• Hirschsprung's disease;
  • genetics;
  • susceptibility gene;
  • pathogenesis


  1. Top of page
  2. Abstract

Hirschsprung's disease (HSCR) is a developmental disorder of the enteric nervous system, which occurs due to the failure of neural crest cells to fully colonize the gut during embryonic development. It is characterized by the absence of the enteric ganglia in a variable length of the intestine. Substantial progress has been made in understanding the genetic basis of HSCR with the help of advanced genetic analysis techniques and animal models. More than 11 genes have been found to be involved in the pathogenesis of HSCR. The RET gene is the most important susceptibility gene involved in HSCR with both coding and non- coding sequence mutations. Due to phenotypic diversity and genetic complexity observed in HSCR, mutational analysis has limited practical value in genetic counseling and clinical practice. In this review, we discuss the progress that has been made in understanding the molecular genetics of HSCR and summarize the currently identified genes as well as interactions between pathways and gene-modifying loci in HSCR. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

Hirschsprung's disease (HSCR), also known as intestinal aganglionosis, is a congenital anomaly of the large intestine arising from a developmental abnormality of the enteric nervous system (ENS). The pathology of HSCR is characterized by the absence of ganglion cells in the myenteric and submucosal plexuses of the distal intestine, resulting in the absence of peristalsis in the affected bowel. This leads to the dilatation and hypertrophy of the proximal large bowel causing symptoms of megacolon. Clinically, according to the length of the aganglionic segment, HSCR can be classified as short-segment HSCR (S-HSCR, 80% of cases) and long-segment HSCR (L-HSCR, 20% of cases). In S-HSCR, the aganglionic segment involves the rectum and the distal sigmoid colon only, whereas in L-HSCR the aganglionic segment extends more proximally. In few L-HSCR cases, the entire colon length may be involved and can be further classified into Total Colonic Aganglionosis (Garver et al., 1985; Badner et al., 1990; Torfs, 1998). The incidence of HSCR differs among populations. Asian population showed the highest incidence by 2.8:10,000 and the Hispanics shows the lowest incidence by 1:10,000 live births. There is significant gender variation in the incidence of HSCR. The male: female ratio is 4:1 among S-HSCR patients and 1:1 among L-HSCR patients (Torfs, 1998). Although HSCR commonly appears as a sporadic trait, 20% of HSCR cases are familial with complex inheritance patterns. In 30% of patients, HSCR is associated with a chromosomal abnormality or multiple congenital anomalies, also known as syndromic HSCR.

The mammalian ENS is composed of numerous distinct neuronal subtypes throughout the gut wall. The majority of the ENS is derived from vagal neural crest cells (NCCs) that emigrate from the neural tube at the level of somites 1–7. At embryonic day (E) 9.0–9.5, the vagal NCCs invade the foregut to become enteric NCCs, which migrate in a rostrocaudal direction to colonize the entire length of the gut (Kapur et al., 1992; Durbec et al., 1996; Gershon, 2010). The colonization of the gut by enteric NCCs requires expression of particular transcription factors and stimulation of specific growth factors. Disruption in these regulatory factors holds the potential for failure of ENS development resulting in HSCR. Sanden et al. (Peters-van der Sanden et al., 1993) showed that the ablation of the neural crest of somites 3–5 resulted in aganglionosis of the hindgut. The embryonic NCCs did not migrate to the intestinal wall, the ENS development was halted and there were no ganglia in the intestinal wall. Li et al. observed the embryonic expression of neural markers in the ENS (Li et al., 2001) and found that the development of colonic ENS has obvious stages. It was demonstrated that HSCR is a developmental disorder caused by a defect in the NCCs migration process. Barriers at different developmental stages of NCCs migration showed different types of clinical HSCR.

It is clear that most, if not all, cases of HSCR result from failure of NCCs to fully colonize the gut during embryonic development, and are associated with one or more genes mutations. Approximately 50% of cases of HSCR have been ascribed to mutations of specific genes involved in early embryonic development. Significant progress has been made in understanding the molecular genetics underlying the pathogenesis of HSCR. Identification of specific gene defect now allows direct genetic analysis, without examining the DNA of any other family member. By analyzing phenotypes with targeted gene mutations, the role of associated genes can be known in the development of ENS. Many genes have been found to be mutated in patients with HSCR. Here, we review progress that has been made in understanding the molecular genetics of HSCR and summarized the currently identified genes in HSCR.


