage of a larger precursor protein.57 These peptides mediate their effects through 2 receptors ET-A, ET-B (coded by EDNRA, EDNRB genes, respectively) that have sequence and structural similarity to the family of G-protein– coupled heptahelical receptors.58 The ET-B receptor is a 442-residue protein expressed in brain, kidney, lung, heart, and endothelial cells.59 Recently, EDNRB also has been shown to be expressed in the human colon, particularly in the myenteric plexus. Both the naturally occurring and induced null mutations in EDN3 and EDNRB make clear that this pathway is intrinsic to the normal, early development of epidermal melanocytes and enteric neurons. The finding of multiple mutations in the EDNRB and EDN3 genes in HSCR has confirmed the developmental importance of these genes, and these mutations suggest that the normal function of the ET-B receptor involves cell differentiation and proliferation. In particular, a missense W276C mutation of the EDNRB gene has been characterized in a large Mennonite pedigree in which HSCR apparently was inherited as a recessive trait, and low penetrant pigmentary defects also were observed.60,61 In contrast to the recessive, fully penetrant defects in rodent models, this human mutation is neither fully dominant nor fully recessive, being present in 74% of the homozygotes and in 21% of the heterozygotes. Because the W276C mutation affects the residual activity of EDNRB, it might explain the dosage effect of this mutation and its incomplete penetrance.62 Recently, homozygous mutations of the endothelin-3 (EDN3) gene on chromosome 20 have been identified in consanguineous HSCR families harboring other malformations of neural crest–derived cells, namely pigmentary anomalies and deafness (Shah-Waardenburg syndrome).63-65 Different investigators65 showed that the EDN3 gene is a rare susceptibility locus in nonsyndromic HSCR (2 of 174 probands in the Bidaud et al series), whereas EDN3 is one of the causative gene of the Shah-Waardenburg syndrome (WS4; HSCR, pigmentary anomaly, deafness). More recently, the molecular defect underlying the dominant megacolon (Dom) mouse model has been identified.62 In particular, a 1-bp insertion of SOX10, a Sry-related transcription factor expressed during embryonic development, has resulted to be responsible for the

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HSCR phenotype recurring in this mouse strain, which is characterized by intestinal aganglionosis and white spotting in the coat.66,67 Mutation search in HSCR patients also has shown in this case that the involvement of this gene is restricted to those showing also pigmentary defects or deafness (Shah-Waardenburg syndrome WS4).67 Hirschsprung’s disease is a multigenic disorder caused by dysfunction of either of 2 principal signalling pathways, the Ret-GDNF and the EDN3-EDNRB receptor systems. Genetic mapping in families and mutation screening of candidate genes, together with the study of several natural and knockout animal models, clearly have shown the involvement of multiple genes in the pathogenesis of HSCR disease. Each of these genes is likely to add, if mutated, a variable contribution to the final disease phenotype and therefore can be regarded as susceptibility or modifier HSCR gene. Such a polygenic model of HSCR inheritance needs further investigations. In addition, the involvement of environmental factors make HSCR a complex genetic disease. RET proto-oncogene is the major disease-causing gene in HSCR. However, RET mutations only account for 50% and 20% of familial and sporadic cases of HSCR, respectively.31 Most RET-mutated HSCR cases are represented by ultralong forms of the disease (74%) without other associated anomalies. The genes of endothelin signalling system (EDNRB, EDN3, and ECE1), when mutated, can be associated with several other neurocristopathies. This fact shows that the development of different neural crest–derived lineages (melanocytes, enteric neuroblasts, and sensory neurons) shares a common regulatory pathway in some crucial phase of neural crest development. It is the case of Waardenburg syndrome type 4 (WS4) or Shah-Waardenburg syndrome, in which HSCR can be associated with deafness or pigmentary defects. WS4 patients do not carry mutations in PAX3, MITF or RET, but are homozygous for mutations in EDNRB or EDN3. SOX 10 also causes a dominant form of WS4.67 SOX 10 is the third gene associated with WS4, and, interestingly, nonsyndromic HSCR does not seem to be caused by SOX10 mutations. On the bases of these clinical observations, it has been hypothesized that SOX10 is required for the survival and development of very early pluripotent crest-derived progenitor cells. Microenvironmental factors also can play a role in the pathogenesis of HSCR. The developmental process of the crest-derived progenitor cells is sensitive to the level of different molecules. The expression deficit of different factors (GDNF, NTN, etc) in the hindgut, in the absence of genetic mutations, could determine a missed activation of the receptor system, causing enteric neuroblast migration arrest and Hirschsprung’s disease.

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