Molecular genetics of Hirschsprung’s disease

Seminars in Pediatric Surgery (2004) 13, 236-248

Molecular genetics of Hirschsprung’s disease Paul K.H. Tam, Mercè Garcia-Barcelo From the Department of Surgery and Genome Research Centre, The University of Hong Kong, Hong Kong, China. Although modern surgical procedures have already achieved a high success rate in the treatment of Hirschsprung’s disease (HSCR), a better understanding of the mechanisms involved in the pathogenesis of this disease should enable doctors to prevent and diagnose it more easily and treat it even more effectively. DNA underlies almost every aspect of human health, both in function and dysfunction. A series of impressive advances in DNA and genetic technologies has yielded a deeper and more comprehensive understanding of the molecular processes underlying diseases. It is becoming clear that genes and gene products interact in networks, rather than in linear pathways. Therefore, understanding how genes and other DNA sequences function together and interact with proteins and environmental factors is paramount to the discovery of the pathways involved in normal processes and in disease pathogenesis. In this respect, the study of the molecular basis of HSCR has made a marked contribution to the understanding of complex diseases. The genetic complexity observed in HSCR can be conceptually understood in the light of the molecular and cellular events that take place during the development of the enteric nervous system (ENS) in the embryonic stage. The ganglion cells of the fully developed ENS are derived from neural crest cells (NCCs) of the vagal, sacral and truncal regions of the neural tube.1 During the colonization process, the NCCs have to adapt to a constantly changing intestinal environment that strongly influences their differentiation into enteric neurons.2 The entire process is regulated by specific molecular signals from within both the neural crest and intestinal environment,3 and the success of the colonization of the gut depends on the synchronization and balAddress reprint requests and correspondence: Paul K.H. Tam, Division of Paediatric Surgery, Department of Surgery, The University of Hong Kong, Queen Mary Hospital, K15, Pokfulam, Hong Kong, P.R. China. E-mail address: [email protected]

1055-8586/$ -see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1053/j.sempedsurg.2004.10.011

ance of the signaling network implicated. DNA alterations in the genes codifying for the signaling molecules may interfere with the colonization process, and consequently represent a primary etiology for HSCR or other neurocristopathies. Since the range of interactions during the ENS development is large, the HSCR phenotype is variable, and may result from single severe mutations in a major gene encoding a crucial molecule or from accumulation of less severe mutations in several genes. The existence of nonclinically affected individuals carrying mutations in major genes invokes a compensatory effect by other genes. HSCR has become a model for a complex of oligogenic and polygenic disorders in which the phenotypes and mode of transmission result from interactions between different genes.

The genetic etiology of Hirschsprung’s disease In 1886, the Danish pediatrician Harald Hirschsprung (1830-1916) described the disease that now bears his name as a cause of constipation in early infancy. The development of a pull-through surgical procedure by Swenson and Bill in 1948 made a major contribution to our understanding of the disease.4 This innovative surgical technique enabled the disease to be treated with considerable success. The result was a far higher survival rate among patients, which created the conditions for the discovery of the familial transmission of the disease and the determination of its genetic nature. Additional evidence for a role of genetic factors in the pathology of HSCR was indicated by (1) an increased risk of recurrence for sibs of affected individuals compared with the population at large; (2) an unbalanced sex-ratio; (3) the association of HSCR with other genetic diseases, including malformation syndromes and chromosomal anomalies; and (4) the existence of several animal models of colonic aganglionosis showing specific mendelian modes of inheritance.

Tam and Garcia-Barcelo Table 1

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Genes involved in HCSR

Gene

% of patients with mutations

Phenotype in mutants

RET GDNF* NRTN* EDNRB EDN3 ECE-1 SOX10 ZFHX1B PHOX2B

⬇50% familial cases; 7%-35% sporadic cases 1 case 1 case ⬇7% ⬍5% 1 case 17 cases ⬍1% 12 cases 3 cases

HSCR either isolated or syndromic HSCR HSCR HSCR or WS4 HSCR or WS4 Syndromic HSCR (craniofacial and cardiac defects) WS4 Syndromic HSCR (mental retardation and facial dysmorphism) Haddad syndrome (CCHS with aganglionosis) Neurobalstoma associated with HSCR

*

Only in conjunction with mutations in RET.