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  2. Abstract

RET Gene and RET/GDNF/GFRα1 Signaling Pathway

The RET gene is located on chromosome 10 band q11.2 and encodes a receptor tyrosine kinase. There are three isoforms of RET: RET51, RET43, and RET9 (Carter et al., 2001). RET is the signaling receptor of a multimolecular complex that binds the growth factors of the glial cell line-derived neurotrophic factor (GDNF) family and the GDNF co-receptor-α (GFRα1) family (Airaksinen et al., 1999; Baloh et al., 2000). The RET/GDNF/GFRα1 signaling pathway plays an important role in the development of ENS. RET has been shown to be associated with papillary thyroid carcinoma (PTC) through chromosomal rearrangements (Fusco et al., 1987). Point mutations of RET were found to be responsible for the inheritance of the medullary thyroid cancer (MTC) and multiple endocrine neoplasia type 2 (MEN2) syndromes (Donghi et al., 1989; Donis-Keller et al., 1993; Mulligan et al., 1993). Furthermore, point mutations in the RET gene were identified in up to 50% of sporadic MTCs (Hofstra et al., 1994). Takahashi et al. reported that HSCR and MEN2A/FMTC co-segregate (Takahashi et al., 1999). Although gain of function mutations in the RET gene may result in MEN2A/FMTC, loss of function mutations have been reported in patients with HSCR. Martucciello et al. (1992) reported a case of total colonic aganglionosis associated with interstitial deletion of the long arm of chromosome 10. Homozygous RET mutations have been demonstrated in mice exhibiting total intestinal aganglionosis and renal agenesis (Schuchardt et al., 1994), suggesting that RET is the susceptibility gene for HSCR. To date more than 100 mutations have been identified encompassing the RET gene, including large-scale deletions, microdeletions, insertions, missense, nonsense, and splicing mutations (Edery et al., 1994; Romeo et al., 1994; Attié et al., 1995; Misha et al., 1995; Hofstra et al., 2000). Mutations in the coding regions of the RET gene account for up to 50% of familial and 7%–35% of sporadic HSCR cases. The majority of HSCR mutations results either in a reduction of dosage of the RET protein or in the loss of RET function (Pasini et al., 1995). Functional consequences of RET mutations in HSCR correlate with their position in the coding sequence and they have been classified into five groups (Plaza-Menacho et al., 2006): Class I mutations lie in a region of single peptide (SP) and cadherin repeat domains (CAD) of the RET protein. Mutations in these extracellular domains impair RET maturation and its translocation at the plasma membrane (Iwashita et al., 1996). Class II mutations lie in the cysteine-rich (CYS) and transmembrane (TM) regions of the RET protein, which markedly reduce RET cell surface expression and impairs proliferation and migration of enteric neurons during embryogenesis (Takahashi et al., 1999). Among these mutations C609, C611, C618, and C620 have a dual impact on RET. On the one hand, they constitutively activate RET receptor leading to uncontrolled proliferation of thyroid C-cells, as seen in MEN2A and FMTC. On the other hand, they result in a marked reduction of RET expression at the plasma membrane leading to apoptosis of enteric neurons, as seen in HSCR (Chappuis-Flament et al., 1998; Arighi et al., 2004). Class III mutations affect the tyrosine kinase (TK) region by partially or completely disrupting the RET catalytic activity (Iwashita et al., 2001). Class IV mutations affect amino acids in the carboxyl-terminal tail and impair the binding of effector or adaptor proteins to RET (Ishiguro et al., 1999). Class V mutations lie in the regulatory sequences and change the expression of the RET gene.

RET may also have a role in promoting apoptosis. In the presence of the ligand, RET produces positive control signal (stimulation) to maintain cell development and survival. In the absence of the ligand, RET produces negative regulation signal (inhibition) and promote apoptosis (Mehlen and Bredesen, 2004). Bordeaux et al. (2000) demonstrated that RET can induce apoptosis in 293T cells. This apoptotic effect of RET was inhibited in the presence of its ligand GDNF. Accordingly, it has been proposed that not only RET mutations and impairment of the signal transduction can cause HSCR, but it may also result from apoptosis of RET-expressing enteric neuroblasts. The mutations were generated by site-directed mutagenesis and these mutant forms transfected into cells to assess effects on GDNF control of RET-induced apoptosis. Apoptosis was found in most transfected cells with the mutant gene regardless of the extent of GDNF. Therefore, the mechanism of RET mutations in HSCR may be composed of two parts, i.e. abnormal cell signal transduction caused by RET mutation leading to the abnormal colonization of NCCs in the intestine, and the negative regulation of the signal induction of the RET ligand GDNF gene to promote apoptosis of neural crest progenitor cells.

RET gene is closely related to the pathogenesis of HSCR. Not only frequencies of RET mutations detected in HSCR cases were higher, but linkage analysis has shown that in 90% of families, HSCR is linked to the RET gene (Bolk et al., 2000). Some non-coding gene mutations in sporadic HSCR have shown link to the RET gene, indicating that non-coding regions of the RET gene also have an important role in HSCR. Although the exact mechanism underlying HSCR is still unclear, it has been demonstrated that RET has a dominant effect in the pathogenesis of HSCR.

