Identification Genetic, Immunologic and Microbial Markers of Hirschsprung Associated Enterocolitis in Children With Hirschsprung Disease

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