In the late 1980s, the locus for Multiple Endocrine Neoplasia (MEN) was assigned to chromosome 10q11.2.5,6 The same locus was also identified as the cause of papillary thyroid carcinoma (PTC)7-9 and familiar medullary thyroid carcinoma (FMTC).10 The cooccurrence of HSCR with multiple endocrine neoplasia type 2 (MEN2) and the observation of a deletion comprising the MEN2 locus in some HSCR patients,11,12 prompted the search for a HSCR gene in that chromosomal region. Linkage analysis using chromosome 10 markers in HSCR families revealed a HSCR locus in the pericentromeric region of chromosome 10.13,14 The observation of a HSCR-phenotype in mice with disrupted Ret gene15 and the detection of RET germline mutations in HSCR families16,17 led to the recognition RET as the cause of HSCR, almost a century after Harald Hirschsprung first described the disease. Subsequent studies led to the identification of several genes implicated in the HSCR phenotype. To date, mutations in nine different genes (RET, GDNF, NRTN, PHOX2B, EDNRB, EDN3, ECE1, SOX10, and ZFHX1B) have been detected in individuals affected with either isolated or syndromic HSCR (Table 1). These genes encode protein members of important interrelated signaling pathways that are critical to the development of enteric ganglia: the GDNF/RET receptor tyrosine kinase, the endothelin type B receptor and SOX10 mediated transcription. Nonetheless, the RET gene is the major gene involved in all forms of HSCR, and its expression is critical for the normal development of the ENS.18,19 Interaction between those signaling pathways could therefore alter the expression of RET and influence the manifestation of the HSCR phenotype.20-24 Similarly, yet unknown loci can modify RET expression and act as a disease promoting or suppressing genes. Common features of the mutations found in the HSCR genes are low and sex dependent penetrance (frequency of individuals carrying a mutation who develop disease as opposed to healthy carriers) and variable expression of the HSCR phenotype (variability in the length of the aganglionic segment and in the manifestation of associated anomalies). These characteristics are consistent with the expression of a HSCR gene subject to modification by other

loci,25,26 perhaps as a result of cross-talk between signaling pathways. In addition, phenotypic/genotypic assessment of the disease has become more difficult since a subset of the observed mutations in HSCR genes is also associated with several additional anomalies (syndromic HSCR). HSCR has a complex pattern of inheritance and manifests with low, sex-dependent penetrance. The male:female ratio (M:F) is approximately 4:1 among patients with short segment aganglionosis (S-HSCR) and approximately 1:1 among patients affected with long segment aganglionosis (L-HSCR). HSCR most commonly presents as an isolated trait (70% of cases), which arises sporadically although it can also be familial (approximately 20% of the cases). Sporadic HSCR patients usually present with short segment aganglionosis and mutations, if detected, are usually de novo. The recurrence risks to sibs of S-HSCR probands ranges between 1.5% and 3.3% while risks to sibs of L-HSCR probands varies from 2.9% to 17.6%.27

RET receptor tyrosine kinase pathway The RET protein functions as a receptor for the Glial cell line-Derived Neurotrophic Factor (GDNF) family. Receptor-ligand interactions are mediated by RET coreceptors (GDNF-family receptors-␣; GFR␣).28,29 Activation of the RET receptor by these neurotrophic factors is essential for the migration and differentiation of specific cell lineages of neural crest origin into enteric neurons.15,30,31 The correct development of the ENS therefore depends on the ability of these neurotrophic factors to activate RET, on the ability of the RET receptor to transduce the signal, and on the competence of the intracellular machinery to elaborate a response (Figures 1 and 2).

RET proto-oncogene and HSCR RET, also known as RET proto-oncogene, is composed of 21 exons that encode the RET receptor tyrosine kinase protein, a cell-surface molecule that transduces signals. Germline mutations in RET are responsible for four unrelated disorders in humans: HSCR, MEN2A, MEN type 2B32

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Figure 1 Schematic drawing showing the RET gene and the RET protein receptor. Exons of RET gene are indicated with numbered boxes and dotted lines indicate the domains of the protein they encode. Different domains of RET receptor are indicated with cylinders. The extracellular part of RET contains cadherin domains (CAD) and the intracellular part of RET contains two tyrosine kinase domains (TK). TMD, transmembrane domain. In brackets, the animo-acid residues.