Transmission disequilibrium test and case–control analysis of sporadic HSCR have revealed that the frequency of the RET gene polymorphisms and haplotypes composition of polymorphic loci is associated with HSCR. Borrego et al. (2003) conducted a population-based study on isolated HSCR patients from Spain and found that RET sequence variants were significantly over-represented in the HSCR patients. Similar findings were reported in a series of HSCR cases from different population bases, Italy, Spain, France, Germany, and the United Kingdom (Lantieri et al., 2006). Fitze et al. used a dual-luciferase assay to evaluate the haplotypes in the RET promoter in 80 HSCR patients. They found a significant difference between the AC haplotype in the HSCR cases (68.8%) compared to the control cases (25%), suggesting the role of RET haplotypes in HSCR (Fitze et al., 2003; Lantieri et al., 2006). Garcia-Barceló et al. (2005b) found HSCR-associated RET promoter SNPs, -5G-A, and -1C-A. Functional analysis of the RET promoter SNPs showed that the HSCR-associated alleles decreased RET transcription.

Emison et al. (2005a) found that a common non-coding RET variant within a conserved enhancer-like sequence in intron 1 was significantly associated with HSCR susceptibility and explained several features of the complex inheritance pattern of HSCR. They thought that RET mutations, whether coding or non-coding, are probably a necessary feature in all HSCR cases. Burzynski et al. (2004) investigated HSCR cases for the RET gene promoter region and intron 1 SNP and found six SNP loci in 55.6% of HSCR patients compared to the 16.2% of controls. The risk of heterozygous haploid in developing HSCR was 2 times higher than the normal population and 20 times higher than the homozygous haploid. In our analysis of the RET intron in the Chinese population with HSCR, we found that a high transmission frequency of the RET+3: T allele led to an increased susceptibility to HSCR (Zhang et al., 2007; Liu et al., 2008). A 5.7-fold and 2.1-fold increase in susceptibility to HSCR in males and females, respectively, was estimated in SNP haplotypes of the RET+3. We also found that the distribution of some other polymorphisms in the intron 1 was significantly different between cases and controls. Among these, two haplotypes were significantly associated with HSCR (Liu et al., 2008). Emison et al. studied 882 probands with HSCR and 1478 first-degree relatives from the US, European, and Chinese families and found that common mutations, individually and together, may contribute to the risk of HSCR. The distribution of RET variants in diverse HSCR patients suggested a “cellular-recessive” genetic model where both RET alleles functions were compromised (Emison et al., 2010). This indicates that non-coding mutation of the RET gene, particularly, in the intron 1 region, play important roles in the pathogenesis of HSCR and is related to the expression level of the RET gene. When the RET gene expression level is decreased, the susceptibility to HSCR will be increased. The effects of RET polymorphisms and haplotypes may be involved in the pathogenic mechanisms of HSCR. Uesaka et al. (2008) showed that when RET expression is reduced to one-third of normal levels, colonic aganglionosis results. It is worth noting that compared to the other races, HSCR-associated RET SNPs and haplotypes are significantly higher in the Chinese population, not only in patients but also in the general population. This could partially explain the higher incidence of HSCR in the Chinese population than Caucasian populations (Emison et al., 2005b).

The GDNF family ligands (GFLs) have been reported to promote the survival of precursor cells during the ENS development (Ruiz-Ferrer et al., 2011). GDNF and GFRα1 homozygous knockout mice have shown severe defects in enteric innervation and renal development, similar to that of RET mutations mice (Pichel et al., 1996; Enomoto et al., 1998). The GDNF mutations in HSCR patients resulted in a significant reduction in the binding affinity to GFRα1. Although none of the GDNF mutations identified so far in HSCR patients are sufficient to cause disease, these mutations may contribute to the pathogenesis of HSCR in conjunction with other genetic lesions (Angrist et al., 1996; Salomon et al., 1996; Eketjäll and Ibáñez, 2002). Therefore, GDNF is considered as a rare susceptibility gene for HSCR (<5%). A heterozygous missense neurturin (NTN) mutation in a large non-consanguineous family including four children affected with a severe aganglionosis phenotype has been reported. The authors thought that the NTN mutation was not sufficient to cause HSCR, because this multiplex family also segregated a RET mutation. It was concluded that this cascade of independent and additive genetic events fits well with the multigenic pattern of inheritance expected in HSCR, and further supports the role of RET ligands in the development of the ENS (Doray et al., 1998). In a recent article, Ruiz et al. analyzed the coding sequence of GDNF family genes in HSCR patients and confirmed the involvement of NTN in HSCR (Ruiz-Ferrer et al., 2011). There was no evidence for any GFRα1 mutations in HSCR patients (Myers et al., 1999). Although GFRα2 variants were found in some patients with HSCR, but there was no indication of GFRα2 involvement in the causation of HSCR (Vanhorne et al., 2001).

