| Objective: Identification of individuals within monozygotic twin(MZT) in the field of forensic DNA typing is still an unresolved problem. This inability causes problems in criminal or paternity cases with a MZT as suspect, or as alleged father. Although there are increasing literatures about genetic differences within MZT pairs, such as copy number variation(CNV), SNP, and DYZ1, it has been concluded that large discordance in DNA is rare within MZT pairs. It is like finding the needle in the haystack to differentiating identical twins by searching for the genetic differences. However, another possible method to distinguishing MZT pairs may be epigenetic markers. A large number of studies have shown that regardless of concordant MZT or discordant MZT, such as difference in obesity, cancer, mental illness, there are differences in genome-wide methylation or specific methylation. In this study, we performed a genome-wide scan for four pairs of concordant MZT using methylated DNA immunoprecipitation(Me DIP) sequencing technology, to find different DNA methylation regionswithin MZT pairs and to get candidate DNA methylation markers for differentiating MZT.Methods: In accordance with the principle of informed consent, peripheral blood samples were collected from four pairs of MZT, each subject signed an informed consent form and filled in the corresponding questionnaire. Genomic DNA was extracted from blood samples and the zygosity of twins was tested by 19 STR loci. The whole genome methylation profile was obtained by using Me DIP sequencing technology. Sequence reads were generated from Illumina Hi Seq 2000, image analysis and base calling were performed using Off-Line Basecaller software(OLB V1.8). After passing Solexa CHASTITY quality filter, the clean reads were aligned to human genome(UCSC HG19) using BOWTIE software(V2.1.0). To quantify the DNA methylation level of any specific region, we used each mapped reads. A methylation score for any region in the genome was defined as number of reads per kb. The DNA methylation status of a specific region was defined as unmethylated if its Me DIP-score was less than 8.31 reads·kb-1, as partially methylated if its Me DIP-score was within 8.31 ~ 374.44 reads·kb-1 and completely methylated if its Me DIP-score is greater than 374.44 reads·kb-1.Results: 1. Whole genome methylation profile 1.1 Cp G Island Methylation ProfileAccording to Me DIP score, among 27841 CGI regions, the number of partially methylated, completely methylatedand unmethylated regions of 8 samples were respectively11324~18050, 4189~5474, 5164~11336, accounting for 40.67%~64.83%, 15.05%~19.66%and18.55%~40.72% of the total CGI regions. For all of the eight samples, about 50% of Cp Gs located in CGI regions were partially methylated, and the completely methylated regions with Me DIP score larger than 374.44 reads·kb-1were less than 20%. These results indicated that the methylation level in CGI across whole genome was not high.Next, we analyzed the Cp G methylation status of different classes of CGI. The results demonstrated that the ratio of completely methylated region in promoter CGI was the lowest, only about 1% to 2%, and the unmethylated region account for the highest proportion, up to 30% to 60%, showing a greatly lower methylation degree in promoter CGI. However, in intragenic CGI, completely methylation reach to 35% to 45%, indicating a higher degree of methylation. The methylation level of intergenic CGI ranks between promoter CGI and intragenic CGI. 1.2 Promoter Methylation ProfileIn the 29402 promoter regions, partially methylated, completely methylated and unmethylated regions were 17213~22432, 69~265, and 6880~11924, accounting for 58.54%~76.29%, 0.23%~0.90% and 23.40%~40.56% of the total number of regions respectively, which showed that the proportion of completely methylated region with Me DIP score greater than 374.44 reads·kb-1 was very low, close to zero, and most promoter regions were in the partially methylated and unmethylated state with lower degree of methylation. 1.3 Gene body methylation profilesThe Ref Seq genes that have more than 3kb in length are chosen for gene body methylation analysis. The gene body region is defined as +2000bp downstream of the transcription start site(TSS) to the transcription termination site(TTS).In the 24157 genebody regions, partially methylated, completely methylated and unmethylated regions were 17166~20850, 33~85, 3273~6917, accounting for 71.06%~86.31%, 0.14%~0.35%, 13.55%~28.63% of the total genebody regions respectively, indicating that the proportion of completely methylated region across genebody was very low, close to zero, and most genebody regions were in the partially methylated and unmethylated state with lower degree of methylation. 2. Differentially Methylated Regions Analysis 2.1 The first layer of screeningTo calculate differentially methylated regions, we performed a Fold Change(FC) filtering between two samples with Me DIP-score. The threshold is FC>= 2.0. According to this criteria, there were totally 22889, 21239, 17926, 25140 DMRs respectively within MZ twin pair #1, #2, #3, and #4. Most of the DMRs were located in CGI, followed by the promoter regions, and only little of them ingenebody. Further analysis of the distribution of DMRs in the different regions of CGI showed that the DMRs in CGI within MZ twin pair mostly occurred in promoter CGI, followed by intergenic CGI, least in intragenic CGI. 2.2 The second layer of screeningIn order to filter out the DMRs with the most different degree, we conducted a second layer of screening according to two standards: 1) the fold change of Me DIP score is 100(FC = 100), 2) at the same time, the Me DIP score of one sample is larger than 8.31. That is to say, the selected DMRs according to the above criteria must be unmethylated in one sample and partially methylated or completely methylated in another sample. We call these filtered DMRs through second layer of screening major DMR(MDMR).Through the second layer of selection, 3766, 2711, 1772, 2387 MDMRs are filtered out within MZ twin pair #1, #2, #3, and #4. Most of the MDMRs are located in CGI, followed by promoter regions, and little of them ingenebody.Further analysis of the distribution of MDMRs in the different regions of CGI showed that the MDMRs in CGI within MZ twin pair mostly occurred in promoter CGI, followed by intergenic CGI, least in intragenic CGI. 2.3 The same MDMRs across the four pairs of MZ twinThirty-eightshared MDMRs across the four pairs of MZ twin were filtered out, which are all located in CGI, including 17 promoter CGI regions, 17 intergenic CGI regions and 4 intragenic CGI regions. The gene-related MDMRs mainly included genes involved in cell proliferation, differentiation, growth and development such as zinc finger protein, PRRX2, RBBP9, and genes involved in G protein signaling such as regulator of G-protein signaling 16.Conclusions: In this study, genome-wide DNA methylation screening was performed for four pairs of concordant MZT using Me DIP sequencing analysis, and a large number of DMRs are found within MZT pairs. Most of the DMRs are located in CGI, especially in promoter CGI and little of them ocuured in intragenic regions. 1772~3766 major DMRs(MDMRs) with the largest different methylation degree are further filtered from DMRs. Finally 38 shared MDMRs by all of four MZT pairs are identified as candidate DNA methylation markers for distingiushing MZT. |
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