Absence Of Enteric Ganglia Research Articles

Literature Review: The Role of RET Gene Mutations in The Emergence of Hirschsprung Disease

Hirschsprung’s disease is a congenital condition characterized by a failure of nerve function along the gastrointestinal (GI) tract, resulting in the absence of enteric ganglia in the most distal segment of the colon. Although the prevalence rate is 1:5000 babies born, Hirschsprung’s disease can have a significant impact on patients, especially in terms of quality of life. One of the main genetic factors often associated with the etiopathogenesis of Hirschsprung’s disease is the RET gene. This study aims to provide scientific insight into advances in the understanding of genetic variants associated with RET gene mutations and their role in causing Hirschsprung disease. The research design used was literature review, articles were collected based on sources derived from Google Scholar, PubMed, and EMBASE which included national and international journals published in the last 10 years. Based on the articles collected, it was found that the RET gene is essential for the growth, migration, and maturation of precursor cells into functional neurons. Mutations in the RET gene can interfere with the normal formation and function of nerve cells, leading to symptoms typical of Hirschsprung’s disease.

Essential Role of BMP4 Signaling in the Avian Ceca in Colorectal Enteric Nervous System Development.

The enteric nervous system (ENS) is principally derived from vagal neural crest cells that migrate caudally along the entire length of the gastrointestinal tract, giving rise to neurons and glial cells in two ganglionated plexuses. Incomplete migration of enteric neural crest-derived cells (ENCDC) leads to Hirschsprung disease, a congenital disorder characterized by the absence of enteric ganglia along variable lengths of the colorectum. Our previous work strongly supported the essential role of the avian ceca, present at the junction of the midgut and hindgut, in hindgut ENS development, since ablation of the cecal buds led to incomplete ENCDC colonization of the hindgut. In situ hybridization shows bone morphogenetic protein-4 (BMP4) is highly expressed in the cecal mesenchyme, leading us to hypothesize that cecal BMP4 is required for hindgut ENS development. To test this, we modulated BMP4 activity using embryonic intestinal organ culture techniques and retroviral infection. We show that overexpression or inhibition of BMP4 in the ceca disrupts hindgut ENS development, with GDNF playing an important regulatory role. Our results suggest that these two important signaling pathways are required for normal ENCDC migration and enteric ganglion formation in the developing hindgut ENS.

Genetics of Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a classical model of enteric neuropathy, occurring in approximately 2-2.8 in 10,000 newborns. It is the commonest form of congenital bowel obstruction and is characterized by the absence of enteric ganglia in distal colon. Recent advances in genome-wide association analysis (GWAS) and next generation sequencing (NGS) studies have led to the discovery of a number of new HSCR candidate genes, thereby providing new insights into the genetic architecture and molecular mechanisms of the disease. Altogether, these findings indicated that genetic heterogeneity, variable penetrance and expressivity, and genetic interaction are the pervasive characteristics of HSCR genetics. In this review, we will provide an update on the genetic landscape of HSCR and discuss how the common and rare variants may act together to modulate the phenotypic manifestation. Translating the genetic findings to genetic risk prediction and to optimize clinical outcomes are undoubtedly the ultimate goals for genetic studies on HSCR. From this perspective, we will further discuss the major obstacles in the clinical translation of these latest genetic findings. Lastly, new measures to address these clinical challenges are suggested to advance precision medicine and to develop novel alternative therapies.

The transition zone in Hirschsprung's bowel contains abnormal hybrid ganglia with characteristics of extrinsic nerves

The aganglionic bowel in short-segment Hirschsprung's disease is characterized both by the absence of enteric ganglia and the presence of extrinsic thickened nerve bundles (TNBs). The relationship between the TNBs and the loss of enteric ganglia is unknown. Previous studies have described decreasing numbers of ganglia with increasing density of TNBs within the transition zone (TZ) between ganglionic and aganglionic gut, and there is some evidence of spatial contact between them in this region. To determine the cellular interactions involved, we have analysed the expression of perineurial markers of TNBs and enteric ganglionic markers for both neural cells and their ensheathing telocytes across four cranio-caudal segments consisting of most proximal ganglionic to most distal aganglionic from pull-through resected colon. We show that in the TZ, enteric ganglia are abnormal, being surrounded by perineurium cells characteristic of TNBs. Furthermore, short processes of ganglionic neurons extend caudally towards the aganglionic region, where telocytes in the TNB are located between the perineurium and nerve fibres into which they project telopodes. Thus, enteric ganglia within the TZ have abnormal structural characteristics, the cellular relationships of which are shared by the TNBs. These findings will help towards elucidation of the cellular mechanisms involved in the aetiology of Hirschsprung's disease.

