Biology of Hirschsprung Disease: Pathomorphological, Histochemical, Immunohistochemical and Genetic (RET Gene) Study of the Enteric Nervous System

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 203 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.

本研究题为《先天性巨结肠（Hirschsprung Disease, HD）的生物学特征：肠神经系统的病理形态学、组织化学、免疫组织化学及RET基因遗传学研究》，是一项针对印度亚人群先天性巨结肠患者的前瞻性横断面研究，于班加罗尔圣约翰医学院病理学系（印度国内先天性巨结肠诊断的国家级转诊中心）开展，研究周期为三年半（2011年11月至2015年6月）。本研究共分为五章，围绕印度人群中HD的病理生物学展开探索，重点聚焦于快速、可靠且易用的诊断手段，并尝试解析HD的分子基础，以深入探究其潜在致病机制。其研究概要如下：
1. 第一项研究题为《微小活检组织的改良双重包埋技术：为组织病理学实验室带来巨大福音》，所设计的快速改良琼脂-石蜡包埋技术，彻底革新了多份微小黏膜及肌层浆膜活检的处理流程——此类活检往往需要精准定向以清晰显示神经丛，尤其针对新生儿采样的标本。该方法简便可靠、易于操作，通过优化标本定向、提升切片质量、缩短检测周转时间，以及减少石蜡块、人力、化学试剂消耗与实验室基础设施投入实现成本效益优化，显著提升了诊断信息质量。改良后的组织块除适用于常规组织化学检测外，同样适配酶组织化学与免疫组织化学检测。
2. 第二项研究题为《改良快速乙酰胆碱酯酶（Acetylcholinesterase, AChE）组织化学法与钙视网膜蛋白（Calretinin）免疫组织化学法在203例先天性巨结肠结直肠活检评估中的对比》，针对福尔马林固定活检标本中的钙视网膜蛋白（一种维生素D依赖性钙结合蛋白，在中枢及外周神经系统中表达）进行检测，并将结果与本系设计的改良快速AChE组织化学法在对应新鲜直肠活检标本（用于HD初诊）的检测结果进行对比。研究证实，钙视网膜蛋白可通过在可疑HD病例的黏膜及黏膜下层固有纤维中呈现颗粒状染色，在福尔马林固定直肠黏膜活检标本中有效排除HD诊断，是可靠的免疫标志物。此外，联合使用AChE与钙视网膜蛋白染色可提升HD的诊断准确性。
3. 第三项研究题为《突触素（Synaptophysin）在先天性巨结肠近端吻合环术中象限神经支配评估中的作用》，针对术中获取的近端吻合环进行神经支配异常的详细评估，以确定其是否适用于拖出术的吻合操作。突触素作为中枢及外周神经系统突触囊泡的特异性标志物、乙酰胆碱酯酶储存囊泡的主要组成成分，亦是重要的神经肌肉接头标志物，可在冰冻切片上通过显示肌纤维层面的突触，间接反映神经节细胞的功能状态，同时通过突触素阳性纤维在固有肌层的分布模式与强度，精准定位神经节-无神经节交界区域，并清晰显示神经节细胞的形态。
4. 第四项研究题为《福尔马林固定直肠活检中先天性巨结肠阳性诊断标志物的探索：七项免疫组织化学标志物的详细研究》，详细阐述了在七项神经标志物（钙视网膜蛋白、胶质纤维酸性蛋白（Glial Fibrillary Acidic Protein, GFAP）、突触素、蛋白基因产物9.5（Protein Gene Product 9.5, PGP 9.5）、CD56、神经丝蛋白（Neurofilament, NF）及S100蛋白（S100 Protein））中，寻找可用于福尔马林固定直肠活检标本HD诊断的阳性标志物的过程。上述标志物均无法特异性染色并区分HD患者的增生肥大神经束与正常神经束及浆膜外神经。尽管CD56与S100无法染色神经节细胞，且对肥大神经束并无特异性，因此均无法作为HD的诊断标志物。至此，针对肠神经系统异常的新型诊断标志物的探索仍在继续，后续可将研究方向转向神经束膜的病理改变相关标志物。
5. 第五项研究题为《印度亚人群先天性巨结肠中RET原癌基因（RET Proto-oncogene）突变的多样性：一项预试验研究》，揭示了RET基因与HD的关联。本预试验对印度亚人群30份病例及对照样本的RET基因外显子10、11、13、14、15及16进行测序，结果在1例长段型HD（Long-segment Hirschsprung Disease, LSHD）患者的第10外显子中发现了新型突变D624N。在短段型HD（Short-segment Hirschsprung Disease, RSHD）及巨结肠类缘病（Transitional Colonic Aganglionosis, TCA）患者中观察到的变异，同样存在于对照组中。然而，本研究纳入的样本量有限且仅扫描了部分外显子，因此未发现其他研究人群中报道的突变。综上，需针对大样本开展全RET基因测序，以强化研究数据并明确其临床相关性。