山西汉族先天性巨结肠症患儿RET和EDNRB基因SNP分析

发布时间：2018-06-23 03:28

本文选题：先天性巨结肠症 + RET基因　；参考：《山西医科大学》2012年硕士论文

【摘要】：背景先天性巨结肠症（Hirschsprung disease，HSCR）又称肠无神经节细胞症（aganglionosis），是典型的肠神经系统发育异常疾病。其主要病理特征为结肠或者直肠的肌层及粘膜下层神经节细胞缺失，肠道神经调节紊乱，以致受累肠段痉挛收缩，其近端结肠代偿性扩张与肥厚，形成巨结肠，从而导致小儿严重的肠梗阻及便秘等消化道症状。目前的研究显示多种基因与HSCR发病有关，主要有来自肠神经系统发育中起主要作用的2个信号转导通路，RET信号通路和内皮素B信号通路。迄今已发现与之相关的基因主要有RET原癌基因（RET proto-oncogen）及内皮素受体B基因（Endothelin receptor B，EDNRB）。研究学者推测基因单核苷酸多态性（single nucleotide polymorphism，SNP）和单倍型可能作为修饰因子存在，增加HSCR患病风险。由于遗传背景等方面的差异，不同的人人群和种族中多态性存在不同。目的研究山西汉族人群先天性巨结肠症患儿RET基因和EDNRB基因常见单核苷酸多态位点与HSCR发病的关系。方法收集2008年1月至2010年12月在山西省儿童医院就诊，经术后病理切片证实的80例汉族先天性巨结肠症患儿，且均为散发性。收集同期山西省儿童医院体检健康儿童80例。经家长及患儿同意后均取静脉血3mL，EDTA抗凝。应用高分辨率熔解曲线技术（HighResolution Melt，HRM）以及PCR产物测序，序列比对的方法，，对山西省80例散发性先天性巨结肠症患儿和80例健康儿童进行RET基因常见多态位点p.A45A，p.V125V，p.A432A，p.G691S，p.L769L，p.S904S以及EDNRB基因1-4号外显子多态位点的分析。结果 1、本课题首次应用高分辨熔解曲线技术并且首次将该技术对山西汉族先天性巨结肠症患儿RET基因单核苷酸多态性进行分析，实验结果如下： RET基因p.A45A位点存在多态性，病例组与对照组相比两组间等位基因差异显著（x~2=35.60，P0.01）。p.V125V位点可能不存在基因多态性。p.A432A位点存在多态性，病例组和对照组中该位点的等位基因分布频率存在显著性差异（x~2=5.668，P=0.017）。p.G691S病例组与对照组两组比较无明显差异（x~2=0.7810，P=0.377）。p.L769L位点存在多态性，病例组和对照组中该位点的等位基因分布频率存在显著差异（x~2=37.458，P0.01）。p.S904S存在多态位点，该位点与疾病无显著相关性（x~2=1.053，P=0.305）。 2、EDNRB基因中在第4外显子发现多态位点c831GA，引起亮氨酸的同义突变（p.L277L）。其中A等位基因频率为0.55，G等位基因频率为0.45。未发现其他已报道突变及多态位点。结论 1、在山西汉族人群中，RET基因2号外显子p.A45A，7号外显子p.A432A，13号外显子p.L769L的基因多态性可能与该地区先天性巨结肠症的发生密切相关（P0.05），11号外显子p.G691S，15号外显子p.S904S在病例组和对照组比较中无明显差异（P0.05），在山西汉族人群中，未发现3号外显子p.V125V多态位点。 2、HSCR可检测到EDNRB基因4号外显子多态性改变（p.L277L），在1-4号外显子中，未发现其他突变及多态位点。
[Abstract]:background
Congenital megacolon (Hirschsprung disease, HSCR), also known as intestinal ganglio cell syndrome (aganglionosis), is a typical dysplasia of the intestinal nervous system. Its main pathological features are the absence of ganglion cells in the myometrium and submucosa of the colon or rectum, the disturbance of the intestinal nerve regulation, and the involvement of the intestinal spasm and contraction of the proximal colon. Compensatory dilatation and hypertrophy, forming a megacolon, leading to severe intestinal obstruction and constipation in children.
Current studies have shown that a variety of genes are associated with HSCR, mainly 2 signal transduction pathways, RET signaling pathways and endothelin B signaling pathways, which are mainly derived from the development of the intestinal nervous system. The genes associated with it have been found to be the RET proto oncogene (RET Proto-oncogen) and the endothelin receptor B gene (Endothelin receptor). B, EDNRB). Researchers have speculated that gene single nucleotide polymorphisms (single nucleotide polymorphism, SNP) and haplotypes may exist as modifying factors to increase the risk of HSCR disease. There are different polymorphisms in different human populations and races due to genetic backgrounds.
objective
Objective to investigate the relationship between RET gene and EDNRB gene single nucleotide polymorphisms and HSCR in children with Hirschsprung disease in Shanxi Han population.
Method
From January 2008 to December 2010 in Shanxi children's Hospital, 80 children with Hirschsprung's disease confirmed by postoperative pathological sections were found to be sporadic. 80 healthy children in the medical examination of Shanxi children's hospital were collected for the same period. After the consent of parents and children, the venous blood 3mL and EDTA anticoagulant were taken. The high resolution fusion curve technique was applied. HighResolution Melt (HRM) and the sequencing of PCR products and sequence alignment were carried out in 80 children with sporadic Hirschsprung's disease and 80 healthy children in Shanxi province. The common polymorphic loci of RET gene p.A45A, p.V125V, p.A432A, p.G691S, p.L769L, p.S904S, and the polymorphism of the polymorphic loci of the EDNRB genes were analyzed.
Result
1, the first use of high resolution fusion curve technique and the first analysis of the single nucleotide polymorphisms of RET gene in children with Hirschsprung's disease in Shanxi is the first time. The results are as follows:
The p.A45A locus of RET gene was polymorphic. Compared with the control group, the difference of the allele between the two groups was significant (x~2=35.60, P0.01).P.V125V loci may not exist polymorphism.P.A432A locus, and there was a significant difference in the frequency of allele distribution in the case group and the control group (x~2=5.668, P=0.017).P.G691S disease. There was no significant difference (x~2=0.7810, P=0.377).P.L769L polymorphism between the two groups and the control group. There was a significant difference in the allele distribution frequency of the loci (x~2=37.458, P0.01).P.S904S in the case group and the control group (x~2=37.458, P0.01), and there was no significant correlation between the loci and the disease (x~2=1.053, P=0.305).
2, the polymorphic loci c831GA was found in the fourth exon of EDNRB gene, causing the synonymous mutation of leucine (p.L277L), of which the A allele frequency was 0.55, and the G allele frequency was 0.45. no other reported mutation and polymorphic loci.
conclusion
1, in the Han population of Shanxi, the gene polymorphism of exon 2 of RET gene, exon 7, exon 7 and exon 13 of exon 13 may be closely related to the occurrence of congenital megacolon in this area (P0.05), 11 exon p.G691S, and exon 15 p.S904S in the case group and the control group (P0.05), in the Han nationality of Shanxi. No polymorphisms of exon 3 were found in p.V125V population.
2, HSCR could detect polymorphisms of exon 4 of EDNRB gene (p.L277L), and no other mutations and polymorphic loci were found in exon 1-4.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R726.5

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