Colostomy Delays Cell Loss in the Brain and Improves Juvenile Survival in a Neonatal Rat Model of Hirschsprung’s Disease

Hirschsprung’s disease is a congenital malformation characterized by the absence of enteric ganglia in the distal intestine and gut obstruction. Our previous study indicates the brain pathology during the disease progression. A subpopulation of Hirschsprung’s disease patients is also associated with anomalies of the central nervous system. In the investigation, we studied a rat model of Hirschsprung’s disease, known as spotting lethal (sl/sl) ETB-/- rats, which carries a spontaneous deletion in endothelin receptor B (human gene name: EDNRB) and manifests a similar phenotype as humans with Hirschsprung’s disease. Homozygous mutant sl/sl rats were successfully rescued from premature death by performing colostomy and dramatically survived to their juvenile age. By the body weight measured, their body growth was not revealed to be significantly different between ETB-/- and wildtype ETB+/+ or heterozygous (+/sl) ETB+/- groups while all underwent the same colostomy. Cell loss was investigated in several brain regions by using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL) in ETB+/+, ETB-/-, and ETB+/- rats. Number of TUNEL-positive cells in the cerebellum and the hippocampus of ETB-/- rats was significantly increased compared with that of the ETB+/+ and ETB+/- rats. TUNEL-positive cells were observed in the molecular layer and granular cell layers of the cerebellum. In contrast, no significant difference in the density of TUNEL-positive cells was revealed in the cerebral cortex. These results suggest that either endothelin receptor B sl mutation or colostomy has predominant lasting effects on the cell survival/loss in the cerebellum and hippocampus of adult ETB-/- rats. Our findings provide the information on cellular changes in the brains of patients with Hirschsprung’s disease due to congenital EDNRB mutation as well as clinically relevant interventions.

A Novel Method for Identifying the Transition Zone in Long-Segment Hirschsprung Disease: Investigating the Muscle Unit to Ganglion Ratio.

Hirschsprung disease (HSCR) is characterised by the absence of enteric ganglia along variable lengths of the distal bowel. Current gold standard treatment involves the surgical resection of the defective, aganglionic bowel. Clear and reliable distinction of the normoganglionated bowel from the transition zone is key for successful resection of the entire defective bowel, and the avoidance of subsequent postoperative complications. However, the intraoperative nature of the tissue analysis and the variability of patient samples, sample preparation, and operator objectivity, make reproducible identification of the transition zone difficult. Here, we have described a novel method for using muscle units as a distinctive landmark for quantifying the density of enteric ganglia in resection specimens from HSCR patients. We show that the muscle unit to ganglion ratio is greater in the transition zone when compared with the proximal, normoganglionated region for long-segment HSCR patients. Patients with short-segment HSCR were also investigated, however, the muscle unit to ganglion ratio was not significantly different in these patients. Immunohistochemical examination of individual ganglia showed that there were no differences in the proportions of either enteric neurons or glial cells through the different regions of the resected colon. In addition, we identified that the size of enteric ganglia was smaller for patients that went on to develop HSCR associated enterocolitis; although the density of ganglia, as determined by the muscle unit to ganglia ratio, was not different when compared with patients that had no further complications. This suggests that subtle changes in the enteric nervous system, even in the “normoganglionated” colon, could be involved in changes in immune function and subsequent bacterial dysbiosis.

Multiple Roles of Ret Signalling During Enteric Neurogenesis.

