To identify demographic, clinical, genetic, immunologic and/or microbial (i.e., fecal stream characterization) risk factors that influence the likelihood of development of the HAEC phenotype in children who carry the diagnosis of HD. The newly formed HAEC Collaborative Research Group (HCRG) will utilize the 4 participating centers in the current consortia and recruit additional centers to enroll children diagnosed with Hirschsprung disease.

1a: To recruit 200 patients with Hirschsprung disease without HAEC.

1b: To recruit 200 patients with Hirschsprung disease and HAEC using standardized diagnostic criteria by collaborating with participating members of the HAEC Collaborative Research Group[1].

1c: To collect clinical and demographic information from well-characterized HD patients both with and without HAEC.

1d: To collect samples blood for DNA for genome wide association study (GWAS) by high throughput SNP technology and mutational analysis of known HSCR genes.

1e: To collect serum samples at the time of recruitment in a subset cohort (n=50 HD only, n=50 HD + HAEC) for serological immune markers known for inflammatory bowel disease (IBD) including ANCA, ASCA, OMPC, I2, and CBir1 and any newly identified markers.

1f: To collect and store fresh fecal specimens for future evaluation by molecular methodologies to determine relative proportions of enteric microflora in a subset cohort (n=50 HD only, n=50 HD + HAEC) of children (<18 years).

1g: To establish a Centralized Data Coordinating Center for data collection, data quality and detailed data analyses (CSMC) and tissue bank (CSMC) to facilitate specimen analysis for this study.

The HAEC risk factor identification will be completed by multivariate logistic regression analysis. Genetic association will be studied for each SNP in the GWAS together with all other potential risk factors. Further analysis will be carried out to evaluate multiple SNPs/genes simultaneously.

Emerging data suggests that the distinct phenotype of HAEC in children with HD may be the result of the complex interplay between host genetics, immune response and environmental triggers. Data suggest that approximately 20-30 percent of patients with HD develop HAEC. Thus, well-designed, multi-disciplinary investigations of genetic, immune and microbial etiologies of HAEC, with sufficient power to detect differences in disease phenotype are critically needed.

The Hirschsprung Disease clinical phenotype of HAEC:

Hirschsprung disease (HD; also known as congenital aganglionic megacolon) affects 1 in 5000 live births [2]. Surgical repair (excision of the aganglionic colon and pull through of the normally ganglionated intestine to the anal canal) is typically performed in infants with HD during the first few months of life [3, 4]. A subgroup of these children with HD will also develop Hirschsprung associated enterocolitis (HAEC), a potentially severe infection of the small bowel and colon[5]. Children who develop HAEC can present with fever, abdominal distention, explosive diarrhea, vomiting, lethargy, and if not diagnosed and treated promptly, can lead to life threatening sepsis and death. HAEC may occur pre- or post-surgery in HD patients. There have been widely varying reports of HAEC incidence ranging from 15-50% pre-surgery to 2-30% post-surgery. A lack of clear definition of HAEC criteria is at least partially responsible for this significant variation in reported incidence[6, 7]. A standardized definition of HAEC was recently developed and published in 2009[1]. The HAEC score consists of the most important clinical diagnostic criteria for HAEC identified from a group of international experts using the Delphi method. Preliminary validation with case scenarios was achieved and the HAEC score can now be used as a standardized and reproducible outcome measure for future studies on children with Hirschsprung disease. Now it is possible to perform rigorous studies of the genetic, immune, and microbial differences in HD patients who develop HAEC compared with those children with HD that have not developed HAEC.

Genetic variation: Currently there are 10 different genes identified (RET, GDNF, NTN, EDNRB, ET3, ECE-1, SOX10, ZFHX1B, PHOX2B, NGR1) in individuals affected with either isolated or syndromic Hirschsprung disease. The RET gene plays a critical role in ENS development and mutations in the RET gene have been found in 7-35% of sporadic cases and approximately 50% of familial cases. EDNRB and ET3 each account for approximately 5% of patients with HD and also are responsible for Shah-Waardenberg Syndrome. Mutations in each of the remaining 7 genes have been found in a small number of isolated and syndromic HD patients[8].

The RET tyrosine kinase signaling pathway plays a major role in ENS migration, along with its ligands GDNF and NTN. The EDNRB G-protein coupled receptor together with its ligand ET3 and endothelin cleavage enzyme ECE-1 forms a separate signaling pathway important in ENS development. The RET and EDNRB signaling pathways appear interact with an additive effect through an as yet to be described mechanism. PHOX2B is a transcriptional regulator of the RET gene and SOX10 appears to be a transcriptional regulator of both RET and EDNRB genes. The complex regulation of the genes in each pathway, combined with the cross-signaling between pathways and the effects on ENS migration is the subject of ongoing investigations[8, 9].

