Value of Y-Chromosome Markers Y Application Advantage Forensic - - PDF document

2012-11-23 1

Understanding the Y chromosome variation by haplogroup and haplotype analyses in Koreans

Kyoung-Jin Shin, Ph.D. Department of Forensic Medicine Yonsei University College of Medicine

Value of Y-Chromosome Markers

Application Advantage

Forensic case work on sexual assault evidence Male-specific amplification (can avoid differential extraction to separate and epithelial cells) Paternity testing Male children can tied to fathers in motherless cases Missing persons investigations Patrilineal male relatives may be used for reference samples

Y

Non-Recombination Portion of Y chromosome (NRY) Human migration and evolutionary studies Lack of recombination enables comparison of male individuals separated by large periods of time Historical and genealogical research Surnames usually retained by males; can make links where paper trail is limited

Y-STRs (short tandem repeats) Y-SNPs (single nucleotide polymorphisms)

7 repeats 8 repeats AATG

2012-11-23 2

Focusing on Y-SNPs

n Increasing requests for information regarding ethnic and

geographical origin from specimens left at a crime scene.

n Globalization of crime suspects and victims n Increase of movement from East-southern population n Increase of excavation of Korean War victims and ancient mummies n Individual identification in mass disaster such as airplane crashes,

tsunamis or terrorist attacks where people from various geographical areas are involved

n Y-haplogroup ß combination of allelic states at SNPs

n Set of haplotypes defined by slowly mutating markers (mainly SNPs)

which have more phylogenetic stability

n Unique event polymorphisms (UEP) record history of Y chromosome

Y chromosome haplogroup

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Y chromosome phylogenetic tree

n In 2008, Karafet TM et al.

• Genome Res 2008;18:830-838

n 20 major clades n 311 haplogroups based on 599 binary

markers

n In 2011, Yan et al.

• Eur J Hum Genet 2011;19:1013-1015

n An updated tree of haplogroup O n Redefined marker KL2, JST002611,

PK4 and P164 A B C D E F G H I J K L M N O P Q R S T

Need for development of Y-SNP typing method according to the revised tree

Relationships between haplogroup and haplotype

n Y-STR haplotype mainly used in forensic field n The Y-STR variability within haplogroup

http://www.yhrd.org 693 population studies from 105 countries 9 loci haplotype (Minimal haplotype): 87440 haplotypes 11 loci haplotype (+ DYS438, DYS439): 59736 haplotypes 12 loci haplotype (+ DYS437): 39349 haplotypes 17 loci haplotype (Yfiler): 27532 haplotypes

Number Name Haplogroup DYS 19 DYS 389I DYS 389II DYS 390 DYS 391 DYS 392 DYS 393 DYS 437 DYS 438 DYS 439 N2341 Khan J2 13 13 30 24 9 14 14 14 12 11 N2350 Khan J2 13 14 29 24 9 15 14 14 12 11 N2351 Khan J2 14 13 29 24 9 14 14 14 12 12 N2984 Khan J2 13 14 30 26 9 15 14 14 12 12 N2990 Khan J2 13 14 30 23 9 14 14 14 12 11

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Relationships between haplogroup and haplotype

n Haplogroup prediction from Y-STR values

The possibility of haplogroup prediction based on the Y-STR haplotype information

Presentation Outline

n Development of multiplex systems for determination of East Asian Y

haplogroups for large reference samples

n Presentation of Y-haplogroup distributions in Koreans n Elucidation

• f

relationships between Y-haplogroups and Y-STR haplotypes in a Korean population

n Evaluation of haplogroup affiliation for Y-STR allele/haplotype n Representation of the substructure within haplogroup n Evaluation of possibility for haplogroup prediction based on Y-STR

haplotypes

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Materials and methods

Multiplex SBE reaction

Multiplex PCR

5’

A T

3’ 5’ 3’ 5’

G C

3’ 5’ 3’ 5’

T A

3’ 5’ 3’

C G

3’ 5’ 5’ 3’

T ddATP

A

3’ 5’

ddGTP C

G

3’ 5’

A

3’ 5’

ddTTP

T

G

3’ 5’

ddCTP

C

G A T C

SAP treatment Electrophoresis Exo-SAP purification SBE reaction

Multiplex allele-specific PCR

M174 78 C 81 T M9 127 G 130 C M231 153 A 156 T PCR Size SNP

5’ 3’

