DOI: 10.7910/dvn/t9duz5
title: Biology of Hirschsprung Disease: Pathomorphological, Histochemical, Immunohistochemical and Genetic (RET Gene) Study of the Enteric Nervous System

description: The present study titled " Biology of Hirschsprung Disease: Pathomorphological, 
Histochemical, Immunohistochemical and Genetic (RET Gene) Study of the Enteric 
Nervous System" is a prospective cross-sectional study on patients from the Indian 
subpopulation with Hirschsprung disease over three and a half years (Nov 2011 to June 2015) 
at the Department of Pathology, St. John’s Medical College, Bangalore - a national referral 
centre for diagnosis of Hirschsprung disease in India. 
The study with five chapters has explored pathobiology of Hirschsprung disease (HD) in the 
Indian context with emphasis on rapid reliable user friendly diagnostic modalities and also 
made an attempt at investigating the molecular basis of the HD to gain insight into its 
possible pathogenic mechanisms. Their summary is as follows: 
1. The rapid modified agar-paraffin block technique designed in the first study titled 
"Improvised double-embedding technique of minute biopsies: A mega boon to 
histopathology laboratory" has revolutionized the processing of multiple minute 
mucosal and seromuscular biopsies which mandate proper orientation to visualize 
neuronal plexuses, especially when sampled from neonates. The simple reliable user 
friendly method has improved the quality of diagnostic information by optimal 
orientation, better quality of sections, faster turnaround time, cost-effectiveness by 
economizing on the number of paraffin blocks, manpower, chemical reagents and 
laboratory infrastructure. The modified tissue blocks are also best suited for enzyme 
and immunohistochemistry in addition to routine histochemistry. 
2. The second study titled "Improvised rapid Acetylcholinesterase histochemistry versus 
calretinin immunohistochemistry in the evaluation of colorectal biopsies for 
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Hirschsprung disease" evaluated calretinin, a Vitamin D dependent calcium binding 
protein expressed in central and peripheral neural system, on formalin fixed biopsies 
and compared the results with the improvised modified rapid AChE histochemistry 
(designed in the Department) on their corresponding fresh rectal biopsies taken for 
the primary diagnosis of HD. Calretinin proved as a reliable immune marker in 
ruling out the diagnosis of HD on formalin fixed rectal mucosal biopsy by 
highlighting granular staining of intrinsic fibres in the mucosa and submucosa in 
suspect cases of HD. The study also proved that the accuracy of diagnosis in 
Hirschsprung disease could be improved by employing both AChE and Calretinin 
stains. 
3. The detailed evaluation of Synaptophysin immunohistochemistry as a labelling 
immunohistochemical method in the third study titled "Role of Synaptophysin in the 
Intra-Operative Assessment of Quadrantic Innervation of the Proximal Doughnut in 
Hirschsprung Disease" assessed proximal doughnut for innervation abnormalities
intraoperatively to find its suitability for anastomosis for pull through surgeries. The 
marker specific for the synaptic vesicles in the central and peripheral nervous system 
and the main constituent of AChE storage compartments, and an important 
neuromuscular junction marker, highlighted the morphology of ganglion cells, 
indirectly reflected their functional status by demonstrating synapses at the level of 
muscle fibers on frozen sections and mapped the ganglionic –aganglionic interface 
with the pattern and intensity of the SY-positive fibre distribution in the muscularis 
propria. 
4. The forth study "The quest for a positive diagnostic marker for Hirschsprung 
Disease in formalin fixed rectal biopsies: A detailed seven marker IHC study"
describes in detail the hunt for a positive diagnostic marker on formalin fixed rectal 
biopsy in the diagnosis for HD from among the panel of seven neural markers 
namely Calretinin, GFAP, Synaptophysin, PGP 9.5, CD 56, NF and S100. None of 
these markers specifically stained and differentiated the hyperplastic-hypertrophic 
nerve bundles of Hirschsprung disease from the normal nerve bundles and extrinsic 
serosal nerves. Though CD 56 and S-100 failed to stain ganglion cells, they were not 
specific for hypertrophic nerve bundles and hence, these markers could not be 
considered as markers for HD. Thus, the quest of a novel marker for abnormal 
enteric nervous system continues with the proposal for the next panel of markers 
which may attempt to highlight pathology in perineurium. 
5. The fifth study "Diversity of RET Proto-oncogene Mutation in an Indian sub 
population of Hirschsprung disease: A Pilot Study " highlights the association of 
RET gene in Hirschsprung disease. Sequencing of six exons namely, 10, 11, 13, 14, 
15 and 16 of RET gene in 30 samples comprising of tests and control of Indian 
subpopulation in this pilot study reports the occurrence of a novel mutation, D624N 
in exon 10 of a single patient with LSHD. The variations seen in RSHD, and TCA 
were also seen in the control group. However, the reported mutations in other study 
population were not seen in the limited samples studied here and this could also
because of limited number of exons scanned. Hence, this calls for complete RET 
gene sequencing of a large sample size to strengthen the data and to study its 
relevance.