Current thought is that while some HD patients have a single mutation that is sufficient to cause HD, many patients likely have weaker mutations in multiple HD susceptibility genes that together are sufficient to cause HD[8, 9]. While there has been intense study of the genetics of HD over many years, there has been no attention given to possible genetic etiologies of HAEC. To date, the influence of genotype on the HAEC phenotype has received little attention, thereby providing additional merit for this proposal.

Immune markers and HAEC phenotypes:

One hypothesis is that HAEC may result from differing immune responses to microbial antigens in HD patients as has been found in patients with inflammatory bowel disease. Antibodies to the E. coli outer-membrane porin C (OmpC), the Pseudomonas fluorescens CD related protein (I2), anti CBiR1 (anti-flagellin), as well as Saccaromyces cerevisiae (ASCA) and autoantigens, perinuclear anti-neutrophil antibody (pANCA) are known to be elevated in the patients diagnosed with Crohn Disease (CD) and ulcerative colitis (UC). Established patterns of elevation of these antibodies serve as very important tools in establishing a diagnosis of IBD, as well as distinguishing patients with UC from those with CD.

By contrast, HAEC is a relatively poorly studied intestinal inflammatory disorder in children with clinical features similar to pediatric IBD. At present little attention has been given to the exploring the similarities in immune responses in HAEC patients to IBD patients. Our aim is to measure a panel of antibodies routinely used for IBD patients, and compare HD patients with and without HAEC. One or more antibody levels may serve as a biomarker for HD patients at risk of developing HAEC.

Microbial factors:

Data suggest that altered gut microbial populations may be partially responsible for the development of HAEC in susceptible HD patients. Most studies have focused on Clostridium difficile, because two groups reported increased frequency of C. diff. toxin positive stools in HD patients with HAEC compared with those without HAEC .[10, 11] Other groups subsequently reported very low frequencies of C. diff. toxin positivity in their patients with HAEC, thereby calling into question the role C. diff. plays in the pathogenesis of HAEC.[12] Other investigators have found E. coli, C. diff. and Cryptosporidium adherent to enterocytes on histological examinations of colon biopsies of patients with HAEC.[5] These findings indicate a breech in the protective mucus gel layer covering the luminal surface of the colon, leading to invasion of the epithelial barrier. Taken together, these studies suggest that the intestine-microbial interaction may be important in the pathogenesis of HAEC.

Moreover, the study of intestine-microbial interactions which lead to development of HAEC might best be undertaken in the pediatric patient whereby influences of host fecal microbial constituents are more easily elucidated. Therefore, research must not fail to identify other critically important pathogens by too narrow a focus on only known pathogens residing in colonic fecal streams in the genetically susceptible host. In addition, the characterization of gut microflora (i.e., fecal streams) and risk assessment of children with HD can optimally be done with a careful evaluation of both components - host and microbe in the index case. Successfully confirming or disproving infectious hypotheses requires that valid diagnostic tools be employed. Systematic investigations into potential infectious etiologies or normal enteric flora triggers of HAEC, therefore appear biologically plausible, and highly justified.

University of Michigan will be conducting metabolomics anaylsis of stool in children with Hirschsprung Disease. The analysis is focused on fatty acids metabolized by bacteria and yeast in the colon. The technique will use a GC-MASS SPEC approach to evaluate for pro-inflammatory vs. anti-inflammatory fatty acids in the stool of children with and or without Hirschsprung Associated Enterocolitis.

Inclusion Criteria:

• 1. Males and females of all ages with a confirmed diagnosis of HD based on standardized histological criteria. Only Males and females ages 0 to 17 with a confirmed diagnosis of HD based on standardized histological criteria will be enrolled at CSMC.
• 2. Able to provide written informed assent if between the ages of 7 and 17. If age 6 and under, able to participate with parental permission.
• 3. Have consented to have specimens tested for genetics, immune responses, stool microflora.

Case Ascertainment:

All patients with a confirmed diagnosis of HD are eligible for enrollment. A diagnosis of HD for this study will require:

• 1)Documented histopathology showing absence of ganglion cells and is consistent with the diagnosis of HD.

Exclusion Criteria:

• 1. Intestinal neuronal dysplasia
• 2. Pseudo-obstruction

Children with Hirschsprung disease under the age of 17 years.