F primer R primer T A G F primer R primer C C F primer R primer T

Multiplex PCR

C T G C A

Electrophoresis

n Multiplex AS-I

Results – Development of multiplex PCR systems

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Haplogroup A,B DE (xD) D C C3 (xC3c, C3d, C3e) C3c C3d C3e G,H,I J1 K (xP, NO) P Q R NO* N O* O1* O1a* O1a1 O1a2 O2* O2a* O2a1 O2b* O2b1 O3* O3a* O3a1 (xO3a1c) O3a1c O3a2* O3a2a O3a2b O3a2c* O3a2c1* O3a2c1a* O3a2c1a1 O3a2c1b A, B DE D C F K O N 1 2 3 a a

M242 P203 Q 1 NO P 2 M110 a b 1 1 1 2 b c c PK4 1 a J1 M267 R M207 a 3 c d e b 1 M134 M174 M231 RPS4Y711 M9 M175 M119 P31 M95 SRY465 47z M122 M324 KL2 P201 JST002611 M7 P164 M117 M159 M89 M217 M48 M407 P53.1 P101 M162 M145 M214

n Multiplex allele-specific PCR assay Multiplex AS-I AS-II

Results – Development of multiplex PCR systems Results – concordance test

Multiplex SBE reactions

All SNPs genotypes were concordant between multiplex SBE reaction and multiplex allele-specific PCR

Multiplex allele-specific PCR

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n Discordance haplogroup O3a2c1a designation between M133 (multiplex

SBE reaction) and M117 (multiplex allele-specific PCR) in one sample

Results – concordance test

SBE reactions Allele-specific PCR M133 - M117 + ATCT deletion Wild

M133 M117 Confirm the allelic state of M133 and M117 by sequencing analysis

Results – distribution of haplogroups

n Distribution of Y haplogroups in 1006 Korean males

21 different haplogroups were identified by 33 Y-SNPs The haplogroup diversity : 0.8830 Discriminatory capacity : 2.1 %

O2b 24.2% O3a2c1a* 12.3% C3 13.4% KL2 JST002611 P164

The phylogenetic position

• f newly redefined markers

KL2, JST002611 and P164 was confirmed

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n Comparing known haplogroup frequencies from other study for Korean

Results – distribution of haplogroups

P > 0.05 P < 0.05 JST002611 Wild Mutant

The mutation of JST002611 is observed in frequently in East Asia. This study reported the presence of the haplogorup O3a1c-JST002611

Results – combined analysis with haplotype

n Non-equivalence between M117 and M133 markers

n Combined haplogroup and haplotypes analyses in 706 samples

with known 22-YSTR genotypes.

n M117 and M133 designate the haplogroup O3a2c1a n Non-equivalence M117(+) M133(-) samples were found. n A median-joining network was constructed to evaluate the

relationships of these samples based on 17 Y-STR haplotype information

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Relationships between haplotypes within haplogroup O3a2c1-M134

17 Y-STR loci (DYS19, DYS388, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS446, DYS456, DYS458, DYS635 and GATA H4)

O3a2c1a M117 + M133 + O3a2c1 M117 - M133 - non-equivalence M117 + M133 -

Allocation

• f

the M133 marker within phylogeny should be considered. More reliable M117 marker for designating haplogroup O3a2c1a

Results – combined analysis with haplotype

Y-STR Gene/haplotype di versity Mutation rate (x10-3)a % Variance Among haplogroups Within haplogroups DYS19 0.7115 1.76 44.82 55.18 DYS385 0.9595 2.09 15.00 85.00 DYS389-I 0.6675 2.44 46.07 53.93 DYS389-II 0.7244 2.60 19.04 80.96 DYS390 0.6733 2.29 44.43 55.57 DYS391 0.2667 3.11 24.87 75.13 DYS392 0.6789 0.68 76.17 23.83 DYS393 0.6281 0.81 66.17 33.83 DYS437 0.4320 2.02 65.59 34.41 DYS438 0.6330 0.33 84.84 15.16 DYS439 0.6329 5.37 12.80 87.20 DYS448 0.7510 0.00 61.27 38.73 DYS456 0.5108 5.59 31.34 68.66 DYS458 0.7779 8.38 10.91 89.09 DYS635 0.6857 5.66 26.24 73.76 GATA H4 0.6115 3.43 38.28 61.72 DYS388 0.4850 0.00 72.33 27.67 DYS446 0.7886 2.71 31.95 68.05 DYS447 0.7520 5.41 42.78 57.22 DYS449 0.8523 18.97 14.86 85.14 DYS464 0.9668 3.99 16.71 83.29

