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Applications of epigenetic methylation in body fluid identification, age determination and phenotyping

Published:October 14, 2019DOI:https://doi.org/10.1016/j.fsigss.2019.10.061

      Abstract

      In this paper, four different applications are presented in forensic epigenetics using bisulfite modified PCR, HRM, and pyrosequencing. The first application involves the development of a multiplex amplification capable of simultaneous analysis of 4 different body fluid/cell types- blood, saliva, sperm, and vaginal epithelia. We have also developed a method for the detection of sperm high-resolution melt analysis (HRM). Two other applications involve a determination of suspect age and a biomarker for determining smoking status.

      Keywords

      1. Introduction

      We have been investigating a variety of applications in forensic epigenetics. These include tissue typing, age determination and behavior (smoking). The value of epigenetic markers is in their connection to gene regulation and the effects of environment [
      • Kayser M.
      • de Knijff P.
      Improving human forensics through advances in genetics, genomics and molecular biology.
      ]. The resultant loci provide a path for forensic analysis. The results demonstrate that epigenetics can provide important information in tissue detection and in phenotyping. Furthermore, this information can be combined with relevant genetic markers to maximize the potential to identify unknown suspects.

      2. Materials and methods

      Epigenetics refers to heritable changes caused by chemical or physical modification of gene expression rather than those caused by alteration of the genetic code itself [
      • Brait M.
      • Sidransky D.
      Cancer epigenetics: above and beyond.
      ]. Epigenetic modifications can occur in various forms, including DNA methylation, chromatic remodeling, post-translational modification of histone proteins, and non-coding RNAs [
      • Lieb J.D.
      • Beck S.
      • Bulyk M.L.
      • et al.
      Applying whole-genome studies of epigenetic regulation to study human disease.
      ]. DNA methylation is the most widely studied epigenetic modification to date, and 5-methylcytosine (5 mC) is the most common form of DNA methylation found in Eukaryotes [
      • Rakyan V.K.
      • Down T.A.
      • Balding D.J.
      • et al.
      Epigenome-wide association studies for common human diseases.
      ,
      • Vidaki A.
      • Daniel B.
      • Court D.S.
      Forensic DNA methylation profiling-potential opportunities and challenges.
      ]. The increased presence of methyl groups in regions of DNA with a significant portion of CpG dinucleotides (CpG islands) is thought to be responsible for chromatin structure changes that can affect the transcription of a gene [
      • Rottach A.
      • Leonhardt H.
      • Spada F.
      DNA methylation-mediated epigenetic control.
      ]. Differentially Methylated Regions (DMRs), a specific form of DNA methylation, are increasingly important in forensics [
      • Frigola J.
      • Song J.
      • Stirzaker C.
      • et al.
      Epigenetic remodeling in colorectal cancer results in coordinate gene suppression across an entire chromosome band.
      ]. The methylation levels in these DMRs have been observed to change due to a multitude of factors including early life experiences, nutrition, aging, pollution, and cell function [
      • Kader F.
      • Ghai M.
      DNA methylation and application in forensic sciences.
      ].
      The most widely used method for discovery of new CpGs is the Infinium MethylationEPIC BeadChip (Illumina, CA), which allows the interrogation of 850,000 CpGs in the human genome, roughly 3% of the total number of CpGs. Methods for whole genome bisulfite sequencing are prohibitively expensive for forensic research laboratories as they require large coverage of each CpG for accuracy. Currently, methylation analysis requires bisulfite conversion of genomic DNA to chemically modify unmethylated cytosines prior to PCR amplification. The methylated cytosines are resistant to chemical modification and remain unchanged. As a result, there is a loss of complexity in the DNA strand, making bisulfite-modified DNA a special template for primer design.

