Mitochondrial DNA and EMPOP

Mitochondrial DNA and EMPOPMitochondrial DNA and EMPOPDr. Walther ParsonInstitute of Legal Medicine

Innsbruck Medical UniversityAustria

Buenos Aires, Argentina, Sept 14th 2009Buenos Aires, Argentina, Sept 14th 2009

Pre-congress educational workshopPre-congress educational workshop

Table of Contents

1) General Introduction

2) Forensic practice of mtDNA typing

3) Forensic application of the mtDNA phylogeny

W. Parson – Mitochondrial DNA and EMPOP

mtDNA research


medical genetics

population genetics

forensic genetics

Mitochondrial DNA


• circular double-stranded molecule• coding region (15 kb) 37 genes• control region (1.1 kb) d-loop• evolutionary rate ~10x of nDNA

MtDNA copy number


MtDNA is present in much higher copy number than nDNA4-5 mtDNA (avg) molecules/mitochondrion (Satoh and Kuroiwa, 1991)up to1000 mitochondria/cell (Robin and Wong, 1988)

500 – 650x more copies of mtDNA than nDNA 150 – 200 mtGE/pg gDNA

MtDNA copy number


This is why mtDNA analysis can bring successful results in stains/ biological specimen, which give only partial or no results with nuclear DNA (STR) typing

Crime cases human IDe.g. Günter Messner

historical casese.g. Romanov family

Maternal inheritance

Mitochondria in a cell/tissue/individual derive from the mitochondria of the fertilized egg – mtDNA passed through the maternal line

Implications:MtDNA results (=haplotypes) identify the entire maternal lineage (not only the individual).MtDNA is inherited along a phylogeny of radiating lineages that spread through human dispersal across the continents.



MtDNA phylogeny

Forster (2004)


MtDNA phylogeny

Kivisild et al (2006)


MtDNA phylogeny

Underhill and Kivisild (2007)

Forensic relevance:huge database (HVS-I and codSNPs) but unknown sample origin

The Genographic Project


www.phylotree.orgvan Oven and Kayser (2009)

continuously updated (Built 5)release historyCollection of mt genomes sequences (5000)Relevance for forensics:Directory of known lineagesDirectory of hg-names


http://www.phylotree.org/

Forensic practice of mtDNA typing


ExtractionQuantitation

MtDNA - specific

Amplification/sequencingControl regionWhole mtDNA genome

Haplotype annotationForensic implications

Interpretation (casework)Database searchesStatistics

DNA Extraction


Guidelines for good laboratory practice (Bär et al 2000)MtDNA extraction

Ph/Chl, Chelex, Silicate, commercial kits, ...Sensitivity of mtDNA typing – contamination

Identification of the two missing Romanov children (Coble et al 2009)Phenol/Chloroform and anorganic extractions followed by purification (Qiagen)

MtDNA quantitation


Often estimated from nDNA or gDNA concentration

Constraints: imprecisetissue dependent differenceslittle information on degradation

mtDNA-specific quantitation methods e.g. Meissner et al (2000)

Andréasson et al (2002)Walker et al (2006)Niederstätter et al (2007)

Modular quantitation assays


MtDNA quantitation in a modular system (Niederstätter et al 2007)parallel determination of nDNA and mtDNAor nDNA/mtDNA quantitation with internal PCR control (IPC)inhibition and degradation sensitive

Quantitative and qualitative values are essential for typing strategy

MtDNA typing methods


AlternativesSSO typing (Stoneking et al 1991)RFLP typing (Torroni et al 1993)PCR-RFLP typing (Torroni et al 1996)SSCP typing (Alonso et al 1996)SBE typing (Tully et al 1996) LSSP-PCR (Baretto et al 1996)High density array (Chee et al 1996)MALDI-TOF (Ross et al 1997)DGGE typing (Steighner et al 1999)

Pyrosequencing (Andreasson et al 2002)dHPLC typing (LaBerge et al 2003)Linear Arrays (Gabriel et al 2003)ESI-TOF (Oberacher et al 2006)454 sequencing (Margulies et al 2005)

OP 26ReSeq Chip (Thieme in press)

Direct sequencingHVS-I / HVS-II sequencing (Piercy et al 1993)CR sequencing (Lutz et al 1998, Brandstätter et al 2004)Full mtDNA sequencing (Ingman et al 2000, Fendt et al 2009)