Diversity, mutation rate and AMOVA analysis for each Y-STR marker and haplotype

Results – combined analysis with haplotype

Y-STR Gene/haplotyp e diversity Mutation rate (x10-3)a % Variance Among haplogroups Within haplogroups 6 Y-STRs 0.9317

• 71.15

28.85 9 Y-STRs 0.9966

• 42.09

57.91 17 Y-STRs 0.9995

• 41.52

58.48 22 Y-STRs 0.9999

• 38.95

61.05 DYS392-DYS393-DYS438-DYS437-DYS448-DYS388

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Results – combined analysis with haplotype

Haplotype (392-393-438-437-448-388) N % Haplogroup YHRD database release 39 DE D C C3 F Q N O 11-13-10-14-17-12 10 55.6 D-M174 88 88 1 1 11-13-10-14-19-12 2 11.1 222 208 3 2 11-13-11-14-17-12 2 11.1 29 28 11-15-10-14-21-12 (or 13) 23 26.4 C3-M217 115 38 11-14-10-14-21-12 (or 13) 19 21.8 2 74 29 11-14-10-14-22-13 12 13.8 45 15 1 11-13-11-14-0-13 4 4.6 11 5 3 14-14-12-14-19-12 7 53.8 Q-M207 20 18 15-14-12-14-19-12 3 23.1 8 5 14-13-10-14-20-13 5 18.5 N-M231 17 14 2 14-13-10-14-19-12 (or 13) 4 14.8 39 27 10 15-13-10-14-19-12 (or 13) 4 14.8 15 14 16-14-11-14-19-12 3 11.1 10 10 14-13-10-14-18-12 3 11.1 60 8 52 Haplotype (392-393-438-437-448-388) N % Haplogroup YHRD database release 39 O O1a O1a1 O2 O2b O2b1 14-13-10-14-18-12 13 68.4 O1a1-P203 52 44 13 1 16-13-10-14-18-12 2 10.5 3 3 2 13-13-10-14-18-12 3 30.0 O2*-P31 15 1 11 13-14-10-14-18-12 2 20.0 45 37 13-13-13-14-18-12 (or 13) 124 80.0 O2b*-SRY465 714 398 398 128 13-13-13-14-18-12 46 64.8 O2b1-47z 714 398 398 128

Results – combined analysis with haplotype

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Haplotype (392-393-438-437-448-388) N % Haplogroup YHRD database release 39 O3 O3a O3a2 O3a2b O3a2c1 O3a2c1a 13-12-10-15-21-12 6 54.5 O3a1-KL2 12 10 2 12-12-10-15-21-12 3 27.3 7 6 13-12-10-14-19-12 24 33.3 O3a1c-JST002611 129 96 10 13-12-10-14-20-12 16 22.2 70 55 16 13-12-10-14-20-13 7 16.7 O3a2*-P201 70 55 16 13-12-10-15-20-13 25 59.5 121 93 77 4 1 13-12-10-15-20-12 3 100 O3a2b-M7 121 93 77 4 1 13-12-9-15-19-12 3 33.3 O3a2c*-P164 4 4 4 13-13-10-15-19-12 2 22.2 22 18 15 12-12-10-15-19-12 36 52.9 O3a2c1-M134 122 122 115 77 12-13-10-15-19-12 9 13.2 30 30 28 20 14-12-11-15-20-10 53 58.9 O3a1c1a-M117 211 211 202 150 110

Results – combined analysis with haplotype

n Relationships between Y-STR variants and haplogroups

n DYS447 unusually short allele n Median-network in haplogroup O3a1-KL2

Results – combined analysis with haplotype

Y-STR Allele Structure Haplogroup N DYS447 18 (TAATA)7TAAAA(TAATA)10TAAAA(TAATA)n O3a1-KL2 2 19 (TAATA)7TAAAA(TAATA)11TAAAA(TAATA)n O3a1-KL2 4

Unusually short allele At DYS447 short allele representing single haplogroup background

