mtDNA haplogroup determination as an additional tool for - PDF document

mtDNA haplogroup determination as an additional tool for authenticating ancient East Asian mtDNA Hwan Young Lee, Ji-Eun Yoo, Myung Jin Park, Ukhee Chung, Chong-Youl Kim, Kyoung-Jin Shin Department of Forensic Medicine, Yonsei University College of Medicine, Seoul, Korea Human Identification Research Center, Yonsei University, Seoul, Korea DNA in Forensics 2006 Merits and limitations of ancient DNA analysis in human genetics � The analysis of ancient DNA is an ideal way to get a direct grip on the past � The vanishingly small traces of DNA fragments left behind in old specimens and the ubiquitous nature of contamination DNA make it very difficult to obtain reliable DNA sequence data from most ancient samples The Nine Criteria by Cooper and Poinar : A priori exclusion of the possibility of contamination A need for the addition of a tenth commandment! DNA in Forensics 2006 1

A posteriori test for the authenticity of ancient mtDNA data � Some indicators can positively exclude or question authenticity of ancient mitochondrial DNA sequencing results � Phylogeographic paradox (or the principle of phylogenetic expectation) � Mosaic structure � Abnormal mutation spectrum HJ Bandelt Eur. J. Hum. Genet. (2005) 13: 1106-1112 DNA in Forensics 2006 Haplogroup determination as a tool for a posteriori authenticity test � East Asian haplogroup determination is efficiently carried out through haplogroup-level coding region SNP analysis and subhaplogroup-level control region sequence analysis � Especially, high incidence of haplogroup-specific mutations in the control region sequence of East Asian mtDNA enables to check the presence of phylogeographic paradox and mosaic structure with ease DNA in Forensics 2006 2

East Asian mtDNA haplogroup determination in Koreans � According to the control region mutation motifs, each obtained haplotype is assigned to appropriate subhaplogroup � Depending on the results, diagnostic coding region 3 multiplexes SNPs are confirmed A M D4 M11 N9 M9 D5 M10 Multiplex-1 M7 M8 G D R9 R B Multiplex-2 D4b D4 D4a D4e D4g D4j D4h Multiplex-3 ( Lee et al. Electrophoresis in press) DNA in Forensics 2006 mtDNA analysis from ancient Korean human remains � 35 museum samples ranged from the Paleolithic age to Goryo dynasty 700 Ky BP BC 8000 BC900 BC18 AD668 AD935 AD1392 Paleolithic Age Neolithic Age Bronze Age Three Kingdoms Shilla Goryo 3 samples 5 samples 6 samples 8 samples 8 samples 4 samples DNA in Forensics 2006 3

Contamination precautions taken to ensure the reliability of results � Physical methods that remove the bone surface and UV irradiation that makes DNA unsuitable for PCR � Isolated laboratory where no post-PCR work has been conducted � Testing of control extracts in parallel with extracts from old specimens � Multiple extractions from the same samples at different times � Quantitation of amplifiable DNA using PCR DNA in Forensics 2006 Control region sequence analysis using small PCR amplicons � Mitochondrial DNA control region sequences were obtained from 8 small overlapping PCR fragments (133 – 177 bp) 73 438 574 16024 16365 340 HV1 HV2 HV3 177 bp 170 bp 167 bp 140 bp 145 bp 133 bp 143 bp M11 M23 S31 143 bp M12 M22 M21 M13 M14 The screening for cross-contamination or sample mix-up was required for assessing authenticity DNA in Forensics 2006 4