MtDNA amplification and sequencing


Separate HVS-I/HVS-II analysis“double-stranded” consensus by forward and reverse sequencing

Frequency of phantom mutations significantly higher beyond length variant regionsBrandstätter et al (2006)

73 3401636516024 HVS-I HVS-II

T16189C

~10-80% ~ 40%

576

31016189

length heteroplasmy, blurred sequence reads> 8C



Separate HVS-I/HVS-II analysisRisk of artificial recombination (mix-up of hypervariable regions between

different samples)

Bandelt et al (2004), Salas et al (2005)

Example South Asia (Sep. 09) – two consecutive samplesH 263G 309.1C 315.1C 489CM 16223T 16256T 16311C 16362C 73G 200G 263G 315.1C

489C is missing

73 3401636516024 HVS-I HVS-II 576

Individual A Individual B



Full CR analysis with overlapping primers

Brandstätter et al (2004)

CR sequencing (“good DNA”)


„AFDIL-protocol“; Irwin et al (2007)

tRNApro HVI HVIIIHVII

16024 44 340116400 438 576

One Amp

SystemF15971 R599

F15971 R16400

F16190 R285

F15 R599

Full CR Amplification

CR Amp01

CR Amp02

CR Amp03

Three Amp

System

F15971 (2X) R599 (2X)

F16190 (2X)

F16450

F155 (2X)

F314 (2X)

R484

R285 (2X)

R16400/16410 (2X)

R16175(Used for CR Amp01)


16024 44 340116400 438 576


16024 44 340116400 438 576

One Amp

SystemF15971 R599

F15971 R16400

F16190 R285

F15 R599

Full CR Amplification

CR Amp01

CR Amp02

CR Amp03

Three Amp

System

F15971 (2X) R599 (2X)

F16190 (2X)

F16450

F155 (2X)

F314 (2X)

R484

R285 (2X)

R16400/16410 (2X)

R16175(Used for CR Amp01)

CR sequencing (“good DNA”)


Control region amplification

Amplicon purification

Cycle sequencing

15900 59916569/116189 309/315

R16 F15851

15851 63916569/116189 309/315 573

PCR 1 F15900

PCR 1 R00599

PCR 2 F15851

PCR 2 R00639

mtDNA control region16569/1

15900 59916569/116189 309/315 573

F15971

15878 64916569/116189 309/315 573Sample including length heteroplasmy in HVS-I and HVS-II

573

F15971R484

F15F314

R159F16268

F29R639

R16

R484F15

F314

F15851R159

F16268F29

R639

Sample without length heteroplasmy

Control region amplification

Amplicon purification

Cycle sequencing

15900 59916569/116189 309/315

R16R16 F15851F15851

15851 63916569/116189 309/315 573

PCR 1 F15900

PCR 1 R00599

PCR 2 F15851

PCR 2 R00639

mtDNA control regionPCR 1 F15900

PCR 1 R00599

PCR 2 F15851

PCR 2 R00639

PCR 1 F15900PCR 1 F15900

PCR 1 R00599PCR 1 R00599

PCR 2 F15851

PCR 2 R00639

mtDNA control region16569/1

15900 59916569/116189 309/315 573

F15971

15878 64916569/116189 309/315 573Sample including length heteroplasmy in HVS-I and HVS-II

573

F15971F15971R484R484

F15F15F314F314

R159R159F16268F16268

F29F29R639R639

R16R16

R484R484F15F15

F314F314

F15851F15851R159R159

F16268F16268F29F29

R639R639

Sample without length heteroplasmy

„EMPOP-protocol“; Parson and Bandelt (2007)

CR sequencing (degraded DNA)


„Midis“ – 282-444bpBerger and Parson (2009)

„Minis“ – 144-237bpEichmann and Parson (2008)

see also Gabriel et al (2001)

Full mtDNA sequencing


8.5kbp amplicons1,000 mtGE

Fendt et al (2009)

1-2kbp ampliconsZimmermann BPoster 122

96 seq primers for full redundant sequence coverage

PCR

Annotation of mtDNA sequences


16311 T>C

MtDNA reference sequences


116569

CRS rCRS

x 3107del3423T

4985A

9559C

11335C

13702C

14766C14368C

14365C14272C

14199T x

x

xx

x

xx

xxx

original Cambridge reference sequenceGenBank: M63933Anderson et al (1981)

revised Cambridge reference sequenceGenBank: NC001807 (mitomap)Andrews et al (1999)

Phylogenetic signature

CR 16024-576

Multiple “Anderson”-sequences!!

