| 研究実績の概要 |
To illustrate the DNA methylation (DNAm) pattern on mitochondrial genome, a total of 18 human ribs (22- to 94-year-old) were collected regardless of the sex and were subjected to DNA extraction and bisulfite conversion. A total of 11 regions of interest (ROIs) were determined and categorized as follows: 2 disease-related SNPs (T8993G and G11778A); 2 rRNAs (12s and 16s rRNAs); 2 transcription-related regions (OLS and minus promoter); as well as 5 encoded genes involving cellular metabolisms (Cyto1&2, CytoB and ND1&5). The abovementioned ROIs were amplified and purified in 18 samples as well as 6 DNAm controls (0% to 100% by a gradient of 20%), which helps to observe the sequencing error. Followed by library preparation, next-generation sequencing was performed. Raw sequencing data were analyzed by bioinformatic tools. Differential DNAm data were extracted then proceeded to statistical analyses (Pearson’s correlation test) on Rstudio/R 4.1.1.
Two ROIs (ND1 and T8893G) were removed from candidates due to poor amplification. The remaining 9 ROIs proceeded to subsequent analyses. However, most targets, except CytoB, ND5 and G11778A, showed a severe read-loss, thus were unable to perform statistical analyses. A positive correlation has been observed in CytoB (R=0.223) whereas G11778A showed a negative correlation of 0.276. ND5 demonstrated a negligible correlation of -0.003 in 18 samples. A general high methylation level has been observed in DNAm controls regardless of their methylation contents. Such unideal results may be caused by poor quality of samples or technical issues.
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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
In this study, a total of 11 ROIs were selected as candidates. However, ND1 and T8993G had encountered difficulties in amplification, where ND1 showed a low amplification efficiency while T8893G had a low specificity that prominently amplified the primer-dimer. Due to this respect, the abovementioned 2 ROIs were removed from candidates. Severe read-losses have been observed in the remaining 9 ROIs. Unfortunately, it cannot be concluded as being due to poor quality of samples or technical issues. For instance, 0% control (unmethylated) was expected to show a methylation level lying in 0 ~ 10%, while universal high methylation levels, usually over 90%, have been found in both low and high DNAm controls. It is highly likely to be associated with PCR byproducts or adaptor-dimers. Sanger sequencing was also performed to ensure all 9 ROIs were properly amplified. Compared with revised Cambridge Reference Sequence (rCRS, NC_012920.1), target ROIs have been identified. Moreover, relatively low correlations have been conformed on CytoB (R=0.223) and G11778A (R=-0.276). ND5 showed a negligible correlation of -0.003. Compared with blood samples, these 3 ROIs demonstrated low correlations with age, though the underlying reasons cannot be determined.
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| 今後の研究の推進方策 |
The future plan can be divided into 2 directions. On the one hand, the blood sample of the same rib donor has also been analyzed using the abovementioned 9 ROIs, resulting in a total of 10 samples (22- to 94 -year-old). Blood samples of 8 rib donors were unavailable owing to high decomposition or skeletonization. This step aims to determine the actual reason that caused the low correlation with age in ribs, whether if it was due to the intrinsic nature of the human mitochondrial genome or simply due to selected DNAm markers. It is currently undergoing data analysis, and proper statistical analyses will be implicated based on obtained sequencing data. On the other hand, a DNA methylation array (Illumina) will also be implicated to screen the bone-specific DNAm markers on the whole epigenome. Since the quality and quantity of initial DNA materials are of extreme importance, skulls from adults were also collected during the autopsy. This project is currently ongoing. Moreover, the detailed explanation of each possible cause of unexpected results have been summarized and will be presented on the 106th Congress of the Japanese Society of Legal Medicine (Nagoya, 2022/06/08 ~ 10).
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