and medullary thyroid carcinoma (MTC). In PTC, RET rearrangements are the most commonly detected genetic alterations. RET is a single-pass membrane protein with two main domains: extracellular (EC) and intracellular (IC). The extracellular domain contains a cadherin-like domain (CAD)33 and a large conserved cystein rich region (CYS). The IC domain contains a typical tyrosine kinase domain (TK) (Figure 1). The RET protein presents three isoforms of 1114 (RET51), 1106 (RET 43) and 1072 (RET 9) amino acids, respectively, with distinct biological roles.34,35 Ligand and coreceptor complexes interact with the EC domain of two RET molecules inducing their homodimerization and tyrosine autophosphorylation. Once phosphorylated, the tyrosine residues (TK domain) serve as high affinity binding sites for various intracellular signaling proteins.36 Activating mutations in the RET gene cause MTC, MEN2A and MEN2B, whereas inactivating mutations lead to Hirschsprung’s disease.37 A large number of RET mutations in HSCR patients constantly reported, and due to the peculiarity of HSCR, it is not always easy to provide biological evidence of their alteration of the RET function. In general, RET gene mutations affecting the RET EC domain alter the folding of the protein impairing its maturation.38,39 RET mutations affecting the RET TK domain result in obliteration of the kinase activity or in interference with the binding to intracellular signaling proteins.39-41 Mutations in exons 20 and 21 of RET may affect the specific isoforms of the RET protein and modify their differential roles in signaling.35 Mutations in any intron/exon boundary affecting the splicing consensus sequences can alter mRNA processing. Remarkably, silent mutations (ie, the mutation does not result in an amino-acid change in the protein) in any of the RET exons can also affect mRNA processing.42 Unlike in MEN 2, RET mutations causing HSCR are, with very few exceptions,25 scattered throughout the gene, affecting indiscriminately any part of the RET protein. However, mutations in the coding regions of RET and other HSCR genes (see below) only account for over 50% of the

familial cases, and between 7% and 35% of the sporadic cases.43-58 Intriguingly, genetic-linkage analyses in HSCR families keep implicating the 10q11 chromosomal region (RET locus) even though no RET coding regions can be found.25,59 This, besides confirming the central role of RET in HSCR, also indicates that mutations in noncoding (regulatory) regions RET remain to be found. Mutations in regulatory regions can alter the DNA binding sites of transcriptional regulators and reduce or abolish the expression of RET. In addition to RET mutations, a possible role in HSCR has been attributed to several single nucleotides polymorphisms (SNPs) of the RET gene since they have been found associated with the disease. Variant alleles (underlined) of the RET coding-region polymorphisms, c135G ⬎ A (A45A; exon 2) and c2307T ⬎ G (L769L; exon 13), are over-represented in HSCR sporadic patients.60-63 The current data indicate that these RET specific SNPs, either singly or in combination (haplotypes), could act as modifiers by modulating the penetrance of mutations in other HSCR genes and possibly of those mutations in the RET gene itself.22,64,65 They might also act as low penetrance alleles conferring risk of or protection against disease, or be in linkage disequilibrium (LD) with a yet unidentified susceptibility locus located in the regulatory regions of the gene.66,67 This possibility has been recently corroborated by the identification of two polymorphisms in the proximal promoter region of RET.68,69 These promoter polymorphisms are in LD with the coding region SNPs. Functional analyses have not yet proved a direct effect of the single SNPs associated with HSCR on the RET function, but it may be the precise combination of SNPs on this chromosomal region (haplotype) that determines regulatory significance.

GDNF family ligands and their receptors (GDNFfamily receptors-␣; GFR␣) The relevance of RET signaling pathway in the ENS development and the phenotypic similarities between Ret, Gdnf and Gfr␣-1 knock-out mice,15,70-72 prompted an ex-

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pathway. It is possible that homozygous mutations in the GDNF family of ligands would be lethal, and that these ligands, particularly GDNF, may have other functions not mediated by RET, and therefore come under selective pressure against mutations.

HSCR genes involved in the transcriptional regulation of RET Because of the key role played by RET in HSCR, mutations in genes known to be involved in the regulation of RET are likely to give rise to phenotypes involving aganglionosis. Not all the genes involved in the regulation process are known and the precise mechanisms of regulation involved also remain unclear. Further investigation on the molecules involved in the regulation of the RET gene is certain to shed more light to the mechanisms underlying HSCR.

