Forensic epigenetics for human body fluid identification. Sohee Cho, Ph.D. and Bruce R. McCord, Ph.D. Florida International University, Miami, FL

Transcription

1 Forensic epigenetics for human body fluid identification Sohee Cho, Ph.D. and Bruce R. McCord, Ph.D. Florida International University, Miami, FL

2 DNA typing of biological materials found at the crime scene Forensic DNA profiling for individual identification Determination of types of biological materials Link suspects and victims to each other and/or to the scenes Include or exclude potential suspects or victims Establish crime scenes Identify weapons Corroborate case circumstances Narrow down the samples for further analysis Sex offender s trial Body fluids founds on the bed

3 What information from the cell types of the crime scene trace? Can support a link between sample donors and physical activities involved in cases Type Forensic relevance Body fluids Blood Violence, human-specific assay Semen Saliva Vaginal secretion Menstrual secretion Organs Brain Head injury Heart, lung Kidney, liver Skeletal muscle Sexual assault, confirmation sampled area Sexual assault e.g. licking, kissing or inoffensive stain Sexual assault, confirmation sampled area Sexual assault or inoffensive alternative scenario Chest injury Abdominal injury Injury Touch Skin Confirmation sampled area Other secretions Sweat Urine Vomit Faeces Confirmation witness report Confirmation sampled area Contains saliva and stomach content, inoffensive scenario Anal sexual assault Sijen. Forensic Sci Int Genet 2015;18:21-

4 Presumptive and confirmatory tests for body fluid identification Chemical, catalytic tests, enzymatic and immunological tests Blood Semen Saliva Vaginal fluid Urine Luminol, Phenolphthalein, SAP, LAP, Starch-iodine, Phadebas, Starch gel/lvalyl-l-leucine, DMAC, Salkowski test, ABAcard, Kastle- Christmas tree Amylose Azure Oestrogen THP, Meyer test stain, receptors Urea NB-test HemeSelect, PSA Virkleret al. Forensic Sci Int 2009;188(1-3):1-17

5 Molecular approach for cell type identification How are different tissues and cells specialized? Messenger RNAs Micro RNAs Epigenetic modification DNA Messenger RNA (mrna) Epigenetic modification Functional protein MicroRNA (mirna)

6 The limitations Marker type Chemical, catalytic, enzymatic and immunological tests Limitations Low specificity Lack of sensitivity Instability of biomolecules assayed Incompatibility with downstream STR analysis Carried out for only one body fluid at a time No statistical confidence associated with any outcome, positive or negative mrna/mirna Additional procedures: extraction of RNA, DNase treatment and cdna preparation Cannot be applied when only DNA extract remains in an old case Variable expression: different per marker, influenced by physiological factors Background signals from spurious transcription No human-specificrna quantification methods Potential of degradation by RNase Sijen. Forensic Sci Int Genet 2015;18:21-

7 Epigenetics one genome Cells and tissues are differentiated by epigenetic mechanisms Histone modification DNA methylation Acetylation, etc. Epigenetic control >200 different types of cells (>200 epigenomes) Image from UNSW Embryology, and Lindroth et al. J Periodontal Implant Sci 2013

8 DNA methylation Methyl residues are covalently bound to the 5 position of cytosine followed by a guanine via DNA methyltansferase (DNMT) forming 5- methylcytosines Observed at CpG dinucleotides (70% of CpGs are methylated in mammals) CpG islands - areas of high CpG density usually mapping to promoter regions Methylation gene silencing

9 What are the advantages of epigenetic DNA methylation typing? The stability of DNA methylation involving covalent boding is high, which permits long term storage - samples over 20 years old can be analyzed Extraction of the sample and recovery of the DNA is the same with standard DNA typing and forensic epigenetics - easily implemented in a forensic laboratory Extracted DNA can be used for multiple purposes including STR amplification

10 Tissue-specific DNA methylation regions (tdmrs) DNA methylation patterns were more consistent between the same tissues from different people than between different tissues from the same individual Intro-individual and inter-individual variations in DNA methylation Byun et al. Hum Mol Genet 2009;18(24):

11 How to detect or measure DNA methylation levels? Standard STR and SNP profiling vs. DNA methylation pattern profiling % of methylation?

