Human variation –DNA profiling with mtDNA

and microsatellitesEric Yap

Defence Medical Research InstituteSingapore Eye Research Institute

COFM Dept, National University of Singaporenmiv3@nus.edu.sg

DNA profiling

• Forensic remains identification• Types of DNA polymorphisms• Methods of genotyping

polymorphisms• Short tandem repeats

(Microsatellites)• Mitochondrial DNA• Osteoarchaeology case study

Jeff Parker,Florida

DNA Profiling for Remains Identification

• Conventional Methods–Personal effects, tattoos–Dermal fingerprints–Dental Xrays

Specimens

• Bloodstain cards

Specimen Processing & Storage

ReceiptDrying,Packaging

Storage

Data entry

QC

Sept 11 - Pentagon & AA77188 casualties (124 Pentagon, 64 AA77)

838 samples to AFDIL(588 bones, 285 soft tissue, 22 teeth, 43 hair)

97% reportable data

182 unique DNA profiles, 177 “by name” DNA report

52 has cards, total 183 reference profiles

177 matched, 178th by dental

188 victims - 178 named = 10 unidentified

5 terrorists with no DNA

reference

5 Americans with DNA references

DNA profiling

• Forensic remains identification• Types of DNA polymorphisms• Methods of genotyping

polymorphisms• Short tandem repeats

(Microsatellites)• Mitochondrial DNA• Osteoarchaeology case study

Types of DNA Polymorphisms

– repeats (may be invariant)• long repeats (SINES, LINES, Alu)

– length (repeat)• minisatellite (variable number of tandem

repeats)• microsatellite (short tandem repeats)

– sequence• minisatellite variant repeats (MVR)• restriction fragment length

polymorphisms• single nucleotide polymorphisms

Almost all transposable elements in mammals fall into one of four classes.

Microsatellites

• Repeat unit–di- (CA)n or (GT)n– triple repeats– tetra-nucleotide, penta, etc

• Ubiquitous, well distributed• Densely mapped in human

genome: 7000+ (pre-sequence)

Biological Significance of STR

• Unstable, highly variable• Conserved across phyla• Associated with disease:

– fragile chromosome syndromes– cancer (MSH, MLH mutations)– degenerative neuromuscular diseases

• Mechanisms– transcriptional regulation & gene expression– structural instability

Applications of STR Genotyping

• Positional cloning/Reverse genetics– linkage studies: genome wide screens–association studies: candidate genes

• Molecular Diagnostics–directly or by linkage

• Individual profiling & Human diversity– remains id, forensics, anthropology

DNA profiling

• Forensic remains identification• Types of DNA polymorphisms• Methods of genotyping

polymorphisms• Short tandem repeats

(Microsatellites)• Mitochondrial DNA• Osteoarchaeology case study

gagagtncttgaaatgttcccaacacacacacactctctctctctcnctctctcacacacacacacacacacacacacacacacacagttttctccctataaaatatgntttccaggcccaatcctctcacaagcatcctagagcagtaaagatttcacatagtgacatcagacttgagtttgattccacctttactattcattcaccatgagactggacaaattagttaacctcagagtctcagtttcctcatctgtgcattgaatataggaaacag

D5S2033

D5S463D5S2099

D5S2090D5S413D5S434

D5S2013

D5S636D5S640

Gene ofPhosphodiesterase A

2 cM

Genetic Map at Phosphodiesteras A gene locus

Sequence of D5S434 markerCA repeatPrimer s

Electrophpresis Gel Separation

DNA Marker

Idiogram of Chromosome 5

Gel electrophoresis & Detection

• Denaturing polyacrylamide gels• Detection

–32P, 33P–Ag–Fluorescence (FAM, HEX, TET + size

standard)• Labelling: Incorporation (Double

strand) vs Primer end-label (Single strand)

D5S2013 D4S3038 D4S1536

D4S3002D3S3606RHO

D4S227 D5S434

High Throughput Genotyping by Multiplexing on Automated Fluorescent Detection System

High-throughput Genotyping. The 11 markers used were labelled with 3 different fluorescent dyes and multiplexed in1 lane. Allele sizing was determined by the Genotyper software.

Multiple Capillary Electrophoresis

Sizing & Analysis

• Size stds (intralane)• Controls (“gel shift”)• Automated sizing, calling of peaks,

alleles• Problems:

–stutter– ‘+A’–allele drop-out

Recent Developments

• Novel genotyping methods–Very high throughput CE–MALDI-TOF Mass Spectrometry

• Single Nucleotide Polymorphisms–300,000 SNPs, Maps–microarray genotyping

Micro Capillary Electrophoresis

1. Load Sample 2. Run analysis 3. See data

DNA profiling

• Forensic remains identification• Types of DNA polymorphisms• Methods of genotyping

polymorphisms• Short tandem repeats

(Microsatellites)• Mitochondrial DNA• Osteoarchaeology case study

Relative Merits of nucDNA with mtDNA

nucDNA

High discriminationOnly 2 copiesBest indirect references -

mother, father, offspring

Sample - several yearsQuick turn-aroundModerate expense

mtDNA

Limited discriminationMultiple copiesOnly indirect references -

maternal lineageSample - decades to

centuriesLengthy turn-aroundHigh expense

STR Markers DNA Profiling Panel and Allele Frequencies for the local population

Rita YongEric Yap

Marker PanelsMarker CODIS FSS Interpol DMRI

Amelogenin X X X XD3S1358 X X X XTH01 X X X XD21S11 X X X XD18S51 X X X XvWA X X X XD8S1179 X X X XTPOX X XFGA X X X XD5S818 X XD13S317 X XD7S820 X XD16S539 X X XCSF1PO X XPenta D XPenta E XD2S1338 XD19S433 X

CODISFBI Laboratory’s Combined DNA Index System, USA.

FSSForensic Science Service, UK.

Marker chromosome Gene Name GenBankLocus

GenBAnkAccess.No.

Repeat Seq

D3S1358 3p 11449919 AGATcomplex

TH01 11p15.5 Human tyrosinehydroxylasegene

HUMTH01 D00269 AATG

D21S11 21q11 – 21q21 HUMD21LOC M84567 TCTAcomplex

D18S51 18q21.3 HUMUT574 L18333X91254

AGAA

Penta D 21q AC001752 AAAGAD5S818 5q23.3 – 32 G08446 AGATD13S317 13q22 – q31 G09017 TATCD7S820 7q11.21 – 22 G08616 GATAD16S539 16q24 – qter G07925 GATACSF1PO 5q33.3 - 34 Human c-fms

proto-oncogenfor CSF-1receptor gene

HUMCSF1PO U63963 AGAT

Penta E 15q AC027004 AAAGAAmelogenin Xp22.1 – 22.3

& YHuman Ychromosomalgene forAmelogenin-likeprotein

HUMAMEL M55419

vWA 12p12 – pter Human vonWillebrandfactor gene

HUMVWFA31 M25858 TCTAcomplex

D8S1179 8q G08710 TCTATPOX 2p23 – 2pter Human thyroid

peroxidase geneHUMTPOX M68651 AATG

FGA 4q28 Humanfibrinogen alphachain gene

HUMFIBRA M64982 TTTCcomplex

DMRI In-house Marker panel

Discrimination Power

• Random Math Probability RMP

RMP = Σfi2i

Where the sum is taken over all I frequencies (counts) observed

• Genetic diversity h

h = (1- Σfi2 )n/(n-1)

Where n is the number of haplotypes

Discrimination Power

Matching Probabilities

AmericanAfrican Caucasian Hispanic Asian

Promega PP16 (15 markers) 7.09 x 10-19 5.46 x 10-18 3.41 x 10-18 2.67 x 10-18

ABI Profiler(2 panels 15 markers) 4.66 x 10-18 2.23 x 10-17

SingaporeanChinese Malay Indian Combined

DMRI Panel (15 markers) 1.16 x 10-17 5.60 x 10-18 2.57 x 10-18 1.01 x 10-18

Quantitative PCR for gene dosage analysis of trisomies

Categories of Disease Causationadapted from Ward RH 1979: Social Biology 27:87-100

Category Examples• Chromosomal

– aneuploidy Down’s syndrome– structural Prader-Willi syndrome

• Single-gene Inborn errors of metabolism• Mitochondrial musculoskeletal/neurodegenerative syn• Multifactorial

– high heritability cleft lip / palate, neural tube defects– low heritability coronary artery disease, asthma etc

• Infectious viral, bacterial, fungal, protozoal pathogens

• Environmental physical (radiation, trauma), chemical (drug, pollutant, occupational), nutritional

Mendelian

Complex

Morbidity of Genetic Disease

TYPE LIFETIME FREQ

• Chromosomal 3.8 per 1000• Single gene 20 per 1000• Multifactorial 646 per 1000

Emory & Rimoin (1997)

Cytogenetic Diagnostic Tests

• Congenital abnormalities, dysmorphic syndromes, short stature

• Delayed development, mental retardation• Recurrent, unexplained miscarriages, subfertility• Ambiguous/abnormal genitalia, amenorrhoea• Down’s (21), Edward’s (18 or E1), Patau’s (13 or D1)

syndrome (advanced maternal age, • Fragile (breakage) chromosomes• Angelman and Prader-Willi syndromes• Haemopoietic neoplasia (NHL, CLL)

46XXfrom VP Johnson, C Christianson

tri21

from VP Johnson, C Christianson

Normal Chr 21 Trisomy

Correlation of Allele distance and Peak height Ratio

0.800

1.000

1.200

1.400

1.600

1.800

2.000

2.200

0 1 2 3 4 5 6 7

Distance of Alleles (bp)

