Analysis of 30 insertion–deletion polymorphisms in the Japanese population using the Investigator DIPplex® kit

Legal Medicine 17 (2015) 467–470

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Short Communication

Analysis of 30 insertion–deletion polymorphisms in the Japanese population using the Investigator DIPplexÒ kit Miya Nunotani, Tetsuya Shiozaki, Noriko Sato, Sayako Kamei, Kanae Takatsu, Tokutaro Hayashi, Masao Ota, Hideki Asamura ⇑ Department of Legal Medicine, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto, Nagano 390-8621, Japan

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Article history: Received 27 May 2015 Received in revised form 2 September 2015 Accepted 8 October 2015 Available online 8 October 2015 Keywords: Investigator DIPplexÒ kit Insertion–deletion polymorphisms (INDELs) Japanese population Degraded DNA

a b s t r a c t Allele frequencies and forensic parameters for 30 insertion–deletion polymorphisms (INDELs) were investigated in a sample of 251 unrelated Japanese individuals using the Investigator DIPplexÒ kit (QIAGEN). The frequency distributions showed no deviations from Hardy–Weinberg equilibrium expectations. The combined powers of discrimination and match probability for the 30 INDELs were 0.9999999998 and 2.67  10 11, respectively. To assess the effectiveness of the kit in typing degraded DNA, an ancient bone sample of a Jomon skeleton was analyzed; most of 30 INDELs and amelogenin were typed successfully. We concluded that the kit offers considerable potential for personal identification from degraded DNA samples due to the small amplicon length and high degree of polymorphisms. Ó 2015 Published by Elsevier Ireland Ltd.

1. Introduction Recent forensic interest has focused on insertion–deletion polymorphisms (INDELs or DIPs) for personal identification. Several authors have reported population genetic data for INDELs since the development of the first commercial kit (QIAGEN, Hilden, Germany) [1–10]. The kit can be used for typing 30 INDELs, and is designed to generate amplicons of less than 160 bp, with the optimal amount of DNA is set low volume (0.2–0.5 ng) according to the manual. Therefore, this technique may be capable of efficiently analyzing degraded samples. In this study, we investigated data obtained from a Japanese population for the 30 INDELs using the commercially available kit, and the allelic frequencies among the Japanese population data were compared with those of other populations. In addition, to assess the effectiveness of the kit in analyzing degraded DNA, we analyzed an ancient bone sample of a Jomon skeleton.

251 unrelated, healthy, adult Japanese individuals (154 males and 97 females). The Jomon skeleton, which was discovered at the Shimekake site in Nagano, Japan, was used as the highly degraded DNA sample. This skeleton is estimated to belong to the Jomon period, approximately 3,300–2,200 years ago [11].

2.2. DNA extraction DNA was extracted from blood or buccal mucosa cells using the QIAamp DNA Blood Mini Kit (QIAGEN). The quantity of DNA was determined using a BioSpec-nano (Shimazu, Kyoto, Japan). In addition, we extracted DNA from the Jomon skeleton according to a previously published protocol [12].

2.3. Polymerase chain reaction (PCR) 2. Materials and methods 2.1. Population This study was approved by the ethics committee of Shinshu University. With informed consent, we collected samples from ⇑ Corresponding author. E-mail address: [email protected] (H. Asamura). http://dx.doi.org/10.1016/j.legalmed.2015.10.002 1344-6223/Ó 2015 Published by Elsevier Ireland Ltd.

PCR amplifications were performed using the Investigator DIPplexÒ kit (QIAGEN) according to the manufacture’s manual. Amplification was carried out using GeneAmp System 9700 (Applied Biosystems, Foster City, CA, USA) in 9600 emulation mode at a final volume of 12.5 ll from the kit and 250 pg of genomic DNA. According to the manual, pre-denaturation was performed at 94 °C for 4 min, followed by 30 cycles of denaturing at 94 °C for 30 s, annealing at 61 °C for 120 s, extension at 72 °C for 75 s, and a final extension at 68 °C for 60 min.

