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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
W. Parson – Mitochondrial DNA and EMPOP
medical genetics
population genetics
forensic genetics
Mitochondrial DNA
W. Parson – Mitochondrial DNA and EMPOP
• 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
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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.
W. Parson – Mitochondrial DNA and EMPOP
W. Parson – Mitochondrial DNA and EMPOP
MtDNA phylogeny
Forster (2004)
W. Parson – Mitochondrial DNA and EMPOP
MtDNA phylogeny
Kivisild et al (2006)
W. Parson – Mitochondrial DNA and EMPOP
MtDNA phylogeny
Underhill and Kivisild (2007)
Forensic relevance:huge database (HVS-I and codSNPs) but unknown sample origin
The Genographic Project
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
Forensic practice of mtDNA typing
W. Parson – Mitochondrial DNA and EMPOP
ExtractionQuantitation
MtDNA - specific
Amplification/sequencingControl regionWhole mtDNA genome
Haplotype annotationForensic implications
Interpretation (casework)Database searchesStatistics
DNA Extraction
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
MtDNA amplification and sequencing
W. Parson – Mitochondrial DNA and EMPOP
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
MtDNA amplification and sequencing
W. Parson – Mitochondrial DNA and EMPOP
Full CR analysis with overlapping primers
Brandstätter et al (2004)
CR sequencing (“good DNA”)
W. Parson – Mitochondrial DNA and EMPOP
„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)
tRNApro HVI HVIIIHVII
16024 44 340116400 438 576
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)
CR sequencing (“good DNA”)
W. Parson – Mitochondrial DNA and EMPOP
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)
W. Parson – Mitochondrial DNA and EMPOP
„Midis“ – 282-444bpBerger and Parson (2009)
„Minis“ – 144-237bpEichmann and Parson (2008)
see also Gabriel et al (2001)
Full mtDNA sequencing
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
16311 T>C
MtDNA reference sequences
W. Parson – Mitochondrial DNA and EMPOP
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)
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
Heteroplasmy
W. Parson – Mitochondrial DNA and EMPOP
BloodSequence (Point)Heteroplasmy
Length Heteroplasmy
Hair 1 Hair 2
Point heteroplasmy
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
Tissue
Cell
Lutz-Bonengel et al (2007)
Cell sorter Microscopic control Low volume PCR Seq / miniseq
96%
4%Lymphocytes
Point heteroplasmy
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – 10 Years of EMPOP in forensic mtDNA analysis
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
W. Parson – 10 Years of EMPOP in forensic mtDNA analysis
EMPOP data management
W. Parson – Mitochondrial DNA and EMPOP
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
W. Parson – Mitochondrial DNA and EMPOP
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
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