Forensic epigenetics for human body fluid identification · stability of DNA methylation ....
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Forensic epigenetics for human body fluid identification Sohee Cho, Ph.D. and Bruce R. McCord, Ph.D. Florida International University, Miami, FL
Transcript of Forensic epigenetics for human body fluid identification · stability of DNA methylation ....
Forensic epigenetics for human body fluid identification
Sohee Cho, Ph.D. and Bruce R. McCord, Ph.D.Florida International University, Miami, FL
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
Body fluids founds on the bedSex offender’s trial
What information from the cell types of the crime scene trace?
Type Forensic relevance
Body fluids Blood Violence, human-specific assay
Semen Sexual assault, confirmation sampled area
Saliva Sexual assault e.g. licking, kissing or inoffensive stain
Vaginal secretion Sexual assault, confirmation sampled area
Menstrual secretion Sexual assault or inoffensive alternative scenario
Organs Brain Head injury
Heart, lung Chest injury
Kidney, liver Abdominal injury
Skeletal muscle Injury
Touch Skin Confirmation sampled area
Other secretions
Sweat Confirmation witness report
Urine Confirmation sampled area
Vomit Contains saliva and stomach content, inoffensive scenario
Faeces Anal sexual assaultSijen. Forensic Sci Int Genet 2015;18:21-32
Can support a link between sample donors and physical activities involved in cases
Presumptive and confirmatory tests for body fluid identification
Chemical, catalytic tests, enzymatic and immunological tests
Blood Semen Saliva Vaginal fluid Urine
Luminol, Phenolphthalein,ABAcard®, Kastle-
Meyer testHemeSelect™,
SAP,LAP,
Christmas tree stain,PSA
Starch-iodine,Phadebas®,
Amylose Azure
Starch gel/L-valyl-L-leucine,
Oestrogenreceptors
DMAC,Salkowski test,
THP,Urea NB-test
Virkler et al. Forensic Sci Int 2009;188(1-3):1-17
Molecular approach for cell type identification
How are different tissues and cells specialized?
– Messenger RNAs– Micro RNAs– Epigenetic
modification
DNA
Epigenetic modification
Messenger RNA (mRNA)
Functionalprotein
MicroRNA (miRNA)
The limitations
Marker type Limitations
Chemical, catalytic, enzymatic and immunological tests
• 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 cDNApreparation
• 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-specific RNA quantification methods• Potential of degradation by RNase
Sijen. Forensic Sci Int Genet 2015;18:21-32
Epigenetics
Image from UNSW Embryology, and Lindroth et al. J Periodontal Implant Sci 2013
>200 different types of cells(>200 epigenomes)
Epigenetic control
one genome
Cells and tissues are differentiated by epigenetic mechanisms
– Histone modification– DNA methylation– Acetylation, etc.
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
https://pubs.niaaa.nih.gov/publications/arcr351/6-16.htm
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
Tissue-specific DNA methylation regions (tDMRs)
Byun et al. Hum Mol Genet 2009;18(24):4808-17
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
How to detect or measure DNA methylation levels?
Standard STR and SNP profiling vs. DNA methylation pattern profiling
% of methylation?
