甄別同卵雙生子的DNA甲基化序列篩選研究
發(fā)布時(shí)間:2018-06-19 12:22
本文選題:同卵雙生子 + DNA甲基化; 參考:《河北醫(yī)科大學(xué)》2017年碩士論文
【摘要】:目的:同卵雙生子(monozygotic twins,MZT)由一個(gè)受精卵分裂形成,DNA序列高度相似,傳統(tǒng)的法醫(yī)學(xué)遺傳標(biāo)記如短串聯(lián)重復(fù)序列(short tandem repeat,STR)、單核苷酸多態(tài)性(single nucleotide polymorphism,SNP)等難以將二者區(qū)分,是為MZT涉嫌犯罪案件中生物檢材身份認(rèn)定的一大難題。大量研究表明MZT之間存在表觀遺傳學(xué)差異。我們前期研究應(yīng)用甲基化免疫共沉淀測(cè)序技術(shù)(Methylated DNA immunoprecipitation,Me DIP),對(duì)5對(duì)表型一致的MZT進(jìn)行全基因組甲基化檢測(cè)分析,從中篩選一些差異甲基化序列,有些分布在Cp G島(Cp G island,CGI),有些序列為非Cp G島序列(non-CGI)。本研究從中選取若干CGI序列和non-CGI序列進(jìn)行大樣本研究,以驗(yàn)證這些序列區(qū)分MZT的效果和能力,為解決甄別MZT個(gè)體難題提供科學(xué)依據(jù)。方法:從本課題組建立的雙生子樣本庫(kù)中選取100對(duì)MZT樣本,應(yīng)用Qiagen公司的QIAamp DNA Blood kit提取血液樣本的DNA。應(yīng)用ZYMO公司的EZ DNA Methylation-Gold Kit對(duì)基因組DNA進(jìn)行重亞硫酸氫鹽轉(zhuǎn)化,以轉(zhuǎn)化后的DNA為模板,擴(kuò)增四個(gè)位于CGI的序列(Cp G island sequence,CGIS)及四個(gè)位于non-CGI的序列。四個(gè)CGI序列分別位于2號(hào)染色體的基因間區(qū)、人類白細(xì)胞抗原B伴隨轉(zhuǎn)錄物3(BAT3)基因啟動(dòng)子區(qū)、氨氯吡嗪脒敏感陽(yáng)離子通道1(ACCN1)基因啟動(dòng)子區(qū)、G蛋白信號(hào)調(diào)節(jié)子16(RGS16)基因啟動(dòng)子區(qū),分別命名為CGIS1、CGIS2、CGIS3、CGIS4。四個(gè)non-CGI序列均位于基因間區(qū)(intergenic sequence,IGS),分別命名為IGS3、IGS4、IGS5、IGS6,其染色體定位分別為:chr1:114870877-114871203、chr12:67213759-67214092、chr11:5539842-5540207、chr1:56877427-56877787。對(duì)擴(kuò)增產(chǎn)物進(jìn)行單鏈純化后行焦磷酸測(cè)序,獲得八條靶序列共40個(gè)Cp G位點(diǎn)甲基化水平的定量數(shù)據(jù)。每個(gè)樣本重復(fù)三次實(shí)驗(yàn)。應(yīng)用Excel、Spearman相關(guān)檢驗(yàn)、t檢驗(yàn)、方差分析、秩和檢驗(yàn)等方法進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果:1八條靶序列的平均DNA甲基化水平1.1四個(gè)CGI序列的平均甲基化水平在所調(diào)查的152個(gè)血液樣本中,CGIS1、CGIS2、CGIS3、CGIS4序列的平均甲基化水平分別為:21.37%、0.41%、5.05%、1.56%。1.2四個(gè)non-CGI序列的平均甲基化水平在所調(diào)查的200個(gè)血液樣本中,IGS3、IGS4、IGS5、IGS6序列的平均甲基化水平分別為88.18%、87.07%、77.45%、87.87%。2雙生子之間甲基化差異分析參照焦磷酸測(cè)序儀用戶手冊(cè),焦磷酸測(cè)序儀靈敏度為5%,在實(shí)驗(yàn)中,如甲基化水平差值在雙生子之間大于5%,則認(rèn)為兩者之間存在差異,反之則認(rèn)為無(wú)差異。