Alignment and notation of mtDNA sequences

365.1C (Th2F8, Thailand, GMI)


rCRS

TA T C CT C C:

455Del

FRE290

Tconsensus

455Del (FRE390, Germany, ILF)

451356.1C

Th2F8

rCRS

C C C A A A CC A ACconsensus

353

Notation with respect to rCRS is straight forward in the majority of haplotypesOnly in homopolymeric regions (e.g. C-tracts) may insertion/deletion events be reported differently – 3’ rule (Bär et al 2000)

Ambiguity in homopolymer stretchesIn some cases the 3 prime positioning rule is not sufficient to unambiguously define a single haplotype, more variants are feasible (e.g. INT028, Spain, Madrid)

Int028

rCRS

16189

16188T 16189C

16188Del 16193.1CA A A A C delCC C C CC C CT

16188 16193.1

TA G


Multiple possible alignments

Wilson et al (2002)

1) Phylogenetic ruleSequences should be aligned with regard to the current knowledge of the phylogeny. In the case of multiple equally plausible solutions, one should strive for maximum (weighted) parsimony. Variants flanking long C tracts, however, are subject to extra conventions in view of extensive length heteroplasmy.

2) C-tract conventionsThe long C tracts of HVS-I and HVS-II should always be scored with 16189C and 310C, respectively, so that phylogenetically subsequent interruptions by novel C to T changes are encoded by the corresponding transition. Length variation of the short A tract preceding 16184 should be notated in terms of transversions.

3) Indel scoringIndels should be placed 3′ with respect to the light strand, unless the phylogeny suggests otherwise.

Alignment in EMPOP

W. Parson – Mitochondrial DNA and EMPOP Bandelt and Parson (2008)

Inconsistent annotation between haplotype and database entries may under-estimate frequency

Alignment and notation of mtDNA sequences


Effect on database searches (EMPOP development 2008-05; N=26.930)

Diff (16184-16194) 16188T 16189C 16188del 16193.1C0 17 0

1 3,524 21

2 21,521 20,333

3 1,852 5,252

Option: query all possible annotations

16181C 16182C 16183C 16189C 16213A 16217C 16242T 16261T 16292T 16301T 16519C61A 62A 73G 183G 263G 309.1C 309.2C 309.3C 315.1C 323N 324N 523Del 524Del

> 900.000 possible alignments tolerating 24+1 mutations

CHN.ASN.000206 (B4g, 24 differences to rCRS)

Option: translate haplotype into string of sequences and compare strings

Database query can be performed in string search mode Query haplotypes and database haplotypes are converted into strings of nucleotidesStrings are compared and query results are reported on the basis of the string search

RationaleThis search mode is useful in cases where multiple sequence alignments are feasible as it avoids that haplotypes are missed in a search due to different notations

It does not replace the presentation and use of the phylogenetic alignment for mtDNA haplotypessee examples EMPOP-Test

String-based mtDNA search


Heteroplasmy


BloodSequence (Point)Heteroplasmy

Length Heteroplasmy

Hair 1 Hair 2

Point heteroplasmy


Point Heteroplasmy

uneven ddNTP incorporation rates – mixture is sequence-specificmixture detection not necessarily quantitativeIUPAC-codevariable in tissues – forensic implications

GEDNAP 32 (2006) Stain 3 – Mixture of hg J1c (25%) and D5a (75%)

16069Y 16266Y 16362Y 228R

Length Heteroplasmy


Length Heteroplasmysemi quantitative detection(hyper)variable in tissuescall dominant type

G16196

A16183C G16196

9C (16193del) ~ 5%10C (rCRS) ~ 25%11C (16193.1C) ~ 50%12C (16193.2C) ~ 20%

T16189C

Source of observed point heteroplasmy


Tissue

Cell

Lutz-Bonengel et al (2007)

Cell sorter Microscopic control Low volume PCR Seq / miniseq

96%

4%Lymphocytes

Point heteroplasmy


Rogaev et al (2009) Coble et al (2009)Ivanov et al (1996)Gill et al (1994)

16169C/T (T2a1a)

Heteroplasmy in hair shafts

W. Parson – Mitochondrial DNA and EMPOPTully et al (2004)

EDNAP study 55 hair shafts by 10 laboratories

7.1

7.2

7.3

7.47.5 Lab A

Lab B

Ref.