The majority of the enteric nervous system is formed by vagal neural crest cells which enter the foregut and migrate rostrocaudally to colonise the entire length of the gastrointestinal tract. Absence of enteric ganglia from the distal colon are the hallmark of Hirschsprung disease, a congenital disorder characterised by severe intestinal dysmotility. Mutations in the receptor tyrosine kinase RET have been identified in approximately 50% of familial cases of Hirschsprung disease but the cellular processes misregulated in this condition remain unclear. By lineage tracing neural crest cells in mice homozygous for a knock-in allele of Ret (Ret51/51), we demonstrate that normal activity of this receptor is required in vivo for the migration of enteric nervous system progenitors throughout the gut. In mutant mice, progenitors of enteric neurons fail to colonise the distal colon, indicating that failure of colonisation of the distal intestine is a major contributing factor for the pathogenesis of Hirschsprung disease. Enteric nervous system progenitors in the ganglionic proximal guts of mutant mice are also characterised by reduced proliferation and differentiation. These findings suggest that the functional abnormalities in Hirschsprung disease result from a combination of colonic aganglionosis and deficits in neuronal circuitry of more proximal gut segments. The reduced neurogenesis in the gut of Ret51/51 mutants was reproduced in the multilineage enteric nervous system progenitors isolated from these animals. Correction of the molecular defects of such progenitors fully restored their neurogenic potential in culture. These observations enhance our understanding of the pathogenesis of Hirschsprung disease and highlight potential approaches for its treatment.

The Emerging Genetic Landscape of Hirschsprung Disease and Its Potential Clinical Applications.

Hirschsprung disease (HSCR) is the leading cause of neonatal functional intestinal obstruction. It is a rare congenital disease with an incidence of one in 3,500–5,000 live births. HSCR is characterized by the absence of enteric ganglia in the distal colon, plausibly due to genetic defects perturbing the normal migration, proliferation, differentiation, and/or survival of the enteric neural crest cells as well as impaired interaction with the enteric progenitor cell niche. Early linkage analyses in Mendelian and syndromic forms of HSCR uncovered variants with large effects in major HSCR genes including RET, EDNRB, and their interacting partners in the same biological pathways. With the advances in genome-wide genotyping and next-generation sequencing technologies, there has been a remarkable progress in understanding of the genetic basis of HSCR in the past few years, with common and rare variants with small to moderate effects being uncovered. The discovery of new HSCR genes such as neuregulin and BACE2 as well as the deeper understanding of the roles and mechanisms of known HSCR genes provided solid evidence that many HSCR cases are in the form of complex polygenic/oligogenic disorder where rare variants act in the sensitized background of HSCR-associated common variants. This review summarizes the roadmap of genetic discoveries of HSCR from the earlier family-based linkage analyses to the recent population-based genome-wide analyses coupled with functional genomics, and how these discoveries facilitated our understanding of the genetic architecture of this complex disease and provide the foundation of clinical translation for precision and stratified medicine.

Correlation analysis of IL-11 polymorphisms and Hirschsprung disease subtype susceptibility in Southern Chinese Children

BackgroundHirschsprung disease (HSCR) is a hereditary defect, which is characterized by the absence of enteric ganglia and is frequently concurrent with Hirschsprung-associated enterocolitis (HAEC). However, the pathogenesis for HSCR is complicated and remains unclear. Recent studies have shown that pro-inflammatory cytokines such as interleukin-11 (IL-11) are involved in the enteric nervous system's progress. It was found that IL-11 SNPs (rs8104023 and rs4252546) are associated with HSCR in the Korean population waiting for replication in an independent cohort. This study evaluated the relationship between IL-11 and the susceptibility of patients to HSCR by performing subphenotype interaction examination, HAEC pre-/post-surgical patient-only association analysis, and independence testing.MethodsIn this study, a cohort consisting of children from Southern China, comprising 1470 cases and 1473 controls, was chosen to examine the relationship between two polymorphisms (rs8104023 and rs4252546 in IL-11) and susceptibility to HSCR by replication research, subphenotype association analysis, and independence testing.ResultsThe results showed that IL-11 gene polymorphisms (rs8104023 and rs4252546) are not associated with the risk of HSCR in the Chinese population. The results of both short-segment and long-segment (S-HSCR and L-HSCR) surgery (3.34 ≤ OR ≤ 4.05, 0.02 ≤ P ≤ 0.04) showed that single nucleotide polymorphisms (SNP) rs8104023 is associated with susceptibility to HAEC.ConclusionsThis study explored the relationship between genetic polymorphisms and susceptibility to HAEC in HSCR subtypes for the first time. These findings should be replicated in a larger and multicentre study.