Figure 2 Schematic representation of the network of interactions that govern the development of the enteric nervous system. Expression of the RET gene (regulated by transcription factors) leads to the formation of the RET protein which functions as a receptor for the Glial cell line-Derived Neurotrophic Factor family (GFL) through coreceptors (GFRA). Activation of the RET receptor by these signaling molecules of the gut environment activates the intracellular machinery necessary for the migration, proliferation and differentiation of specific cell lineages of neural crest origin into enteric neurons. Similarly, the EDNRB receptor is activated by EDN3 and initiates a series of events that will also regulate the development of the enteric nervous system in conjunction with the RET pathway. Double head arrows indicated interrelation between pathways. Question marks represent yet unidentified molecules members of the network (see “Modifying Genes and Interaction between Signaling Pathways” section in the text).

tensive mutation screening of the genes encoding the GDNF family of ligands—GDNF, Neurturin (NTN) artemin (ARTN) and persepin (PSPN)—and of the genes encoding their coreceptors (GFR-␣1-4) and adaptors. However, mutations in those genes have been identified in a very small number of patients and generally cosegregate with mutations in RET.73-77 None of the of mutations so far identified in the GDNF gene are likely per se to result in HSCR, though they can contribute to disease via interaction with other susceptibility loci.78,79 Surprisingly, RET is virtually the only target of HSCR mutations in the RET signaling

SOX10 The SOX10 gene encodes a transcriptional regulator whose expression is essential for the development of cells in the neural crest cell lineage, including melanocites and enteric neurons. During the development of the ENS, the SOX10 protein physically interacts and cooperates with another transcriptional regulator (PAX3) to activate transcription of RET.31,80,81 Sox10 has recently been found to genetically and functionally interact with Ednrb in mice.82,83 The significance of the SOX10 gene in HSCR was revealed almost accidentally, through the study of a mouse model of HSCR (Dom) fortuitously generated at the Jackson Laboratory.84 The molecular defect in Dom mice was a mutation in the Sox10 gene.85-88 Heterozygous Dom mice presented with distal colonic aganglionosis and localized hypomelanosis of the skin and hair (features similar to those in Waardenburg’s syndrome), which indicated that neural crest-derived melanocytes and enteric neurons were affected in Sox10 mutants. Dom homozygous mice were embryonic lethal. In humans, 17 heterozygous SOX10 mutations have been identified in patients with Waardenburg-Shah syndrome (WS4; HSCR type 2). Patients with WS4 display a combination of the features of Waardenburg syndrome (wide bridge of the nose, pigmentary abnormalities, and cochlear deafness) and Hirschsprung’s disease.85,89-95 SOX10 mutations also lead to Yemenite deaf-blind hypopigmentation syndrome and other myelin deficiencies.96 The majority of mutations in the human SOX10 result in truncation of the SOX10 protein. Most truncated proteins have reduced transcriptional activity and a dominant negative effect.97,98 To date, no SOX10 mutations have been found in patients presenting aganglionosis as an isolated trait. SOX10 is therefore unlikely to be major gene in isolated HSCR. Interestingly, despite absence of genetic mutations, abnormal SOX10 gene expression can be observed in aganglionic intestine of isolate HSCR patients.99

