甄别同卵双生子的DNA甲基化序列筛选研究

发布时间：2018-06-19 12:22

本文选题：同卵双生子 + DNA甲基化　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:同卵双生子(monozygotic twins,MZT)由一个受精卵分裂形成,DNA序列高度相似,传统的法医学遗传标记如短串联重复序列(short tandem repeat,STR)、单核苷酸多态性(single nucleotide polymorphism,SNP)等难以将二者区分,是为MZT涉嫌犯罪案件中生物检材身份认定的一大难题。大量研究表明MZT之间存在表观遗传学差异。我们前期研究应用甲基化免疫共沉淀测序技术(Methylated DNA immunoprecipitation,Me DIP),对5对表型一致的MZT进行全基因组甲基化检测分析,从中筛选一些差异甲基化序列,有些分布在Cp G岛(Cp G island,CGI),有些序列为非Cp G岛序列(non-CGI)。本研究从中选取若干CGI序列和non-CGI序列进行大样本研究,以验证这些序列区分MZT的效果和能力,为解决甄别MZT个体难题提供科学依据。方法:从本课题组建立的双生子样本库中选取100对MZT样本,应用Qiagen公司的QIAamp DNA Blood kit提取血液样本的DNA。应用ZYMO公司的EZ DNA Methylation-Gold Kit对基因组DNA进行重亚硫酸氢盐转化,以转化后的DNA为模板,扩增四个位于CGI的序列(Cp G island sequence,CGIS)及四个位于non-CGI的序列。四个CGI序列分别位于2号染色体的基因间区、人类白细胞抗原B伴随转录物3(BAT3)基因启动子区、氨氯吡嗪脒敏感阳离子通道1(ACCN1)基因启动子区、G蛋白信号调节子16(RGS16)基因启动子区,分别命名为CGIS1、CGIS2、CGIS3、CGIS4。四个non-CGI序列均位于基因间区(intergenic sequence,IGS),分别命名为IGS3、IGS4、IGS5、IGS6,其染色体定位分别为:chr1:114870877-114871203、chr12:67213759-67214092、chr11:5539842-5540207、chr1:56877427-56877787。对扩增产物进行单链纯化后行焦磷酸测序,获得八条靶序列共40个Cp G位点甲基化水平的定量数据。每个样本重复三次实验。应用Excel、Spearman相关检验、t检验、方差分析、秩和检验等方法进行统计学分析。结果:1八条靶序列的平均DNA甲基化水平1.1四个CGI序列的平均甲基化水平在所调查的152个血液样本中,CGIS1、CGIS2、CGIS3、CGIS4序列的平均甲基化水平分别为:21.37%、0.41%、5.05%、1.56%。1.2四个non-CGI序列的平均甲基化水平在所调查的200个血液样本中,IGS3、IGS4、IGS5、IGS6序列的平均甲基化水平分别为88.18%、87.07%、77.45%、87.87%。2双生子之间甲基化差异分析参照焦磷酸测序仪用户手册,焦磷酸测序仪灵敏度为5%,在实验中,如甲基化水平差值在双生子之间大于5%,则认为两者之间存在差异,反之则认为无差异。2.1各序列双生子间差异分析在所检测的76对MZT中,CGIS1序列在MZT之间甲基化水平有差异的有6对,占MZT样本量的7.89%,表明CGIS1序列4个Cp G位点区分MZT的鉴别能力为7.89%;在所检测的100对MZT中,IGS3、IGS4、IGS5、IGS6序列在MZT之间甲基化水平有差异的分别有37对、7对、48对、44对,四个non-CGI序列累积能够区分81对MZT,占MZT样本量的81.00%,表明四个non-CGI序列16个Cp G位点区分MZT的累积鉴别能力为81.00%。对CGIS1序列和四个non-CGI序列区分MZT的能力进行c2检验比较分析,发现和non-CGI序列区分MZT的能力高于CGIS1序列区分MZT的能力(P0.05)。2.2双生子之间甲基化差异与年龄的相关性分析探讨双生子间甲基化的差异与年龄的相关性,结果表明,在MZT之间,IGS6序列的DNA甲基化差异与年龄呈较弱的负相关,相关系数为-0.304,也就是,随着年龄增大,MZT之间的甲基化差异逐渐减小。其余序列甲基化水平在MZT之间的差异与年龄均无明显相关性。3甲基化与年龄、性别、民族、吸烟的相关性3.1年龄对四个non-CGI序列甲基化的影响Spearman秩相关分析结果表明,IGS3、IGS4两条靶序列的甲基化程度均与年龄呈弱相关,相关系数分别为-0.161、-0.384。其余两条序列甲基化程度与年龄无相关性。以10岁为年龄间隔,将样本分为6个年龄组:0-10岁、11-20岁、21-30岁、31-40岁、41-50岁及50岁。比较各年龄组间甲基化水平的差异,分析得出IGS4序列在年龄组间甲基化水平存在显著差异。其余三条序列在各年龄组间甲基化程度无显著差异。3.2性别对四个non-CGI序列甲基化的影响通过比较不同性别间的甲基化水平,结果表明,在所检测的四个non-CGI序列中,甲基化程度在不同性别之间无显著差异。3.3民族对四个non-CGI序列甲基化的影响样本志愿者来自六个民族:汉族、哈尼族、拉祜族、壮族、回族及彝族,由于后四组样本较少,故本研究只分析汉族和哈尼族样本的甲基化水平差异,发现IGS3、IGS6序列的甲基化水平在汉族与哈尼族之间表现出显著差异。其余序列未表现出显著性差异。3.4吸烟对四个non-CGI序列甲基化的影响IGS4序列的Cp G2、Cp G4位点及IGS6序列的Cp G3位点甲基化程度,吸烟组和不吸烟组表现出显著差异,其余序列的甲基化水平在吸烟组与不吸烟组之间无明显差异。结论:1对四个CGI候选序列的样本研究结果表明,该四个CGI序列的甲基化程度非常低,在检测的76对MZT样本中,CGIS1序列区分MZT的效能仅有7.89%,提示CGI区域DNA甲基化可能并不是区分MZT的理想区域。2对四个non-CGI候选序列的大样本研究结果表明,该四个位于non-CGI的基因间区序列甲基化程度较高,且区分MZT的能力也较高,累积区分效能达81%,可作为区分MZT的候选序列,提示位于non-CGI的基因间区序列是甄别MZT较为理想的序列。