RET compound inheritance in Chinese patients with Hirschsprung disease: lack of penetrance from insufficient gene dysfunction.

Hirschsprung disease (HSCR) is a neurocristopathy characterized by the absence of enteric ganglia along variable lengths of the intestine. Genetic defects play a major role in HSCR pathogenesis with nearly 50% of patients having a structural or regulatory deficiency in the major susceptibility gene RET. However, complete molecular defects remain poorly characterized in most patients. Here, we performed detailed genetic, molecular, and populational investigations of rare null mutations and modifiers at the RET locus. We first verified the pathogenicity of three RET splice site mutants (c.1879 + 1G > A, c.2607 + 5G > A and c.2608-3C > G) at the RNA level. We also identified significantly higher risk allele (genotype) frequencies, and their over-transmission, from unaffected parents to affected offspring of three functionally independent enhancer variants (rs2506030, rs7069590 and rs2435357, with odd ratios (OR) of 2.09, 2.71 and 7.59, respectively, P < 0.001). These three common variants are in significant (P < 4.64 × 10-186) linkage disequilibrium in the Han Chinese population with ~ 60% of them carrying at least one copy and > 10% with two copies. We show that RET compound inheritance of rare and common variants prevails in 64% (seven out of 11) of Chinese HSCR families. This study supports the idea that common RET variants can modify the penetrance of rare null RET mutations in HSCR, and the combined high susceptibility allele dosage may constitute the unique raised "risk baseline" among the Chinese population.

Sequence characterization of RET in 117 Chinese Hirschsprung disease families identifies a large burden of de novo and parental mosaic mutations

BackgroundHirschsprung disease (HSCR) is an inherited congenital disorder characterized by the absence of enteric ganglia in the distal part of the gut. RET is the major causative gene and contains > 80% of all known disease-causing mutations.ResultsTo determine the incidence of RET pathogenic variants, be they Mendelian inherited, mosaic in parents or true de novo variants (DNVs) in 117 Chinese families, we used high-coverage NGS and droplet digital polymerase chain reaction (ddPCR) to identify 15 (12.8%) unique RET coding variants (7 are novel); one was inherited from a heterozygous unaffected mother, 11 were DNVs (73.3%), and 3 full heterozygotes were inherited from parental mosaicism (2 paternal, 1 maternal): two clinically unaffected parents were identified by NGS and confirmed by ddPCR, with mutant allele frequency (13–27%) that was the highest in hair, lowest in urine and similar in blood and saliva. An extremely low-level paternal mosaicism (0.03%) was detected by ddPCR in blood. Six positive-controls were examined to compare the mosaicism detection limit and sensitivity of NGS, amplicon-based deep sequencing and ddPCR.ConclusionOur findings expand the clinical and molecular spectrum of RET variants in HSCR and reveal a high frequency of RET DNVs in the Chinese population.

Measurement of novel intestinal secretory and barrier pathways and effects of proteases.

The epithelial lining of the gastrointestinal (GI) tract in conjunction with the enteric nervous system (ENS) plays an important role in mediating solute absorption and secretion. A dysregulated ionic movement across the epithelium can result in GI diseases that manifest as either watery diarrhea or constipation. Hirschsprung disease is an example of an ENS disorder characterized by absence of enteric ganglia in distal gut resulting in obstructive phenotype. Receptor rearranged during transfection (RET) gene variants are the most commonly recognized genetic associations with Hirschsprung disease. In this issue of Neurogastroenterology and Motility, Russell et al demonstrate that RET mediates colonic ion transport through modulation of cholinergic nerves. They go on to show inhibition of RET can attenuate accelerated transit in a rat model. Normalizing secretory and absorptive defects has been an attractive therapeutic strategy. In addition to the intrinsic regulation of secretory processes, luminal mediators like bile acids, short-chain fatty acids, and proteases can affect both secretion and barrier function of the intestinal epithelium. Elevated levels of proteases have been identified in a wide range of GI diseases including irritable bowel syndrome. Proteases are known to cause visceral hypersensitivity and barrier disruption in vitro and in animal models. The goals of this review are to describe fundamental concepts related to intestinal epithelial secretion, the utility of Ussing chambers to measure ionic mechanisms and to discuss examples of novel signaling pathways; namely the RET signaling cascade in secretomotor neurons and effects of luminal proteases on barrier and ionic secretion.