Discrimination of C / 5-mC using sodium bisulfite conversion
Sodium bisulfite conversion
Highest degree of resolution of the methylation status of a given sample
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):1059-64
Application of high resolution melt (HRM) PCR method
Methylation differences are examined with real-time PCR/high resolution melt curve analysis (HRM)
Figures from Francisco Bizouarn, BIO-RAD
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
High resolution melt (HRM) PCR for DNA methylation analysis
Bisulfite treated DNA with all CpG-sites methylated will have higher Tm than if non-methylated
5’--C--C--C--C--3’3’--G--G--G--G--5’
5’--C--C--C--C--3’3’--G--G--G--G--5’
5’--U--U--C--U--3’ 5’--C--C--C--C--3’
5’--U--U--C--U--3’3’--A--A--G--A--5’
5’--C--C--C--C--3’3’--G--G--G--G--5’
Genomic DNA
Bisulfite treatment
PCR (1st cycle)
PCR (amplification)
5’--T--T--C--T--3’3’--A--A--G--A--5’
UnmethylatedPredominantly T=A bound
5’--C--C--C--C--3’3’--G--G--G--G--5’
MethylatedPredominantly G=C bound
m
m
m
m m m m
m m m m
m m m m
HRM analysis of DNA methylation to discriminate semen in biological stains
saliva
semen blood
Antunes. et al. Anal Biochem 2016;494:40-5
Hypomethylation of semen is expected to result in a melt curve with a lower melting temperature
Joana Antunes
No humic acid (HA) (thin lines) HA added before bisulfite conversion (dashed lines) HA added after bisulfite conversion (thick lines)
saliva
semen blood
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
Body fluid-specific DNA methylation analysis using pyrosequencing method
DNA extraction & quantification
Bisulfite conversion
PCR
Pyrosequencing
Data analysis
Exploring markers& assay design
EpiTect Fast Bisulfite Conversion Kit (QIAGEN)
PyroMark PCR kit (QIAGEN)
PyroMark Q24 Advanced /Q48 AutoPrep system (QIAGEN)
Identified CpG loci based on large scale epigenetic arraysPyroMark Assay Design SW (QIAGEN)
EZ1 Advanced & EZ1DNA investigator Kit (QIAGEN)
Body fluid ID assay design for pyrosequencing
Bisulfite conversion
PCR
Pyrosequencing
Data analysis
Exploring markers& assay design
DNA extraction & quantification
Biotinylated
One of the PCR primers is biotinylated Sequencing primer anneals to single-strand DNA
before pyrosequencing reaction
Genome-wide array
Target CpG loci-specific analysis
DNA extraction & quantification
DNA extraction– EZ1 Advanced & EZ1 DNA investigator Kit
(QIAGEN)– Phenol chloroform extraction method DNA quantification
– Alu-sequence quantification based on real-time PCR
Bisulfite conversion
PCR
Pyrosequencing
Data analysis
Exploring markers& assay design
DNA extraction & quantification
Bisulfite treatment & PCR amplification
Bisulfite conversion
PCR
Pyrosequencing
Data analysis
DNA extraction & quantification
Exploring markers& assay design
Zilberman et al. Development 2007;134(22):3959-65
Pyrosequencing platforms - PyroMark Q24
PyroMark Q24/Q24 Advanced
Template preparation
Pyrosequencing
Bisulfite conversion
PCR
Pyrosequencing
Data analysis
PCR primer
PCR primer
Sequencing primer
Region of interest
Streptavidin coated magnetic beads for DNA capture
DNA extraction & quantification
Exploring markers& assay design
< Single strand preparation >
Pyrosequencing platforms - PyroMark Q48
PyroMark Q48 Autoprep
Bisulfite conversion
PCR
Pyrosequencing
Data analysis
Template preparation, anneal sequencing primer,
pyrosequencing(+ washing cartridges)
Pyrosequencer that handles all of the mixing and preparation for each sample - just load the reagent cartridges, samples, and go!
DNA extraction & quantification
Exploring markers& assay design
Analysis of DNA methylation dataSequence to analyze
Dispensation order (generated by software)Negative controls
Madi et al. Electrophoresis 2012;33:1736-1745
Blood (hypermethylation) Semen (hypomethylation)
[C signal intensity]
[T signal + C signal]Met % = X 100
Bisulfite conversion
PCR
Pyrosequencing
Data analysis
DNA extraction & quantification
Exploring markers& assay design
95% 97% 100% 100% 84% 2% 4% 6% 4% 3%
Bisulfite control(Negative control)
The markers identified based on pyrosequencing
BCAS4 and ZC3H12DIdentified in 2012
cg06379435Identified in 2015
PFN3AIdentified in 2016
The markers identified based on pyrosequencing (cont.)
