International Journal of Legal Medicine

, Volume 133, Issue 2, pp 335–345 | Cite as

Identification of potential markers of fatal hypothermia by a body temperature-dependent gene expression assay

  • Takahiro UmeharaEmail author
  • Takehiko Murase
  • Yuki Abe
  • Hiromi Yamashita
  • Yoshinori Shibaike
  • Shinichiro Kagawa
  • Takuma Yamamoto
  • Kazuya Ikematsu
Original Article


Diagnosis of fatal hypothermia is considered to be difficult in forensic practice and even if findings due to cold exposure are evident, cold exposure is not necessarily a direct cause of death. Identification of useful molecular markers for the diagnosis of fatal hypothermia has not been successful. In this study, to identify novel molecular markers that inform the diagnosis of fatal hypothermia, we focused on skeletal muscle, which plays a role in cold-induced thermogenesis in mammals. We made rat models of mild, moderate, and severe hypothermia and performed body temperature-dependent gene expression analysis in the iliopsoas muscle using next-generation sequencing (NGS). NGS showed that after severe hypothermia, the expression levels of 91 mRNAs were more than double those in mild and moderate hypothermia and control animals. Gene ontology (GO) analysis indicated that these mRNAs are involved in a number of biological processes, including response to stress and lipids, and cellular response to hypoxia. The expression of four genes [connective tissue growth factor (Ctgf), JunB proto-oncogene, AP-1 transcription factor subunit (Junb), nuclear receptor subfamily 4, group A, member 1 (Nr4a1), and Syndecan 4 (Sdc4)] and the level of one protein (CTGF) were induced only by severe hypothermia. These genes and protein are involved in muscle regeneration, tissue repair, and lipid metabolism. These results indicate that heat production to maintain body temperature in a process leading to fatal hypothermia might be performed by the iliopsoas muscle, and that Ctgf, Junb, Nr4a1, and Sdc4 genes are potential diagnostic markers for fatal hypothermia.


Iliopsoas muscle Body temperature-dependent gene expression analysis Thermogenesis Marker gene 



We thank Jeremy Allen, PhD, from Edanz Group ( for editing the draft of this manuscript.

Funding information

This work was supported in part by the Japan Society for the Promotion of Science (Grant-in-Aid for Scientific Research C, 16K09210).

Compliance with ethical standards

The Animal Care Committee of Nagasaki University approved this research protocol (approval number 1606081312).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

414_2018_1888_Fig8_ESM.png (681 kb)
Figure. 1

In the severe hypothermia group, the expression levels of 77 mRNAs were less than half those in control, mild and moderate hypothermia groups. The heatmap illustrates profiles of 77 mRNAs from the iliopsoas muscle of control, mild, moderate and severe hypothermia rats. Quantification and log2 calculations were performed using Deseq, and normalization for each gene was performed using the median of all samples. (PNG 680 kb)

414_2018_1888_MOESM1_ESM.tiff (1.5 mb)
High resolution image (TIFF 1521 kb)
414_2018_1888_Fig9_ESM.png (826 kb)
Figure. 2

A. In the moderate hypothermia group, 67 mRNAs showed a more than twofold difference in expression level compared with levels in control, mild and severe hypothermia groups. B. In the mild hypothermia group, 28 mRNAs showed a more than twofold difference in expression level compared with levels in control, moderate and severe hypothermia groups. The heatmap illustrates profiles of 67 and 28 mRNAs from the iliopsoas muscle of control, mild, moderate and severe hypothermia rats. Quantification and log2 calculations were performed using Deseq, and normalization for each gene was performed using the median of all samples. (PNG 826 kb)

414_2018_1888_MOESM2_ESM.tiff (1.5 mb)
High resolution image (TIFF 1521 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Division of Forensic Pathology and Science, Unit of Social Medicine, Course of Medical and Dental Sciences, Graduate School of Biomedical SciencesNagasaki University School of MedicineNagasakiJapan
  2. 2.Center for Forensic Pathology and ScienceNagasaki University Graduate School of Biomedical SciencesNagasakiJapan

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