Diagnosing and Managing Hirschsprung Disease in the Newborn

Hirschsprung disease is characterized by the absence of enteric ganglia in the distal bowel and is caused by the failure of enteric neural crest–derived cells to complete their craniocaudal migration along the intestine during embryogenesis. Neonates typically present with failure to pass meconium within the first 48 hours after birth. Diagnosis relies on rectal biopsy to document the presence of aganglionosis, and management requires surgical resection of the aganglionic segment. Despite advances in the diagnosis and management of the disease, patients remain at risk for long-term gastrointestinal morbidity, including obstructive symptoms, fecal incontinence, and enterocolitis, with negative implications on quality of life. Neuronal stem cell–based therapy is being explored as a novel treatment for this disease.

Identification of Variants in RET and IHH Pathway Members in a Large Family With History of Hirschsprung Disease

Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by absence of enteric ganglia in the distal part of the gut. Variants in ret proto-oncogene (RET) have been associated with up to 50% of familial and 35% of sporadic cases. We searched for variants that affect disease risk in a large, multigenerational family with history of HSCR in a linkage region previously associated with the disease (4q31.3-q32.3) and exome wide. We performed exome sequencing analyses of a family in the Netherlands with 5 members diagnosed with HSCR and 2 members diagnosed with functional constipation. We initially focused on variants in genes located in 4q31.3-q32.3; however, we also performed an exome-wide analysis in which known HSCR or HSCR-associated gene variants predicted to be deleterious were prioritized for further analysis. Candidate genes were expressed in HEK293, COS-7, and Neuro-2a cells and analyzed by luciferase and immunoblot assays. Morpholinos were designed to target exons of candidate genes and injected into 1-cell stage zebrafish embryos. Embryos were allowed to develop and stained for enteric neurons. Within the linkage region, we identified 1 putative splice variant in the lipopolysaccharide responsive beige-like anchor protein gene (LRBA). Functional assays could not confirm its predicted effect on messenger RNA splicing or on expression of the mab-21 like 2 gene (MAB21L2), which is embedded in LRBA. Zebrafish that developed following injection of the lrba morpholino had a shortened body axis and subtle gut morphological defects, but no significant reduction in number of enteric neurons compared with controls. Outside the linkage region, members of 1 branch of the family carried a previously unidentified RET variant or an in-frame deletion in the glial cell line derived neurotrophic factor gene (GDNF), which encodes a ligand of RET. This deletion was located 6 base pairs before the last codon. We also found variants in the Indian hedgehog gene (IHH) and its mediator, the transcription factor GLI family zinc finger 3 (GLI3). When expressed in cells, the RET-P399L variant disrupted protein glycosylation and had altered phosphorylation following activation by GDNF. The deletion in GDNF prevented secretion of its gene product, reducing RET activation, and the IHH-Q51K variant reduced expression of the transcription factor GLI1. Injection of morpholinos that target ihhreduced the number of enteric neurons to 13% ± 1.4% of control zebrafish. In a study of a large family with history of HSCR, we identified variants in LRBA, RET, the gene encoding the RET ligand (GDNF), IHH, and a gene encoding a mediator of IHH signaling (GLI3). These variants altered functions of the gene products when expressed in cells and knockout of ihh reduced the number of enteric neurons in the zebrafish gut.

Association of NRG1 and AUTS2 genetic polymorphisms with Hirschsprung disease in a South Chinese population.

Hirschsprung disease (HSCR) is a genetic disorder characterized by the absence of enteric ganglia. There are more than 15 genes identified as contributed to HSCR by family‐based or population‐based approaches. However, these findings were not fulfilled to explain the heritability of most sporadic cases. In this study, using 1470 HSCR and 1473 control subjects in South Chinese population, we replicated two variants in NRG1 (rs16879552, P = 1.05E‐04 and rs7835688, P = 1.19E‐07), and further clarified the two replicated SNPs were more essential for patients with short‐segment aganglionosis (SHSCR) (P = 2.37E‐05). We also tried to replicate the most prominent signal (rs7785360) in AUTS2, which was a potential susceptibility gene with HSCR. In our results, in terms of individual association, marginal effect was observed to affect the HSCR patients following recessive model (P = 0.089). Noteworthy, significant intergenic synergistic effect between rs16879552 (NRG1) and rs7785360 (AUTS2) was identified through cross‐validation by logistic regression (P = 2.45E‐03, OR = 1.53) and multifactor dimensionality reduction (MDR, P < 0.0001, OR = 1.77). Significant correlation was observed between expression of these two genes in the normal segments of the colons (P = 0.018), together with differential expression of these genes between aganglionic colonic segments and normal colonic segments of the HSCR patients (P value for AUTS2 <0.0001, P value for NRG1 = 0.0243). Although functional evaluation is required, we supply new evidence for the NRG1 to HSCR and raised up a new susceptibility gene AUTS2 to a specific symptom for the disease.

Genome-wide association study of Hirschsprung disease detects a novel low-frequency variant at the RET locus.

Hirschsprung disease (HSCR) is a congenital disorder with a population incidence of ~1/5000 live births, defined by an absence of enteric ganglia along variable lengths of the colon. HSCR genome-wide association studies (GWAS) have found common associated variants at RET, SEMA3, and NRG1, but they still fail to explain all of its heritability. To enhance gene discovery, we performed a GWAS of 170 cases identified from the Danish nationwide pathology registry with 4717 controls, based on 6.2 million variants imputed from the haplotype reference consortium panel. We found a novel low-frequency variant (rs144432435), which, when conditioning on the lead RET single-nucleotide polymorphism (SNP), was of genome-wide significance in the discovery analysis. This conditional association signal was replicated in a Swedish HSCR cohort with discovery plus replication meta-analysis conditional odds ratio of 6.6 (P = 7.7 × 10-10; 322 cases and 4893 controls). The conditional signal was, however, not replicated in two HSCR cohorts from USA and Finland, leading to the hypothesis that rs144432435 tags a rare haplotype present in Denmark and Sweden. Using the genome-wide complex trait analysis method, we estimated the SNP heritability of HSCR to be 88%, close to estimates based on classical family studies. Moreover, by using Lasso (least absolute shrinkage and selection operator) regression we were able to construct a genetic HSCR predictor with a area under the receiver operator characteristics curve of 76% in an independent validation set. In conclusion, we combined the largest collection of sporadic Hirschsprung cases to date (586 cases) to further elucidate HSCR's genetic architecture.

Reduced cell proliferation and increased apoptosis in the hippocampal formation in a rat model of Hirschsprung's disease

Hirschsprung's disease (HSCR) is a congenital malformation characterized by the absence of enteric ganglia in the distal intestine and gut obstruction. Some HSCR patients also have associated neurological symptoms. We studied a rat model of HSCR, also known as spotting lethal (sl/sl) rat, which carries a spontaneous deletion in the gene of endothelin receptor B (EDNRB) and a similar phenotype as humans with HSCR. We focused on the changes in cell proliferation and apoptosis in the hippocampal formation of the sl/sl rat. Proliferating cells in wildtype (+/+), heterozygous (+/sl) and homozygous (sl/sl) rats were labelled by intraperitoneal injection of 5-bromo-2′-deoxyuridine (BrdU) at postnatal day 2. The density of proliferating cells in the CA1 and CA3 regions of the hippocampus and dentate gyrus of sl/sl rats was significantly reduced compared to +/+ rats. The effect of EDNRB mutation on cell apoptosis was examined by using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling assay. This showed that the density of apoptotic cells in the hippocampal formation, particularly in the CA1 region of sl/sl rats, was significantly increased compared to +/+ rats. The expression of brain derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) was measured with ELISA in the hippocampal formation, but no difference was revealed between genotypes. These results suggest that EDNRB mutation reduces cell proliferation and increases apoptosis in the hippocampal formation of the sl/sl rat, but does not alter the levels of BDNF and GDNF. Our findings provide an insight into the cellular changes in the brains of HSCR patients caused by EDNRB mutation.

Genetic variants of IL-11 associated with risk of Hirschsprung disease.

