International Journal of Legal Medicine

, Volume 126, Issue 1, pp 55–62 | Cite as

Potential forensic application of DNA methylation profiling to body fluid identification

  • Hwan Young Lee
  • Myung Jin Park
  • Ajin Choi
  • Ja Hyun An
  • Woo Ick Yang
  • Kyoung-Jin Shin
Original Article


DNA analysis of various body fluid stains at crime scenes facilitates the identification of individuals but does not currently determine the type and origin of the biological material. Recent advances in whole genome epigenetic analysis indicate that chromosome pieces called tDMRs (tissue-specific differentially methylated regions) show different DNA methylation profiles according to the type of cell or tissue. We examined the potential of tissue-specific differential DNA methylation for body fluid identification. Five tDMRs for the genes DACT1, USP49, HOXA4, PFN3, and PRMT2 were selected, and DNA methylation profiles for these tDMRs were produced by bisulfite sequencing using pooled DNA from blood, saliva, semen, menstrual blood, and vaginal fluid. The tDMRs for DACT1 and USP49 showed semen-specific hypomethylation, and the tDMRs for HOXA4, PFN3, and PRMT2 displayed varying degrees of methylation according to the type of body fluid. Preliminary tests using methylation-specific PCR for the DACT1 and USP49 tDMRs showed that these two markers could be used successfully to identify semen samples including sperm cells. Body fluid-specific differential DNA methylation may be a promising indicator for body fluid identification. Because DNA methylation profiling uses the same biological source of DNA for individual identification profiling, the determination of more body fluid-specific tDMRs and the development of convenient tDMR analysis methods will facilitate the broad implementation of body fluid identification in forensic casework.


Body fluid identification DNA methylation Tissue-specific differentially methylated region 



This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009–0073496 and 2010–0005208).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

414_2011_569_MOESM1_ESM.pdf (151 kb)
ESM 1 (PDF 150 kb)
414_2011_569_MOESM2_ESM.pdf (483 kb)
ESM. 2 (PDF 482 kb)


