Epigenetic age signatures in the forensically relevant body fluid of semen: a preliminary study.

To date, DNA methylation has been regarded as the most promising age-predictive biomarker. In support of this, several researchers have reported age predictive models based on the use of blood or even across a broad spectrum of tissues. However, there have been no publications that report epigenetic age signatures from semen, one of the most forensically relevant body fluids. In genome-wide DNA methylation profiles of 36 body fluids including blood, saliva, and semen, the previous age predictive models showed considerable prediction accuracy in blood and saliva but not in semen. Therefore, we selected CpG sites, whose methylation levels are strongly correlated with age in 12 semen profiles obtained from individuals of different ages, and investigated DNA methylation changes at these CpGs in 68 additional semen samples obtained from individuals aged 20 to 73 years using methylation SNaPshot reaction. Among the selected age-related CpG candidates, outstanding age correlation was obtained at cg06304190 in the TTC7B gene. Interestingly, the region around the TTC7B gene has been reported to show age-related DNA methylation alteration in the sperm methylome of 2 samples collected from individuals at certain time intervals. The age-predictive linear regression model trained with 3 CpGs (cg06304190 in the TTC7B gene, cg06979108 in the NOX4 gene and cg12837463) showed a high correlation between the predicted age and the chronological age, with an average absolute difference of approximately 5 years. These selected epigenetic age signatures are expected to be useful for considerably accurate age estimation in the forensically relevant body fluid of semen. However, because the findings were limited by small sample size, it will be necessary to further evaluate the age correlation of the selected CpGs and to encourage further investigation.

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

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.

Forensic interlaboratory evaluation of the ForFLUID kit for vaginal fluids identification.

Identification of vaginal fluids is an important step in the process of sexual assaults confirmation. Advances in both microbiology and molecular biology defined technical approaches allowing the discrimination of body fluids. These protocols are based on the identification of specific bacterial communities by microfloraDNA (mfDNA) amplification. A multiplex real time-PCR assay (ForFLUID kit) has been developed for identifying biological fluids and for discrimination among vaginal, oral and fecal samples. In order to test its efficacy and reliability of the assay in the identification of vaginal fluids, an interlaboratory evaluation has been performed on homogeneous vaginal swabs. All the involved laboratories were able to correctly recognize all the vaginal swabs, and no false positives were identified when the assay was applied on non-vaginal samples. The assay represents an useful molecular tool that can be easily adopted by forensic geneticists involved in vaginal fluid identification.

Body fluid identification by integrated analysis of DNA methylation and body fluid-specific microbial DNA.

Identification of body fluids found at crime scenes provides important information that can support a link between sample donors and actual criminal acts. Previous studies have reported that DNA methylation analysis at several tissue-specific differentially methylated regions (tDMRs) enables successful identification of semen, and the detection of certain bacterial DNA can allow for identification of saliva and vaginal fluid. In the present study, a method for detecting bacterial DNA was integrated into a previously reported multiplex methylation-sensitive restriction enzyme-polymerase chain reaction. The developed multiplex PCR was modified by the addition of a new semen-specific marker and by including amplicons for the 16S ribosomal RNA gene of saliva- and vaginal fluid-specific bacteria to improve the efficacy to detect a specific type of body fluid. Using the developed multiplex system, semen was distinguishable by unmethylation at the USP49, DACT1, and PFN3 tDMRs and by hypermethylation at L81528, and saliva could be identified by detection of saliva-specific bacteria, Veillonella atypica and/or Streptococcus salivarius. Additionally, vaginal fluid and menstrual blood were differentiated from other body fluids by hypomethylation at the PFN3 tDMR and the presence of vaginal fluid-specific bacteria, Lactobacillus crispatus and/or Lactobacillus gasseri. Because the developed multiplex system uses the same biological source of DNA for individual identification profiling and simultaneously analyses various types of body fluid in one PCR reaction, this method will facilitate more efficient body fluid identification in forensic casework.

DNA methylation-specific multiplex assays for body fluid identification.

Recent advances in whole-genome epigenetic analysis indicate that chromosome segments called tissue-specific differentially methylated regions (tDMRs) show different DNA methylation profiles according to cell or tissue type. Therefore, body fluid-specific differential DNA methylation is a promising indicator for body fluid identification. However, DNA methylation patterns are susceptible to change in response to environmental factors and aging. Therefore, we investigated age-related methylation changes in semen-specific tDMRs using body fluids from young and elderly men. After confirming the stability of the body fluid-specific DNA methylation profile over time, two different multiplex PCR systems were constructed using methylation-sensitive restriction enzyme PCR and methylation SNaPshot, in order to analyze the methylation status of specific CpG sites from the USP49, DACT1, PRMT2, and PFN3 tDMRs. Both multiplex systems could successfully identify semen with spermatozoa and could differentiate menstrual blood and vaginal fluids from blood and saliva. Although including more markers for body fluid identification might be necessary, this study adds to the support that body fluid identification by DNA methylation profiles could be a valuable tool for forensic analysis of body fluids.

Potential forensic application of DNA methylation profiling to body fluid identification.

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.