2.1各序列雙生子間差異分析在所檢測(cè)的76對(duì)MZT中,CGIS1序列在MZT之間甲基化水平有差異的有6對(duì),占MZT樣本量的7.89%,表明CGIS1序列4個(gè)Cp G位點(diǎn)區(qū)分MZT的鑒別能力為7.89%;在所檢測(cè)的100對(duì)MZT中,IGS3、IGS4、IGS5、IGS6序列在MZT之間甲基化水平有差異的分別有37對(duì)、7對(duì)、48對(duì)、44對(duì),四個(gè)non-CGI序列累積能夠區(qū)分81對(duì)MZT,占MZT樣本量的81.00%,表明四個(gè)non-CGI序列16個(gè)Cp G位點(diǎn)區(qū)分MZT的累積鑒別能力為81.00%。對(duì)CGIS1序列和四個(gè)non-CGI序列區(qū)分MZT的能力進(jìn)行c2檢驗(yàn)比較分析,發(fā)現(xiàn)和non-CGI序列區(qū)分MZT的能力高于CGIS1序列區(qū)分MZT的能力(P0.05)。2.2雙生子之間甲基化差異與年齡的相關(guān)性分析探討雙生子間甲基化的差異與年齡的相關(guān)性,結(jié)果表明,在MZT之間,IGS6序列的DNA甲基化差異與年齡呈較弱的負(fù)相關(guān),相關(guān)系數(shù)為-0.304,也就是,隨著年齡增大,MZT之間的甲基化差異逐漸減小。其余序列甲基化水平在MZT之間的差異與年齡均無(wú)明顯相關(guān)性。3甲基化與年齡、性別、民族、吸煙的相關(guān)性3.1年齡對(duì)四個(gè)non-CGI序列甲基化的影響Spearman秩相關(guān)分析結(jié)果表明,IGS3、IGS4兩條靶序列的甲基化程度均與年齡呈弱相關(guān),相關(guān)系數(shù)分別為-0.161、-0.384。其余兩條序列甲基化程度與年齡無(wú)相關(guān)性。以10歲為年齡間隔,將樣本分為6個(gè)年齡組:0-10歲、11-20歲、21-30歲、31-40歲、41-50歲及50歲。比較各年齡組間甲基化水平的差異,分析得出IGS4序列在年齡組間甲基化水平存在顯著差異。其余三條序列在各年齡組間甲基化程度無(wú)顯著差異。3.2性別對(duì)四個(gè)non-CGI序列甲基化的影響通過(guò)比較不同性別間的甲基化水平,結(jié)果表明,在所檢測(cè)的四個(gè)non-CGI序列中,甲基化程度在不同性別之間無(wú)顯著差異。3.3民族對(duì)四個(gè)non-CGI序列甲基化的影響樣本志愿者來(lái)自六個(gè)民族:漢族、哈尼族、拉祜族、壯族、回族及彝族,由于后四組樣本較少,故本研究只分析漢族和哈尼族樣本的甲基化水平差異,發(fā)現(xiàn)IGS3、IGS6序列的甲基化水平在漢族與哈尼族之間表現(xiàn)出顯著差異。其余序列未表現(xiàn)出顯著性差異。3.4吸煙對(duì)四個(gè)non-CGI序列甲基化的影響IGS4序列的Cp G2、Cp G4位點(diǎn)及IGS6序列的Cp G3位點(diǎn)甲基化程度,吸煙組和不吸煙組表現(xiàn)出顯著差異,其余序列的甲基化水平在吸煙組與不吸煙組之間無(wú)明顯差異。結(jié)論:1對(duì)四個(gè)CGI候選序列的樣本研究結(jié)果表明,該四個(gè)CGI序列的甲基化程度非常低,在檢測(cè)的76對(duì)MZT樣本中,CGIS1序列區(qū)分MZT的效能僅有7.89%,提示CGI區(qū)域DNA甲基化可能并不是區(qū)分MZT的理想?yún)^(qū)域。2對(duì)四個(gè)non-CGI候選序列的大樣本研究結(jié)果表明,該四個(gè)位于non-CGI的基因間區(qū)序列甲基化程度較高,且區(qū)分MZT的能力也較高,累積區(qū)分效能達(dá)81%,可作為區(qū)分MZT的候選序列,提示位于non-CGI的基因間區(qū)序列是甄別MZT較為理想的序列。
[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.
【學(xué)位授予單位】:河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】:碩士
【學(xué)位授予年份】:2017
【分類號(hào)】:D919.4
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