  1. 1.
    Virkler K, Lednev IK (2009) Analysis of body fluids for forensic purposes: from laboratory testing to non-destructive rapid confirmatory identification at a crime scene. Forensic Sci Int 188:1–17PubMedCrossRefGoogle Scholar
  2. 2.
    Bauer M, Polzin S, Patzelt D (2003) Quantification of RNA degradation by semi-quantitative duplex and competitive RT-PCR: a possible indicator of the age of bloodstains? Forensic Sci Int 138:94–103PubMedCrossRefGoogle Scholar
  3. 3.
    Bauer M, Patzelt D (2002) Evaluation of mRNA markers for the identification of menstrual blood. J Forensic Sci 47:1278–1282PubMedGoogle Scholar
  4. 4.
    Juusola J, Ballantyne J (2003) Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identification. Forensic Sci Int 135:85–96PubMedCrossRefGoogle Scholar
  5. 5.
    Juusola J, Ballantyne J (2005) Multiplex mRNA profiling for the identification of body fluids. Forensic Sci Int 152:1–12PubMedCrossRefGoogle Scholar
  6. 6.
    Nussbaumer C, Gharehbaghi-Schnell E, Korschineck I (2006) Messenger RNA profiling: a novel method for body fluid identification by real-time PCR. Forensic Sci Int 157:181–186PubMedCrossRefGoogle Scholar
  7. 7.
    Juusola J, Ballantyne J (2007) mRNA profiling for body fluid identification by multiplex quantitative RT-PCR. J Forensic Sci 52:1252–1262PubMedGoogle Scholar
  8. 8.
    Zubakov D, Hanekamp E, Kokshoorn M, van Ijcken W, Kayser M (2008) Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples. Int J Leg Med 122:135–142CrossRefGoogle Scholar
  9. 9.
    Hanson EK, Lubenow H, Ballantyne J (2009) Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs. Anal Biochem 387:303–314PubMedCrossRefGoogle Scholar
  10. 10.
    Setzer M, Juusola J, Ballantyne J (2008) Recovery and stability of RNA in vaginal swabs and blood, semen, and saliva stains. J Forensic Sci 53:296–305PubMedCrossRefGoogle Scholar
  11. 11.
    Zubakov D, Kokshoorn M, Kloosterman A, Kayser M (2009) New markers for old stains: stable mRNA markers for blood and saliva identification from up to 16-year-old stains. Int J Leg Med 123:71–74CrossRefGoogle Scholar
  12. 12.
    Holliday R, Pugh JE (1975) DNA modification mechanisms and gene activity during development. Science 187:226–232PubMedCrossRefGoogle Scholar
  13. 13.
    Bird AP (1986) CpG-rich islands and the function of DNA methylation. Nature 321:209–213PubMedCrossRefGoogle Scholar
  14. 14.
    Russo VEA, Martienssen RA, Riggs AD (1996) Epigenetic mechanisms of gene regulation. Cold Spring Harbor Laboratory Press, PlainviewGoogle Scholar
  15. 15.
    Tost J (ed) (2008) Epigenetics. Caister Academic Press, NorfolkGoogle Scholar
  16. 16.
    Ohgane J, Yagi S, Shiota K (2008) Epigenetics: the DNA methylation profile of tissue-dependent and differentially methylated regions in cells. Placenta 29:S29–S35PubMedCrossRefGoogle Scholar
  17. 17.
    Christensen BC, Houseman EA, Marsit CJ, Zheng S, Wrensch MR, Wiemels JL, Nelson HH, Karagas MR, Padbury JF, Bueno R, Sugarbaker DJ, Yeh RF, Wiencke JK, Kelsey KT (2009) Aging and environmental exposures alter tissue-specific DNA methylation dependent upon CpG island context. PLoS Genet 5:e1000602PubMedCrossRefGoogle Scholar
  18. 18.
    Schilling E, Rehli M (2007) Global, comparative analysis of tissue-specific promoter CpG methylation. Genomics 90:314–323PubMedCrossRefGoogle Scholar
  19. 19.
    Kitamura E, Igarashi J, Morohashi A, Hida N, Oinuma T, Nemoto N, Song F, Ghosh S, Held WA, Yoshida-Noro C, Nagase H (2007) Analysis of tissue-specific differentially methylated regions (TDMs) in humans. Genomics 89:326–337PubMedCrossRefGoogle Scholar
  20. 20.
    Igarashi J, Muroi S, Kawashima H, Wang X, Shinojima Y, Kitamura E, Oinuma T, Nemoto N, Song F, Ghosh S, Held WA, Nagase H (2008) Quantitative analysis of human tissue-specific differences in methylation. Biochem Biophys Res Commun 376:658–664PubMedCrossRefGoogle Scholar
  21. 21.
    Song F, Mahmood S, Ghosh S, Liang P, Smiraglia DJ, Nagase H, Held WA (2009) Tissue specific differentially methylated regions (TDMR): changes in DNA methylation during development. Genomics 93:130–139PubMedCrossRefGoogle Scholar
  22. 22.
    Song F, Smith JF, Kimura MT, Morrow AD, Matsuyama T, Nagase H, Held WA (2005) Association of tissue-specific differentially methylated regions (TDMs) with differential gene expression. Proc Natl Acad Sci USA 102:3336–3341PubMedCrossRefGoogle Scholar
  23. 23.
    Illingworth R, Kerr A, Desousa D, Jørgensen H, Ellis P, Stalker J, Jackson D, Clee C, Plumb R, Rogers J, Humphray S, Cox T, Langford C, Bird A (2008) A novel CpG island set identifies tissue-specific methylation at developmental gene loci. PLoS Biol 6:e22PubMedCrossRefGoogle Scholar
  24. 24.
    Doi A, Park IH, Wen B, Murakami P, Aryee MJ, Irizarry R, Herb B, Ladd-Acosta C, Rho J, Loewer S, Miller J, Schlaeger T, Daley GQ, Feinberg AP (2009) Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts. Nat Genet 41:1350–1353PubMedCrossRefGoogle Scholar
  25. 25.
    Li LC, Dahiya R (2002) MethPrimer: designing primers for methylation PCRs. Bioinformatics 18:1427–1431PubMedCrossRefGoogle Scholar
  26. 26.
    Bock C, Reither S, Mikeska T, Paulsen M, Walter J, Lengauer T (2005) BiQ Analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing. Bioinformatics 21:4067–4068PubMedCrossRefGoogle Scholar
  27. 27.
    Rohde C, Zhang Y, Jurkowski TP, Stamerjohanns H, Reinhardt R, Jeltsch A (2008) Bisulfite sequencing Data Presentation and Compilation (BDPC) web server—a useful tool for DNA methylation analysis. Nucleic Acids Res 36:e34PubMedCrossRefGoogle Scholar
  28. 28.
    Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386PubMedGoogle Scholar
  29. 29.
    Bitu CC, De Souza Setubal Destro MF, Lopes MA, Jorge J, Graner E, Coletta RD (2008) Dysregulated expression of Hox genes in oral squamous cell carcinomas. Oral presentation #17 at 2008 International Association of Oral Pathologists, San Francisco, USAGoogle Scholar
  30. 30.
    Byun HM, Siegmund KD, Pan F, Weisenberger DJ, Kanel G, Laird PW, Yang AS (2009) Epigenetic profiling of somatic tissues from human autopsy specimens identifies tissue- and individual-specific DNA methylation patterns. Hum Mol Genet 18:4808–4817PubMedCrossRefGoogle Scholar
  31. 31.
    Sugimoto K, Koh E, Sin HS, Maeda Y, Narimoto K, Izumi K, Kobori Y, Kitamura E, Nagase H, Yoshida A, Namiki M (2009) Tissue-specific differentially methylated regions of the human VASA gene are potentially associated with maturation arrest phenotype in the testis. J Hum Genet 54:450–456PubMedCrossRefGoogle Scholar
  32. 32.
    Frumkin D, Wasserstrom A, Budowle B, Davidson A (2010) DNA methylation-based forensic tissue identification. Forensic Sci Int Genet. doi: 10.1016/j.fsigen.2010.12.001 Google Scholar
  33. 33.
    Naito E, Dewa K, Fukuda M, Sumi H, Wakabayashi Y, Umetsu K, Yuasa I, Yamanouchi H (2003) Novel paternity testing by distinguishing parental alleles at a VNTR locus in the differentially methylated region upstream of the human H19 gene. J Forensic Sci 48:1275–1279PubMedGoogle Scholar
  34. 34.
    Nakayashiki N, Kanetake J, Aoki Y (2004) A parent-of-origine detectable polymorphism in the hypermethylated region upstream of the human H19 gene. Int J Leg Med 118:158–161CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Hwan Young Lee
    • 1
    • 2
  • Myung Jin Park
    • 1
  • Ajin Choi
    • 1
  • Ja Hyun An
    • 1
  • Woo Ick Yang
    • 1
  • Kyoung-Jin Shin
    • 1
    • 2
  1. 1.Department of Forensic Medicine and Brain Korea 21 Project for Medical ScienceYonsei University College of MedicineSeoulSouth Korea
  2. 2.Human Identification Research CenterYonsei UniversitySeoulSouth Korea

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