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n Relationships between Y-STR variants and haplogroups

n DYS449 atypical allele

Y-STR Allele Structure Haplogroup N DYS449 25 (26)

(TTTC)12-tctctctcctcctc-(tttc)2-N4-(tttc)2-N12-cttc- (TTTC)14

J1-M267 1 27.2

(TTTC)3TC(TTTC)10-tctctctcctcctc-(tttc)2-N4-(tttc)2-N12-cttc-(TTTC)14

O2b1-47z 1 28.2

(TTTC)3TT(TTTC)11-tctctctcctcctc-(tttc)2-N4-(tttc)2-N12-cttc-(TTTC)14

C3-M217 1 29.2

(TTTC)3TC(TTTC)11-tctctctcctcctc-(tttc)2-N4-(tttc)2-N12-cttc-(TTTC)15

O2b1-47z 1 30.1 (30)

(TTTC)16-tctctctcctcctc-(tttc)2-N4-(tttc)2-N12-ctttc-(TTTC)14

O3a1c-JST002611 1 30.2

(TTTC)16-tctctctcctcctc-(tttc)2-N4-(tttc)2-N12-cttc-(TTTC)10TT(TTTC)4

O1a1-P203 12 42 (33)

(TTTC)15-tctctctcctcctc-[(tttc)2-N4-(tttc)2-N12-cttc]2-(TTTC)18

O3*-M122 1

n Relationships between haplotypes within haplogroup O1a1-P203

Allele 30.2 At DYS449, allele 30.2 representing single haplogroup background

Results – combined analysis with haplotype

n Relationships between Y-STR variants and haplogroups

n DYS464 variants n Median-network in haplogroup N-M231

Y-STR Allele Structure Haplogroup N DYS464 12.3 (CCTT)7CTT(CCTT)5 O3a2*-P201 1 14.3 (CCTT)3CTT(CCTT)11 N-M231 5

At DYS464, allele 14.3 did not form district cluster Allele 14.3

Results – combined analysis with haplotype

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n Relationships between Y-STR variants and haplogroups

n DYS448 null allele AZFa q11.21

q11.221 q11.222 q11.223q11.23

AZFb

AZFc

DYS 464 DYS 464 DYS 464

sY1201 sY1161 sY1191 sY1291 sY1206 sY1206 sY1161

DYS 464 DYS 448

p11.2 p11.31 p11.32

50f2/C

u2 b1 u3 u1 t1 t2 b2 g1 r1 r2 b3 g2 r3 r4 g3 b4

b1/b3 deletion polymorphic 50f2/C deletion

DYS448 DYS464 Type of deletion in AZFc region Offspring Haplogroup N Null 11-12-17 polymorphic 50f2/C deletion n.k C3-M217 3 Null 14-16 b1/b3 deletion Y O3a2*-P201 2 Null 11-17 b1/b3 deletion Y C3-M217 1

Results – combined analysis with haplotype

n Relationship between haplotypes carrying DYS448 null allele

50f2/C deletion sample b1/b3 deletion sample 50f2/C deletion mutation belong to haplogroup C3-M217

n The allele-specific PCR assays were optimized for simultaneous

detection of Y-SNPs followed by fragment analysis on an automatic DNA analyzer like general forensic STR typing method and useful for simple and rapid identification of the haplogroups in large number of samples.

n A total of 21 differenct haplogroups were identified by 33 Y-SNPs

using the multiplex allele-specific PCR, multiplex SBE reactions as well as additional monoplex SBE reactions.

n Phylogenetic non-equivalence was found between SNPs M117 and

M133, suggesting that the position of the M133 should be corrected.

Summary

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n

DYS392-DYS393-DYS438-DYS437-DYS448-DYS388, which markers are showed the relatively slower mutation rates, could be used for rough major haplogroup prediction through matching the haplotypes with known haplogroups in the reference database.

n

The DYS447, DYS449 and DYS464 markers have a higher mutation rate, their atypical alleles seem to reflect an independent single mutation event, thereby acting like binary markers.

n

Our finding and the dataset

• f

Y-STR/Y-SNP provide useful information for inferring haplogroup background as well as for forensics, population genetics and resolving male genealogies in East Asia.

Summary

n This presentation material was prepared by Myung Jin

Park in the course of her doctoral dissertation.

Acknowledgement