The genetics of Hirschsprung disease

Gastroenterol Clin N Am 32 (2003) 819–837

The genetics of Hirschsprung disease Douglas R. Stewart, MDa, Daniel von Allmen, MDb,* a

The Children’s Hospital of Philadelphia, 34th & Civic Center Boulevard, Philadelphia, PA 19104, USA b Division of Pediatric Surgery, University of North Carolina–Chapel Hill, Chapel Hill, North Carolina 27599, USA

Harald Hirschsprung, a Danish pediatrician (1830–1916), first reported in 1888 two unrelated boys who died from severe constipation and abdominal distension resulting from congenital megacolon [1]. The etiology of the condition that bears his name, also called aganglionic megacolon, was not recognized until 1940, when Whitehouse and Kernahan [2] reported the absence of intramural ganglion cells of the myenteric (Auerbach’s) and submucosal (Meissner’s) plexuses. In 1948 Swenson and Bill [3] developed the life-saving surgical procedure that spared patients an (historically) invariably premature death. Patients’ survival and reproduction revealed the previously unsuspected familial transmission of Hirschsprung disease (HSCR) [4]. In 1967 Passarge reported 63 families with HSCR [5]. Although cases of autosomal recessive and autosomal dominant (usually with decreased penetrance) inheritance were reported, it soon became clear that HSCR did not always follow the rules of Mendelian (or monogenic) inheritance [6]. By the mid-1980s it was apparent that non-syndromic HSCR was a multi-factorial disorder [7]. As HSCR developed as a genetic disease, an understanding of the underlying neurologic derangement also emerged. The first step occurred with the development of rectal suction biopsies that used histochemical staining for acetylcholinesterase (AchE) [8]. Benish’s 1975 paper defining a neurocristopathy as a syndrome involving neural crest cells introduced a useful concept in understanding the pathogenesis of HSCR [9]. A neurocristopathy is a syndrome or tumor that arises secondary to an abnormality in neural crest (NC) cell development. The NC is a multipotent embryonic structure located at the junction of neural and epidermal ectoderm in the neural folds of the neurula-stage embryo [10]. At precise

* Corresponding author. E-mail address: [email protected] (D. von Allmen). 0889-8553/03/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0889-8553(03)00051-7

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times in development the NC releases cells that migrate to target sites, settle and differentiate into a variety of end organs and tissues. In the case of HSCR, the enteric nervous system (ENS) is inadequately formed secondary to the arrest, in the 5th to 12th week of development, of NC cell emigration to the hindgut [11]. As a model system, HSCR has been of long-standing interest to gastroenterologists, surgeons, embryologists and geneticists. In the last decade, HSCR has emerged as a model of a ‘‘complex trait,’’ [12] a term applied to diseases in which there is clearly familial aggregation, but where Mendel’s laws do not seem to apply. Although monogenic disorders can be ‘‘complex,’’ the usage typically applies to oligogenic or polygenic diseases [13–15]. In HSCR, oligogenic inheritance applies in most cases (usually short segment disease) [16]. However, even within families with apparent monogenic inheritance of HSCR (usually long segment disease), the phenotype severity can be broad among family members, a finding seen in many Mendelian disorders, and a clue that ‘‘monogenic’’ disorders are more complex than previously thought [17]. High throughput genotyping and modified genetic linkage techniques, developed in the last decade, have made complex traits more amenable to genetic analysis. Application of these techniques in combination with mouse models to HSCR has uncovered not only previously unsuspected genes and chromosomal loci, but also entire signaling pathways and gene–gene interactions [18,19]. This recent work promotes HSCR as the model for the analysis of other complex traits. Such sophisticated genetic analysis stands to teach us not just about the pathogenetics of HSCR, but also potentially much about neurocristopathies, ENS and autonomic nervous system biology.

Epidemiology Hirschsprung disease is common, with a population incidence of 1/5000. The largest and most recent survey, with near-complete ascertainment of cases, comes from the California Birth Defects Monitoring Program (1983– 1997) and found an incidence of 1.5/10,000 live births in whites, 2.1/10,000 live births in African-Americans, 1.0/10,000 live births in Hispanics and 2.8/ 10,000 live births in Asians [20]. It has been long recognized that males are more commonly affected than females. The California study confirms these findings and demonstrated male:female ratios of 3.3, 3.4, 3.0, 4.4:1 among whites, African-Americans, Hispanics and Asians, respectively. The reason for these skewed ratios is unclear; no X-linked loci have been described. If the index patient is female, the proportion of affected siblings is higher. This presumably reflects the greater contribution of genetic factors required for manifestation of the disease in females, a paradox known as the Carter effect [21].

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Hirschsprung disease has traditionally been divided into two types: short segment (S-HSCR) and long segment (L-HSCR). The internal anal sphincter is the inferior limit in both types. In the more common S-HSCR (80% of cases) the aganglionic segment is contained below the splenic flexure. L-HSCR (20% of cases) is typically defined as aganglionosis including and beyond the splenic flexure. Rarer but instructive variants of HSCR include total colonic aganglionosis (TCA, 3%–8% of cases), total intestinal HSCR (whole bowel involvement) and ‘‘ultra-short segment’’ HSCR involving the distal rectum [22,23]. When congenital aganglionosis is present with other congenital abnormalities (30% of cases), it is considered syndromic HSCR. For this reason, all patients with HSCR should be evaluated by an experienced dysmorphologist, as recurrence risks vary considerably between syndromic and non-syndromic HSCR, and among specific syndromes. Non-syndromic HSCR (70% of cases) refers to the disease as an isolated trait. Among syndromic cases, approximately 40% (12% of total) are secondary to chromosomal abnormalities and the remaining 60% (18% of the total) have multiple congenital anomalies or recognized genetic syndromes [24]. Although a full appraisal of the various syndromes associated with HSCR is beyond the scope of this review, a handful bear mentioning given their importance in elucidating the genetic etiology (complete discussion can be found in Chakravarti and Lyonnet [41]).

Syndromic Hirschsprung disease Chromosomal anomalies play an important role in dissecting the genetic burden of many diseases as individual patients with a specific chromosomal deletion, duplication or translocation can give a clue to an involved gene’s location. The most common (2%–10% of all ascertained cases; 90% of all chromosome cases) chromosomal anomaly in HSCR is trisomy-21 (Down syndrome) [4,7,20,24–26]. The number of males affected (5.5 to 10.5: 1 male: female) and the percentage of S-HSCR (85%) is even greater than in sporadic HSCR [24]. The reason for this imbalance is unknown. Presumably a chromosome 21 gene ‘‘dosage’’ disruption is to blame for the phenotype in trisomy-21, however only one locus on chromosome 21 in one study has been linked to HSCR [27]. Mutations in RET, endothelin receptor B (EDNRB) and glial cell line-derived neutrophic factor (GDNF), key genes in non-syndromic HSCR, have been reported in patients with trisomy-21 and HSCR [28,29]. A smaller percentage of chromosomal anomalies include cytogenetically-visible interstitial deletions. These are a prime source of candidate gene loci in many syndromes. Interstitial deletions or translocations of chromosomes 10q11.2, 13q22.1-32.1 and 2q22-23 all lead to the mapping and identification of RET, EDNRB and SIP1 (Smad interacting protein-1, also known as ZFHX1B), respectively [30–36].

