SEMA3D, SEMA3A, SEMA3C, ZEB2, NRG1, ERBB, VRK2/FANCL, LRBA, IHH, GLI3, ACSS2, ADAMST17, ENO3, FAM213A, SH3PXD2A, SLC27A4, UBR4, DENND3, NCLN, NUP98, TBATA, UFD1L, TBX2, SLC8A1, MAPK8 and

GATA2 (refs. 22,83–88). Many of these genes encode proteins involved in the GDNF–RET signalling pathway and endothelin receptor type B signalling pathway, or encode transcription factors that regulate these developmental pathways89–91.

The RET signalling pathway was the first to be implicated in HSCR pathogenesis, and is still the most studied92. Studies have found >100 mutationsinRET,andRET variantsarethemostfrequentlyobservedin patientswithHSCR89.RETvariantsaccountfor15–35%ofsporadicHSCR and~50%offamilialHSCR93.RETmutationsaremorecommonlyfoundin long-segment HSCR than in short-segment HSCR83,85. Loss-of-function RET mutations are the most frequently observed, although activating missensemutationshavebeendescribed.VariantsinGDNF,eitheralone or in combination with variants in RET can lead to HSCR occurrence.

EDNRB was the second gene found to be associated with HSCR40. In addition, mutations in SOX10, ZFXH1, GATA2 and PHOX2B (encoding transcription factors) have an indirect involvement in the development of HSCR. Finally, EDNRB has an epistatic interaction with RET5, and both genes are regulated by SOX10 and GATA2 in a dose-dependent manner, which influences their expression levels.

Variants, partial penetrance and epigenetics. HSCR has a complex genetic basis involving diverse genetic variations such as predisposing haplotypes, rare coding variants and large copy number variants. These genetic variations can be coding or non-coding and can act additively, and single-nucleotide polymorphisms may alsobeinvolved. The combination of rare and common variants seems to give a higher risk than rare or common variants only22. Common forms of HSCR are hypothesized to be determined by common variants with small functional effects, whereas rare variants with a stronger phenotype determine rare forms of HSCR. Additionally, functional small effects of non-codingvariantsareamplifiedinageneregulatorynetwork94.Inthis way, common variants of transcription factors worksynergistically and enhance the disrupting effects of RET variants95.

HSCR is recognized as a multifactorial polygenic disorder with a variablepatternofinheritance.Mutationsinknowngenesdonotalways manifest in disease owing to partial penetrance, which is why even a high-risk genetic profile is associated with a risk of clinically manifest HSCRofonly1in120(ref.22).ClinicalmanifestationofthetypicalHSCR phenotype depend on other epigenetic regulation factors, and studies have shown, for instance, that the RET promoter is susceptible to methylation96. However, epigenetic changes and post-transcriptional regulation of NCCs are also related to other mechanisms, such as modification of histone molecules and microRNA expression97.

Disease models

Several disease models are available that can be used to understand HSCRpathogeneticmechanisms64.Zebrafisharecommonlyusedowing to their quick development and high number of offspring53. However, zebrafish lack certain components of the ENS found in mammals, such as discrete ganglia and myenteric and submucosal plexuses. Several models of HSCR can be found in mammals including mice, rats and horses. In mice, HSCR models include the lethal spotted mouse, the piebald lethal mouse and the dominant megacolon mouse, which exhibit distal aganglionosis owing to mutations in Ednrb, Edn3 and Sox10, respectively98. Several research groups have created transgenic mouse models of HSCR involving various pathways, such as the

RET–GDNF pathway, the EDNRB–EDN3 pathway, SOX10, PHOX2B, collagen VI, DDX3Y, cell adhesion molecules, hedgehog proteins and bone morphogenetic proteins64,66,98,99. Additionally, chemically induced models of aganglionosis have been used, including benzalkonium chloride-induced irreversible depolarization and cell death in the myenteric plexus100 and teratogenic exposure to aberrant vitamin A and/or retinoic acid, which is thought to interfere with retinoic acid signalling during ENS development101.

Diagnosis, screening and prevention

Clinical presentation

Approximately 80% of patients with HSCR are diagnosed during the neonatal period. The typical signs include distal intestinal obstruction, including delayed passage of meconium beyond 24 h, abdominal distension, feeding difficulties and bilious vomiting1. However, not all signs might be present, especially in patients with total colonic aganglionosis in which the colon is not distended. In such infants, meconium passage may not be delayed, and the infant may have regular bowel movements or even diarrhoea, indicative of HSCR-associated enterocolitis (HAEC)19,102,103.

HAEC is an inflammatory and infectious disorder and the main cause of morbidity and mortality in HSCR104,105. Classic signs of HAEC include fever, lethargy, vomiting, diarrhoea, rectal bleeding and abdominal distension105. HAEC can be the initial presentation ofHSCR,especiallyinneonateswithDownsyndromeandlong-segment HSCR104. Therefore, HSCR should be suspected in a full-term neonate who develops enterocolitis, as up to 20% of these patients may have underlying HSCR106,107.

Neonates with the most severe HSCR present with bowel perforation, which can be multifactorial, linked to extensive bowel distension due to obstruction, bowel ischaemia due to enterocolitis and/or secondary to a rectal washout108. Up to 7% of neonates with HSCR develop this severe life-threatening complication while awaiting diagnosis or surgery108.

Diagnosis

During clinical examination and history taking, attention should also be given to associated malformations and family history. A family history of HSCR is reported in 8% of all patients with HSCR and increases to 20–50% in those with total colonic aganglionosis109.

In an infant presenting with the typical symptoms of HSCR, a plain abdominal radiography will usually show dilated bowel loops throughout the abdomen, indicating distal bowel obstruction with little or no air in the lower part of the pelvis (Fig. 1). A contrast enema might be helpful to suggest HSCR and rule out differential diagnoses such as meconium plug syndrome, meconium ileus or intestinal atresia110. The characteristic feature of HSCR is the presence of a change in bowel size between the normally innervated, proximal dilated bowel and the distal narrow, aganglionic colon, suggesting the start of the transition zone (Fig. 1). The rectosigmoid index, calculated by dividing the widest diameter of the rectum by the widest diameter of the sigmoid colon, is considered abnormal if the value is <1, and pathognomonic for recto­ sigmoid HSCR111. Irregular, sawtooth contractions of the aganglionic colorectum may also be observed. However, these findings may be absent, especially in infants with total colonic aganglionosis, in whom contrast enema may show a microcolonin 24%, ashort ‘question-mark’- shaped colon in 18%, or surprisingly, even a normal-shaped colon in 50%112 (Fig. 1). The mean sensitivity and specificity of contrast enema for HSCR diagnosis have been estimated to be 70% and 83%113.