EDNRB/EDN3 Genes and Signaling Pathway

The EDNRB gene is located on the chromosome 13q22. The gene encodes a 442 residue protein of heptahelical receptors, also known as G-protein-coupled receptors containing seven-transmembrane domains. The extracellular regions of receptors and transmembrane domains are involved in ligand binding, whereas the intracellular domains are involved in G-protein-mediated intracellular signaling pathways (Pingault et al., 2010). EDNRB and its ligand EDN3 play an essential role in the normal development of two neural crest-derived cell lineages, enteric neurons, and melanocytes in mice and humans (Lee et al., 2003). Their critical role in melanocytes and enteric development has been demonstrated via targeted disruption of the mouse genes Edn3 and Ednrb. Targeted disruption of Ednrb resulted in an autosomal recessive phenotype with white spotting and aganglionic megacolon. By phenotypic non-complementation and molecular analysis, it has been demonstrated that Ednrb is allelic to two other hypopigmentation alleles in mouse (piebald, s, hypomorphic mutation; and piebald-lethal, sl, gene deletion) (Hosoda et al., 1994). In addition, it has also been demonstrated that the targeted deletion of the gene encoding Edn3 is allelic to the mouse white spotting and aganglionosis mutation (lethal-spotting, ls, missense mutation) (Baynash et al., 1994). Although the phenotypes of mice with mutations of EDNRB and EDN3 seemed to be similar, the differences were apparent. EDNRB knockout (Ednrb-/-) and piebald-lethal mice were almost completely devoid of coat color with variable ranges, usually developed megacolon and did not survive to adult. However, mice with mutation in Edn3 (Edn3-/-, lethal-spotting) showed pigmentation over 20%–30% of their bodies, and at least 15% survived into adulthood. These finding implicates that the EDNRB/EDN3 signaling pathway plays an important role in the development of neural crest-derived cells (McCallion and Chakravarti, 2001).

The Mennonite families (community) are known as genetically closed population with high incidence of HSCR. Most affected individuals in this family presented with HSCR, but some have features of Waardenburg syndrome type 4 (WS4). A susceptibility locus on 13q22 has been identified by linkage analysis performed on the Mennonite family, and a missense mutation (W276C) in the EDNRB gene was confirmed to be associated with HSCR. The mutation was dosage sensitive, in that W276C homozygotes and heterozygotes had a 74% and a 21% risk, respectively, of developing HSCR (Puffenberger et al., 1994). It can be considered that homozygotes have a high probability of developing severe phenotypes, while heterozygotes may, in some instances, present one or more features of HSCR with low-penetrance. To date, more than 20 different EDNRB mutations, including large-scale deletions have been identified in sporadic and familial HSCR, accounting for 5% of the HSCR patients (Auricchio et al., 1996). These different mutations were found scattered along the protein, resulting in impaired ligand binding and reduced transduction signal. A research performed on EDN3 mutations showed similar observations as in EDNRB. Two homozygous mutations of EDN3 were identified in WS4 patients with HSCR (Edery et al., 1996; Hofstra et al., 1996; Bidaud et al., 1997). Some novel heterozygous mutations of EDN3 were also identified in patients with isolated HSCR. Endothelin converting enzyme 1 (ECE1) is involved in the proteolytic processing of EDN3 to biologically active peptides. ECE1-deficient mice lack enteric neurons and epidermal/choroidal melanocytes, reproducing the phenotype of Edn3 and Ednrb knockout mice (Yanagisawa et al., 1998). Hofstra et al. described involvement of the ECE1 gene in HSCR. The patient had skip-lesions HSCR, cardiac defects, and autonomic dysfunction (Hofstra et al., 1999).

Studies have found that the mutations of the EDNRB/EDN3 genes tends to give rise to S-HSCR, whereas RET mutations appear to play a major role in L-HSCR. This may be due to the relevant regulation of both EDNRB/EDN3 and RET in NCCs at different times. EDNRB is expressed primarily in migrating NCCs, whereas EDN3 is expressed in the hindgut mesoderm and at high levels in the cecum and proximal colon. This pattern of expression suggests that EDN3-EDNRB signaling is involved in regulating the normal migration of NCCs. Therefore, EDN3-EDNRB mutations are believed to occur in NCCs migrating toward the hindgut and the hindgut is mainly affected, as often seen in S-HSCR. Now, it is well known that the EDN3-EDNRB signaling pathway in the enteric nerve cells is essential during the colonization process of the small intestine and colon (Barlow et al., 2003). RET mutations in the ENS occurred throughout the entire length of the intestine, as often seen in L-HSCR. It is estimated that RET mutations account for approximately 50% of HSCR cases and EDNRB mutations account for approximately 5%, while S-HSCR occurs in about 25% of RET-caused cases and in more than 95% of EDNRB-related cases (Chakravarti, 1996).

