• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to project page

2021 Fiscal Year Annual Research Report

The Development of Age Estimation Model for Unidentified Individuals Based on Mitochondrial DNA Methylation Changes

Research Project

Project/Area Number 21J23562
Allocation TypeSingle-year Grants
Research InstitutionTohoku University

Principal Investigator

GUAN XUETING  東北大学, 医学系研究科, 特別研究員(DC1)

Project Period (FY) 2021-04-28 – 2024-03-31
KeywordsDNA methylation / Age estimation / Mitochondrial epigenome / Post-mortem samples / Forensic epigenetics
Outline of Annual Research Achievements

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.

Current Status of Research Progress
Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

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.

Strategy for Future Research Activity

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).

  • Research Products

    (4 results)

All 2022 2021

All Journal Article (2 results) (of which Peer Reviewed: 2 results) Presentation (2 results)

  • [Journal Article] Age-related DNA methylation analysis for forensic age estimation using post-mortem blood samples from Japanese individuals2021

    • Author(s)
      Guan X.、Ohuchi T.、Hashiyada M.、Funayama M.
    • Journal Title

      Legal Medicine

      Volume: 53 Pages: -

    • DOI

      10.1016/j.legalmed.2021.101917

    • Peer Reviewed
  • [Journal Article] Analysis of Autopsy Cases of Unknown Cause of Death in Psychotropic Drug Users2021

    • Author(s)
      X.Guan, T.Ohuchi, K. Usui, Y. Igari, T. Ogawara, M. Hashiyada, A. Usui and M. Funayama
    • Journal Title

      The Research and Practice in Forensic Medicine

      Volume: 64 Pages: 159-165

    • Peer Reviewed
  • [Presentation] Report on Age Estimation Based on Mitochondrial DNA Methylation Level Using Unidentified Human Ribs2022

    • Author(s)
      GUAN XUETING
    • Organizer
      第106次日本法医学会学術全国集会
  • [Presentation] 向精神薬服用者の死因不明剖検例の検討2021

    • Author(s)
      GUAN XUETING
    • Organizer
      第105次日本法医学会学術全国集会

URL: 

Published: 2022-12-28   Modified: 2023-08-01  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi