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Haplotype and mutation analysis for newly suggested Y-STRs in Korean father-son pairs

Yu Na Oh, Hwan Young Lee, Eun Young Lee, Eun Hey Kim,

Woo Ick Yang, Kyoung-Jin Shin

Department of Forensic Medicine

Yonsei University College of Medicine

Y chromosomal STRs in forensics

u Useful for the investigation of sexual assault cases and deficiency paternity testing of male offspring

u 17 Y-STR loci included in the AmpFℓSTR®Yfiler™ PCR amplification kit commonly used

u Continuous efforts to achieve higher discrimination led to adding new markers to the Yfiler 17 Y-STRs

u Reliable estimates of haplotype and mutation analysis required for non-erroneous interpretation of the genetic profiles

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Newly suggested Y-STR loci

Commercial kit17 STRs +

DYS576DYS481DYS549DYS533DYS570DYS643

17 STRs +

DYS627*DYS576DYS460DYS518*DYS449*DYS570DYS533DYF387S1*DYS481* RMY-STR

DYS460

DYS627

DYS518

DYS449

DYF387S1

DYS391

DYS385a/b

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Aim of the study

v Development of Y-STR multiplex PCR system analyzing

Ø New single copy Y-STR from PowerPlex Y23

Ø Rapidly mutating (RM) Y-STR from Yfiler plus simultaneously

üFinal amplicon size < 350 bp

üSensitivity able to genotype of 100 pg of genomic DNA

üSpecificity to analyze mixed DNA with [M:F=1:1000] ratio

üConstruction of allelic ladder for accurate genotyping

Haplotype and mutation analysis in Koreans

Method (1)

u Collect information and design optimal primer set for 14 Y-STRsü 6 Y-STRs for PowerPlex Y23: DYS481, DYS533, DYS549, DYS643,

DYS570 and DYS576

ü 4 RM Y-STRs: DYF387S1, DYS449, DYS518 and DYS627

ü DYS460, DYS391 and DYS385a/b

u Development of multiplex PCR system (Euplex Y14)

u Construction of allelic ladder, bin set and panel for genotyping

u Checking mutation on primer binding area using 100 Korean male samples

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PCR component Volume

dH20 5.4

Gold ST*R 10× Buffer 1× 1.0

5× Primer Mix 2.0

AmpliTaq Gold (5 U/uL) 2.5 U 0.6

Template DNA (1 ng/uL) 1.0

SUM 10.0

95℃ 11 min

94℃ 20 sec

60℃ 90 sec × 30 cycles

72℃ 60 sec

60℃ 45 min

4℃ forever

u Reaction Mixture u Thermal Cycle

u Multiplex PCR test

- Check unexpected primer interference

- Adjusting intervals between adjacent loci

- Remove incomplete adenylation

- Peak height balancing

Optimizing multiplex PCR system

Allelic size range of 14 Y-STR loci

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Electropherogram of 2800M standard DNA

Construction of allelic ladder

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Sensitivity and specificity test

[100 pg of 007 DNA][1 : 1000 mixture](100 pg of 007 DNA : 100ng of female DNA)

u Identification of mutations in father-son pairsl 363 Korean father-son haplotype transfers in 351 families were analyzed

using the developed multiplex PCR system (Euplex Y14).

l All pairs with a mutation were analyzed using the PowerPlex®16 HS

System (Promega) to confirm biological paternity.

l The protocol was approved by Institutional Review Board of Severance

Hospital at Yonsei University.

u Statistical analysis

l Haplotype frequencies and haplotype diversities were estimated according

to the Nei’s formula.

l Mutation rates were calculated as the number of mutations divided by the

number of allele transmissions.

Method (2)

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11 Y-STRs in Euplex Y14 Yfiler 17 Y-STRs

Locus Gene Diversity Locus Gene Diversity

387S 0.94566 385 0.96117

518 0.86134 458 0.79300

449 0.85635 448 0.76855

627 0.83279 389-II 0.72558

570 0.79689 19 0.70232

481 0.79407 392 0.68090

643 0.78997 390 0.67704

576 0.77436 635(C4) 0.67196

460 0.64943 389-I 0.66919

549 0.60645 439 0.65008

533 0.50740 393 0.63028

H4 0.60200

438 0.59038

456 0.53843

437 0.44182

391 0.24991

Gene diversity

Haplotype diversity

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Distinctive tri-allelic pattern at DYF387S1

Micro-variant on DYS449, 518, 570, 627 loci

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Gene diversities and mutation estimates for 28 Y-STRs

Mutations observed at more than one loci

Sample Locus Allele Type of mutation

CG29-F CG29-S 481 24 23 Loss (-1)

449 35 36 Gain (+1)

DJ022-F DJ022-S 576 18 17 Loss (-1)

385 13,20 13,19 Loss (-1)

DJ064-F DJ064-S

627 19 20 Gain (+1)

481 28 29 Gain (+1)

635(C4) 21 22 Gain (+1)

SN012-F SN012-S 576 20 19 Loss (-1)

635(C4) 22 21 Loss (-1)

SN109-F SN109-S 387S1 37,38 37,39 Gain (+1)

464 12,14,15,16 12,14,15,17 Gain (+1)

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Conclusions

v Newly suggested 11 Y-STRs with high gene and haplotype diversity values could be successfully used for male DNA analysis in Koreans.

v Twenty nine single step mutations were observed and two father-son pairs have mutations at two different loci at 11 Y-STRs with locus-specific mutation rates up to 1.93ⅹ10-2.

v DYS449, DYS576 and DYS627 loci showed mutation rate estimates over 1%. However, the mutation rate estimates at DYF387S1, DYS518 and DYS570 were considerably lower in Koreans.

Acknowledgements

v This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MEST) (No. 2012-0009833)

Yu Na Oh Eun Young Lee Hwan Young Lee