240 PHOX2B The paired mesoderm homeobox 2b gene (PHOX2) encodes a transcription factor which is involved in the development of several noradrenergic neuron populations. In mice homozygous disruption of the Phox2b gene results in absence enteric ganglia, a feature reminiscent of HSCR.100 Furthermore, there is no Ret expression in Phox2b mutant embryos indicating that regulation of Ret by Phox2b could account for the failure of the ENS to develop.101,102 However, the details of the mechanism by which the Phox2b protein modulates the Ret gene are not yet known. All these observations make the PHOX2B a candidate gene for HSCR. Interestingly, a chromosomal alteration involving the deletion of the PHOX2B locus has been described in a patient with syndromic HSCR (developmental delay, severe hypotonia, facial dysmorphism, and short segment aganglionosis).103 This suggests that PHOX2B haploinsufficiency may predispose to HSCR. A PHOX2B SNP has also been found significantly associated with isolated HSCR.104 PHOX2B represents the first gene for which germline mutations predispose to neuroblastoma105,106 and is the major locus for congenital central hypoventilation syndrome (CCHS, Ondine’s curse).107-110 Intriguingly, both neuroblastoma and CCHS are frequently associated with HSCR.111-123 In particular, 25 to 30% of the CCHS patients are affected with aganglionosis (Haddad syndrome) and some of them harbor mutations in RET, GDNF and in the brain-derived neurotrophic factor gene (BDNF).124-127 Taken together these data support the contribution of PHOX2B to HSCR. ZFHX1B (previously SIP1) The observation of a translocation involving the ZFHX1B locus (2q22) in a patient with HSCR disease, microcephaly, mental retardation, epilepsy and characteristic facial appearance, led to the first documentation of ZFHX1B involvement in this syndromic form of HSCR.128,129 Other chromosomal abnormalities involving 2q22 and mutations in the ZFHX1B gene are being described in patients with similar clinical features (recently named Mowat-Wilson syndrome) although not all of them present with aganglionosis.130-135 ZFHX1B encodes a transcriptional repressor (SIP1, Smad interacting protein 1), which interacts with several members of the Smad family. Some Smad proteins act as transducers in signaling cascades critical to embryogenesis. It is not yet know how mutations in ZFHX1B may result in defects of the ENS, although it is tempting to speculate on a possible functional link between SIP1 and the signaling pathways currently known to be essential for the ENS development. As in SOX10, no mutations have been found in patients with isolated HSCR, and therefore ZFHX1B is unlikely to be a major gene in nonsyndromic HSCR.

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Endothelin type B receptor pathway The endothelins are a family of three signaling peptides (EDN1, EDN2 and EDN3) that can act on two subtypes of G protein-coupled receptors, termed endothelin-A and endothelin-B receptors (EDNRA and EDNRB) (Figure 2). The endothelins are synthesized as much larger proteins which are cleaved by the endothelin converting enzyme (ECE-1) to produce an active peptide. The human genes coding for these proteins are EDN1, EDN2, EDN3, EDNRA and EDNRB, respectively. The involvement of the endothelin type B receptor pathway in the pathology of HSCR was demonstrated when a targeted disruption of the mouse endothelin-B receptor gene (Ednrb) resulted in an autosomal recessive phenotype with aganglionic megacolon and white spotting of the coat. A targeted disruption of the mouse endothelin-3 ligand (Edn3) gene produced a similar recessive phenotype of megacolon and white coat spotting. These findings indicated an essential role for the members of the EDNRB pathway in the development of two neural crest-derived cell lineages, enteric neurons and epidermal melanocytes.136,137 It has recently been demonstrated that EDNRB signaling not only regulates migration of the ENS progenitors but can also modulate the response of neural crest cells to GDNF.2,23 This demonstration provides evidence of interaction between two different pathways implicated in the pathogenesis of HSCR.

EDNRB gene and HSCR The occurrence of multiple cases of both isolated HSCR and WS4 in an inbreed Old Order Mennonite community138 facilitated the mapping of another major HSCR susceptibility gene to the chromosomal region 13q22.139 The gene identified on 13q22 was the Endothelin type B receptor (EDNRB) which had a mutation that resulted in the W276C amino-acid change in the protein.140 In contrast to the recessive, fully penetrant defects of the rodent model, this human mutation was neither fully dominant not fully recessive. The homozygous W276C mutation (CC) was more penetrant than the heterozygous (WC), and that penetrance was sex dependent. Individuals homozygous (CC) presented with WS4 features, while those with the heterozygous mutation (WC) presented with isolated HSCR. Furthermore, some family members with no W279C mutation were clinically affected. This implied the presence of additional predisposing genes among this closely related group. In fact, a genetic modifier of HSCR found among the group was mapped to chromosome 21q21.139 Subsequent EDNRB mutation analyses conducted on both isolated HSCR and WS4 patients revealed other EDNRB mutations with similar genetic behavior to W279C. Homozygous EDNRB mutations were associated with WS4141,142 and heterozygous mutations with isolated HSCR.57,58,143-148 Overall, EDNRB mutations account for 5% of the isolated HSCR phenotype. Functional analyses of EDNRB missense mutations showed

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Molecular Genetics of Hirschsprung’s Disease

impairment of the intracellular signaling.145,149,150 Unlike RET mutations, EDNRB mutations are mainly inherited from unaffected parents, and mainly associated with shortsegment aganglionosis.