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Patients with additional congenital anomalies but without cytogenetically visible anomalies may belong to one of three possible categories: neurocristopathy syndromes, non-neurocristopathy syndromes with HSCR variably featured, and non-syndromic HSCR with isolated anomalies. The distinction is critical, as prognosis and counseling for recurrence risk vary significantly among categories. Accurate phenotyping is also key in any study attempting to discern the pathogenetic mechanism of HSCR. These considerations emphasize the role for a thorough examination and family history by a knowledgeable dysmorphologist. Neurocristopathies These encompass an array of syndromes with seemingly unrelated disparate clinical anomalies that belie the pluripotenital nature of neural crest cells. Characterization and study of these syndromes by geneticists has lead to a recognition of the unique role of neural crest cells and their key role in embryogenesis of neuronal, endocrine and paraendorine tissues, the conotruncal region of the heart and craniofacial skeleton and pigment cells [10]. Of particular interest to the gastroenterologist is the formation of the enteric nervous system, which is derived like other branches of the peripheral nervous system, from neural crest cells [37]. Thus, identification of specific genes involved in HSCR offers a handle to understand the growth, development and derangement of the ENS, and by extension, the autonomic nervous system. The ENS has been called ‘‘the second brain’’ [38]. It contains 100 million neurons (more than the spinal cord), the same classes of neurotransmitters found in the central nervous system (CNS) and reflex arcs that function independently of the CNS [39,40]. Anatomically it contains two concentric rings, the outer myenteric plexus and in the inner submucosal plexus and associated connecting neural structures in the bowel wall. Although it is in communication with the CNS by way of parasympathetic and sympathetic neurons, it is able to independently process and integrate gut wall dilatation, muscle contractility and secretion, all without CNS input [41]. Briefly, the ENS is derived from NC cells migrating during the 5th to 12th week of gestation from the vagal neural crest (somites 1–5), the anterior trunk NC (somites 6–7) and the sacral neural crest (posterior to somite 24) [11]. In addition NC cells from the sacral neural tube (posterior to somite 28) colonize the hindgut. The submucosal ganglia derived from the vagal crest forms first and the outer myenteric ganglia form from cells migrating along blood vessels from these submucosal ganglia. Failure of the vagal derived NC cells to colonize the hindgut results in failure of hindgut ENS development. Clearly, careful orchestration of sacral and vagal NC cells is needed for the sacral enteric NC contribution to the ENS [37]. Given NC cells’ role in the development of neuronal, endocrine, paraendocrine, cono-truncal structures and pigmentary cells, it should not

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come as a surprise that anomalies of these tissues are seen in HSCR. These neurocristopathies include: Shah–Waardenburg syndrome Shah–Waardenburg syndrome (OMIM 277580, Online Mendelian Inheritance in Man, http://www.ncbi.nlm.nih.gov/Omim) is also known as HSCR-Waardenburg or Waardenburg syndrome type 4. Waardenburg syndrome is the most common condition combining pigmentary anomalies (white forelock, extensive depigmentation of the skin, premature graying, heterochromic irides) and sensorineural deafness (1/50,000 live births, 2%– 3% of all congenital deafness). Mutations in the EDNRB in a Mennonite community with this phenotype was first described in 1994 [42]. Individuals homozygous for the mutation had a 74% risk of HSCR; heterozygotes had a 21% risk, suggesting the role of other genes. A more severely affected patient with Shah–Waardenburg with mental retardation, cerebellar ataxia and autonomic dysfunction was heterozygous for a mutation in the SOX10 gene. Other patients with more classical features of Shah–Waardenburg have also been reported with SOX10 mutations. Interestingly, a patient with chronic pseudoobstruction and Shah–Waardenburg features has also been reported with a SOX10 mutation [43–48]. Congenital central hypoventilation syndrome Congenital central hypoventilation syndrome (Ondine’s curse, OMIM 209880) is a rare disorder of the autonomic nervous system whose primary feature is an abnormal ventilatory response to hypoxia and hypercapnia. Other autonomic symptoms include esophageal motility abnormalities, profuse sweating, decreased body temperature and insufficient tear production. Neural crest cell derived tumors have been described [49–52]. Haddad syndrome is the combination of CCH and HSCR; unlike sporadic HSCR, L-HSCR and TCA are more frequent and the sex ratio is equal [53,54]. Like sporadic HSCR, mutations in RET, GDNF and EDN3 have been described in both CCHS and Haddad syndrome [55]. Multiple endocrine neoplasia 2 Multiple endocrine neoplasia 2 (MEN2), perhaps the best-known neurocristopathy, is the inherited autosomal dominant cancer syndrome MEN2. There are three types: familial medullary thyroid carcinoma (FMTC), MEN2A (medullary thyroid carcinoma, pheochromocytoma, parathyroid hyperplasia), and MEN2B (features of MEN2A plus hyperganglionosis of the himdgut, marfanoid habitus and oral neuromas). Only FMTC and MEN2A have been associated with HSCR [56–63]. All of the MEN2A syndromes are associated with germline mutations of RET. Unlike