SOX10 Gene

The SOX10 gene encodes a 466-amino acid transcription factor that contains a high mobility group (HMG) DNA-binding domain and a C-terminal transactivation domain. SOX10 exerts its function through binding to the promoters or enhancers of its target genes, alone or in association with other transcription factors. It is a key transcription factor during neural crest-derived cells migration and differentiation (Lee et al., 2000; Kelsh, 2006). Interest in the functional role of SOX10 intensified with the discovery that the neurocristopathy phenotypes in the Dominant megacolon (Dom) mice are linked to a SOX10 mutation. The SOX10 gene mutations have been identified in the heterozygous Dom mice with enteric aganglionosis and hypopigmentation, while homozygous SOX10 Dom mutant mice were embryonic lethal. In addition, mice lacking SOX10 expression were shown to reduce the number of neural precursor cells, indicating that SOX10 plays an important role in maintaining the functional status of neural progenitor cells in the intestine (Paratore et al., 2002). Pingault et al. showed that patients with WS4 had mutations in SOX10, whereas no mutations were detected in patients with HSCR alone. These mutations were likely to result in haploinsufficiency of the SOX10 product (Pingault et al., 1998), indicating that SOX10 is the major susceptibility gene for WS4. Only one case of SOX10 mutation has been reported to date to be associated to isolated HSCR patient (Avencia et al., 2010). Therefore, SOX10 is unlikely to be a major gene in the pathogenesis of isolated HSCR and its mutation in syndromic HSCR does not exceed in more than 5% of HSCR cases (McCallion et al., 2003).


The ZFHX1B gene encodes Smad-interacting protein-1 (SMADIP1 or SIP1), a transcriptional repressor involved in the transforming growth factor-β signaling pathway. It is a highly evolutionarily conserved gene, widely expressed in embryological development (Dastot-Le Moal et al., 2007). Mutant mice which have lost ZFHX1B protein in their NCCs were shown to display specific abnormalities in melanocyte development as well as defects in the peripheral nervous system of the gastrointestinal tract and loss of vagal NCCs (Van de Putte et al., 2007). Mowat-Wilson Syndrome (MWS) is a multiple congenital anomaly characterized by distinct facial features, intellectual deficiency, and epilepsy, and is sometimes associated with HSCR. Wakamatsu et al. reported nonsense mutations of the ZFHX1B gene in patients with MWS. These mutations represented null alleles, suggesting that haploinsufficiency for ZFHX1B is sufficient to cause MWS phenotype (Wakamatsu et al., 2001). So far, more than 110 different mutations have been described in the ZFHX1B gene. Nonsense mutations accounted for approximately 41% of the known punctual mutations and were localized mainly in exon 8 (Dastot-Le Moal et al., 2007). No ZFHX1B mutations have been reported to be associated to isolated HSCR patients, indicating that ZFHX1B is a susceptibility gene for syndromic HSCR (Amiel et al., 2001).


The PHOX2B gene is located on chromosome 4p12 and encodes a 314-amino acid paired box homeodomain transcription factor that is expressed in the developing hindbrain and peripheral nervous system. The homozygous disruption of the PHOX2B gene has been reported to result in the absence of enteric ganglia, a feature which is reminiscent of HSCR (Elworthy et al., 2005). Amiel et al. (2003) reported that PHOX2B is the primary disease locus in congenital central hypoventilation syndrome (CCHS). They found that mutations in PHOX2B not only exist in isolated cases of CCHS but also in individuals with a more complex neural crest involvement including CCHS and HSCR (Haddad syndrome). Fitze et al. (2008) found no PHOX2B mutations in the HSCR population, but RET variants were identified in CCHS/HSCR group, which were similar in location and frequency to the HSCR group. They proposed that the combined CCHS and HSCR phenotype was not associated solely with PHOX2B mutations. A mutated PHOX2B protein may interfere with the RET protein or RET expression resulting in HSCR. PHOX2B SNPs were also found to be associated with the pathogenesis of isolated HSCR and interaction between RET and PHOX2B SNPs has shown to increase the risk for HSCR (Garcia-Barceló et al., 2003; Liu et al., 2009).