Coding region SNP scoring using three PCR multiplexes � Small amplicon sizes of the three PCR multiplexes enabled SNP score to be successfully analyzed in old skeletal remains 100 bp 115 bp 130 bp 145 bp 160 bp D5 M10 Multiplex I M11 D4 N9 M9 M A G Multiplex II M7 M8 R9 B R D D4 D4e D4b Multiplex III D4J D4a D4h D4g DNA in Forensics 2006 mtDNA haplogroup determination by the control region mutation motifs Control Region Sequence Sample Relative dating Haplogroup nt16024-nt16365 nt073-nt340 nt438-nt548 KO-02 † Paleolithic Age B4b1 16136-16182C-16183C-16189-16217- 73-199-202-207-263-309.1C-309.2C- 499 16284-16357 315.1C KO-06 † Three Kingdoms D4e1 16092-16187-16223-16362 73-94-263-315.1C 489 KO-07 Goryo F1a1 16129-16162-16172-16304 73-249d-263-309.1C-315.1C 523d-524d KO-08 † Goryo D6 16183C-16189-16223-16274-16362 73-263-309.1C-309.2C-315.1C 489 KO-09 Goryo A5c 16126-16129-16213-16223-16290- 73-152-235-263-309.1C-315.1C 16319 KO-18 † Goryo N9a1 16129-16189-16223-16257A-16261 73-150-263-309.1C-309.2C-315.1C KO-28 † Neolithic Age B4f 16168-16172-16183C-16189-16217- 73-200-257-263-309.1C-315.1C 16249-16266-16325 KO-29 Neolithic Age D4c 16223-16245-16362 73-263-315.1C 489 KO-30 Paleolithic Age G3a 16223-16274-16325-16362 73-143-152-263-309.1C-315.1C 489 KO-32 † Neolithic Age B4b1 16136-16183C-16189-16217-16284N 73-199-202-207-263-309.1C-315.1C 499 KO-34 Bronze Age D4 16223-16362 73-152-263-315.1C 489-523d- 524d KO-35 Bronze Age D4c 16223-16224-16245-16292-16362 73-146-263-315.1C 489 *Determined haplogroups and haplogroup-specific control region mutation motifs are indicated in blue and red, respectively † Haplotypes which have partial but articulate sequences DNA in Forensics 2006 5

Confirmation of the haplogroup- specific coding region SNPs � Diagnostic coding region SNPs were confirmed using monoplex SNaPshot, multiplex SNaPshot or sequencing Coding Region Sample Haplogroup M1 SNP A KO-02 B4b1 9bp del KO-06 D4e1 3010, 14668, 11215 B KO-07 F1a1 3970 M2 KO-08 D6 4883, 12654 KO-09 A5c 8794 KO-32 KO-18 N9a1 5417 KO-09 M3 D4e KO-28 B4f 9bp del KO-29 D4c 3010, 14668 KO-06 KO-30 G3a 4833 KO-32 B4b1 9bp del M3 D4 KO-34 D4 3010, 14668 KO-29 KO-35 D4c 3010, 14668 DNA in Forensics 2006 mtDNA haplogroup determination in ancient Korean human remains � Contamination from the investigators was excluded on the basis of mtDNA sequence comparison results � 12 of 35 mtDNAs were successfully assigned to appropriate East Asian mtDNA haplogroups or subhaplogroups � No compound haplotype � No mosaic structure � No abnormal mutation DNA in Forensics 2006 6

mtDNA analysis in samples from geographically distant region � A 800 year-old skeletal remain sample was obtained from East Mongolia � mtDNA haplogroup could be determined by the control region sequence analysis and the coding region SNP analysis � Haplogroup-directed database comparison was performed in 1192 East Asian mtDNA database 10400T HV1-HV2-HV3 region sequence M Multiplex I 16129-16152-16179-16192- 16223 -16362 9824C 73-263-315.1C 489 M7 Multiplex II DNA in Forensics 2006 Haplogroup-directed database comparisons to reveal mtDNA origin � Sequence variations shown in the skeletal remain were detected in the Ulchi of the Lower Amur, not in 1192 East Asian mtDNA database. This supports the sample authenticity HV1 sequence variation Skeletal remain found in Mongolia 16129-16152-16179 -16192- 16223-16362 M7 observed in Ulchi 16129-16152-16179 -16189- 16223-16362 Starikovskaya EB et al. Ann. Hum. Genet. (2005) 69:67-89 DNA in Forensics 2006 7

Concluding remarks � East Asian haplogroup determination and haplogroup- directed database comparison can be efficiently used for the detection of presence of phylogeographic paradox, mosaic structure and abnormal mutation in mtDNA control region sequences � We suggest mtDNA haplogroup determination and haplogroup-directed database comparison as an additional tool for authenticating ancient East Asian mtDNA besides rigid adherence to the 9 criteria suggested by Cooper and Poinar. DNA in Forensics 2006 Acknowledgement � to our lab members and to Koguryo Research Foundation for research fund support DNA in Forensics 2006 8