印记基因H19上游高甲基化区SNPs多态性研究

目的建立简单、高效的DNA甲基化标记和SNPs联合检测技术，并用于H19基因上游高甲基化区两组SNPs群体遗传学检测。方法用PCR—DGGE技术对232例武汉汉族无关个体H19基因上游启动子区H19FR1和H19FR2单倍型进行检测；同时选用两种甲基化敏感的限制酶（msRE）HpaⅡ和HhaⅠ，检测H19FR等位基因亲代来源。结果H19FR1区检出5种单倍型、9种表型组合，其个体识别能力（DP）、多态性信息含量（PIC）和非父排除率（PE）分别为0．803、0．58和0．322；H19FR2区检出2种单倍型、3种表型组合，其DP、PIC和PE值分别为0．626、0．37和0．162。测序结果显示，片段H19FR1含有a7342g、a7357g和g7547a3个SNPs与1个g7351c点突变；H19FR2仅含aS097g1个SNP。msREHpaⅡ或HhaⅠ可消化个体母源等位基因，PDP-DGGE分析仅能检测到父源等位基因。结论PDP-DGGE是一种简单、灵敏、高效的DNA甲基化标记和SNPs联合分析技术，其在进行多态性分型同时还可以确定等位基因的亲代来源，具有较高的法医学应用价值。

A study on SNIP polymorphisms in the hypermethylated region upstream of the human H19 gene

Objective To establish a simple and high-performance analytical technique for detecting DNA methylation markers and SNPs simultaneously,and obtain the population genetics data of some SNPs in the hypermethylated region upstream of the human H19 gene.Methods The haplotypes of H19FR1 and H19FR2 which located in the promoter region upstream of the human H19 gene were investigated from 232 unrelated Chinese individuals living in Wuhan by means of PCR and subsequent denaturing gradient gel electrophoresis(DGGE).Based on the methylation status of the genomic DNA,selective detection of the parental alleles for H19FRs was examined by using the methylation-sensitive restriction enzyme(msRE) Hpa II or Hha I.Results Five haplotypes and nine phenotypes were observed for H19FR1 in Chinese Han population in Wuhan,and the power of discrimination(DP),polymorphism information content(PIC) and probability of paternity exclusion(PE) were 0.803,0.58 and 0.322 respectively.For the H19FR2,two haplotypes and three phenotyes were detected,and the DP,PIC and PE were 0.626,0.37 and 0.162 respectively.Sequencing results showed that there were 3 SNPs,a7342g,a7357g and g7547a,and one g7351c point mutation in H19FR1.In the H19FR2,there was only one SNP,a8097g.The msRE,HpaⅡ or HhaⅠ,could digest the maternal allele,and only a single band derived from the paternal allele was detected by post-digestion PCR-DGGE(PDP-DGGE) technique.Conclusion PDP-DGGE is a simple,sensitive and effective technique for analyzing DNA methylation status and SNPs simultaneously,and can be used for discriminating the parental origin of alleles.