L1CAM Gene

The L1CAM gene, located on chromosome Xq28, is a member of the immunoglobulin gene superfamily of neural cell adhesion molecules. It is a major susceptibility gene for X-linked hydrocephalus (XLH). Mutations in the L1CAM gene have been identified in human HSCR cases associated with XLH (Nakakimura et al., 2008). Although L1CAM is involved in the proper migration of neural components and it may modify the effects of a HSCR-associated gene to cause intestinal aganglionosis (Parisi et al., 2002), L1CAM mutations alone do not account for HSCR (Jackson et al., 2009).

KIAA1279 Gene

The KIAA1279 gene is located on chromosome 10q22.1 and encodes a protein with two tetratrico peptide repeats. Homozygous mutations in KIAA1279 have been identified in a large consanguineous Moroccan family with Goldberg-Shprintzen megacolon syndrome (GOSHS). All patients in this family had bilateral generalized polymicrogyria in addition to HSCR, establishing the importance of KIAA1279 in both enteric and central nervous system development (Brooks et al., 2005). Recently, KIAA1279 has been implicated in the regulation of neuronal microtubules organization, and in axonal growth and maintenance by interacting with SCG10, a membrane-associated neuronal protein. Also, SCG10 has been previously identified as a down-regulated gene in a RET mouse model for HSCR. So, interaction between KIAA1279 and SCG10 may influence the neuronal development and could cause HSCR in some patients (Lyons et al., 2008; Alves et al., 2010).

TCF4 Gene

The TCF4 gene is located on chromosome 18q21.1. It is highly expressed throughout the developing human central nervous system and sclerotomal components of the somites (de Pontual et al., 2009). Pitt-Hopkins syndrome (PHS) is a rarely reported syndrome characterized by mental retardation, wide mouth, and intermittent hyperventilation. PHS is also one of the syndromes associated with HSCR. Amiel et al. found heterozygous missense mutations in TCF4 in patients with PHS (Amiel et al., 2007). Zweier et al. showed that HSCR in patients with PHS might be explained by altered development of noradrenergic derivatives (Zweier et al., 2007). They thought haploinsufficiency as the disease-causing mechanism (Zweier et al., 2008).

Today, mutations in more than 11 different genes have been implicated in the pathogenesis of HSCR (Table 1). Many of these genes are associated with syndromic HSCR.

Table 1. Gene mutations and animals models associated with HSCR
GeneLocusPhenotypeInheritanceMutationModel organisms (mouse)
  1. AD, autosomal dominant; AR, autosomal recessive; RLX, recessive linked to X chromosome; CF, craniofacial; MR, mental retardation.

RET10q11.2HSCR/MEN2AD, incomplete penetranceHeterozygotesKnockout
EDNRB13q22HSCR/WS4AD/ARHetero/homozygotesKnockout, sl
EDN320q13HSCR/WS4AD/ARHetero/homozygotesKnockout, ls
ECE11p36HSCR,CF andADHeterozygotesKnockout
  Cardiac defect   
SOX1022q13WS4/PCTHADHeterozygotesKnockout, Dom
L1CAMXq28MR hydrocephalusrlxHeterozygotes
RMRP9p21Skeletal dysplasiaARHomozygotes 
  With immunodeficiency (CHH)   
  Syndrome (SLO)   
Locus 29q31HSCRAD, incomplete penetrance 
Locus 319q12HSCRNon-Mendelian 
Locus 416q23WS syndromeNon-Mendelian 
Locus 54q31.3-32.3HSCRAD, incomplete penetrance 


  1. Top of page
  2. Abstract

As described above, the successful colonization of the gut by the ENS precursors depends on a coordinated and balanced network of interacting molecules. However, the question of how these interacting molecules and signaling pathways are involved in the normal ENS development still remains to be answered.

A whole-genome association scan was carried out in 43 Mennonite family trios with HSCR and a significant association between the transmission of the EDNRB Trp276Cys mutation and an HSCR-susceptibility RET haplotype was found. The combination of these two genotypes increased the penetrance of the Trp276Cys mutation and the risk of HSCR (Carrasquillo et al., 2002). These findings implied the existence of interactions between RET and EDNRB signaling pathways. Transgenic animal model also confirmed that the coordinated activity of the RET and EDNRB signaling pathways controlled neurogenesis throughout the murine intestine (Barlow et al., 2003).

EDN3 and GDNF seemed to have a synergistic effect on the proliferation of the undifferentiated ENS progenitors and an antagonistic effect on the migration of differentiated ENS cells (Tam et al., 2009). Activation of the receptor tyrosine kinase RET by GDNF is required for the directional migration of ENS progenitors toward and within the gut wall (Natarajan et al., 2002). GDNF availability determines enteric neuron number by controlling ENS precursor proliferation (Gianino et al., 2003). Interactions between the RET and EDNRB signaling systems have been demonstrated to control ENS development throughout the intestine, providing evidence for the coordinated and balanced interactions between these signaling pathways (Barlow et al., 2003).