12 Discrimination of C / 5-mC using sodium bisulfite conversion Sodium bisulfite conversion Highest degree of resolution of the methylation status of a given sample

13 Detection methods of bisulfite-treated DNA Bisulfite sequencing A qualitative and quantitative approach to identify 5-methylcytosine at single base pair resolution EpiTYPER using mass spectrometry Bisulfite Amp using T7-promoter tagged-primer base specific cleavage MS Methylation-specific PCR/qPCR Using two primer pairs which are detectable methylated and unmethylated DNA, respectively Correa et al. Inmunologia 2012;31:97-105, Van den Boom et al. Methods Mol Biol 2009;507:207-27, Zhang et al. Lab Chip 2009;9(8):

14 Application of high resolution melt (HRM) PCR method Methylation differences are examined with real-time PCR/high resolution melt curve analysis (HRM) After real-time PCR amplification, a melt curve is performed in presence of a DNA binding fluorescence dye Melting temperature (Tm) depends on nucleotide content and length Distinguish products based on their Tms Figures from Francisco Bizouarn, BIO-RAD

15 High resolution melt (HRM) PCR for DNA methylation analysis m 5 --C--C--C--C G--G--G--G--5 Genomic DNA m m m m 5 --C--C--C--C G--G--G--G--5 Bisulfite treatment m m m m m 5 --U--U--C--U C--C--C--C--3 Bisulfite treated DNA with all CpG-sites methylated will have higher Tm than if non-methylated m 5 --U--U--C--U A--A--G--A--5 PCR (1 st cycle) m m m m 5 --C--C--C--C G--G--G--G--5 PCR (amplification) 5 --T--T--C--T A--A--G--A--5 Unmethylated Predominantly T=A bound 5 --C--C--C--C G--G--G--G--5 Methylated Predominantly G=C bound

16 HRM analysis of DNA methylation to discriminate semen in biological stains semen Joana Antunes blood saliva semen blood saliva Hypomethylation of semen is expected to result in a melt curve with a lower melting temperature No humicacid (HA) (thin lines) HA added before bisulfite conversion (dashed lines) HA added after bisulfite conversion (thick lines) Antunes. et al. Anal Biochem 2016;494:40-5

17 Pyrosequencing Based on sequencing-by-synthesis principle Stepwise synthesis of DNA by addition of nucleotides Enzyme cascade generates a light signal upon incorporation of nucleotides Figures from Qiagen website

18 Body fluid-specific DNA methylation analysis using pyrosequencing method Exploring markers & assay design Identified CpG loci based on large scale epigenetic arrays PyroMark Assay Design SW (QIAGEN) DNA extraction & quantification EZ1 Advanced & EZ1DNA investigator Kit (QIAGEN) Bisulfite conversion EpiTect Fast Bisulfite Conversion Kit (QIAGEN) PCR PyroMark PCR kit (QIAGEN) Pyrosequencing PyroMark Q24 Advanced /Q48 AutoPrep system (QIAGEN) Data analysis

19 Body fluid ID assay design for pyrosequencing Exploring markers & assay design DNA extraction & quantification Bisulfite conversion Genome-wide array Target CpG loci-specific analysis PCR Biotinylated Pyrosequencing Data analysis One of the PCR primers is biotinylated Sequencing primer anneals to single-strand DNA before pyrosequencing reaction

20 DNA extraction & quantification Exploring markers & assay design DNA extraction & quantification Bisulfite conversion DNA extraction EZ1 Advanced & EZ1 DNA investigator Kit (QIAGEN) Phenol chloroform extraction method DNA quantification Alu-sequence quantification based on real-time PCR PCR Pyrosequencing Data analysis

21 Bisulfite treatment & PCR amplification Exploring markers & assay design DNA extraction & quantification Bisulfite conversion PCR Pyrosequencing Data analysis Zilbermanet al. Development 2007;134(22):

22 Pyrosequencing platforms - PyroMark Q24 Exploring markers & assay design PyroMark Q24/Q24 Advanced DNA extraction & quantification Bisulfite conversion PCR primer Sequencing primer Region of interest < Single strand preparation > PCR primer Streptavidin coated magnetic beads for DNA capture PCR Template preparation Pyrosequencing Data analysis Pyrosequencing

23 Pyrosequencing platforms - PyroMark Q48 Exploring markers & assay design PyroMark Q48 Autoprep DNA extraction & quantification Template preparation, anneal sequencing primer, pyrosequencing (+ washing cartridges) Bisulfite conversion PCR Pyrosequencing Pyrosequencer that handles all of the mixing and preparation for each sample - just load the reagent cartridges, samples, and go! Data analysis

24 Analysis of DNA methylation data Exploring markers & assay design Sequence to analyze Bisulfite control (Negative control) DNA extraction & quantification Bisulfite conversion PCR Pyrosequencing Negative controls Blood (hypermethylation) Dispensation order (generated by software) [C signal intensity] Met % = [T signal + C signal] X 100 Semen (hypomethylation) 95% 97% 100% 100% 84% 2% 4% 6% 4% 3% Data analysis Madi et al. Electrophoresis 2012;33:

25 The markers identified based on pyrosequencing BCAS4 and ZC3H12D Identified in 2012 cg Identified in 2015 PFN3A Identified in 2016

26 The markers identified based on pyrosequencing (cont.) C20orf117 - hypermethylated in blood Eckhardt et al. - hypermethylated in lymphocytes and hypomethylated in sperm and skin cells BCAS4 - hypermethylatedin saliva Eckhardt et al. - hypermethylated in sperm with an 80% methylation level, but we found this loci to be hypermethylated in saliva Madi et al. Electrophoresis 2012;33:

27 The markers identified based on pyrosequencing (cont.) ZC3H12D (MCPIP4) - hypomethylated in sperm The total zinc content in semen from mammals is high, and zinc has been found to be critical to spermatogenesis. (Sørensen et al. Mol Hum Reprod. 1999;5(4):331-7) FGF7 - hypermethylated in sperm fibroblast growth factor 7, keratinocyte growth factor Madi et al. Electrophoresis 2012;33:

28 PFN3 A - intermediate level of methylation in vaginal epithelia Lee et al. (2010) - tdmr named PFN3 presented an overall different DNA methylation level for vaginal fluid Nine out of ten CpGs are useful to discriminate vaginal epithelia Tested for specificity, sensitivity, and mixtures Antuneset al. Electrophoresis 2016;37(21):

29 cg loci - a useful loci for blood marker Bioinformatic work from Dr. Balamurgen (University of Southern Mississippi) identified cg as a useful blood marker. Pyrosequencing identified 5 CPG sites Blood (n=10) Skin (n=7) Semen (n=9) Saliva (n=10) Vaginal Epithelia (n=10) 0 CpG 1 CpG 2 CpG 3 CpG 4 CpG 5 Blood markers were explored using epigenetic microarray data (Park et al. Forensic Sci Int Genet 2014;13:147-53)

30 Development of multiplex PCR for body fluid identification using pyrosequencing Singleplexreaction Multiplex reaction saliva marker blood marker semen marker vaginal epithelia marker 4 body fluid markers

31 The first attempt at a multiplex cg (blood marker) Incorrect peak height ratios 3 : 1 : 1 : 0 1 : 1 : 3 : 0 : 1 BCAS4 (saliva marker) Non-specific peaks where there should be nothing ZC3H12D (semen marker) Signals too low for data interpretation and baseline drift PFN3A (vaginal epi. marker) This slide was kindly provided by Quentin Gauthier from our lab

32 What is causing the issues? Pyrosequencing with BCAS4 (saliva marker) sequencing primer 400 BCAS4 PCR Product cg PCR Product PFN3A PCR Product ZC3H12D PCR Product Lack of Stringency Sequencing primer binding to incorrect PCR product causes peak height imbalances that make the multiplex results unusable This slide was kindly provided by Quentin Gauthier from our lab

33 Balancing forward and reverse PCR primers in duplex and triplex Examples of duplex PCR primer sets Sequencing primer A ZC3H12D + BCAS4 ZC3H12D B C ZC3H12D + cg ZC3H12D + PFN3A A B C Examples of triplex D D E F PCR primer set ZC3H12D + BCAS4 + cg Sequencing primer BCAS4 cg ZC3H12D E F

34 Balancing forward and reverse PCR primers in quadplex cg (blood marker) ZC3H12D (semen marker) BCAS4 (saliva marker) PFN3A (vaginal epi. marker)

35 Major issues PFN3A, vaginal epithelia marker Replace to a new marker, VE_8 Developed to distinguish saliva and vaginal epithelia Expected to be hypomethylated in vaginal epithelia Interference in BCAS4 pyrosequencing Add formamide to the sequencing primers Use the unmethylated PCR primer as a sequencing primer Redesign sequencing primers Modify the sequence to analyze

36 Reconfigured multiplex analysis (in progress) cg (blood marker) ZC3H12D (semen marker) VE_8 (vaginal epi. marker) BCAS4 (saliva marker) Testing is still ongoing for optimization in the multiplex

37 DNA methylation levels in four body fluids on multiplex PCR BCAS4 (saliva marker) CpG1 CpG2 CpG3 CpG4 Singleplex-literature Saliva Blood Vaginal Epi Semen VE_8 (vaginal epi. Marker) CpG1 CpG2 CpG3 CpG Cg (blood marker) CpG1 CpG2 CpG3 CpG4 CpG5 Blood-Literature Saliva Blood Vaginal Epi Semen ZC3H12D (semen marker) CpG1 CpG2 CpG3 CpG4 CpG5 VE-Joana's Saliva Blood Vaginal Epi Semen semen-literature Saliva Blood Vaginal Epi Semen

38 Summary Forensic epigenetics for body fluid identification is a promising application in forensic casework. Multiple genetic loci were identified for discriminating saliva, blood, semen (or sperm) and vaginal epithelia. A multiplex PCR approach to identify the different types of body fluids was developed with a new marker for vaginal epithelia. Pyrosequencing is a useful technique for measuring DNA methylation in forensic laboratories, especially based on the autoprep system.

39 Acknowledgements Research Advisor Dr. Bruce R. McCord, PhD Epigenetics Group Quentin Gauthier, MSFS Hussain Alghanim, MSFS Joana Antunes, PhD Collaborators QIAGEN Inc. Major support for this research provided by award no NE-BX-0001 from the National Institute of Justice. Points of view in the presentation are those of the authors and do not necessarily represent the official view of the US Department of Justice. McCord DNA Research Group

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