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Table 1 Allele frequencies and forensic parameters of the 30 INDELs in 251 Japanese individuals. INDELs

DIP

DIP +

Ho

He

PIC

PD

MP

PE

TPI

HWE

HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD

0.562 0.452 0.629 0.323 0.582 0.910 0.574 0.478 0.078 0.568 0.375 0.133 0.482 0.576 0.404 0.614 0.727 0.616 0.484 0.763 0.639 0.155 0.086 0.436 0.645 0.649 0.295 0.695 0.795 0.309

0.474 0.548 0.371 0.677 0.418 0.090 0.426 0.522 0.922 0.432 0.625 0.867 0.518 0.424 0.596 0.386 0.273 0.384 0.516 0.237 0.361 0.845 0.914 0.564 0.355 0.351 0.705 0.305 0.205 0.691

0.5498 0.5060 0.4542 0.3984 0.5100 0.1633 0.4940 0.5020 0.1474 0.5299 0.5179 0.2112 0.5187 0.4900 0.5219 0.4781 0.3546 0.4980 0.5219 0.3624 0.4140 0.2630 0.1633 0.5538 0.4940 0.4143 0.4064 0.4502 0.3147 0.4581

0.4997 0.4964 0.4674 0.4380 0.4876 0.1635 0.4901 0.5000 0.1436 0.4918 0.4694 0.2312 0.5004 0.4895 0.4827 0.4752 0.3977 0.4742 0.5005 0.3147 0.4620 0.2390 0.1570 0.4929 0.4586 0.4563 0.4166 0.4246 0.3268 0.4277

0.37 0.37 0.36 0.34 0.37 0.15 0.37 0.37 0.13 0.37 0.36 0.20 0.37 0.37 0.37 0.36 0.32 0.36 0.37 0.30 0.35 0.23 0.14 0.37 0.35 0.35 0.33 0.33 0.27 0.34

0.595 0.617 0.611 0.597 0.607 0.287 0.617 0.623 0.258 0.600 0.584 0.376 0.574 0.618 0.595 0.609 0.563 0.599 0.613 0.528 0.619 0.416 0.280 0.586 0.586 0.612 0.574 0.570 0.492 0.570

0.405 0.383 0.389 0.403 0.393 0.713 0.383 0.377 0.742 0.400 0.416 0.624 0.426 0.382 0.405 0.391 0.437 0.401 0.387 0.472 0.381 0.584 0.720 0.414 0.414 0.388 0.426 0.430 0.508 0.430

0.235 0.193 0.150 0.133 0.196 0.021 0.182 0.189 0.017 0.215 0.204 0.033 0.269 0.179 0.207 0.169 0.089 0.186 0.207 0.070 0.120 0.041 0.021 0.239 0.182 0.123 0.118 0.148 0.070 0.153

1.11 1.01 0.92 0.83 1.02 0.60 0.99 1.00 0.59 1.06 1.04 0.63 1.20 0.98 1.05 0.96 0.77 1.00 1.05 0.73 0.85 0.66 0.60 1.12 0.99 0.85 0.84 0.91 0.73 0.92

0.1337 0.8021 0.6832 0.1979 0.5167 1.0000 1.0000 1.0000 1.0000 0.2528 0.1122 0.1697 0.0116 1.0000 0.2414 1.0000 0.1148 0.5039 0.5303 0.0568 0.1035 0.1516 1.0000 0.0573 0.2759 0.1696 0.7613 0.3785 0.5613 0.3031

77 45 131 70 6 111 58 56 118 92 93 99 88 101 67 83 114 48 124 122 125 64 81 136 133 97 40 128 39 84

DIP; DIP : deletion; DIP +: insertion; Ho: observed heterozygosity; He: expected heterozygosity; PIC: polymorphic information contents; PD: power of discrimination; MP: matching probability; PE: power of exclusion; TPI: typical paternity index; HWE: Hardy–Weinberg equilibrium.

Table 2 Comparison between the Japanese population and other Asian and European populations for 30 INDELs using the chi-square test.

HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD HLD

77 45 131 70 6 111 58 56 118 92 93 99 88 101 67 83 114 48 124 122 125 64 81 136 133 97 40 128 39 84

South Korea [1]

Chinese [2]

Taiwanese [3]

Danish [4]

Spanish [5]

Basque [5]

Northeast-Italian [6]

Czech [7]

German [8]

Finnish [9]

0.652 0.141 0.721 0.116 0.641 0.269 0.001 0.829 0.042 0.691 0.486 0.007 0.732 0.761 0.002 0.714 0.591 0.781 0.161 0.306 0.847 0.606 0.021 0.528 0.456 0.445 0.004 0.118 0.000 0.011

0.901 0.000 0.003 0.000 0.001 0.000 0.357 0.021 0.000 0.037 0.024 0.000 0.033 0.000 0.278 0.026 0.007 0.149 0.364 0.000 0.000 0.004 0.000 0.322 0.011 0.674 0.000 0.022 0.279 0.566

