Experimental Evaluation Of Etiological Factor Of Hirschsprung Disease

Hirschsprung disease (HD, aganglionic megacolon) is the main genetic cause of functional intestinal obstruction with an incidence of 1/4000 live births. This developmental disorder is a neurocristopathy and is characterised by the absence of the enteric ganglia along a variable length of the intestine. The treatment of HSCR is surgical. After careful preoperative management, the principle is to place the normal bowel at the anus and to release the tonic contraction of the internal anal sphincter. Since the initial protocol of Swenson described in 1948, a series of operative approaches have been developed, such as the Soave and Duhamel procedures. A one stage procedure is possible when diagnosis is made early, before colonic dilatation. Otherwise, a primary colostomy is required. Fistula or stenosis of the anastomosis and enterocolitis are the main short term complications. Long term complications include chronic constipation (10-15%) and soiling. Laparoscopic techniques have recently been proposed in HSCR surgery. Mortality is under 6% since the 1980s and may be related to short term complications or caused by the associated malformations.Part I. Expression of CAMs in Hirschsprung'diseaseIn this study, we investigated the expression of CAMs in ganglionic(NG) and aganglionic(AG) segments of HD in order to understand the role of CAM-FGFR signaling in the pathogenesis of HD. Methods: The specimens of 16 cases was divided into NG group and AG group respectively according to haematoxylin and eosin staining and acetylcholinesterase histochemistry and then the CAMs expression was carried out by immunohischemistry-morphometric analyses. Results: NCAM immunoreactivity was observed in the submucous and myenteric plexus in NG bowel and in hypertrophied nerve trunks in AG bowel and in the smooth muscle. Numerous NCAM-positive fine fibres were present in the smooth-muscle layers in NG bowel. Whereas only a few were present in smooth-muscle in AG bowel, and so did the N-cadherin immunoreactivity. Conclusion: The markedly decreased expression of CAMs on nerve fibres within the muscle of AG bowel suggests that CAM-FGFR signaling is altered in HD, resulting in failure of enteric neuroblast migration.Part II. Association between the RET proto-oncogene polymorphismsand Hirschsprung disease in Chinese Han populationIn this study, we established the genetic background of exon2, exon13, exon11 and exon15 polymorphisms of RET proto-oncogene in Chinese population and study the possible involvement of RET proto-oncogene in the etiology of Hirschsprung disease (HD). Methods: The genotype and allele frequencies of RET proto-oncogene polymorphisms were identified by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPS) in 94 HD patients and 122 control subjects. Results: The genotype and allele frequencies of exon2 in control were AA 0.17, AG 0.72,GG 0.11, A 0.53, G 0.47 in control, and in HD were AA 0.61, AG 0.35,GG 0.04 , A 0.78, G0.22 exon13: GG0.30, GT0.52, TT0.18, G0.56, T 0.44 respectively in control, and in HD were GG 0.49, GT 0.36, TT 0.15, G 0.67, T 0.33. There were significant differences between HD and control about two polymorphisms. exon11: AA 0.05, AG 0.16, GG 0.79, A 0.13, G 0.87 in control and in HD were AA 0.02, AG 0.14, GG 0.84 , A 0.09, G 0.91, the differences was not found between two group about this site. And exon15 were all of CC genotype in spite of control or HD. Conclusion: These data provided evidences for contributions of exon2 and exon13 polymorphisms of RET proto-oncogene to susceptibility to HD. Part III . A promoter single nucleotide polymorphism -6G>A in the ret proto-oncogene affected the expression of RETThe activation of the RET signaling pathway during embryogenesis is a crucial prerequisite for a directional migration of enteric nervous system progenitor cells. Loss-of-function germline mutations of the RET ptoto-oncogene are reported in familial and sporadic cases of Hirschsprung disease with a variable frequency. Furthermore, variants of several RET polymorphisms are over- or under-represented in Hirschsprung disease populations.The sequence variants of the gene promoter that might impair its expression or determine anomalous gene regulation—thus predisposing to Hirschsprung disease—we analyzed the RET basal promoter region by PCR and sequencing. The -6G>A polymorphism was found in Chinese Han population residing in Hubei. To further confirm whether the promoter single nucleotide polymorphism affects the expression of RET directly, the Dual-Luciferase Reporter Assay System was used. A DNA fragment of the promoter region of the RET gene was amplified by the PCR method. The template used genomic DNA from the -6GG or -6AA. The promoter DNA fragment was introduced into PGL3-basic vector upstream the firely luciferase DNA. Thus, the recombinant vector G-PGL3 or A-PGL3 was obtained. The IMR32 cell was transiently transfected with the recombinant plasmid in vitro. Luciferase activities were measured using dual-luciferase reporter assay reagent according to the instruction in the kit. The results indicated the relative Luciferase activity of the -6A allele was lower than that of the -6G allele. From the results, we suggest that the -6G>A promoter polymorphism is a independent gene variation that can affect the expression or function of the protein products and plays a role in predisposition to Hirschsprung disease. In conclusion, this present study was aimed with finding or further understanding the genetic or other factors of Hirschsprung disease. We firstly studied the expression of CAMs in ganglionic(NG) and aganglionic(AG) segments of HD in order to understand the role of CAM-FGFR signaling in the pathogenesis of HD. Secondly, investigating the susceptibility of the four polymorphisms in the RET gene to Hirschsprung disease. The data provided evidences for contributions of exon2 and exon13 polymorphisms of RET proto-oncogene to susceptibility to Hirschsprung disease. Lastly, we found that there is a single nucleotide polymorphism -6G>A in Chinese Han population, and suggested the promoter polymorphism -6G>A in RET gene was a functional gene variation in Hirschsprung disease.