16093C 16129A 16162G 16172C 16234Y 16304C 73G 249DEL 263G 309.1C 315.1C (hg F1a1)

Donor‘s haplotype (blood)

ResultsDifferent segregation of 16234Yat varying ratiosAlso at 16093 and HV2 stretch16129 transition in one hair16195 PHP in one hair segment16304 PHP in one hair segment

0

5

10

15

20

25

30

35

40

45

1602

4

1605

116

078

1610

516

132

1615

9

1618

616

213

1624

016

267

1629

416

321

1634

816

375

1640

216

429

1645

616

482

1650

916

536

1656

3 21 48 77 104

131

158

185

212

239

269

297

322

349

378

405

433

460

490

519

553

Num

ber o

f Obs

erva

tions

16093

16519

146

152

1618316189 195

204

16192215

Evaluation of PHP in 5.015 samples

W. Parson – Mitochondrial DNA and EMPOP Irwin et al (2009)

6% (311) of sampled individuals display PHP at 114 positions in the CR97% (302) single, 7 (2%) double and 1 (<1%) triple observationsSignificant correlation (p < 0.001) between heteroplasmy hotspots and substitution

hotspots determined from the same datasetOf the 27 sites at which PHP was observed 3 or more times:

21 were among the top 50 fastest evolving sites5 were within the top 100 fastest evolving sites2 sites 214 and 215 – NOT evolutionary hotspots (OH)

Average evolutionary rate of positions at which we observe PHP is 6.4 times greater than the average mutation rate over the entire CR

Sites at which PHP observed more than once are 11X “faster” than the average rate

Occurrence of heteroplasmy


Individuals are by and large homoplasmic despite:The presence of billions of mtDNA moleculesReplication that is not tied to mitotic or meiotic cell divisionsA mutation rate that is ~10X nuclear DNA rate

Why isn’t heteroplasmy ubiquitous?

Mechanisms of mtDNA heteroplasmy

W. Parson – Mitochondrial DNA and EMPOP Chinnery et al (2000)

Prevention of Müller’s Ratchet

W. Parson – Mitochondrial DNA and EMPOP Bergstrom and Pritchard (2000)

Population level

Intra-Individual level

“…a tight germline bottleneck reduces the long-term damage due to Müller’s ratchet”

Interpretation of mtDNA evidence


Guidelines (2000-2003) define

Exclusionwhen there are more than 2 differences between 2 sequences or“unequivocally different” including tissue specificity and “mutation rates”

Cannot Excludewhen sequences are identical (except for PHP and LHP in HVS-II C-tract)

Inconclusivefor one difference between two sequences

ISFG (Bär et al 2000), EDNAP (Tully et al 2001), SWGDAM (Budowle et al 2003)

Interpretation of mtDNA evidence


Endorsements:Tissue specificity

HairMuscle

Mutation ratesDifferences at phylogenetically stable positions provide more evidence for exclusion than differences at known hotspots

Insertions and deletionsDiscrimination between hotspot indels and stable insertion and deletion events

ack. Franz Neuhuber (Salzburg, Austria)

Casework example


HVS-I HVS-II HVS-III

16024

16365 73

340

16519C

263G

315.1C

453C

x x x xSuspect

16519C

263G

315.1C309.1C

x x xxVictim

16299G

x

146C

x

x x x

16519C

263G

315.1C

453C

x7 of 10 hair shafts

Database search


EMPOP 1.0 West Eurasian HVS-I/II: N=4476; CR: N=2242

a 95% upper bound confidence intervalb Balding and Nichols size bias correction (Balding and Nichols 1994)