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the haploinsufficiency seen in HSCR, the RET mutations in MEN2 are gain-of-function. This has raised the question of whether all patients with HSCR should be screened for mutations in RET to rule out any cancer predisposition [55]. Non-neurocristopathies Hirschsprung disease is also seen, with variable frequency, in other genetic syndromes. The reader is referred to Chakravarti et al [41] for a complete accounting. These syndromes, while rare, are often characteristic to a clinical geneticist and typically include dysmorphic features, skeletal dysplasias, heart defects, and other congenital anomalies. Examples include Goldberg–Shprintzen syndrome (OMIM 235730; cleft palate, mental retardation, hypotonia, dysmorphia), HSCR with limb anomalies (several distinct syndromes, OMIM 235750, 235760, 604211, 306980), BRESEK syndrome (OMIM 300404; brain abnormalities, retardation, ectodermal dysplasia, skeletal malformation, ear and eye anomalies, kidney dysplasia), Bardet-Biedl syndrome (OMIM 209900; pigmentary retinopathy, obesity, hypogenitalism, mild mental retardation, postaxial polydactyly), Kauffman– McKusick (OMIM 236700; hydrometrocolpos, postaxial polydactyly, congential heart defect), and Smith–Lemli–Opitz (OMIM 270400; a disorder of cholesterol metabolism with growth retardation, microcephaly, mental retardation, hypospadias, 2,3 toe syndactyly, and dysmorphia). Syndromic involvement must be distinguished from the presence of occasional isolated anomalies. These can be divided into two groups: regional anomalies secondary to local bowel dilatation (renal obstruction) and more distant anomalies. The incidence of additional isolated anomalies in sporadic HSCR is between 5% and 30% [64–67]. Regional anomalies include colonic, rectal, and anal atresia [68,69]. Renal anomalies like megalonephrosis, hydronephrosis, and megalocystis are also ascribed to HSCR bowel obstruction [41]. Anomalies presumably not related to the bowel wall include cardiovascular defects (ASD and VSD in non-trisomy 21 patients), renal and genital anomalies, skeletal and limb anomalies (polydactyly), deafness and autonomic dysfunction. Meckel diverticulum, pyloric stenosis, small bowel atresia, and inguinal hernia have all been reported [70– 72]. Whether these isolated anomalies are simply coincidental or provide insight into the underlying pathogenesis of HSCR is difficult to determine, given the infrequency of any particular defect. The recurrence risk and prognosis of syndromic HSCR (including HSCR associated with chromosomal abnormalities) will depend on the recurrence risk of the associated syndrome. Typically, these syndromes follow Mendelian laws of inheritance, with predicted recurrences of up to 25% (for autosomal recessive conditions) and 50% (for autosomal dominant conditions). Decreased penetrance may reduce these figures. The recurrence risk of HSCR within a syndrome is not well studied. However, given the

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relatively good outcome of surgically treated HSCR, prognostication should consider those features in a syndrome (mental retardation, severe heart malformation) less amenable to treatment.

