| 研究課題/領域番号 |
21J23562
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| 配分区分 | 補助金 |
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| 研究機関 | 東北大学 |
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研究代表者 |
GUAN XUETING 東北大学, 医学系研究科, 特別研究員(DC1)
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| 研究期間 (年度) |
2021-04-28 – 2024-03-31
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| キーワード | DNA methylation / Age estimation / Mitochondrial genome / Post-mortem samples / Forensic epigenetics |
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| 研究実績の概要 |
To further investigate the age-related DNA methylation pattern of mitochondrial genome, a total number of 10 blood samples (22-94 y.o) was collected from the same rib donor if available. All blood samples were subjected to DNA extraction, quantification and bisulfite conversion. The amplification of 9 regions of interests (ROIs), which have been analyzed with 18 rib samples, was performed then sequenced on Miseq platform (Illumina). Two methylated controls(0% and 100%) were also sequenced to ensure the quality of sequencing. Raw data were trimmed on Ubuntu, the alignment and differential DNA methylation calling were performed by CLC-Genomics Workbench 11 (Qiagen).
As a result, the quality of sequencing has improved with a mean depth of 3010 reads at each ROI. Two ROIs (OLS and CytoB) showed weak correlations with aging. One CpG site at Cyto2 showed a positive correlation of 0.74 with aging, followed by 12S(R=0.587), G11778A(R=0.534), Promoter(-)(R=0.334)and 16S(R=0.331). While one CpG site at ND5 showed a negative correlation of 0.587 with aging, followed by Cyto1(R=-0.495). Owing to small sample size, 42 blood samples (21-94 y.o) were collected and proceeded to the sequencing of 9 ROIs followed by the same workflow as mentioned above. Unexpectedly, moderate methylation levels (40%-60%) were observed with most samples, leading to negligible correlations with increasing age.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
As reported above, unideal sequencing results have been observed frequently in 42 additional blood samples. It is well accepted that mitochondrial genome is depleted of DNA methylation contents, which are generally lower than 20%. We have obtained unexpected DNA methylation callings, resulting in 40% to 60% on the starting of the mitochondrial genome, especially OLS, 12S and 16S. It is highly possible that such results were caused by incomplete bisulfited conversion, giving rise to the overestimation of methylation levels on the ROIs. Unlike the helix-coiled structure of double-stranded DNA in the genome, mitochondrial DNA is circle double-stranded, denoting as heavy and light strands. This unique structure also allows certain secondary structures to be formed, like super-coil and etc. Such secondary structures may block the access of the unmethylated cytosine during bisulfite treatment, leaving the unmethylated cytosine as cytosine instead of converting it to uracil. This would eventually lead to overestimation of DNA methylation levels on the targets, thus masking the actual correlations. This is not a widely discussed topic, re-analysis of half of the 42 blood samples is currently ongoing.
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| 今後の研究の推進方策 |
To enrich the knowledge of differential DNA methylated patterns between different types of samples, bones and blood samples, collected from 12 adult donors (22-78 y.o), were also analyzed with Infinium Methylation EPIC Microarray, Illumina. This microarray covers 863904 CpG as well as 2932 non-classical CNGs (N denotes any types of nucleotides) over the whole genome in humans and is able to detect and compare the differential DNA methylation levels among different types of samples. In current study, 12 samples were roughly divided with four categories, denoting as blood-young, blood-old, bone-young and bone-old. Donors that were younger than 50-year-old were assigned to the young group, otherwise to the old group. Statistical and comparative analyses were also performed. Overlapped probes and areas were recorded and extracted if they passed the threshold (adjusted p-value: 6.782e-08). A total number of 9 probes met the requirement before-mentioned, as common CpG sites for both bones and blood samples. Further research will be focused on evaluating the underlying correlation between these CpGs and two types of samples. The initial observation will be reported on the 107th Congress of the Japanese Society of Legal Medicine.
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