Ratio

of A

ve. P

eak

Heig

ht

0.800

0.900

1.000

1.100

1.200

1.300

1.400

1.500

1.600

1.700

0 2 4 6 8 10 12

No. of Allele Repeats (per 4 bp)R

atio

of A

ve. P

eak

Hei

ght

D21S11 D21S1412

Chr 21 TrisomyPeak Ht Characteristic of genotype

Marker GenotypePeak Ht Ratio

D21S11 221 225

0.686 1

D21S1412 406 410 414

1.225 1.185 1

D21S1413 156 176

0.816 1

D21S1411 284 300

0.576 1

Conclusion - Utility in trisomy diagnosis

DNA profiling

• Forensic remains identification• Types of DNA polymorphisms• Methods of genotyping

polymorphisms• Short tandem repeats

(Microsatellites)• Mitochondrial DNA• Osteoarchaeology case study

Mitochondrial DNA

• 16 569 bp, circular double strand DNA

• 37 genes :

> 2 rRNAs,

> 22 tRNAs &

> 13 mRNAs that encode 12 polypeptides for OXPHOS

Control region

16024 73 340 57616365OH HSP

LSP

tRNApro HV1 VR1 HV2 VR2 tRNAphe

HV1 sequence of a Chinese sample

HV2 sequence of a Chinese sample

Polymorphisms between Cambridge reference sequence (Anderson et al 1981) & a local Chinese sample

M tD N Aposition

R ef S eq L oca l C h in esesam ple

F ragm en t

16086 T C H V 1

16129 G A H V 1

16192 C T H V 1

16223 C T H V 1

16297 T C H V 1

73 A G H V 2

150 C T H V 2

199 T C H V 2

263 A G H V 2

311 - In sertion C H V 2

verify with reverse sequences

C R SN I S T _ 9 9 4 7 A - C R

1 4 6 - C R2 0 6 - C R

N I S T _ C H R - C R1 5 2 - C R

1 7 3 - C R1 9 0 - C R2 3 4 - C R

1 8 0 - C R2 0 4 - C R

2 0 8 - C R2 4 7 - C R

2 0 9 - C RQ L 0 2 9 9 1 1 3 - C R

1 6 0 - C R1 9 6 - C R

2 4 0 - C R1 6 8 - C R

Q L 0 2 9 9 1 1 6 - C R1 9 4 - C R

2 3 6 - C R1 7 4 - C R

2 4 2 - C R2 1 4 - C R

1 6 7 - C R1 8 3 - C R

9 2 9 - C R1 3 0 - C RQ L 0 2 9 9 1 3 6 - C R

1 5 5 - C R2 0 3 - C R

2 0 5 - C R1 5 0 - C R

2 4 1 - C R1 7 2 - C R

2 1 5 - C R2 4 6 - C R

1 8 6 - C R1 8 8 - C R

1 8 4 - C R2 4 8 - C R

1 6 2 - C R1 6 6 - C R

1 9 1 - C R2 0 1 - C R

2 3 9 - C R1 7 0 - C R

2 4 4 - C R1 8 1 - C R

1 9 7 - C R1 4 7 - C R

1 7 1 - C R2 1 7 - C R

2 4 9 - C R2 0 7 - C R

1 4 8 - C R1 7 7 - C R

1 5 4 - C R2 1 1 - C R

1 9 2 - C R1 9 3 - C R2 4 3 - C R

R i t a - C R2 5 2 - C R

1 5 1 - C R1 5 7 - C R

2 1 0 - C R2 4 5 - C R

1 5 9 - C R2 0 2 - C R

2 3 8 - C R1 7 8 - C R

1 4 9 - C R1 7 6 - C R

1 6 3 - C R1 6 1 - C R

2 1 6 - C R2 3 5 - C R

1 8 9 - C R2 5 0 - C R

2 1 2 - C R2 0 0 - C R

1 5 3 - C R2 1 8 - C R

1 5 6 - C R2 1 9 - C R

1 6 9 - C R1 8 5 - C R

1 6 4 - C RQ L 0 2 9 9 1 1 5 - C R

2 1 3 - C R1 3 6 - C R

1 9 5 - C R1 6 5 - C R

1 7 5 - C R2 3 7 - C R

1 9 8 - C R1 7 9 - C R

1 8 2 - C R2 5 1 - C R

CRSNIST_CHR

NIST_9947A

136QL136

Hapltype no.Occurance1 33 2

88 1

QC:

• 2 NIST ref samples produced concordant seq.

• Internal std (QL136) reproduced seq

CR region = 1197 bp

Random Match Probability = 0.0116

Genetic Diversity = 0.9987114

Phylogenetic Analysis of mt DNA CR region of 101 DNAs

Relative Merits of nucDNA with mtDNA

nucDNA

High discriminationOnly 2 copiesBest indirect references -

mother, father, offspring

Sample - several yearsQuick turn-aroundModerate expense

mtDNA

Limited discriminationMultiple copiesOnly indirect references -

maternal lineageSample - decades to

centuriesLengthy turn-aroundHigh expense

DNA profiling

• Forensic remains identification• Types of DNA polymorphisms• Methods of genotyping

polymorphisms• Short tandem repeats

(Microsatellites)• Mitochondrial DNA• Osteoarchaeology case study