Non-syndromic Hirschprung disease The study of recurrence risk in non-syndromic HSCR has led to important insights into the genetics of long- and short-segment disease. Early studies recognized that the recurrence risk to relatives were much higher than the general population [4,5], however, more specific predictions were not possible. More accurate recurrence risks are possible based on gender and length of aganglionosis [7]. The average recurrence risk in siblings is 3% to 4% but can be much higher (approximately 23%). This corresponds to relative risks many hundreds times greater than the general population. Segregation analysis by Badnor et al [16] on similar data were elemental to understanding the particular genetic model at work in the different types of HSCR. Their study used uniform classification and near-complete ascertainment of all primary relatives. With L-HSCR, heritability (defined as the proportion of the total phenotypic variance in a trait that is caused by the additive effects of genes [73]) was 100% whereas with S-HSCR heritability was between 80% to 95%. These percentages suggest an absence or near-absence of environmental effects on the phenotype. Thus, by segregation analysis, the most likely model in L-HSCR and colonic segment (TCA) HSCR was of autosomal dominance. In S-HSCR, the most likely models were either multifactorial inheritance or segregation of a common recessive gene. The robustness of these results were confirmed by re-analysis of the data by considering aganglionosis in discrete segments of the gastrointestinal tract [16,41]. Based on these analyses, L-HSCR and TCA are best viewed as genetically heterogeneous autosomal dominant conditions with reduced penetrance secondary to ‘‘modifier genes.’’ The likelihood the phenotype will emerge depends on mutations, or perhaps just polymorphisms, in other genes. How these ‘‘modifier genes’’ work is poorly understood. S-HSCR is best considered an oligogenic disease. This means that the interaction of several genes is necessary for the phenotype. In many cases a mutation in coding DNA has not been found. Presumably variants in non-coding (intronic or other regulatory regions) DNA account for a significant percentage of the alleles. Recent papers have shed important light on the exact nature of the particular genes or loci and how they interact [18,19]. Short- or long-HSCR, and the genetic methods used to dissect their inheritance, now serve as models for approaching other complex traits. Other commentators have extended the model even further, noting that the relationship between

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implicated genes resembles a robust scale-free network [12,15]. Thus far, eight genes have been implicated in both short- and long-HSCR, and the interaction among them is only beginning to be understood. However, although the number of genes involved in the network is large, for an individual patient, the number of genes involved will be small, and thus will represent a unique phenotype. A similar situation will apply to patients with diabetes mellitus, hypertension, cancer, and coronary heart disease. Thus understanding and identifying not only the particular gene involved but also how they interact in a biological network at a fundamental level will permit translation of genotype information into predictions about phenotype [12]. The eight genes involved in HSCR are all involved with the early development of the enteric nervous system. Two genes, RET and EDNRB, account for 50% and 5%, respectively, of cases of HSCR in the general population. Disease associated mutations in the other six genes (EDN3, ECE1, SOX10, GDNF, NRTN and ZFHX1B) are rarer and in some cases have been documented in only one family. However, they must act through RET- and EDNRB-mediated pathways [41]. Additional loci have been found recently, although the specific gene or genes at these loci remain unknown [18,19]. Specific details about the eight known genes follow. RET signaling pathway In both short and long segment HSCR, the RET signaling pathway plays a critical role. RET was the first HSCR susceptibility gene identified [74,75]. It is a proto-oncogene 1114 amino acid transmembrane receptor with a cadherin-like extra-cellular domain, a cysteine-rich region and an intracellular tyrosine kinase domain. RET (‘‘rearranged in tumors’’) is expressed in the developing central and peripheral nervous system. In MEN2A (which occasionally features HSCR), where RET was first identified, mutations primarily appear in exons 10 and 11. In MEN2B (in which HSCR has not been reported), the mutation is in exon 16. In HSCR, the identified mutations occur throughout the gene [41]. In the cancer-susceptibility syndromes, the mutations appear to be activating and cause constitutive dimerization of the receptor and subsequent transformation [76]. In HSCR mutations are inactivating and lead to misfolding or failure to transport the protein to the cell surface [77–80]. This effectively results in half the usual dose of functioning protein, a situation known as haploinsufficiency. Further proof that haploinsufficiency is the underlying mechanism comes from patients with HSCR with outright deletions of RET. Clearly, half of the wild type RET is not sufficient for normal enteric development. GDNF is a member of the TGF-b superfamily and is the ligand for RET. Gdnf ÿ/ÿ mice display intestinal aganglionosis and renal agenesis [81–83], which prompted Angrist et al [84] to screen 106 unrelated HSCR patients for mutations in GDNF. One missense mutation was found in a patient with

