Introduction of forensic genetics

Molecular medicine -

Genetic analysis of molecular evidences

Pádár Zsolt

„Traces” - Death Valley, CA 2007 / 10 / 11

… there are things in LIFE …

… and there are BAD things in LIFE …

… some we do …

… some other do to us …

… some, they suppose that we did …

… and there are EXTREMELY BAD things of

LIFE …

… and things, that we tought could only

happend to others …

What a heck is the forensic genetics ?

Transmission geneticsMolecular geneticsPopulation geneticsEcological geneticsQuantitative geneticsNurture geneticsMedical geneticsCytogeneticsMitochondrial geneticsGenomics…

forward geneticsreverse geneticsevolution genetics molecular taxonomyepigeneticspharmacogeneticsforensic geneticspathogeneticsbioinformaticsgenetic engineering…

JurisprudenceJurisprudenceCriminalisticsCriminalisticsEthicsEthicsMedical Medical sciencessciencesVeterinary Veterinary sciencessciences

2008. 11. 1. / 2009. 05. 04.

Forensic geneticsGenetics and lawThe traits of the children are not only the sum of parent’s…„The science is only a method, but not the truth itself”

Applied scienceObjective examination of biological traces using methodo-

logy of molecular genetics regarding the given crimeTools for confirmation or rejection of criminal hypotheses

Bilateral relation to basic sciencesApplication and modification of methods in molecular

geneticsModification of requirements concerning basic sciencesProviding of new scientific resultsImpact on evolution of society and economy, effects to

community and peoples

How does it work …?Human forensics

Crimeshomicide, robbery, rape …

Paternitygenealogy

Sudden deathsudden cardiac death (SCD)

sudden arrhythmic death syndrome

sudden infant death syndrome (SIDS)

Mass disastersvictim’s identification

How does it work …

Nonhuman forensicsAnimal forensics

dog, cat, wildlife …cruelty, attack, poaching …

entomologytime, area

Forensic botany & fungileaves, seeds …pollens

Microorganismssoil identification

Analysis of genetic polymorphismsTemperature sensitive expression of a recessive gene

TYR gene ► tyrosinase enzyme ► synthesis of melanineDefective tyrosinase ► loss of function at normal body temperature

The first step - The crime scene …

„CSI University” ☺

Complex analysis - „DNA investigation”

IdentificationDetermination of origin - where the result’s coming from?

Individualization – common source of identitynuclear DNA, mitochondrial DNA

Extension of the target materials and markersGenetic and technical variability and limitations

quantity – sensitivity, threshold, reproducibilityquality – degradation, contamination, assurancereaction complexity - stochasticsgenetic complexity - pigmentation

Requirements …The forensic genetic investigation is not scientific research or experiment but examination using scientific methods…

Sensitivity, reliability, reproducibility, verification

Technology, methodologyScientific criteria, statisticsEthical criteria

ResponsibilityQuality assurance

… some, they suppose that we did …

Milestones in forensic genetics …

LandsteinerJeffreysMullis

Targets …Human and nonhuman DNA

Nuclear DNAAutosomes

Repetitive markers (VNTR’s, STR’s)Single nucleotide polymorphisms (SNP’s)

Sex chromosomesRepetitive markers (VNTR’s, STR’s)Single nucleotide polymorphisms (SNP’s)

Mitochondrial DNACytochrome(b)Control region (HVI, HVII, CR)SNP’s

PCR

Short Tandem Repeats (STRs)

AATG AATG AATGFluorescent

dye label

7 repeats

8 repeats

the repeat region is variable between samples while the flanking regions where PCR primers bind are constant

Homozygote = both alleles are the same length

Heterozygote = alleles differ and can be resolved from one another

Primer positions define PCR product size

Principles of sample separation and detection

Sample Detection

CCD Panel

ColorSeparation

Ar+ LASER (488 nm)

Fluorescence ABI Prism spectrograph

Capillary or Gel Lane

Size Separation

Labeled DNA fragments (PCR products)

Detection region

Principles of fluorerescent detection

Laboratory process …

Short Tandem Repeats (STRs)

13 STR loci with chromosomal positions

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGAVWA

AMEL

AMEL

amelogenin

D19

D3

D8

TH01

VWA D21FGA

D16D18 D2

amelogeninD19

D3D8 TH01

VWA D21

FGA

D16D18 D2

Two

diff

eren

tind

ivid

uals

DNA Size (base pairs)

Results obtained in less than 5 hours with a spot of blood the size of a pinhead

probability of a random match: ~1 in 3 trillion

Human identity testing with multiplex STRs

Simultaneous Analysis of 10 STRs and Gender ID

AmpFlSTR® SGM Plus™ kit

Multiplex - 9 autosomal loci

15 autosomal loci …

… from a mosquito ?

Upps! Here I am !!!