Phenotype analysis of SOX10, EDNRB, and SOX10 and EDN3 double mutants showed that a coordinated and balanced interaction between these molecules is required for the normal ENS development. The partial loss of EDNRB in SOX10 heterozygous mice impaired colonization of the gut by enteric crest cells at all stages observed, while double mutants presented with a severe increase in white spotting and more severe ENS defects (Stanchina et al., 2006). SOX10 was also shown to interact with RET (Lang and Epstein, 2003).

HSCR has been found in the aforementioned genes, although evidence is building up that HSCR is a multigenic congenital malformation in the majority of HSCR patients. Non-coding mutations in the RET gene have also contributed to the risk of HSCR in combination with other genes or loci. Many studies have demonstrated genetic modifiers of HSCR phenotype. The genetic modifiers may reflect interactions among genes already known to underlie HSCR. Carrasquillo et al. conducted a genome-wide association study in 43 Mennonite family trios using 2,083 microsatellites and SNPs. They identified susceptibility loci at 10q11, 13q22, and 16q23 and showed that the gene at 10q11 was RET and the gene at 13q22 was EDNRB. The authors suggested that genetic interactions between mutations in RET and EDNRB is an underlying mechanism for HSCR (Carrasquillo et al., 2002). Bolk et al. (2000) performed linkage analysis in 12 cases of familial HSCR and found that six families harbored severe RET mutations and the six remaining families (five RET-linked families and one RET-unlinked family) revealed a new susceptibility locus on chromosome 9q31. These remaining six families were correlated with the 9q31 locus. Tang et al. performed fine mapping of HSCR loci in 9q31 and identified two different HSCR-associated genes, i.e. SVEP1 and IKBKAP. The association of IKBKAP was stronger in Chinese HSCR patients with RET mutations, while SVEP1 SNPs were found to be associated with Dutch HSCR patients (Tang et al., 2010). Brooks et al. reported a multigenerational Dutch family with isolated HSCR. Analysis of the RET gene revealed neither linkage nor mutations. A genome-wide linkage analysis was performed and revealed suggestive linkage to a region on 4q31-q32. Their results suggested the existence of a new susceptibility locus for HSCR at 4q31-q32. Considering the low penetrance of disease in this family, the 4q locus may be necessary but not sufficient to cause HSCR in the absence of modifying loci elsewhere in the genome (Brooks et al., 2006). Gabriel et al. conducted a genome scan in families with S-HSCR and identified susceptibility loci at 3p21, 10q11, and 19q12. The gene at 10q11 appeared to be RET, supporting its crucial role in all forms of HSCR. However, coding sequence mutations in RET were present in only 40% of families linked to 10q11, suggesting the importance of non-coding variation. They concluded that the action of these loci seemed to be necessary and sufficient to manifest S-HSCR (Gabriel et al., 2002). Garcia-Barceló et al. (2008a) provided additional evidence for the susceptibility locus on chromosome 3p21 in a Chinese population. They proposed that a susceptibility locus for HSCR could lie on chromosome 3p21. However, more evidence is needed to confirm these findings.

Garcia-Barceló et al. analysis of 172 sporadic Chinese HSCR patients revealed the presence of HSCR-associated RET promoter SNPs. These SNPs overlapped a TTF-1 binding site and TTF-1-activated RET transcription was decreased by the HSCR-associated SNPs. They also studied the NK2 Homeobox 1 gene (NKX2-1) as a HSCR locus and found that NKX2-1 mutations could contribute to HSCR by affecting RET expression through defective interactions with other transcription factors (Garcia-Barceló et al., 2005, 2008b). Genes identified by genome-wide screens were likely to provide an even richer source of new possible HSCR loci. Garcia-Barceló et al. (2009) carried out genome-wide association study and identified NGR1 as a HSCR susceptibility locus. The study found strong associations for two SNPs located in intron 1 of NGR1. NGR1 were shown to increase the risk of HSCR in the presence of RET, indicating interactions between these two genes. Recently, a total of 13 different heterozygous mutations were identified in the sequenced NRG1 exons. It was found that not only common, but also rare variants of the NRG1 gene contributed to HSCR. These findings strengthen the role of NRG1 in the pathogenesis of HSCR (Tang et al., 2012).

These studies emphasize the central role of different genes in the pathogenesis of HSCR and also indicate that HSCR susceptibility genes may lie in the effects of different gene modifiers.


