SNRPN基因rs220030位点的分型及甲基化亲缘相关性

Genotyping and Parental Related Methylation of SNRPN Gene rs220030

目的建立变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)技术与焦磷酸测序技术对小核核糖核蛋白多肽N(small nuclear ribonucleoprotein polypeptide N,SNRPN)基因rs220030位点的分型方法。建立应用焦磷酸测序技术分析CpG甲基化状态的方法,探讨rs220030位点用于亲缘等位基因判定的可行性。方法应用DGGE技术对97例上海地区汉族家系血样rs220030位点进行分型,同时应用焦磷酸测序技术对其中25例血液来源的家系样本的rs220030位点分型,并对两种方法在SNP分型结果上进行比较。通过重亚硫酸盐修饰联合焦磷酸测序技术分析随机2组家系样本rs220030位点上游CpG甲基化状态,判断甲基化是否有亲缘相关性。结果经DGGE检测97例家系血样rs220030位点分型结果为C纯合子20例,T纯合子29例,C/T杂合子48例。经焦磷酸测序检测25例血液来源的家系样本结果与DGGE检测结果一致。经重亚硫酸盐修饰联合焦磷酸测序技术分析,2组血液来源的家系子代的rs220030位点上游CpG甲基化状态均与母亲较相似。结论相比DGGE技术,焦磷酸测序技术更精确、方便,适合大样本、高通量SNP分型。重亚硫酸盐修饰联合焦磷酸测序技术可以精确分析CpG甲基化状态。rs220030位点可用于亲缘等位基因判定。

Objective To establish two methods by denaturing gradient gel electrophoresis （DGGE） and py- rosequencing for genotyping rs220030 （a SNP in the promoter region of small nuclear ribonucleoprotein polypeptide N, SNRPN）. To establish an analytical technique for detecting CpG methylation status by py- rosequencing and to further investigate the feasibility of applying rs220030 to the determination of parental origin allele. Methods The rs220030 of 97 blood samples from individuals of Shanghai Han population were genotyped by DGGE, meanwhile the rs220030 of 25 blood samples of them were geno- typed by pyrosequencing to compare the two methods in genotyping SNP. Pyrosequencing united bisulfite conversion method was applied to detect CpG methylation status of region upstream rs220030 of two random blood genealogical samples and investigate whether the methylation status was parental related. Results The rs220030 genotyping results of 97 blood samples detected by DGGE were 20 C homozygote, 29 T homozygote, and 48 C/T heterozygote. Twenty-five blood samples genotyped by pyrosequencing showed the same result with DGGE. The CpG methylation status of region upstream rs220030 of the child was similar to the mother. Conclusion Compared with DGGE, pyrosequencing is more accurate, convenient, and suitable for large samples and high throughput SNP genotyping. Pyrosequencing united bisulfite conversion can be used to detect CpG methylation status precisely. It is feasible to apply rs220030 to parental origin allele determination.