Increasing interest in the Y-chromosome…Increasing interest in the Y-chromosome…

STR MarkersDYS19

DYS389I/II

DYS390

DYS391

DYS392

DYS393

DYS385

YCAII

DYS437

DYS438

DYS439

Y

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Human Genome

1 2 3 4 5 6 7 8 9 10 11 12

13 14 15 16 17 18 19 20 21 22 X Y

17 Y-chromosome loci - haplotype

The talking envelope …the victim was brutally murdered…inconsistent confessions of suspects…

primitive personality…„but I didn’t do that …”less information, more doubts …

… but one envelope at crime scene …nuclear DNA analysis …

The fingerprint…

The fingerprint - resultsautosomal lociY-STR loci

inclusion

Single Nucleotide Polymorphisms (SNP’s)

Millions of SNP in genomein every 500-1000 base pairspoint mutation in genes or/and regulator regions

Relation to phenotypeLineages of ancestry (Y-SNP’s)Diagnostics

multi-factorial polygene diseases

T T C A A G Template

Primer

Primers - tailed different lengths

SNaPshotTM Multiplex Kit:one base extension

C G

Primer extensionand termination

TA

T

C

A

GA AT

GC

CG

G

T

C Colour = genotype

Length = lociElectrophoresis

Automatic analysis

The mitochondrial genomecircular DNACambridge Reference Sequence~ thousand copy / cellno histones associationhigh mutation rate

at D-loop during ~ 9000 year 1%susceptibility to free oxygen radicals

diseases, aging no excision repair system

no repair only selectionbottleneck effect

heteroplasmyrecombination within the same mitochondrion - no recombinant variance like nuclear DNA maternal inheritance - matrilineage

geographically determinedgeological segregation of the populations regarding fixed mutations at non coding regions and the maternal inheritance

The control region

Control Region (CR) –D(isplacement)-loop

~1120 base pairsorigin of replication opens hypervariable regions

HV1, HV2 and HV3lower selection pressuresource of variations is the mutation

Sequence DatabasesEMPOP

Control Region (D-loop)

1/16569

cyt b

ND5ND6

ND4

ND4L

ND3

COIIIATP6

ATP8 COII

12S rRNS

16S rRNS

ND1

ND2

COI

OH

OL

HV1 HV216024 16365 73 340

16024 576

„16 569” bp

Forensic point of viewThe mtDNA has lower discrimination power than nuDNA

The mtDNA identifies not individual but haplotype of maternal lineage

on the other handThe level of diversity fairly high it’s a high probable, that two randomly selected individual have different mtDNAhaplotype

this probability is usually over 95 % - independently of populationsThe 2/3 part of the detected haplotypes from new samples -are newThe common (frequency over 1%) haplotypes are rareThere are many thousands of haplotypes the relative frequencies of haplotypes are low

Terminal (Sanger) sequencingdNTP and ddNTPdifferent fragment sizedye labelled primersordye labelled nucleotides

dideoxi inhibits !

D-loop sequence - forensic tools of matrilineage identification

Assembling the sequence of the questionable sample to reference sequence CRS (e.g. 16071-16140)

Different nucleotides at same position provide the mtDNAhaplotype of the given sample

Evidence(Q)

16093C 16129A

Reference(R)

16093C 16129A

mtDNA haplotypes

Genealogy

3 AFRICAN - 9 EUROPEAN - 7 ASIAN MAIN HAPLOGROUPS

Couple murdering …

2004 – a married couple is missing personal belongings, comb, toothbrush, cap, cup

2006 – NN male body murderedbroken piece of hair from a car

morphological similarity to hairs from comb2007 – cranium, mandible2008 – reference samples♀ child and ♂ brother

Whose are the cranium and the broken hair?

Cranial remains

Results of sequencingcombbonehairchildbrother

Results

exclusion

Cytochrome b gene

14747 → 15886 position(1140 base pairs)

Cytochrome b-c1 complex

Coding sequences:

- lower mutation rates,- synonym mutations

Genetic drift - sequence differences among species

Phylogenetic analyses

Cytochrome b gene sequence – a forensictool of species identification

14747 → 15886 position (1140 basepair) PCR: 358 bp fragment

Standard deviation approx. 15-20%

SNP a cytochrome b génben (Canis f.)

The expert opinion …Answers and interpretations …

Population geneticsRepresentative populations

Statistical geneticsIndividualizationGenealogy, relationship

Errors …Technical reasons

Contaminations, incompatibilityPersonal reasons

Proficiency

Hypothesis testing (Bayes)Hypothesis of prosecution (Hp):E.g.: The putative origin of bloodstain from crime scene is -

the suspect (XY)

Hypothesis of defense (Hd):E.g.: The putative origin of bloodstain from crime scene is -not the suspect, but unknown person (NN)

Bayesian-theory (test of probability)

Pr: probabilityE: evidenceI: informationH: hypothesisp: prosecutiond: defense

I)E,Pr(HI)E,Pr(H

I),HPr(EI),HPr(E

I)Pr(HI)Pr(H

d

p

d

p

d

p=×

priorprobability

(PrPR)

LikehoodRatio

(genetic evidence)

posterior

probability(PoPR)

The Likehood RatioWhen the origin of biological trace is a given person and the analytical error is excluded, Pr(E|Hp,I)=1.

the Likehood Ratio (LR) is – in a simplified form –the reciprocal value of frequency of the given genetic profile

I),HPr(G1

I),H,GPr(G1

I),HPr(EI),HPr(E

LRdcdscd

p≈==

Matching Probability Frequency of genetic profile

Pr: probabilityE: evidenceG: genetic profileI: informationH: hypothesisp: prosecutiond: defensec: crime scenes: suspect

Thank you for your attention!

„Genetically modified Christmas tree” - Amboy, CA