[Abstract]:Objective: monozygotic twins (MZT) is divided by a fertilized egg, and the DNA sequence is highly similar. The traditional forensic genetic markers such as short tandem repeat, STR, single nucleotide polymorphisms (single nucleotide polymorphism, SNP) are difficult to distinguish between the two. A large number of studies have shown that there is an epigenetic difference between MZT. We used the methylation immunoprecipitation sequencing technology (Methylated DNA immunoprecipitation, Me DIP) to analyze 5 pairs of phenotypic MZT methylation, and select some differential methylation sequences. Some of them are distributed in Cp G Island (Cp G Island, CGI) and some sequences are non Cp G Island sequence (non-CGI). A number of CGI sequences and non-CGI sequences are selected from this study to verify the effect and ability of these sequences to distinguish the MZT. 100 pairs of MZT samples are selected, and the DNA. DNA Blood kit of Qiagen company is used to extract the DNA. from the blood samples, and the EZ DNA Methylation-Gold Kit of ZYMO company is used to convert the hydrogen sulphate into the genome. Four CGI sequences are located in the intergenic region of chromosome 2, the human leukocyte antigen B is accompanied by the promoter region of the transcription 3 (BAT3) gene, the promoter region of the amamoxamidine sensitive cation channel 1 (ACCN1) gene, and the promoter region of the G protein regulator 16 (RGS16) gene, named CGIS1, CGIS2, CGIS3, CGIS4. four non-CGI sequences, respectively. In the intergenic region (intergenic sequence, IGS), named IGS3, IGS4, IGS5, IGS6 respectively, their chromosomal location was chr1:114870877-114871203, chr12:67213759-67214092, chr11:5539842-5540207, and chr1:56877427-56877787. after single strand purification of the amplified products by pyrosequencing, and eight target sequences were obtained with 40 Cp locus methyl groups. Quantitative data of the level. Three experiments were repeated in each sample. Excel, Spearman correlation test, t test, variance analysis, rank sum test and other methods were used for statistical analysis. Results: average DNA methylation level of 1 eight target sequences 1.1 four CGI sequences in 152 blood samples, CGIS1, CGIS2, CGIS3 The average methylation levels of CGIS4 sequences are 21.37%, 0.41%, 5.05%, and 1.56%.1.2 four non-CGI sequences with average methylation levels in the 200 blood samples investigated. The average methylation levels of the IGS3, IGS4, IGS5, IGS6 sequences are 88.18%, 87.07%, 77.45%, and the methylation difference analysis between the twins of 87.87%.2 is sequenced by pyrosequencing. The sensitivity of the pyrosequencing instrument is 5%. In the experiment, in the experiment, if the difference of the methylation level is greater than 5% between the twins, it is considered that there is a difference between the two. On the contrary, the difference analysis between the two offspring of the.2.1 sequences in the 76 pairs of the detected 76 pairs of MZT is that there are 6 pairs of differences in the methylation level between the CGIS1 sequence and MZT, accounting for MZT. The 7.89% of the sample size indicates that the 4 Cp G loci of CGIS1 sequence distinguishes MZT from 7.89%; in the 100 pairs of MZT, IGS3, IGS4, IGS5, and IGS6 sequences are 37 pairs, 7 pairs, 48 pairs, 44 pairs, and four non-CGI sequences can distinguish 81 pairs of MZT, indicating four sequence. The cumulative discrimination ability of 16 Cp G loci differentiating MZT was compared with the ability of 81.00%. to distinguish MZT between the CGIS1 sequence and the four non-CGI sequence. It was found that the ability to