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a known RET mutation and malrotation of the gut and prompted the authors to speculate the GDNF is a minor (\ 5%) contributor to human HSCR. Other investigators have found GDNF mutations in a small number of HSCR patients (including one with trisomy-21) and confirmed Angrist’s suspicion that mutations in GDNF are neither necessary nor sufficient to cause HSCR [28,85]. GDNF is proteolytically cleaved to form a 134 amino acid protein that homodimerizes. GDNF activates RET only in the presence of glycosylphosphatidylinositol (GPI) co-receptors, of which four have been described (GFRA1-4) [86,87]. No mutations have been reported in these co-receptors, although Gfra ÿ/ÿ mice are phenotypically similar to Ret and Gdnf ÿ/ÿ mice [28,85]. In addition, three other growth factor ligands (neurturin, artemin and persephin) support RET. One family with aganglionosis extending to the small bowel (and a RET mutation) has been reported with a heterozygous neurturin mutation in a highly conserved region [88]. Endothelin signaling pathway EDNRB, ligand endothelin 3 (EDN3), and associated enzyme endothelin converting enzyme (ECE1) constitute the other major pathway in HSCR. Approximately 5% of patients with HSCR have mutations (as heterozygotes) in EDNRB or EDN3 [74]. How EDNRB and RET interact received greater clarification in a recent study on HSCR, and EDNRB has emerged as one of the first ‘‘modifier’’ genes to be identified [19]. The endothelin pathway has a vasoconstrictive effect and was first studied in its role in hypertension. EDNRB and its related type A receptor, EDNRA are G-protein coupled heptahelical receptors that transduce signals from the 21 amino acid endothelin (EDN1, -2, -3) [89,90]. EDNRB, 443 amino acids, is expressed in the myenteric plexus, mucosal layer, ganglia, and vessels of the submucous of the colon, among other tissues [91,92]. EDNRB’s role in HSCR emerged from several lines of evidence. Linkage to 13q22 in an Old Order Amish population with multiple cases of HSCR has been found in two studies [19,27]. Several patients have been reported with de novo interstitial deletions of 13q22 [32,33,93]. Lastly, comparative mapping of the piebald-lethal gene (a murine model of aganglionosis) revealed homology to EDNRB, located at 13q22 [94]. Confirmation of EDNRB’s particular role arrived with identification of an EDNRB missense mutation (Trp276Cys) in the Mennonite population [42]. Like RET, haploinsufficiency seems to be the cause in HSCR [95]. However, only 87% of affected individuals carry the mutation and the phenotype (pigmentary defects like those seen in Shah–Waardenburg) is penetrant in 74% of homozygotes. In heterozygotes (where only HSCR is seen, and not the pigmentary defects) penetrance is only 21% [42]. EDNRB’s interaction with RET will be discussed in the section ‘‘Genetic interactions.’’

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Mice mutant for EDN3 also display a phenotype similar to Shah– Waardenburg [96]. Humans with Shah–Waardenburg and isolated HSCR have been reported with mutations in EDN3 [97–100]. Further evidence of the role of the endothelin signaling pathway in neural crest-derived neuron development stems from the identification of a mutation in EDN3 in a patient with isolated congenital central hypoventilation syndrome (Ondine’s curse) [50]. ECE1 is involved in proteolytic processing of endothelin-1, -2 and -3. A patient with skip-lesion HSCR, cardiac defects, craniofacial and hand abnormalities, and autonomic dysfunction had a mutation in amino acid position 742, which is in the vicinity of the active site of ECE1 [101].

