先天性巨结肠与RET基因c135位点单核苷酸多态性相关性分析

目的确定国人陕西地区正常汉族人群中是否存在RET基因c135位点单核苷酸多态性及基因频率,研究先天性巨结肠患儿该位点基因型与表型的相关性,探讨其病因。方法整体抽样随机选取40例陕西西安地区健康汉族献血者血样进行DNA抽提。对16例病理诊断证实为先天性巨结肠患儿狭窄段肠组织标本进行DNA提取(均为陕西地区患儿,汉族,散发性)。PCR扩增RET基因外显子2(含c135片段),长度300 bp,产物纯化后直接测序,并用相关软件及统计学分析。结果陕西地区正常汉族人群存在RET基因c135位点单核苷酸多态性A/G,A、G基因频率分别为0.475、0.525,各等位基因频率接近,G等位基因频率稍占优势。病例组16例A、G基因频率分别为0.844,0.156。病例组基因型以突变型AA(13/16)为主。对照组与病例组存在明显差异(P<0.001)。A等位基因与疾病关联强度OR=5.97,OR95%CI(2.24,15.92),OR值在0.05水平上有显著性。结论国人陕西地区汉族人群RET位点存在单核苷酸多态性A/G,c135G>A。先天性巨结肠表型与该位点A基因型明显相关。RET基因c135位点单核苷酸多态性纯合突变型AA在先天性巨结肠患儿中过高表达。

The relationship between Hirschsprung disease and single nucleotide polymorphisms of c135 in RET proto-oncogene

Objective To make sure if there is single nucleotide polymorphisms(SNP) of c135 in RET proto-oncogene in Han ethnic group of Shaan Xi,China and investigate the relationship between Hirschsprung disease(HSCR) and SNP of RET proto-oncogene c135.Methods A total of 40 blood samples were collected from healthy donors through cluster sampling in Xi'an,Shaan Xi province.Sixteen tissues in aganglionic segments of HSCR were studied(they were all sporadic in Han ethnic group of Shaan Xi province of China and diagnosed by pathology).DNA was isolated from above samples and PCR amplified.The fragment of products were 300 case pairs(bp) which located in RET exon2 including c135.All of fragments were purified and directly sequenced and analyzed by related software and statistical methods.Results There really existed the single nucleotide polymorphisms of c135 A/G of RET in controls.A,G allele frequency was 0.475 and 0.525,respectively,and there was no distinctive dominant genotype.In patient group,A,G allele frequency was 0.844 and 0.156,respectively.c135A genotype was dominant in patients.There was significant statistical difference between patients and controls in RET c135A/G(P<0.001).By calculating odds ratios 5.97 and 95% Mantel-haenszel confident interval(2.24,15.92),it was concluded that there was relationship between the disease and the c135A genotype of RET.Conclusion Single nucleotide polymorphisms of c135 A/G of RET was confirmed in Han ethnic group in Shaan Xi.The allele frequency is c135G>A.There is relationship between the disease and the c135 A genotype of RET.c135A genotype in the RET proto-oncogene are over-expressed in patients with Hirschsprung disease.