Madi et al. Electrophoresis 2012;33:1736-1745
C20orf117 - hypermethylated in blood
– Eckhardt et al. - hypermethylated in lymphocytes and hypomethylated in sperm and skin cells
BCAS4 - hypermethylated in saliva– Eckhardt et al. - hypermethylated in sperm
with an 80% methylation level, but we found this loci to be hypermethylated in saliva
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)
Madi et al. Electrophoresis 2012;33:1736-1745
FGF7 - hypermethylated in sperm
– fibroblast growth factor 7, keratinocyte growth factor
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
Antunes et al. Electrophoresis 2016;37(21):2751-2758
cg0679435 loci - a useful loci for blood marker
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20
40
60
80
100
CpG 1 CpG 2 CpG 3 CpG 4 CpG 5
Blood (n=10) Saliva (n=10)
Skin (n=7) Vaginal Epithelia (n=10)
Semen (n=9)
Blood markers were explored using epigenetic microarray data(Park et al. Forensic Sci Int Genet 2014;13:147-53)
Bioinformatic work from Dr. Balamurgen (University of Southern Mississippi) identified cg0679435 as a useful blood marker. Pyrosequencing identified 5 CPG sites.
Development of multiplex PCR for body fluid identification using pyrosequencing
saliva marker
blood marker
semen marker
vaginal epitheliamarker
Singleplexreaction Multiplex reaction
4 body fluidmarkers
The first attempt at a multiplex
cg0679435(blood marker)
BCAS4(saliva marker)
ZC3H12D(semen marker)
PFN3A(vaginal epi. marker)
Incorrect peak height ratios
3 : 1 : 1 : 0 1 : 1 : 3 : 0 : 1
Non-specific peaks where there should be nothing
Signals too low for data interpretation and baseline drift
This slide was kindly provided by Quentin Gauthier from our lab
What is causing the issues?
Lack of Stringency
Sequencing primer binding to incorrect PCR product causes peak height imbalances that make the multiplex results unusable
BCAS4 PCR Product
cg0679435 PCR Product
PFN3A PCR Product
ZC3H12D PCR Product
400
30
200
10
Pyrosequencing with BCAS4 (saliva marker) sequencing primer
This slide was kindly provided by Quentin Gauthier from our lab
Balancing forward and reverse PCR primers in duplex and triplex
PCR primer sets Sequencing primer
A ZC3H12D + BCAS4 ZC3H12D
B ZC3H12D + cg0679435
C ZC3H12D + PFN3A
PCR primer set Sequencing primer
D ZC3H12D + BCAS4 + cg0679435
BCAS4
E cg0679435
F ZC3H12D
A
B
C
D
E
F
Examples of duplex
Examples of triplex
Balancing forward and reverse PCR primers in quadplex
cg0679435(blood marker)
ZC3H12D(semen marker)
BCAS4(saliva marker)
PFN3A(vaginal epi. marker)
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
Reconfigured multiplex analysis (in progress)
Testing is still ongoing for optimization in the multiplex…
cg0679435(blood marker)
ZC3H12D(semen marker)
VE_8(vaginal epi. marker)
BCAS4(saliva marker)
DNA methylation levels in four body fluids on multiplex PCR
0102030405060708090
100
CpG1 CpG2 CpG3 CpG4
BCAS4(saliva marker)
Singleplex-literature Saliva Blood Vaginal Epi Semen
0102030405060708090
100
CpG1 CpG2 CpG3 CpG4 CpG5
Cg0679435(blood marker)
Blood-Literature Saliva Blood Vaginal Epi Semen
0102030405060708090
100
CpG1 CpG2 CpG3 CpG4
VE_8(vaginal epi. Marker)
VE-Joana's Saliva Blood Vaginal Epi Semen
0102030405060708090
100
CpG1 CpG2 CpG3 CpG4 CpG5
ZC3H12D(semen marker)
semen-literature Saliva Blood Vaginal Epi Semen
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.
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. 2017-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
