DNA Methylation as a Biomarker for Body Fluid Identification

Rania Gomaa, Jawad Salehi, Shalini Behl


Currently, available identification techniques for forensic samples are either enzyme or protein based, which can be subjected to degradation, thus limiting its storage potentials. Epigenetic changes arising due to DNA methylation and histone acetylation can be used for body fluid identification. Markers DACT1, USP49, ZC3H12D, FGF7, cg23521140, cg17610929, chromosome 4 (25287119–25287254), chromosome 11 (72085678–72085798, 57171095–57171236, 1493401–1493538), and chromosome 19 (47395505–47395651) are currently being used for semen identification. Markers cg26107890, cg20691722, cg01774894 and cg14991487 are used to differentiate saliva and vaginal secretions from other body fluids. However, such markers show overlapping methylation pattern. This review article aimed to highlight the feasibility of using DNA methylation of certain genetic markers in body fluid identification and its implications for forensic investigations. The reviewed articles have employed molecular genetics techniques such as Bisulfite sequencing PCR (BSP), methylation specific PCR (MSP), Pyrosequencing, Combined Bisulfite Restriction Analysis (COBRA), Methylation-sensitive Single Nucleotide Primer Extension (SNuPE), and Multiplex SNaPshot Microarray. Bioinformatics software such as MATLAB and BiQ Analyzer has been used. Biological fluids have different methylation patterns and thus, this difference can be used to identify the nature of the biological fluid found at the crime scene. Using DNA methylation to identify the body fluids gives accurate results without consumption of the trace evidence and requires a minute amount of DNA for analysis. Recent studies have incorporated next-generation sequencing aiming to find out more reliable markers that can differentiate between different body fluids. Nonetheless, new DNA methylation markers are yet to be discovered to accurately differentiate between saliva and vaginal secretions with high confidence. Epigenetic changes are dynamic and it is important to find stable DNA sequences that can be used as biomarkers.

Keywords: Forensic Science; DNA analysis; Methylation; body fluid; identification;  Pyrosequencing; DACT1; USP49; ZC3H12D; FGF7.


Forensic Science; DNA analysis; Methylation; body fluid identification; Pyrosequencing; DACT1; USP49; ZC3H12D; FGF7

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Frumkin D, Wasserstrom A, Budowle B, Davidson A. DNA methylation-based forensic tissue identification. Forensic Sci Int Genet. 2011;5(5):517-24.https://doi.org/10.1016/j.fsigen.2010.12.001, PMid:21196138

Hedman J, Dalin E, Rasmusson B, Ansell R. Evaluation of amylase testing as a tool for saliva screening of crime scene trace swabs. Forensic Sci Int Genet. 2011;5(3):194-8. https://doi.org/10.1016/j.fsigen.2010.03.003, PMid:20457099

Madi T, Balamurugan K, Bombardi R, Duncan G, McCord B. The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing. Electrophoresis. 2012;33(12):1736-45. https://doi.org/10.1002/elps.201100711, PMid:22740462

Greenfield A and Sloan MA. Identification of biological fluids and stains in: James SH, Nordby JJ, Bell S, editors. Forensic science: an introduction to scientific and investigative techniques. CRC press; 2002 Aug 29.

Vidaki A, Daniel B, Court DS. Forensic DNA methylation profiling—Potential opportunities and challenges. Forensic Sci Int Genet. 2013;7(5):499-507. https://doi.org/10.1016/j.fsigen.2013.05.004, PMid:23948320

Tammen SA, Friso S, Choi SW. Epigenetics: the link between nature and nurture. Mol Aspects Med. 2013;34(4):753-64. https://doi.org/10.1016/j.mam.2012.07.018, PMid:22906839 PMCid:PMC3515707

Maresca A, Zaffagnini M, Caporali L, Carelli V, Zanna C. DNA methyltransferase 1 mutations and mitochondrial pathology: is mtDNA methylated?. Front Genet. 2015;6:90. https://doi.org/10.3389/fgene.2015.00090, PMid:25815005 PMCid:PMC4357308

Espada J, Esteller M. DNA methylation and the functional organization of the nuclear compartment. InSeminars in cell & developmental biology 2010 Apr 30 (Vol. 21, No. 2, pp. 238-246). Academic Press.