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  2. Abstract

The molecular genetics pertaining to HSCR clearly have an important utility in understanding the disease recurrence risks in families. Genotype–phenotype correlation in patients with syndromic HSCR is sometimes sufficient to suggest mutation in a particular susceptibility gene. For example, XLH is associated with the L1CAM gene and GOSHS is associated with the KIAA1279 gene. However, considerable phenotypic overlap exists among syndromic HSCR patients, and different mutations in the same gene may cause similar phenotypes. Therefore, a complete clinical evaluation and family history should be collected.

Carter (1969) thought that multifactorial diseases had different trait from Mendelian diseases. First, the underlying disease allele is polymorphic. Second, recurrence risks in relatives of a proband depend inversely on population incidence, increasing with greater severity and being greater for a less frequently affected class. Finally, recurrence risks vary across families, even for the same genetic relationship. According to Carter's paradox, isolated HSCR is a complex multifactoral disease with low, sex-dependent penetrance and variable expression according to the length of the aganglionic segment and has a high sibling recurrence risk.

Challenges remain in the application of current understanding of HSCR genetics to clinical practice. HSCR exhibits variable penetrance even in families with the same genetic variants, reflecting the influence of environmental factors and genetic modifiers. Genetic modifiers can affect the phenotypic outcome of a given genotype by interacting with the genes. Because of these uncertain and variable factors, gene mutation testing has limited value in clinical practice. However, there are some exceptions such as MEN2A/FMTC-associated RET mutations were characterized in exons 10 and 11.

The treatment of HSCR is surgical. However, stem cell therapy has also seemed to show therapeutic potential for HSCR (Theocharatos and Kenny, 2008). It is reported that neural stem cells can colonize in the embryonic gut and give rise to enteric ganglion cells. Nevertheless there is still a long way to go before successfully colonizing stem cells in the mature gut and establishing functional connections.


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  2. Abstract

The causative genes have been identified in no more than 50% of familial, 7%–35% of sporadic, and 10% of syndromic HSCR cases, suggesting that other susceptibility genes remain to be identified.

To identify unknown susceptibility genes and locus, genome-wide association studies (GWAS) employing common SNPs seems to be a useful method. GWAS have successfully revealed numerous susceptibility genes for common diseases. NGR1 has been identified as a HSCR susceptibility locus by GWAS (Garcia-Barceló et al., 2009).

It is assumed that risk alleles with large effect size may be rare in frequency and hard to detect by GWAS employing common SNPs. The use of high-throughput sequencing technologies to locate rare variants of larger effect may aid in the complete resolution of this trait. Since the cost for whole genome resequencing for a read depth sufficient to identify variants with a high accuracy is still expensive, it is not easy to resequence the whole genome of a large number of individuals (Shoji, 2010). Therefore, whole exome sequencing is a good compromise for larger numbers of samples.

Although many technologies were used to identify susceptibility genes, these genes are still less important in HSCR, because a small number of patients account for mutation of these genes. RET is the major susceptibility gene in HSCR and the emphasis on RET study should switch from mutation scan and association analysis to regulatory mechanisms underlying gene expression.

HSCR-associated alleles affect the activity of the RET promoter. Functional analysis of the RET promoter SNPs in the context of additional 5′ regulatory regions demonstrated that the HSCR-associated alleles decrease RET transcription. These SNPs overlap a TTF-1 binding site and TTF-1-activated RET transcription is also decreased by the HSCR-associated SNPs (Garcia-Barceló et al., 2005b). Sribudiani et al. reported that binding of the transcription factors NXF, ARNT2, and SIM2 to RET depends on the RET polymorphism of Enh2 (rs2506004) and affects RET expression and the development of HSCR (Sribudiani et al., 2011).

AU-rich elements (AREs) are found in the 3′UTR region. AREs and AUUUA motifs have been shown in many cases to mediate mRNA destabilization. The SNP rs3026785 is located in a U-rich region near the last AUUUA sequence, and may affect the secondary structure and the stability of RET mRNA. The study of regulatory mechanisms of such SNPs can be more essential than the association analysis of SNPs.

miRNAs are small non-coding RNAs of approximately 21–24 nucleotides, which interact with specific target mRNAs in the 3′UTR region, resulting in translational repression followed by mRNA deadenylation and degradation.

Several studies demonstrate interconnections between microRNAs and AREs (von and Gallouzi, 2008). Some miRNA target sequences are predicted or have been demonstrated within AUEs (Robins and Press, 2005). It suggested that AREs and miRNAs cooperatively regulate the mRNA expression. We found significant difference of two SNPs distribution in RET 3′UTR between cases and controls (data not published), which might exert a role in posttranscriptional regulation of mRNA.

In conclusion, although important advances have been made in understanding the genetic basis of HSCR, there remain significant challenges including, identifying the unknown susceptibility genes and loci in HSCR, and exploring how these genes interact in the ENS development and pathology of HSCR.


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