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Epigenetics – a New Perspective for Improving Forensic Sciencep g

Hwan Young Lee, Ph.D.Department of Forensic MedicineYonsei University College of Medicine

Forensic cases -- matching suspect with evidence

Paternity testing -- identifying father

Current Forensic DNA Typing

Paternity testing identifying father

Involves generation of DNA profiles usually with the same genetic

markers and then MATCHING TO REFERENCE SAMPLE

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Challenges in Crime Scene Investigation

Technical challenges in forensic STR profiling

Degraded DNA Degraded DNA

Low amount of DNA

Increasing discrimination power

Familiar searching….

Important challenges remain for the use of genetic and Important challenges remain for the use of genetic and

epigenetic approaches in forensic investigations

Kayser M and de Kniff P, Nat Rev Genet (2011)

Future of Crime Scene Investigation

The ability to infer biogeographic ancestry and external

visible characteristics from DNA

Eye (IrisPlex), hair and skin color

Freckles

Stature

Hair morphology

Male baldness, etc.

http://meglikestodraw.tumblr.com/Kayser M and de Kniff P, Nat Rev Genet (2011)

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Body fluid identification can provide information linking

sample donors with actual criminal acts

Future of Crime Scene Investigation

Detailed reconstruction of crime events by determining

the time of sample donation and sample age

Kayser M and de Kniff P, Nat Rev Genet (2011)

Body fluids founds on the bedSex offender’s trial

Genetics and Epigenetics

Epi (on top of or in addition to) + Genetics

Epigenetics is the study of heritable changes in gene

expression or cellular phenotype caused by mechanisms

other than changes in the underlying DNA sequence

Genome – Epigenome, Genetic code – Epigenetic code

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What is a Epigenome?

DNA methylation

Histone modification Histone modification

MicroRNA

http://learn.genetics.utah.edu/content/epigenetics/

Methyl tag Acetyl tag

DNA Packaging

Felsenfeld G and Groudine M, Nature (2003)

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Gene Control by the Epigenome

http://learn.genetics.utah.edu/content/epigenetics/

Gene Control by the Epigenome

Epigenome is flexible and epigenetic tags react signals, e.g.,

diet, stress, etc

http://learn.genetics.utah.edu/content/epigenetics/

Methyl tag

Acetyl tag

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Epigenetic Tags and Cellular Memory

A cell’s epigenetic profile – a collection of tags that tell

genes whether to be on or off – is the sum of the signals itg g

has received during its lifetime

Epigenetic profiles are specific to tissue, age and

environmental factors

http://learn.genetics.utah.edu/content/epigenetics/

DNA Methylation

DNA methylation is the addition of a methyl group to the

DNA base cytosine followed by a guanine (5' CG 3')DNA base cytosine followed by a guanine (5 CG 3 )

Cytosine 5-Methyl Cytosine

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tDMRs and Body Fluid Identification

Chromosome pieces called tDMRs (tissue-specific

differentially methylated regions) show different DNAdifferentially methylated regions) show different DNA

methylation profiles according to the type of cell or tissue.

Tissue UCSC location (Mar. 2006) CGI Gene Function References

Testis chr14:58182690–58182995 cpgi50 DACT1 Dapper 1 isoform 2 Genomics. 89:326

Testis chr6:41881884–41882111 cpgi46 USP49 Ubiquitin carboxyl-terminal hydrolase 49 Genomics. 89:326

Table. Genomic information for candidate tDMRs for body fluid identification

Blood chr7:27135995–27136879 cpgi87 HOX44 Homeobox protein Hox-A4 PLoS Biol. 6:e22

Blood chr5:176758438–176760564 cpgi82 PFN3 Profilin-3 PLoS Biol. 6:e22

Blood chr21:46905647–46905874 cpgi55 PRMT2 Protein arginine N-methyltransferase 2 PLoS Biol. 6:e22