EDN3 and ECE-1 genes Because of the HSCR-like phenotype presented by mice with disruption of the Edn3 HSCR patients are being screened for mutations in the human EDN3. To the best of our knowledge, very few EDN3 mutations in have been characterized in HSCR patients. With very few exceptions,87 heterozygous EDN3 mutations are associated with isolated HSCR151,152 and homozygous mutations with WS4.153,154 Another knock-out mouse model again provided evidence of the involvement of another member of the EDNRB pathway in HSCR.155 The gene encodes the endothelin converting enzyme (ECE-1), which cleaves the large inactive endothelins into smaller 21-amino-acid active endothelins. Mice carrying an ECE-1 null mutation (no protein is synthesized) present with craniofacial and cardiac abnormalities, absence of melanocytes and absence of the enteric neurons in the distal gut. This phenotype is similar to that presented by mice with mutations in the genes encoding other members of the EDNRB pathway (EDN3 and EDNRB). To date, only one mutation in the ECE-1 gene has been found and the patient was affected with a syndromic form of HSCR.156 As a general rule, severe homozygous mutations in genes involved in the EDNRB pathway are mainly associated with WS4 (absence of enteric ganglia and epidermal melanocytes, both neural crest cell derivatives). The association of homozygous mutations with WS4 and heterozygous mutations with isolated HSCR may indicate that melanocytes and enteric ganglia differ in sensitivity to the varying levels of EDNRB signaling.20 The restriction of aganglionosis to the distal colon in mice with EBNRB and EDN3 deficiency and the fact that HSCR patients with mutations in these genes mainly present with short segment aganglionosis indicated that the EDNRB signaling pathway is only required during the late states of the colonization of the colon.157,158 However, it has recently been shown that the EDNRB signaling pathway is required for the colonization of both colon and small bowel.23 The colonization process is subject to distinct spatial and temporal signaling requirements and at some stage, EDNRB signaling enhances the ability of the neural crest cells to migrate into the distal bowel.2

Modifying genes and interaction between signaling pathways It has already been stressed that the successful colonization of the gut by the ENS precursors depends on a coordinated

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and balanced network of interacting molecules (Figure 2). Conceivably, there should be a functional and genetic link among these molecules for them to interact. The mechanisms underlying these interactions may help to explain the complexity of the HSCR phenotype and resolve puzzling genetic observations, eg, that in some cases more than one mutated gene is needed to produce the phenotype, while (conversely) healthy individuals exist with mutations in HSCR genes. Interaction between pathways requires not only coordination among the pathway members but also with those molecules that mediate their interaction. The RET and EDNRB signaling pathways were initially thought to be biochemically independent. However, the case of an HSCR patient with a weak mutation in both RET and EDNRB whose healthy parents were heterozygous for one mutation implies interaction between these two pathways.42 Some reports have also hinted the possibility of cross-talk between G-protein-coupled-receptors (ie, EDNRB) and tyrosine kinase receptors (ie, RET).159 The genetic interaction between mutations in RET and EDNRB in HSCR was verified in 2002 in an association study conducted on 43 Mennonite family trios segregating the W276C mutation in the EDNRB described above.22 The study demonstrated the join transmission of both, the W276C mutation in EDNRB and HSCR-associated haplotypes in RET in affected individuals. The combination of these two genotypes increased the penetrance of the mutation and therefore the risk to disease. Genetic interaction between RET and EDNRB pathways has also been demonstrated in mice.22-24 The fact that two mutated genes are needed for the manifestation of the disease also implies functional interaction. EDN3 and GDNF seem to have a synergistic effect on the proliferation of the undifferentiated ENS progenitors and an antagonistic effect on the migration of differentiated enteric ganglia. It appears that EDN3 has a variable distribution along the developing gut with differential effects on processes regulated by the RET. It has been firmly established that the interaction between these signaling pathways controls ENS development throughout the intestine.2,23 However, both pathways have to be integrated by additional molecules or “mediators.” Protein kinase A has been suggested as a key component of the molecular mechanisms that mediate and link the RET and EDNRB signaling pathways23 Also, in mice, Sox10 has been shown to act on both Ret and EdnrB genes80-83 demonstrating its contribution to the network that produces variation between patients with aganglionosis. Further investigation is needed to identify these molecules that act as mediators between those pathways. This means that genes involved in the development of the enteric nervous system still await discovery, and that DNA alterations in multiple genes have the potential to combine and hinder a phenotype. Obviously, defective functioning of these still unknown “mediators” could modify the outcome of the development of the ENS. RET and EDNRB are central to the genesis of HSCR but little is yet known of the influence of variation in multiple genes or genetic background. Un-