      3. Results and discussion

      3.1 Body fluid type

      Several studies have been conducted in our laboratory for the determination of body fluid type using bisulfite conversion followed by amplification and pyrosequencing. Madi et al. found four genes – C20orf117, ZC3H12D, BCAS4, and FGF7 – capable of discerning whether DNA came from either blood, saliva, semen or epithelial tissue [
      • Madi T.
      • Balamurugan K.
      • Bombardi R.
      • et al.
      The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing.
      ]. Later, Antunes et al. utilized ZC3H12D for differentiating semen from blood and saliva using high-resolution melt analysis [
      • Antunes J.
      • Silva D.S.B.S.
      • Balamurugan K.
      • et al.
      High-resolution melt analysis of DNA methylation to discriminate semen in biological stains.
      ]. A separate study by Silva et al. examined markers such as CG06379435 for identifying blood, and a developmental validation of these markers was conducted [
      • Silva D.S.B.S.
      • Antunes J.
      • Balamurugan K.
      • et al.
      Developmental validation studies of epigenetic DNA methylation markers for the detection of blood, semen and saliva samples.
      ]. Most recently, four markers – BCAS4 for saliva, cg06379435 for blood, VE_8 for vaginal epithelia, and ZC3H12D for semen – were combined into a single multiplex assay and analyzed with cluster analysis [
      • Gauthier Q.T.
      • Cho S.
      • Carmel J.H.
      • et al.
      Development of a body fluid identification multiplex via DNA methylation analysis.
      ]. Validation studies performed on ZC3H12D, BCAS4 and cg06379435 showed consistency for detecting low concentrations of DNA varying from 0.1 to 10 ng. Markers were tested on different species with results for certain non-human primates, however all other species were negative. The loci were also effective when inhibitors were present or degraded by heat. Blood and semen samples as old as 20 years were typable. Overall, these validation studies demonstrated the robustness of DNA methylation markers for forensic investigation [
      • Silva D.S.B.S.
      • Antunes J.
      • Balamurugan K.
      • et al.
      Developmental validation studies of epigenetic DNA methylation markers for the detection of blood, semen and saliva samples.
      ,
      • Gauthier Q.T.
      • Cho S.
      • Carmel J.H.
      • et al.
      Development of a body fluid identification multiplex via DNA methylation analysis.
      ].

      3.2 qPCR data

      Following bisulfite modified PCR, GC content differs between methylated and unmethylated loci. For example, when the ZC3H12D locus is amplified from bisulfite modified DNA from semen, the GC content is lower when compared with DNA from saliva or blood [
      • Antunes J.
      • Silva D.S.B.S.
      • Balamurugan K.
      • et al.
      High-resolution melt analysis of DNA methylation to discriminate semen in biological stains.
      ]. High-resolution melt analysis can be used to distinguish body fluids that present large differences in methylation such as ZC3H12D [
      • Silva D.S.B.S.
      • Antunes J.
      • Balamurugan K.
      • et al.
      Developmental validation studies of epigenetic DNA methylation markers for the detection of blood, semen and saliva samples.
      ]. Briefly, DNA is amplified in the presence of an intercalating dyes and when PCR is complete, the temperature is increased gradually while fluorescence is measured. Amplicons that result from methylated DNA will contain more cytosines than those resulting from unmethylated DNA, thus melting at higher temperatures. The plot of the first derivative of fluorescence change over temperature produces peaks at temperatures that reflect the methylation of the amplified DNA. Because HRM analysis only requires a real-time PCR instrument with melt curve capacity, it presents some advantages in comparison to other methods. We have found this procedure to be useful in detecting of the presence of semen (sperm) in forensic DNA extracts [
      • Silva D.S.B.S.
      • Antunes J.
      • Balamurugan K.
      • et al.
      Developmental validation studies of epigenetic DNA methylation markers for the detection of blood, semen and saliva samples.
      ].

