Genome-wide methylation profiling and a multiplex construction for the identification of body fluids using epigenetic markers

https://doi.org/10.1016/j.fsigen.2015.03.002Get rights and content

Highlights

  • Forty two body fluid samples were analyzed by the Illumina BeadChip array.

  • A total of 64 CpGs were selected as body fluid-specific marker candidates.

  • A multiplex methylation SNaPshot reaction was constructed using 8 CpG sites.

  • The multiplex assay differentiates between blood, saliva, semen and vaginal fluid.

  • The multiplex produced successful methylation profiles in aged or mixed samples.

Abstract

The identification of body fluids found at crime scenes can contribute to solving crimes by providing important insights into crime scene reconstruction. In the present study, body fluid-specific epigenetic marker candidates were identified from genome-wide DNA methylation profiling of 42 body fluid samples including blood, saliva, semen, vaginal fluid and menstrual blood using the Illumina Infinium HumanMethylation450 BeadChip array. A total of 64 CpG sites were selected as body fluid-specific marker candidates by having more than 20% discrepancy in DNA methylation status between a certain type of body fluid and other types of body fluids and to have methylation or unmethylation pattern only in a particular type of body fluid. From further locus-specific methylation analysis in additional samples, 1 to 3 CpG sites were selected for each body fluid. Then, a multiplex methylation SNaPshot reaction was constructed to analyze methylation status of 8 body fluid-specific CpG sites. The developed multiplex reaction positively identifies blood, saliva, semen and the body fluid which originates from female reproductive organ in one reaction, and produces successful DNA methylation profiles in aged or mixed samples. Although it remains to be investigated whether this approach is more sensitive, more practical than RNA- or peptide-based assays and whether it can be successfully applied to forensic casework, the results of the present study will be useful for the forensic investigators dealing with body fluid samples.

Introduction

Besides providing information on donor identity, body fluids found at the crime scenes play an important role in forensic investigations [1]. Determination of the type and origin of body fluids can provide important clues for crime scene reconstruction by linking donors and actual criminal acts. However, most currently used catalytic, enzymatic, and immunologic tests for forensic body fluid identification suffer from several limitations, such as low specificity, lack of sensitivity, sample destruction, instability of biomolecules being assayed or incompatibility with downstream individual identification [2].

In recent years, tissue-specific RNA expression and DNA methylation have been suggested as a promising new tool to overcome these limitations and to distinguish between different types of body fluids [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]. Especially, recent approaches based on tissue-specific mRNA and miRNA expression have proven to be useful because of their high specificity, ability to be analyzed in multiplex, and unexpected long-term stability [3], [4], [5], [6], [7], [8], [9], [10], [11]. However, particular care and effort to inactivate ubiquitously present ribonucleases still prevents forensic experts from routinely integrating RNA analysis into a forensic casework scheme. On the other hand, the DNA methylation-based method uses the same biological source of DNA for personal identification profiling; thus, special training is not required for an assay. Moreover, body fluid-specific differential DNA methylation showed high specificity and multiplex analysis was also possible [13], [14], [15], [16], [17], [18]. Nevertheless, there is a need to identify more body fluid-specific DNA methylation markers. Many semen-specific markers have been identified, but markers specific to blood, saliva, vaginal fluid, menstrual blood, or skin have been reported only in a few publications and require more validation [12], [14], [18].

Meanwhile, rapid advances in epigenetics make genome-wide DNA methylation profiling accessible to many researchers. For example, Illumina’s HumanMethylation450 BeadChip array provides DNA methylation profiles at more than 450,000 CpG sites using only a small amount of DNA. Therefore, DNA methylation profiling for various types of body fluids using this technique would enable the identification of significant DNA methylation markers that are useful for forensic body fluid identification.

Therefore, we produced genome-wide DNA methylation profiles of blood, saliva, semen, vaginal fluid and menstrual blood using Illumina’s HumanMethylation450 BeadChip array and selected several body fluid-specific DNA methylation marker candidates. Then, we developed a multiplex methylation SNaPshot to analyze DNA methylation profiles at the selected body fluid-specific CpG sites. Body fluid specificity and forensic applicability of the multiplex system were also tested with various samples.

Section snippets

Samples

Blood, saliva, and semen from 84 males and blood, saliva, vaginal fluid, and menstrual blood from 16 female volunteers were collected using procedures approved by the Institutional Review Board of Severance Hospital, Yonsei University in Seoul, Korea. The 100 donors provided written informed consent after the goals, and procedures of the study were explained. Blood was collected in a syringe, and 200 μL aliquots were stored frozen. Saliva and freshly ejaculated semen were collected in a

HumanMethylation450 BeadChip array analysis

We analyzed HumanMethylation450 BeadChip array to obtain genome-wide DNA methylation profiles of 42 body fluid samples including 12 samples each from blood, saliva and semen, and 3 samples each from vaginal fluid and menstrual blood. To exclude the influence of age, analyses for blood, saliva and semen were performed with 36 samples from 18 male donors aged 20–59. Blood donors’ age was 24, 27, 28, 33, 37, 38, 41, 43, 48, 50, 57 and 59 years, saliva donors’ age was 20, 24, 27, 29, 32, 37, 38,

Conclusion

We analyzed the DNA methylation profile of 42 body fluid samples including venous blood, saliva, semen, vaginal fluid and menstrual blood using the Illumina’s Infinium Human Methylation450 BeadChip array and selected 64 body fluid-specific CpG marker candidates. Through further validation using targeted bisulfite sequencing, we confirmed that a subset of CpG markers showed body fluid-specific differential DNA methylation and could be useful biomarkers in forensic body fluid identification. The

Conflict of interest statement

The authors declare that they have no conflict of interest.

Acknowledgements

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 (NRF-2012R1A1A2007031) and a grant from the Ministry of Public Administration and Security through the National Forensic Service (Grant nos.1315000435 and 1315000544).

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