Hirschsprung disease (HSCR) is a congenital and heterogeneous disorder characterized by the absence of enteric ganglia during enteric nervous system (ENS) development. Our recent genome-wide association study has identified a variant (rs6509940) of interleukin-11 (IL-11) as a potential susceptible locus for HSCR. As interleukins play important roles in the ENS, we further studied associations with HSCR of nine common single nucleotide polymorphisms (SNPs) on IL-11. Biopsy specimens or surgical materials from all patients that were tested for histological examination based on the absence of the enteric ganglia were collected. A total of nine SNPs on IL-11 were genotyped in 187 HSCR patients and 283 unaffected controls using TaqMan genotyping assay. Combined analysis revealed that several SNPs (minimum p=1.57×10(-7) ) showed statistically significant associations with HSCR, even after Bonferroni correction (pcorr =1.73×10(-6) for the SNP). Moreover, the most common haplotype was strongly associated with HSCR (pcorr =2.20×10(-6) ). In further analysis among three HSCR subtypes (short segment, S-HSCR; long segment, L-HSCR; total colonic aganglionosis, TCA) based on the extent of aganglionic segment, the result showed a different association pattern depending on the subtypes (minimum pcorr =6.12×10(-5) for rs6509940 in S-HSCR; but no significant SNP in L-HSCR and TCA). Although further replication in a larger cohort and functional evaluations are needed, our findings suggest that genetic variations of IL-11 may be associated with the risk of HSCR and/or the mechanisms related to ENS development.

Gut Epithelium‐derived Sonic Hedgehog Regulates the Extracellular Matrix During Formation of the Intestinal Nervous System

The enteric nervous system (ENS) is principally derived from vagal-level neural crest cells that migrate rostrocaudally along the entire length of the gastrointestinal tract, giving rise to neurons and glial cells in two ganglionated plexuses. Incomplete migration of vagal-derived enteric neural crest cells (ENCCs) leads to Hirschsprung disease (HD), a congenital disorder characterized by the absence of enteric ganglia along variable lengths of the distal intestine. Inductive interactions between gut epithelium and mesenchyme have been suggested to regulate the migration and differentiation of ENCCs. However, little is known about the function of epithelial derived factors, such as Sonic hedgehog (Shh), how they influence hindgut mesenchyme derived factors and how they regulate extracellular matrix expression during ENS development. Hindgut from 6 day old chicken embryo was cultured in the presence of Shh protein or Shh overexpressing RCAS-virus. In presence of Shh the hindgut is aganglionic, while in the presence of Shh inhibitor large, ectopic ganglia developed. Shh treatment strongly induced the expression of extracellular matrix versican and collagen type IX, whereas cyclopamine reduced the expression pattern of these inhibitory matrix molecules. These results indicate that versican and collagen IX is a candidate for mediating the effects of Shh on ENCC migration. Shh also inhibited the proliferation and promoted the differentiation of ENCCs. Abnormalities of NCC migration and extracellular pattern formation are characteristic of two human intestinal disorders, HD and intestinal neuronal dysplasia. Our results support an essential role for epithelial-mesenchymal interactions in these aspects of ENS development.

Prospective identification and culture of rat enteric neural stem cells (ENSCs).

Hirschprung's disease (HD), a very common congenital abnormality in children, occurs mainly due to the congenital developmental defect of the enteric nervous system. The absence of enteric ganglia from the distal gut due to deletion in gut colonization by neural crest progenitor cells may lead to HD. The capacity to identify and isolate the enteric neuronal precursor cells from developing and mature tissues would enable the development of cell replacement therapies for HD. However, a mature method to culture these cells is a challenge. The present study aimed to propose a method to culture enteric neural stem cells (ENSCs) from the DsRed transgenic fetal rat gut. The culture medium used contained 15% chicken embryo extract, basic fibroblast growth factor, and epidermal growth factor. ENSCs were cultured from embryonic day 18 in DsRed transgenic rat. Under inverted microscope and fluorescence staining, ENSCs proliferated to form small cell clusters on the second day of culture. The neurospheres-like structure were suspended in the medium, and there were some filaments between the adherent cells from day 3 to day 6 of the culture. The neurospheres were formed by ENSCs on day 8 of the culture. Network-like connections were formed between the adherent cells and differentiated cells after adding 10% FBS. The differentiated cells were positive for neurofilament and glial fibrillary acidic protein antibodies. The present study established a method to isolate and culture ENSCs from E18 DsRed transgenic rats in the terminal stage of embryonic development. This study would offer a way to obtain plenty of cells for the future research on the transplantation of HD.