      3.3 Phenotyping and age determination

      Forensic DNA phenotyping is emerging as a new tool that utilizes different biomarkers to infer externally visible characteristics from DNA evidence [
      • Kayser M.
      • de Knijff P.
      Improving human forensics through advances in genetics, genomics and molecular biology.
      ]. For example, finding a suitable DNA methylation marker to determine the smoking status could predict the individual’s lifestyle. In addition, as individuals age, physical appearance can narrow down the potential identity of a suspect associated with the DNA evidence [
      • Vidaki A.
      • Daniel B.
      • Court D.S.
      Forensic DNA methylation profiling-potential opportunities and challenges.
      ].
      Tobacco smoking is considered one of the most powerful environmental factors that cause DNA methylation changes [
      • Zeilinger S.
      • Kühnel B.
      • Klopp N.
      • et al.
      Tobacco smoking leads to extensive genome-wide changes in DNA methylation.
      ]. Several epigenome-wide association studies (EWASs) in the effect of smoking reported numbers of candidate loci that show differentially methylated patterns among different smoking habits in different genes including AHRR, ALPP2, GFI1, and MYO1F [
      • Zeilinger S.
      • Kühnel B.
      • Klopp N.
      • et al.
      Tobacco smoking leads to extensive genome-wide changes in DNA methylation.
      ,
      • Monick M.M.
      • Beach S.R.
      • Plume J.
      • et al.
      Coordinated changes in AHRR methylation in lymphoblasts and pulmonary macrophages from smokers.
      ,
      • Shenker N.S.
      • Polidoro S.
      • van Veldhoven K.
      • et al.
      Epigenome-wide association study in the European prospective investigation into Cancer and Nutrition (EPIC-Turin) identifies novel genetic loci associated with smoking.
      ,
      • Elliott H.R.
      • Tillin T.
      • McArdle W.L.
      • et al.
      Differences in smoking associated DNA methylation patterns in South Asians and Europeans.
      ]. In our lab, a broad range of CpG sites located around six genomic loci were investigated [
      • Alghanim H.
      • Wu W.
      • McCord B.
      DNA methylation assay based on pyrosequencing for determination of smoking status.
      ]. An assay was developed that composed of four CpGs at AHRR and could determine the smoking status utilizing a singleplex bisulfite modified PCR followed by pyrosequencing. Based on multinomial linear regression analysis, the 4-CpG assay provided high accuracy correctly predicting 90.0% of current smokers, 66.7% of former smokers, and 84.9% of never smokers in blood. In saliva, the method correctly predicted 86.9% of current smokers, 54.5% of former smokers, and 77.8% of never smokers [
      • Alghanim H.
      • Wu W.
      • McCord B.
      DNA methylation assay based on pyrosequencing for determination of smoking status.
      ].
      Many efforts recently have been targeting DNA methylation-based methods to estimate chronological age of individuals connected with a crime. One approach is to identify a large set of CpG sites that show linear correlation with aging based on EWASs [
      • Hannum G.
      • Guinney J.
      • Zhao L.
      • et al.
      Genome-wide methylation profiles reveal quantitative views of human aging rates.
      ,
      • Horvath S.
      DNA methylation age of human tissues and cell types.
      ]. Other studies have examined the use of more specific subsets of age-related CpG sites ranging from 1 to 5 CpGs [
      • Bocklandt S.
      • Lin W.
      • Sehl M.E.
      • et al.
      Epigenetic predictor of age.
      ,
      • Zbieć-Piekarska R.
      • Spólnicka M.
      • Kupiec T.
      • et al.
      Development of a forensically useful age prediction method based on DNA methylation analysis.
      ,
      • Alghanim H.
      • Antunes J.
      • Silva D.S.B.S.
      • et al.
      Detection and evaluation of DNA methylation markers found at SCGN and KLF14 loci to estimate human age.
      ]. In our lab, similar approach was taken by identifying small set of CpG sites located at SCGN and KLF14 [
      • Alghanim H.
      • Antunes J.
      • Silva D.S.B.S.
      • et al.
      Detection and evaluation of DNA methylation markers found at SCGN and KLF14 loci to estimate human age.
      ]. Age-predictor models for saliva and blood were developed from CpGs in SCGN and KLF14 using a multivariate linear regression analysis. In particular, the single-locus model is very cost effective to estimate age especially for younger subjects (≤40 years) being able to predict age (±8 years) with an accuracy of 78.9% and a mean absolute deviation of 5.1 years in the validation set [
      • Alghanim H.
      • Antunes J.
      • Silva D.S.B.S.
      • et al.
      Detection and evaluation of DNA methylation markers found at SCGN and KLF14 loci to estimate human age.
      ].

      4. Conclusions

      Epigenetic markers have proven to be an exceptionally useful technique for forensic applications. They provide results with high specificity and sensitivity for body fluid identification, age and smoking status. Promising results from these markers would fit nicely within the workflow of current forensic DNA laboratories because they are quantitative and can be used for processing trace evidence samples on serological examination. The need for the development of more genetic markers, specifically epigenetic methylation markers for different phenotypic traits, is critical for investigators when an unknown suspect’s DNA is found on a crime scene.

      Declaration of Competing Interest

      The authors gratefully acknowledge material support from QIAGEN. The authors declare no other conflict of interest.

      Acknowledgements

      Portions of this study were supported from award # 2017-NE-BX-0001 , from the National Institute of Justice , USA. Points of view in the document are those of the authors and do not necessarily represent the official view of the U.S. Department of Justice. We also acknowledge the Dubai Police for funding and thank Wensong Wu, Sohee Cho, Justin Carmel, Clarice Alho, and Deborah Silva for their scientific contributions to this work.

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