Patterson K, Molloy L, Qu W, Clark S. DNA methylation: bisulphite modification and analysis. J Vis Exp. 2011;(56):3170. https://doi.org/10.3791/3170

Lokk K, Modhukur V, Rajashekar B, Märtens K, Mägi R, Kolde R, Koltšina M, Nilsson TK, Vilo J, Salumets A, Tõnisson N. DNA methylome profiling of human tissues identifies global and tissue-specific methylation patterns. Genome Biol. 2014;15(4):3248. https://doi.org/10.1186/gb-2014-15-4-r54, PMid:24690455 PMCid:PMC4053947

Fan S, Zhang X. CpG island methylation pattern in different human tissues and its correlation with gene expression. Biochem Biophys Res Commun. 2009;383(4):421-5. https://doi.org/10.1016/j.bbrc.2009.04.023, PMid:19364493

Naito E, Dewa K, Yamanouchi H, Takagi S, Kominami R. Sex determination using the hypomethylation of a human macro-satellite DXZ4 in female cells. Nucleic Acids Res. 1993;21(10):2533-4. https://doi.org/10.1093/nar/21.10.2533, PMid:7685086 PMCid:PMC309572

Ghosh S, Yates AJ, Frühwald MC, Miecznikowski JC, Plass C, Smiraglia D. Tissue specific DNA methylation of CpG islands in normal human adult somatic tissues distinguishes neural from non-neural tissues. Epigenetics. 2010;5(6):527-38. https://doi.org/10.4161/epi.5.6.12228, PMid:20505344 PMCid:PMC3322498

Manjegowda MC, Gupta PS, Limaye AM. Hyper-methylation of the upstream CpG island shore is a likely mechanism of GPER1 silencing in breast cancer cells. Gene. 2017;614:65-73. https://doi.org/10.1016/j.gene.2017.03.006, PMid:28286086

Kumar SR, Bryan JN, Esebua M, Amos-Landgraf J, May TJ. Testis specific Y-like 5: gene expression, methylation and implications for drug sensitivity in prostate carcinoma. BMC cancer. 2017;17(1):158. https://doi.org/10.1186/s12885-017-3134-7, PMid:28235398 PMCid:PMC5326500

Perri F, Longo F, Giuliano M, Sabbatino F, Favia G, Ionna F, Addeo R, Scarpati GD, Di Lorenzo G, Pisconti S. Epigenetic control of gene expression: Potential implications for cancer treatment. Crit Rev Oncol Hematol. 2017;111:166-72. https://doi.org/10.1016/j.critrevonc.2017.01.020, PMid:28259291

Lim DH, Maher ER. DNA methylation: a form of epigenetic control of gene expression. The Obstetrician & Gynaecologist. 2010;12(1):37-42. https://doi.org/10.1576/toag.

Nazarenko MS, Markov AV, Lebedev IN, Freidin MB, Sleptcov AA, Koroleva IA, Frolov AV, Popov VA, Barbarash OL, Puzyrev VP. A comparison of genome-wide DNA methylation patterns between different vascular tissues from patients with coronary heart disease. PLoS One. 2015;10(4):e0122601. https://doi.org/10.1371/journal.pone.0122601, PMid:25856389 PMCid:PMC4391864

Bird A. DNA methylation patterns and epigenetic memory. Genes Dev. 2002;16(1):6-21. https://doi.org/10.1101/gad.947102, PMid:11782440

Rando OJ, Verstrepen KJ. Timescales of genetic and epigenetic inheritance. Cell. 2007;128(4):655-68. https://doi.org/10.1016/j.cell.2007.01.023, PMid:17320504

Haas C, Klesser B, Maake C, Bär W, Kratzer A. mRNA profiling for body fluid identification by reverse transcription endpoint PCR and realtime PCR. Forensic Sci Int Genet. 2009;3(2):80-8. https://doi.org/10.1016/j.fsigen.2008.11.003, PMid:19215876

Sijen T. Molecular approaches for forensic cell type identification: on mRNA, miRNA, DNA methylation and microbial markers. Forensic Sci Int Genet. 2015;18:21-32. https://doi.org/10.1016/j.fsigen.2014.11.015, PMid:25488609

Boks MP, Derks EM, Weisenberger DJ, Strengman E, Janson E, Sommer IE, Kahn RS, Ophoff RA. The relationship of DNA methylation with age, gender and genotype in twins and healthy controls. PLoS One. 2009;4(8):e6767. https://doi.org/10.1371/journal.pone.0006767, PMid:19774229 PMCid:PMC2747671

Fraser HB, Lam LL, Neumann SM, Kobor MS. Population-specificity of human DNA methylation. Genome Biol. 2012;13(2):R8. https://doi.org/10.1186/gb-2012-13-2-r8, PMid:22322129 PMCid:PMC3334571

Zhang FF, Cardarelli R, Carroll J, Fulda KG, Kaur M, Gonzalez K, Vishwanatha JK, Santella RM, Morabia A. Significant differences in global genomic DNA methylation by gender and race/ethnicity in peripheral blood. Epigenet. 2011;6(5):623-9. https://doi.org/10.4161/epi.6.5.15335, PMCid:PMC3230547

Darst RP, Pardo CE, Ai L, Brown KD, Kladde MP. Bisulfite sequencing of DNA. Curr Protoc Mol Biol. 2010; 91:7.9:7.9.1–7.9.17.