distinguish MZT from non-CGI sequence was higher than that of CGIS1 sequence to differentiate MZT. The correlation between the difference of methylation and age showed that the difference of DNA methylation in IGS6 sequences was negatively correlated with age between MZT, and the correlation coefficient was -0.304, that is, the difference of methylation between MZT decreased with age. The difference between the other sequence methylation levels between MZT was not significantly correlated with age. The correlation between sexual.3 methylation and age, sex, nationality, smoking and the correlation of 3.1 age to four non-CGI sequence methylation Spearman rank correlation analysis showed that the degree of methylation of IGS3, IGS4 two target sequences was weakly correlated with age, the correlation coefficient was -0.161, and the degree of methylation of the other two sequences of -0.384. was not related to age. At the age of 10, the samples were divided into 6 age groups: 0-10, 11-20, 21-30, 31-40, 41-50 and 50. The methylation levels of IGS4 sequences were significantly different between age groups. There was no significant difference in the methylation of.3.2 between the other three sequences in the age groups. The effect of methylation on the methylation of the four non-CGI sequences was compared with the level of methylation between different homosexual groups. The results showed that there was no significant difference in methylation between different sexes in the four non-CGI sequences detected by.3.3. The effect of.3.3 on methylation of four non-CGI sequences came from six ethnic groups: Han, Hani, and Lahu, The Zhuang, Hui and Yi people, because of the lower four groups, only analyzed the differences in the level of methylation in the Han and Hani samples, and found that the level of methylation in the IGS3 and IGS6 sequences showed significant differences between the Han and Hani ethnic groups. The other sequences did not show the effect of significant difference.3.4 smoking on the methylation of the four non-CGI sequences I The degree of methylation of Cp G2, Cp G4 site and Cp G3 site of IGS6 sequence in the GS4 sequence showed significant difference between smoking and non smoking groups. The methylation level of the other sequences was not significantly different between the smoking group and the non smoking group. Conclusion: the results of 1 pairs of four CGI candidate sequences showed that the methylation degree of the four CGI sequences was very high. Low, in the 76 pairs of MZT samples detected, the CGIS1 sequence distinguishes MZT from only 7.89%, suggesting that DNA methylation in the CGI region may not be a large sample study of the ideal region of MZT for the four non-CGI candidate sequences, indicating that the four intergenic region sequences of the non-CGI are highly methylation, and the ability to distinguish MZT is also higher. The cumulative discrimination efficiency is 81%, which can be used as a candidate sequence to distinguish MZT. It suggests that the intergenic region sequence located in non-CGI is an ideal sequence for screening MZT.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：D919.4

【参考文献】