Detection Modification Polymorphism on DNA Methylation and its application to Forensic Investigation

对11p15.5上印迹结构域中含有H19(母系表达)和IGF2基因(父系表达)SNPs的正常外周血DNA甲基化状态的多样性和亲本等位基因的选择性检测进行了研究。首先，我们检测了H19和IGF2基因中已知的SNPs，以确定检测方法和寻找等位基因分布。此外，采用聚合酶链式反应-单链构象多态性、ARMS-聚合酶链式反应和聚合酶链式反应-凝胶电泳法分析了H19FR的单倍型，发现了3种主要的单倍型，等位基因频率分别为0.5177、0.3409和0.1414。作为识别亲本来源的另一种策略，基因组DNA用甲基化敏感的限制性内切酶处理，然后进行H19FR或H19SNP分型。因此，在这两个系统中具有杂合子基因的DNA样本都显示出来自甲基化父亲来源的单一等位基因。然而，在对H19 SNP进行同样的操作后，IGF2 SNP只显示了27%的母亲等位基因。该方法适用于H19基因上游区域的SNPs，有望成为法医学鉴定亲本来源的有用工具。

Regions containing SNPs in H19 (maternal expression) and IGF2 gene (paternal expression), which are involved in an imprinting domain on 11p15.5, were investigated concerning existence of diversity on methylation status and selective detection of parental allele in DNA from normal peripheral blood.At first, we examined known SNPs in H19 and IGF2 gene in order to determine detection methods and find allele distributions. In addition, a haplotype polymorphism composed of three SNPs close to H19 SNP, named H19FR, was analyzed by PCR-SSCP, ARMS PCR and PCR-CDGE, and three major haplotypes with allele frequencies of 0.5177, 0.3409 and 0.1414 were observed.After treatment of genomic DNA with bisulfite reagent, a methylated allele of H19FR with heterozygous genotype was selectively amplified by methylation-specific PCR. However, polymorphic methylation status in CpGs was not observed in this study.As another strategy for recognizing parental origin, genomic DNA was treated with methylation-sensitive restriction enzyme and followed by H19FR or H19 SNP typing. As a result, DNA sample having heterozygous genotype in either system showed a single allele derived from methylated paternal origin. The IGF2 SNP revealed, however, only 27 % of maternal allele after the same procedure for H19 SNPs. It is assumable that this method applied for SNPs in upstream region of H19 will be a useful tool for forensic examination to discriminate parental origin.