16519C 263G 315.1C

CR (16024-576) funcorr UPCI a p (N+2)b

hotspots as individual differences 29/2242 1,293 1,761 1,381

ignoring hot spots 81/2242 3,613 4,385 3,699

HVS-I (16024-16365) HVS-II (73-340)

hotspots as individual differences 139/4476 3,105 3,614 3,149

ignoring hot spots 318/4476 7,105 7,857 7,146

Casework example


HVS-I HVS-II HVS-III

16024

16365 73

340

16519C

263G

315.1C

453C

x x x xSuspect

16519C

263G

315.1C309.1C

x x xxVictim

16299G

x

146C

x

x x x

16519C

263G

315.1C

453C

x7 of 10 hair shafts

Database search


EMPOP 1.0 West Eurasian HVS-I/II: N=4476; CR: N=2242

a Upper bound confidence intervalb Balding and Nichols size bias correction (Balding and Nichols 1994)c Confidence limit from zero proportion (Holland and Parsons 1999)

16519C 263G 315.1C 453C

CR (16024-576) funcorr UPCI a p (N+2)b UPCI a CL 0 propc

hotspots as individual differences 0/2242 n.a. n.a. 0,0089 0,213 0,134

ignoring hot spots 1/2242 0,045 0,132 0,134 0,285

mtDNA screening tools

W. Parson – 10 Years of EMPOP in forensic mtDNA analysis

Brandstätter et al (2006)Brandstätter et al (2003)

Casework example Madrid (Lourdes Prieto Solla):Hairs on victim (HV1/2) 73 263 309+C 315+CSuspect (HV1/2) 73 263 309+C 315+C

CR 16519CR 523/4 del

6776 (hg H3)3992 (hg H4)

MtDNA Databasing efforts


High quality mtDNA databases essential for forensicsBody of population dataList of “confirmed mutations” – etalon datasets (forensic data in EMPOP)Estimate mutation ratesEstimate heteroplasmy ratesCalibrate network filters

Haplotypes should cover entire CRDouble sequence strand coverageInclude codR SNPs (if necessary) for hg-affiliationEssential to know the geographic and social background of

samples

%Hg N C SA2* 14,14 9,33 10,87A2l 4,04 2,07 -A2m 3,03 2,07 -B2* 11,11 7,25 2,17B2b - 2,07 2,17B2e - - 6,52C1b* 17,17 5,18 2,17C1b6 - 2,07 6,52C1c 2,02 2,07 -C1d 2,02 3,63 4,35D1* 4,04 5,18 2,17D1e - 1,55 28,26D1f 1,01 4,15 -D1g 4,04 - -D4c2 1,01 - -D4h3a 2,02 1,55 4,35H 12,12 18,13 15,22HV0 1,01 4,66 4,35R*,R0*,R0a - 2,07 -U 11,11 10,88 2,17JT 7,07 10,88 4,35I,N1b,W,X 1,01 3,63 2,17M1a1 - 0,52 -L0,L1,L2,L3e 2,02 1,04 2,17

100,00 100,00 100,00

Argentinean mtDNA databasing project

Bobillo et al (2009)

A2*A2lA2mB2*B2bB2eC1b*C1b6C1cC1dD1*D1eD1fD1gD4c2D4h3aHHV0R*,R0*,R0aUJTI,N1b,W,XM1a1L0,L1,L2,L3e


EMPOP database

EMPOP is a global mtDNA database (www.empop.org)Collaborative effort between forensic and population genetic labs

First release launched October 2006Forensic data (4,527)

Literature data (646)

Second release pending (~10,000)Publication strategiesNew anonymization requirements


http://www.empop.org/

EMPOP data management


a not yet searchable in EMPOPb important additional information

Forensic data Literature data

Raw data full double strand information no / partial / single strand sequences

EMPOP QC yes yes

Point heteroplasmy yes no / no full availablility

Length heteroplasmy yes / dominant type no / no full availablility

Region CR / HVS-I and HVS-II all possible rangescodR SNPs / full genomesa codR SNPs / full genomesa

For forensic queries yes yesb

Etalon dataset yes no

Network filter calibration yes no

EMPOP 1 Data Reanalysis


Intention:Harmonization of LHP calls across contributions from different laboratories

Forensic/literature data (N=5,173)

Polymorphism change 2 haplotypes

Alignment change 7 haplotypes

Harmonization of LHP 17 haplotypes

Reading frame change 2 haplotypes

Documents

Mitochondrial DNA and EMPOP