Other genes: SOX 10 and SIP1 Recognition of SOX10’s involvement in HSCR arose, like EDNRB and EDN3, from a de novo mouse mutation. The murine dominant megacolon (Dom) is a model of Shah–Waardenburg in heterozygotes; homozygous Dom mutations are embryonic lethal [102]. Dom heterozygotes display regional deficiencies of neural crest-derived enteric ganglia in the distal colon. Dom is homologous to human SOX10 (SRY-like HMG box transcription factor, chromosome 22q13) [103]. Haploinsufficiency appears to be the mechanism for the small number of individuals with familial and syndromic Shah–Waardenburg syndrome, some of whom have neurologic variants with autonomic dysregulation, demyelination, or leukodystrophy [43,48]. One unusual patient with a SOX10 mutation with deafness and a peripheral neuropathy with hypomyelination had chronic intestinal pseudo-obstruction, but no HSCR [47]. Mutation analysis in such rare patients serves as an example of garnering insight into the pathogenesis of common entities like chronic intestinal pseudo-obstruction. SIP1, a transcription factor, was reported to be mutated in patients with HSCR, mental retardation and a unique facies in 2001 [36]. Subsequently additional patients with large deletions or truncating mutations were identified, suggesting that haploinsufficiency was the mechanism [35,104]. Patients without HSCR, but with a similar facies and mental retardation, have also been reported [105].

Genetic interactions We noted previously that RET is implicated in both S- and L-HSCR, and that S-HSCR follows an oligogenic model and L-HSCR follows an autosomal dominant model, with ‘‘modifier genes’’ regulating the expression of the phenotype. The most recent and exciting work on HSCR has examined how genetic elements (genes or loci) interact to yield the HSCR phenotype [18,19].

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Bolk Gabriel et al [18] performed a genome scan with 371 markers on 67 distinct affected sib pairs (ASPs). Genome scans use many evenly spaced markers (either microsatellites or single nucleotide polymorphisms, SNPs) across the genome and statistical methods to look for evidence of linkage to common genomic elements. In complex traits, where large multi-generational families with multiple affected members (typical kindreds in classical linkage studies) are rare, ASP analysis is a modified linkage technique that quantifies the degree of shared alleles at a marker locus or loci under investigation. This is done by IBD (identical-by-descent) analysis, which quantifies the number of shared alleles (0,1,2) between siblings, as inherited from the parents. The genome scan, performed on ASPs with S-HSCR from the general population, revealed three susceptibility loci, at chromosomes 3p21, 10q11, and 19q12. The gene at 10q11 is likely to be RET, however coding sequence mutations were present in only 40%. Segregation at 10q11 alone was not sufficient to explain the inheritance of S-HSCR. Inheritance of susceptibility alleles from all three loci is needed for the S-HSCR phenotype (although inheritance of susceptibility alleles from RET and either 3p21 or 19q12 cannot be absolutely refuted). This fits a multiplicative model of inheritance, an observation that accords with previous studies [16]. The authors also observed a skewed parental transmission of susceptibility alleles of RET (but not from the other two loci): 21 maternal and 6 paternal transmissions. Such distorted transmission may explain the unusual sex ratio of 4:1 males: females long observed in HSCR [106]. The success in identifying just three loci in a robust recapitulation of oligogenic inheritance of S-HSCR launches non-sydromic HSCR, and the genetic tools to dissect it, as a model approach in the investigation of other complex traits [106]. A similar approach using a whole genome association scan was undertaken by Carrasquillo et al [19] in 36 families in an inbred Old Order Mennonite population, where the incidence of HSCR at 1/500 is approximately 10 times greater than that of the general population. A whole genome association scan uses markers (microsatellites of SNPs) and looks for evidence of linkage disequilibrium in affected family members. The unit of analysis is an affected individual and his or her parents (a ‘‘trio’’). Trios are typically more common than ASPs. Linkage disequilibrium (also known as allelic association) is a special type of very tight linkage that extends over much shorter (\ 30 kilobases) distances than traditional linkage. SNPs, by the nature of their plentitude throughout the genome, permit precise testing of particular alleles. In the study, 2083 loci spanning all 46 chromosomes were tested with microsatellites and SNPs. Susceptibility loci were identified at chromosomes 10q11, 13q22 and 16q23. RET and ENDRB are the genes at 13q22 and 10q11, respectively. The gene at locus 16q23 is currently unknown. RET was then fine-mapped with additional markers. Haplotypes were then created in the 59 and 39 ends, based on areas of L.D. One particular haplotype at each end (‘‘59a’’ and ‘‘39a’’) seemed to be more commonly transmitted to affected individuals