Lee HY et al. Int J Legal Med (2012)

Analysis of DNA Methylation

Met Met

Sodium bisulfite conversion

CpG

CpG

CpG

UpG

CpG

TpG

Sodium bisulfite PCR Sequencing

SBE

MSP

MSRE-PCR

Methylation-sensitive restriction enzyme

PCR product

No PCR product

PCRCpG

CpG

CpG

CpG

Met Met

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DNA methylation profiles for the DACT1, USP49,HOXA4, PRMT2 and PFN3 tDMRs in pooled DNA

l f bl d li l bl d d

Differential DNA Methylation

: DACT1 : USP49

samples from blood, saliva, semen, menstrual blood andvaginal fluid

Lee HY et al. Int J Legal Med (2012)

DNA methylation profiles for the DACT1, USP49,HOXA4, PRMT2 and PFN3 tDMRs in pooled DNA

l f bl d li l bl d d

Differential DNA Methylation

: DACT1 : USP49: HOXA4 : PRMT2

samples from blood, saliva, semen, menstrual blood andvaginal fluid

Lee HY et al. Int J Legal Med (2012)

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DNA methylation profiles for the DACT1, USP49,HOXA4, PRMT2 and PFN3 tDMRs in pooled DNA

l f bl d li l bl d d

Differential DNA Methylation

: DACT1 : USP49: HOXA4 : PRMT2: PFN3

samples from blood, saliva, semen, menstrual blood andvaginal fluid

Lee HY et al. Int J Legal Med (2012)

DNA Methylation and Aging

The DACT1, USP49 and PRMT2 tDMRs in pooled DNAsamples from blood, saliva and semen obtained from 20

( 30 ) d 15 ld ( 50 )young (< 30 y) and 15 old (> 50 y) men

: DACT1 : USP49

: PRMT2

An JH et al. Int J Legal Med in press

: PRMT2

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Methylation-Specific Multiplex Assays

MSRE-PCR Methylation-SNaPshot

Blood

Saliva

Menstrual blood

An JH et al. Int J Legal Med in press

Vaginal fluid

Semen

DNA Methylation and Age Estimation

The methylation of three sites—in the promoters of the

EDARADD NPTX2 and TOM1L1 genes—is linear withEDARADD, NPTX2 and TOM1L1 genes is linear with

age over a range of five decades in saliva.

Percentage methylation versus age for three markers, (a) EDARADD, (b) NPTX2 and (c) TOM1L1

Bocklandt et al. PLoS One (2011)

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Age Estimation Using Body Fluids

TOM1L1SPAG7

ALDH1A1 WISP2 EDARADD

20sTOM1L1

SPAG7ALDH1A1 WISP2 EDARADD

20s

DNA Methylation in Saliva DNA Methylation in Semen

20s

30s

50s

50s

20s

50s

50s

DNA Methylation in Skin EpidermisTOM1L1

SPAG7ALDH1A1 WISP2 EDARADD

20s

30s

60s

Discrimination Between Identical Twins

Identical twins exhibit differences in DNA methylationprofile

Fraga MF et al. Proc Natl Acad Sci U S A (2005), Zhao SM et al. Fa Yi Xue Za Zhi (2011)

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Detection of Fabricated DNA Evidence

Natural and artificial DNA are distinguishable based on

methylation analysis of a set of genomic loci: in artificialy y g

DNA, all loci are unmethylated

Bisulfite sequencing

Natural

Frumkin D et al. Forensic Sci Int Genet (2010), New York Times- Aug 17, 2009

Artificial

Current View of Human Epigenome

DNA methylation

Histone modification Histone modification

Small RNAs

Microarray

Next Generation Sequencing…

Schones DE and Zhao K, Nat Rev Genet (2008)

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The Future of Forensic DNA Typing

Genetics, Genomics, Epigenetics, Epigenomics, Epitranscriptomics…

Thank you for your attention!

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