242 known loci (that could encode protein members of the RET and EDNRB pathways) can modify RET expression and act as a disease promoting or suppressing genes (modifiers). Therefore, the poor genotype–phenotype correlation in HSCR is due to the effects of multiple genes combined, including HSCR known genes and still unknown loci. A linkage study conducted on 12 HSCR families enriched for L-HSCR demonstrated linkage to RET in all but one family. However, 6 out the 11 families linked to RET, also showed linkage to the 9q31 locus. Interestingly, no severe RET mutations were detected in those 6 families. It was concluded that these 6 families harbored “weak” RET DNA alterations and that the effect of the 9q31 locus was required to produce the phenotype.160 A genome-wide scan on 49 S-HSCR families detected linkage to three chromosomal regions, 10q12, 3p21 and 19q12. The 10q21 region corresponded to RET although causative RET mutations were present in only 40% of the linked families, again stressing the importance of variations in noncoding regions. Further analyses of the data demonstrated oligogenic inheritance of S-HSCR. The action of RET (10q12) and 3q21 and/or 19q12 seemed to be necessary and sufficient for the manifestation of S-HSCR. Since S-HSCR did not segregate in the absence of RET, the authors suggested that 3q21 and 19q12 loci are RET-dependent and, therefore, modifiers of the RET expression.25 These studies emphasize the central role of RET in all forms of HSCR (short and long) and explained the nonmendelian inheritance of the disease. Also, they suggest that the genetics of long-segment Hirschsprung disease and short-segment Hirschsprung disease may depend on the effects of the different RET modifiers.

Seminars in Pediatric Surgery, Vol 13, No 4, November 2004 gene (2q33-35) are associated with congenital limbs malformations165 and other skeleton dysplasias,166 some of which are also seen in some HSCR patients.27 Nevertheless, IHH mutation analysis in over 60 HSCR patients with no mutations in the HSCR genes described so far, reveal no coding region mutations that could account for the disease.167 Similar results were obtained for the human L1CAM gene. This gene encodes the L1 cell adhesion molecule involved in the development of the nervous system.168 Mutations in L1CAM are linked to a recessive form of congenital hydrocephalus.169 The detection of L1CAM mutations in individuals with congenital hydrocephalus and HSCR170,171 and the fact that L1CAM maps to chromosome Xq28, (which could account for the higher penetrance of HSCR in males) encouraged the mutational screening of this gene in isolated HSCR patients.172 Although no coding region mutations were identified, it was hypothesized that L1CAMmediated cell adhesion may be important for the ability of ganglion cell precursors to populate the gut, and that the L1CAM gene could modify the effects of a Hirschsprung disease-associated gene to cause intestinal aganglionosis. The characterization of the gene network directing the development of the ENS may help in the identification of additional HSCR candidate genes. These genes may play a minor role in the expression of the disease, and they are likely to be modifiers of the already known HSCR major genes.

Genetic counseling Other HSCR candidate genes Mouse models and syndromes associated with HSCR provide insights into the genes that may share a common signaling pathway involved in the development of the ENS and therefore in the pathogenesis of HSCR. It is therefore logical to study the possible contribution of these genes to HSCR. This can be illustrated by the example posed by the Indian hedgehog gene (IHH). The Hedgehog gene family encodes a group of secreted signaling molecules that are essential for growth and patterning of many different body parts of vertebrate and invertebrate embryos.161 In particular, Ihh signaling controls growth of bones162,163 and is required for the proper development of the ENS and the intestinal stem cell proliferation and differentiation.164 Ihh mutant mice present with dilated colon with abnormally thin wall, and enteric neurons missing from parts of the small intestine and from the dilated regions of the colon.164 This HSCR-like phenotype is observed with an incomplete penetrance of 50%, which suggests that interaction with other factors will be required for full expression of the phenotype.164 These features are strikingly similar to those observed in HSCR patients. In humans, mutations in the IHH