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than other haplotypes. Since RET and EDNRB are not syntenic (on the same chromosome), their transmission should be independent, unless there is a synergistic interaction between them. Such an interaction between EDNRB, ‘‘59a’’ and ‘‘39a’’ were observed. Thus the authors conclude that ‘‘genetic interaction between each of the RET LD domains and EDNRB, and possibly an interaction between the RET and EDNRB biochemical pathways, may form the basis for HSCR’’ A model based on the data can then estimate penetrance, based on which particular RET haplotypes are coinherited with the EDNRB Trp276Cys mutation. An extensive search at the RET locus did not detect variants that altered the function or production of RET, again suggesting the presence of non-coding mutations in distal regulatory regions. A murine model intercrossing Ret-null and EDNRB hypomorphic piebald alleles confirmed the interaction of Ednrb and Ret in creating the HSCR phenotype. Lessons from the genetic analysis of Hirschsprung disease HSCR has been investigated for over 100 years, but the pace of discovery and understanding is quickening. Although the application of sophisticated genetic tools is recent, several important lessons have emerged. The importance of genome-wide scans This technique, used so effectively by Bolk Gabriel et al and Carrasquillo et al [18,19], places markers agnostic to gene location to uncover shared genomic elements. In both cases putative candidate genes were refuted, and the significance of others (RET, EDNRB) confirmed. Most importantly, genome-wide scans uncover previously unsuspected genes or loci (the loci chromosomes 3p21, 19q12 and 16q23). Such scans postulate that certain genes and their pathways, previously thought to be independent, may interact to produce the trait of interest. This reverses the traditional candidate gene approach, where examining involved pathways creates a list of putative genes. Genome-wide scans now offer the ability to generate candidate pathways and interactions, based on the genes and loci uncovered. Indeed, the question is not ‘‘what do pathways tell us about genetics’’ but ‘‘what does genetics tell us about pathways?’’ Role of non-coding variants Both the Bolk Gabriel and Carrasquillo papers detected coding DNA variants in genes of interest in only a fraction of affected patients. This failure suggests the presence of mutations in distal regulatory regions. Finding these regions belie the importance of understanding gene expression in complex traits. Identifying those genetic variants that vary gene expression is a future challenge. The search is already underway [107]. In

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complex traits changes in mRNA could be more significant than protein polymorphisms. Emergence of genotype–phenotype predictions Success in phenotype prediction will certainly potentiate genetics’ role in clinical medicine. Currently our ability correlate phenotype with a particular genotype, in HSCR and many ‘‘simple’’ monogenic diseases, is poor [13– 15,17]. However, with better understanding of the variants that control gene expression, ‘‘modifier genes,’’ susceptibility haplotypes and gene–gene interactions (and in the further future, proteomic networks) this predictive power will improve. Genetics should be able to predict not just the phenotype (length of aganglionosis) but also prognosis following surgery. This is currently an area of active research in both HSCR and other diseases [108–112]. HSCR and other diseases of gastrointestinal motility Understanding the genetics of HSCR will naturally expand our understanding of other neurocristopathies and also of the enteric nervous system and autonomic system biology. As other disorders of GI motility are investigated, genetics may be able to resolve certain clinical questions. For example, isolated hypoganglionosis without aganglionosis has been reported as a primary cause of intestinal pseudo-obstruction. Is such hypoganglionosis merely a forme-fruste of HSCR, or a result from an entirely different pathogenetic mechanism [113]? Can irritable bowel syndrome or severe constipation be related to specific mutations, polymorphisms or haplotypes? How might an understanding of derangements of the ENS be translated to understanding derangements of the CNS? Clearly, we should anticipate improved prognostication, counseling and hopefully, therapies, with future genetic insights.

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