Shiraishi M, Hayatsu H. High-speed conversion of cytosine to uracil in bisulfite genomic sequencing analysis of DNA methylation. DNA res. 2004;11(6):409-15. https://doi.org/10.1093/dnares/11.6.409, PMid:15871463

Lee HY, Lee SD, Shin KJ. Forensic DNA methylation profiling from evidence material for investigative leads. BMB reports. 2016;49(7):359. https://doi.org/10.5483/BMBRep.2016.49.7.070, PMid:27099236 PMCid:PMC5032003

Park JL, Kwon OH, Kim JH, Yoo HS, Lee HC, Woo KM, Kim SY, Lee SH, Kim YS. Identification of body fluid-specific DNA methylation markers for use in forensic science. Forensic Sci Int Genet. 2014;13:147-53. https://doi.org/10.1016/j.fsigen.2014.07.011, PMid:25128690

Lee HY, An JH, Jung SE, Oh YN, Lee EY, Choi A, Yang WI, Shin KJ. Genome-wide methylation profiling and a multiplex construction for the identification of body fluids using epigenetic markers. Forensic Sci Int Genet. 2015;17:17-24. https://doi.org/10.1016/j.fsigen.2015.03.002, PMid:25796047

Forat S, Huettel B, Reinhardt R, Fimmers R, Haidl G, Denschlag D, Olek K. Correction: Methylation Markers for the Identification of Body Fluids and Tissues from Forensic Trace Evidence. PLoS One. 2016;11(5):e0156472. https://doi.org/10.1371/journal.pone.0156472, PMid:27219355 PMCid:PMC4878790

Wasserstrom A, Frumkin D, Davidson A, Shpitzen M, Herman Y, Gafny R. Demonstration of DSI-semen—a novel DNA methylation-based forensic semen identification assay. Forensic Sci Int Genet. 2013;7(1):136-42. https://doi.org/10.1016/j.fsigen.2012.08.009, PMid:22944353

Li Y, Tollefsbol TO. DNA methylation detection: bisulfite genomic sequencing analysis. Epigenetics Protocols. 2011:11-21. [Part of the Methods in Molecular Biology book series (MIMB, volume 791].

Vidaki A, Ballard D, Aliferi A, Miller TH, Barron LP, Court DS. DNA methylation-based forensic age prediction using artificial neural networks and next generation sequencing. Forensic Sci Int Genet. 2017;28:225-36. https://doi.org/10.1016/j.fsigen.2017.02.009, PMid:28254385 PMCid:PMC5392537

Shen L, Waterland RA. Methods of DNA methylation analysis. Curr Opin Clin Nutr Metab Care. 2007 Sep 1;10(5):576-81. https://doi.org/10.1097/MCO.0b013e3282bf6f43, PMid:17693740

Bock C. Epigenetic biomarker development. Epigenom. 2009; 1(1): 99–110. https://doi.org/10.2217/epi.09.6, PMid:22122639

Lee HY, Park MJ, Choi A, An JH, Yang WI, Shin KJ. Potential forensic application of DNA methylation profiling to body fluid identification. Int J Legal Med. 2012;126(1):55-62. https://doi.org/10.1007/s00414-011-0569-2, PMid:21626087

LaRue BL, King JL, Budowle B. A validation study of the Nucleix DSI-Semen kit—a methylation-based assay for semen identification. Int J Legal Med. 2013;127(2):299-308. https://doi.org/10.1007/s00414-012-0760-0, PMid:22895803

Eckhardt F, Lewin J, Cortese R, Rakyan VK, Attwood J, Burger M, Burton J, Cox TV, Davies R, Down TA, Haefliger C. DNA methylation profiling of human chromosomes 6, 20 and 22. Nat genet. 2006;38(12):1378-85. https://doi.org/10.1038/ng1909, PMid:17072317 PMCid:PMC3082778

Wu HC, Wang Q, Chung WK, Andrulis IL, Daly MB, John EM, Keegan TH, Knight J, Bradbury AR, Kappil MA, Gurvich I. Correlation of DNA methylation levels in blood and saliva DNA in young girls of the LEGACY Girls study. Epigenet. 2014;9(7):929-33. https://doi.org/10.4161/epi.28902, PMid:24756002 PMCid:PMC4143407

Choi A, Shin KJ, Yang WI, Lee HY. Body fluid identification by integrated analysis of DNA methylation and body fluid-specific microbial DNA. Int J Legal Med. 2014;128(1):33-41. https://doi.org/10.1007/s00414-013-0918-4, PMid:24052059

DOI: http://dx.doi.org/10.26735/16586794.2017.001


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