In isolated HSCR, a relatively precise recurrence risk tailored to individual families could be provided based on the estimates provided by Badner.173 The highest recurrence risk is for a male sib of a female proband with L-HSCR.27 Nonetheless, the reduced penetrance of the HSCR mutations makes it difficult to rationally predict and assess the risk to disease. Genetic testing is only performed on a research basis, and due to the advances of the surgical management of HSCR, its utility is questionable. As outlined earlier, mutations in the RET proto-oncogene are also the underlying cause of the inherited cancer syndromes MEN2A and MEN2B and FMTC. In HSCR these mutations are dispersed throughout the gene, while in MEN2A and FMTC, they are clustered in the cysteine codons of the RET extracellular domain (exons 10 and 11). Although HSCR and MEN2A are two different entities, occasionally they cosegregate in some families,174-181 and affected individuals carry a single mutation in exons 10 or 11. Importantly, RET mutations identical to those found in MEN2A have been detected in HSCR patients with no clinical symptoms of MEN2A.182,183 That means that some HSCR patients may be exposed to a highly increased risk of tumors. Where HSCR patients carry these tumor-specific mutations, exploration of the family history of MEN2A and

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periodic screening for tumors is advisable. In combined MEN2A/HSCR disease families, RET gene testing, tumor screening, and prophylactic thyroidectomy are also warranted.

Future directions The major breakthrough in the study of the pathogenesis of HSCR has been the demonstration of the genetic and functional interaction between the RET and EDNRB signaling pathways and their establishment as key players in the development of the ENS. These important findings should lead to the study of the complete gene network that makes the genesis of the ENS possible. Whole genome maps have recently been produced for several key organisms (including humans). The challenge now is to use this newly available genomic information firstly to elucidate normal gene function, and then to determine how alterations cause disease in humans. In the past researchers studied one or a few genes at a time. Nowadays, with the advent of new high-throughput technologies, it is possible to study all the genes expressed in a particular tissue, organ, or tumor, and to establish how thousands of genes and proteins work together. Systematic studies of gene and protein function—functional genomics— on a hitherto-unimagined scale will be the focus of biological explorations for decades to come. For obvious reasons, conducting gene or protein expression analyses for the study of HSCR and other human diseases that result from gene dysfunction during development is not feasible. However, mice can provide access to the study of human genes and proteins that have an equivalent in mice (orthologues). This kind of gene-expression data should help to determine the developmental stages at which different genes come into play. This information, supplemented with similar data obtained from mutated mice, could be used to monitor gene expression changes resulting from the inactivated genes. The knowledge thus gained, if applied to embryonic gut tissue, may enable new genes to be identified and clarify the unknown regulatory mechanisms governing ENS development. However, spatio-temporal patterns of expression should be linked to the morphological and physiological changes of the multiple cell types forming the gut, and knowledge of the roles of individual genes in specific developmental pathways should be linked with the differences between normal and abnormal development. While this may provide a general picture of the genes and proteins active at a given time point during development, the analysis of the embryonic gut as a whole to attempt to determine which genes and proteins are associated with a particular cell type poses major problems. Scientists have therefore resorted to isolating individual cell types by using techniques such as laser capture microdissection184 or by making use of stem cell research. For instance, this has allowed the study of the specific migratory

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requirements of the gut neural crest stem cells (NCSCs).2,23,185 High-throughput technologies applied to individual cells types can provide their characteristic gene and protein expression profile. Use of these approaches has also provided major breakthroughs in the molecular studies of the HSCR pathogenesis19,186,187 and importantly, in novel therapeutic resources. If the behavior of the gut neural crest stem cells (NCSCs) is better understood, it may be possible to treat HSCR by transplanting NCSCs directly into the aganglionic gut. It will be fascinating to determine whether these NCSCs are able to mature and engraft in the forming gut and enhance bowel motility, and further research of this kind should yield new therapeutic approaches.

Acknowledgment The authors would like to thank Dr. David Wilmshurst, technical writer at the University of Hong Kong, for his comments on the manuscript and the Research Grant Council for support (HKU7392/04M).

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Tam and Garcia-Barcelo

Molecular Genetics of Hirschsprung’s Disease

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