Forensic genetic analysis of nine miniSTR loci in the Korean population

Legal Medicine 11 (2009) 209–212

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Forensic genetic analysis of nine miniSTR loci in the Korean population Myun Soo Han a,1, Yang Seop Kim b,1, Han Jun Jin b, Jong Jin Kim a, Kyoung Don Kwak a, Jong Eun Lee c, Joon Myong Song d, Wook Kim b,* a

DNA Analysis Section, National Institute of Scientific Investigation, Seoul 158-097, South Korea Department of Biological Sciences, Dankook University, Cheonan 330-714, Republic of Korea c DNA Link, Yonsei University Milk Building 2F, Seoul 120-110, South Korea d Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, South Korea b

a r t i c l e

i n f o

Article history: Received 28 July 2008 Received in revised form 31 January 2009 Accepted 25 March 2009 Available online 6 May 2009 Keywords: MiniSTR D1S1677 D2S441 D4S2364 D10S1248 D12S391 D14S1434 D16S3253 D20S161 D22S1045 Forensic genetics Koreans

a b s t r a c t Nine miniSTR loci were analyzed in 191 unrelated individuals from Korea using three multiplex PCR systems (multiplex I: D1S1677, D2S441 and D4S2364; multiplex II: D10S1248, D14S1434 and D22S1045; multiplex III: D12S391, D16S3253 and D20S161). Due to the short PCR amplicons (<145 bp), miniSTR systems can effectively be used in forensic analysis with highly degraded DNAs. Allele frequencies and forensic parameters were calculated to evaluate their usefulness in forensic casework. The Exact Test demonstrated that all loci surveyed here were found to be no deviation from Hardy–Weinberg equilibrium, except two miniSTR markers (D4S2364 and D16S3253). When we compared the distribution of genetic variation of six miniSTR markers (D1S1677, D2S441, D4S2364, D10S1248, D14S1434 and D22S1045), the Exact Test revealed significant differences (P < 0.05) between the Korean sample studied here and almost all of other samples of East Asian and European populations. The combined probability of match calculated from nine miniSTR loci was 1.28  10 8, which is high degree of polymorphism. Thus, the miniSTR system, combined with other valuable miniSTR markers, may be suitable for recovering useful information in analyzing degraded DNA samples. Ó 2009 Elsevier Ireland Ltd. All rights reserved.

Population: We studied 191 healthy Koreans selected at random (and therefore likely to be unrelated) from Cheonan in Korea. Informed consent was obtained from all the donors before collecting their blood or buccal cells. Extraction: Genomic DNA was extracted from whole blood samples by the standard method of phenol–chloroform–isoamyl alcohol extraction [1], or from buccal cells according to Richards et al. [2]. PCR: Three triplex PCRs were performed with the same primer sets designed by Coble and Butler [3] and Asamura et al. [4]. Changes were made to the fluorescent dye labeled to accommodate subsequent PCR fragment analysis detection. The forward primers for D4S2364, D10S1248 and D16S3253 were labeled with 6FAM, D2S441, D14S1434 and D20S161 with HEX, and D1S1677, 12S391 and D22S1045 with NED. Each PCR multiplex was carried out in a total volume of 10 ll containing 1 ng genomic DNA, 1 PCR Gold buffer (Applied Biosystems, Foster City, CA, USA), 1.0 U * Corresponding author. Tel./fax: +82 41 550 3441. E-mail address: [email protected] (W. Kim). 1 These authors contributed equally to this work. 1344-6223/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2009.03.012

of AmpliTaq GoldÒ DNA polymerase (Applied Biosystems, Foster City, CA, USA), 1.5 mM MgCl2, 200 lM of each dNTP, and similar primer concentration as published [3,4]. Thermal cycling was conducted on a GeneAmpÒ PCR System 9700 (Applied Biosystems, Foster City, CA, USA) with slight modification in PCR condition [3,4]. Initial denaturation was carried out at 95 °C for 10 min, followed by 30 cycles of 94 °C for 30 s, 55 °C (except for multiplex III: 58 °C) for 30 s, 72 °C for 45 s, and final extension of 60 °C for 45 min. Detection and genotyping: The PCR products were mixed with GeneScan 500 ROX Size Standard (Applied Biosystems, Foster City, CA, USA). Capillary electrophoresis was performed on the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The results were analyzed using GeneScanÒ 3.1.2 software (Applied Biosystems, Foster City, CA, USA). Allele designations were determined using Genotyper 2.5 software (Applied Biosystems, Foster City, CA, USA) by comparison with sequenced allelic ladders. The allelic ladders for multiplex I, II and III were created using combination of individual templates, which represent the range for alleles observed in this study. The allele nomenclatures for five miniSTR loci (D4S2364, D1S1677, D10S1248, TM

TM

TM

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Table 1 Genetic characteristics of nine miniSTR loci examined in the present study. Locus

9948 DNA allele

Allele spread

Allele size range (bp)

Allele correctiona

Repeat motif

Multiplex I D1S1677 D2S441 D4S2364

13,14 11,12 9,10

10–17 10–15 7–10

80–108 81–101 67–79

+1 repeat – 1 repeat

[TTCC]n [TCTA]n [GAAT][GGAT][GAAT]n

Multiplex II D10S1248 D14S1434 D22S1045

12,15 13,14 16,18

11–17 10–16 11–19

91–115 73–97 85–109

1 repeat 4 repeats +3 repeats

[GGAA]n [CTGT]m[CTAT]n [ATT]n

Multiplex III D12S391 D16S3253 D20S161

18,24 10,11 17,18

15–25 6–16 15–21

97–137 75–115 100–124

a

[AGAT]m[AGAC]n[AGAT] [TAGA]n [TAGA]l[TAGG]m[TAGA]n

The corrected allele repeats. The allele nomenclatures were different from Coble and Butler [3].

D14S1434 and D22S1045) were corrected based on the sequencing analysis results of observed alleles at each locus (Table 1). At least two different homozygous samples for each miniSTR markers were sequenced to confirm the allele nomenclature as the same procedure described in Chung et al. [5]. Results: See Tables 1–3 and Fig. 1. Quality: Commercial DNA standard 9948 (Promega Corporation, Madison, WI, USA) was genotyped as standard reference. We have used sequenced allelic ladders, and a concordant study was performed to ensure reproducible and accurate genotyping

by a re-genotyping procedure using more than 20 samples for each multiplex. Analysis of data: General goodness-of-fit test of v2-test and Gtest (likelihood ratio test) were used to assess Hardy–Weinberg equilibrium (HWE). Possible divergence from HWE was also examined by the Exact Test [6]. These statistical analyses were performed with the PowerMarker v3.25 [7] and ARLEQUIN Version 3.11, http://lgb.unige.ch/arlequin, developed by S. Schneider, D. Roessli and L. Excoffier. Important parameters of forensic paternity testing were calculated using the PowerStats v1.2 computer program (Promega, Web site, http://www.promega.com.geneticid-

Table 2 Allele frequencies and statistical parameters for nine miniSTR loci in the Korean population (n = 191). Allele

D1S1677

D2S441

D4S2364

D10S1248

D14S1434

D22S1045

D12S391

D16S3253

D20S161

6 7 8 9 10 11 11.3 12 13 14 15 16 17 18 19 19.3 20 21 22 23 24 25 Hobsa Hexpb v2-test (P) G-test (P) Exact Test (P) PMd PDe PICf PPEg

– – – – 0.005 0.005 – 0.013 0.115 0.476 0.306 0.073 0.005 – – – – – – – – – 0.660 0.670 0.756 0.791 0.234 0.182 0.818 0.610 0.384

– – – – 0.157 0.560 0.016 0.131 0.055 0.073 0.008 – – – – – – – – – – – 0.636 0.602 0.654 0.747 0.524 0.171 0.829 0.600 0.293

– 0.026 0.100 0.432 0.442 – – – – – – – – – – – – – – – – – 0.607 0.733 0.000 0.000 0.000 0.337 0.663 0.530 0.481

– – – – – 0.005 – 0.055 0.377 0.225 0.236 0.086 0.016 – – – – – – – – – 0.741 0.764 0.719 0.823 0.880 0.110 0.890 0.700 0.535

– – – – 0.107 0.165 – 0.021 0.249 0.445 0.011 0.003 – – – – – – – – – – 0.701 0.665 0.968 0.909 0.881 0.129 0.871 0.660 0.376

– – – – – 0.189 – 0.005 – 0.005 0.327 0.238 0.207 0.024 0.005 – – – – – – – 0.757 0.775 0.119 0.554 0.192 0.105 0.895 0.720 0.553

– – – – – – – – – – 0.011 0.005 0.113 0.309 0.209 0.005 0.152 0.097 0.060 0.024 0.013 0.003 0.811 0.806 0.999 0.995 0.941 0.063 0.937 0.790 0.611

0.052 – 0.131 0.053 0.343 0.374 – 0.016 0.011 0.018 – 0.003 – – – – – – – – – – 0.719 0.608 0.000 0.000 0.000 0.138 0.862 0.670 0.300

– – – – – – – – – – 0.042 0.170 0.390 0.189 0.118 – 0.090 0.003 – – – – 0.760 0.748 0.256 0.314 0.118 0.094 0.906 0.730 0.508

a b c d e f g

c

Hobs: observed heterozygosity. Hexp: expected heterozygosity. Exact Test (Monte Carlo method). PM, probability of match. PD, power of discrimination. PIC, polymorphism information content. PPE, paternity power of exclusion.

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M.S. Han et al. / Legal Medicine 11 (2009) 209–212 Table 3 Cavalli-Sforza’s cord genetic distance (4D) values among eight Eurasian populations using six miniSTR markers only. Population (1) (2) (3) (4) (5) (6) (7) (8) a b c d e

a

Korean Japaneseb Han chinesec Korean chinesec Chinese singaporeand Malay singaporeand Indian singaporeand Spanishe

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

0.0000 0.0075 0.0080 0.0110 0.0061 0.0143 0.0171 0.0231

– 0.0000 0.0070 0.0070 0.0047 0.0114 0.0150 0.0185

– – 0.0000 0.0074 0.0057 0.0152 0.0159 0.0228

– – – 0.0000 0.0078 0.0135 0.0129 0.0180

– – – – 0.0000 0.0060 0.0135 0.0180

– – – – – 0.0000 0.0109 0.0136

– – – – – – 0.0000 0.0141

– – – – – – – 0.0000

Present study. Asamura et al. [11]. Bai et al. [12]. Yong et al. [13]. Martin et al. [14].

Fig. 1. Multidimensional scaling plot of Cavalli-Sforza’s cord genetic distances (4D); stress = 0.13. European is represented by open square, East Asian populations by open diamonds and Korean by closed diamond.

tools) [8]. We used the PHYLIP v3.57c [9] to compute genetic distances for use in distance matrix programs based on allele frequencies. Genetic distance was estimated by Cavalli-Sforza’s cord genetic distance (4D) [10]. The genetic distance values were also displayed as a multidimensional scaling (MDS) plot, using SPSS 12.0. Other remarks: Exact Test demonstrated that no significant deviations from the Hardy–Weinberg equilibrium were observed, except for D4S2364 and D16S3253 (Table 2), showing a similar figure to previous result from a Korean population [5]. Thus, larger sample sizes are necessary to understand the cause of deviation from the equilibrium model at the two loci surveyed here. The calculated parameters showed D12S391 to be the most polymorphic marker. In contrast, D4S2364 marker revealed to be the lowest variation from the nine miniSTR loci studied. Almost all of the other seven miniSTRs appear to have relatively high level of heterozygosity values. The combined probability of match (PM) from nine miniSTR loci was estimated to be 1.28  10 8, which is high degree of polymorphism. The main advantage of this system is short PCR product size that provides an effective tool for recovering useful information in analyzing degraded DNA samples. When we compared the distribution of genetic variation of six miniSTR markers

(D1S1677, D2S441, D4S2364, D10S1248, D14S1434 and D22S1045), no statistically significant difference in their allele frequencies was found between the present sample and previously reported Korean sample [5]. However, statistical analysis examined by the Exact Test using the ARLEQUIN Version 3.11 revealed significant differences (P < 0.05) in the distribution of six miniSTR allele frequencies between the Korean sample and almost all of other samples of East Asian and European populations (Table 3). This finding is consistent with an earlier comparison of allele distribution of six miniSTR loci between a Korean ethnic population in China and Japanese population and three Singapore populations [11–13]. Population division comparison based on 4D measures (Table 3) is also listed in Fig. 1 as MDS plot, which displayed quite population differentiation in the distribution of six miniSTR variation between the present Korean population and other Eurasian populations.

Acknowledgements We would like to thank all volunteers for proving DNA samples. This work was supported by a grant from the Korean Science Engi-

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neering Foundation (KOSEF, M10740040002-07N4004-00210), Republic of Korea. References [1] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory, Cold Spring Harbor; 1989. [2] Richards B, Skoletsky J, Shuber AP, Balfour R, Stern RC, Dorkin HL, et al. Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs. Hum Mol Genet 1993;2:159–63. [3] Coble MD, Butler JM. Characterization of new miniSTR loci to aid analysis of degraded DNA. J Forensic Sci 2005;50:43–53. [4] Asamura H, Fujimori S, Ota M, Fukushima H. MiniSTR multiplex systems based on non-CODIS loci for analysis of degraded DNA samples. Forensic Sci Int 2007;173:7–15. [5] Chung U, Shin KJ, Park MJ, Kim NY, Yang WI, Cho SH, et al. Population data of nine miniSTR loci in Koreans. Forensic Sci Int 2007;168:e51–3. [6] Guo SW, Thompson EA. Performing the Exact Test of Hardy–Weinberg proportion for multiple alleles. Biometrics 1992;48:361–72.

[7] Liu K, Muse SV. PowerMarker: integrated analysis environment for genetic marker data. Bioinformatics 2005;21:2128–9. [8] Tereba A. Tools for analysis of population statistics. Profiles DNA 1999;2: 14–6. [9] Felsenstein J. PHYLIP (phylogeny inference package) version 3.57c. Seattle: Department of Genetics, University of Washington; 1995. [10] Cavalli-Sforza LL, Bodmer MF. The genetics of human populations. San Francisco: Freeman WH; 1971. [11] Asamura H, Uchida R, Takayanagi K, Ota M, Fukushima H. Allele frequencies of the six miniSTR loci in a population from Japan. Int J Legal Med 2006;120:182–4. [12] Bai R, Shi M, Yu X, Lv J, Tu Y. Allele frequencies for six miniSTR loci of two ethnic populations in China. Forensic Sci Int 2007;168:e25–8. [13] Yong RY, Gan LS, Coble MD, Yap EP. Allele frequencies of six miniSTR loci of three ethnic populations in Singapore. Forensic Sci Int 2007;166: 240–3. [14] Martín P, García O, Albarrán C, García P, Yurrebaso I, Alonso A. Allele frequencies of six miniSTR loci (D10S1248, D14S1434, D22S1045, D4S2364, D2S441 and D1S1677) in a Spanish population. Forensic Sci Int 2007;169:252–4.