0.641 0.001 0.036 0.609 0.006 0.002 0.769 0.011 0.411 0.348 0.221 0.348 0.441 0.347 0.093 0.366 0.711 0.170 0.608 0.026 0.002 0.651 0.000 0.001 0.945 0.302 0.082 0.217 0.317 0.269

0.004 0.359 0.001 0.000 0.018 0.000 0.003 0.005 0.000 0.418 0.662 0.000 0.279 0.214 0.967 0.000 0.021 0.000 0.374 0.000 0.000 0.000 0.000 0.121 0.000 0.000 0.000 0.003 0.000 0.000

0.125 0.790 0.005 0.150 0.060 0.000 0.014 0.000 0.000 0.256 0.011 0.000 0.045 0.057 0.503 0.004 0.000 0.000 0.005 0.000 0.024 0.000 0.000 0.875 0.000 0.000 0.000 0.001 0.000 0.055

0.009 0.345 0.794 0.567 0.015 0.000 0.000 0.061 0.000 0.756 0.004 0.000 0.020 0.857 0.010 0.001 0.255 0.003 0.001 0.030 0.020 0.000 0.000 0.006 0.000 0.000 0.000 0.000 0.096 0.024

0.201 0.133 0.000 0.000 0.076 0.000 0.067 0.001 0.000 0.405 0.015 0.000 0.833 0.509 0.583 0.000 0.000 0.000 0.000 0.000 0.111 0.000 0.000 0.190 0.000 0.000 0.000 0.000 0.000 0.000

0.175 0.964 0.000 0.000 0.223 0.000 0.000 0.004 0.000 0.924 0.166 0.000 0.085 0.021 0.021 0.332 0.181 0.006 0.000 0.000 0.000 0.000 0.000 0.037 0.000 0.001 0.000 0.004 0.010 0.000

0.051 0.643 0.000 0.002 0.004 0.000 0.056 0.003 0.000 0.694 0.089 0.000 0.102 0.115 0.018 0.869 0.021 0.000 0.016 0.000 0.000 0.000 0.000 0.121 0.000 0.000 0.000 0.000 0.000 0.023

0.105 0.341 0.000 0.000 0.000 0.000 0.010 0.009 0.000 0.610 0.646 0.000 0.299 0.003 0.917 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.027 0.000 0.001 0.000 0.000 0.000 0.000

Significant differentiation after Bonferroni correction (p < 0.005).

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Fig. 1. Electropherogram of the DNA typing results of a Jomon sample. (a) AmpFLSTR IdentifilerÒ Plus PCR Amplification Kit. (b) Investigator DIPplexÒ kit.

2.4. INDELs typing and statistical analysis Electrophoresis was performed using an ABI PRISM 3130-Avant Genetic Analyzer (Applied Biosystems), following the manufacturer recommendations. Data were analyzed using GeneMapper ID 3.2.1 software (Applied Biosystems). Allele frequencies, observed heterozygosity (Ho), polymorphic information contents

(PIC), power of discrimination (PD), matching probability (MP), power of exclusion (PE), and typical paternity index (TPI) were calculated using the PowerStats v12 software package (Promega, Madison, WI, USA). Expected heterozygosity (He) and Hardy– Weinberg equilibrium (HWE) were determined using GENEPOP v4.2.

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2.5. Quality control Analysis of the DNA polymorphisms was conducted following ISFG recommendations [13]. 2.6. Analysis of highly degraded DNA sample We analyzed the degraded DNA samples to determine whether the system were capable of effectively analyzing degraded DNA. Amplification was carried out using the Investigator DIPplexÒ kit at a final volume of 12.5 ll from the kit and 1 ll of DNA solution from the Jomon skeleton (the PCR cycle number was 30 cycles). On the other hand, we performed analysis with the AmpflSTRÒ IdentifierÒ plus PCR Amplification kit (Applied Biosystems). 4 ll of DNA solution from the Jomon skeleton was applied to the reaction mixture for the PCR (final volume 25 ll), and the PCR cycle number was 30 cycles. 3. Results and discussion Table 1 shows the allele frequencies and forensic statistical parameters of 30 INDELs in the Japanese population. The genotype frequency distributions showed no significant deviation from HWE by an exact test (p = 0.0017, after Bonferroni’s correction for multiple testing). Ho values ranged from 0.1474 (HLD 118) to 0.5538 (HLD 136), with a mean value of 0.422. The PD values ranged from 0.258 (HLD 118) to 0.623 (HLD 56), and 24 of the 30 INDELs showed PD values greater than 0.5. The combined PD and MP values for the 30 INDELs were 0.9999999998 and 2.67  10 11, respectively. The allelic frequencies for the 30 INDELs in the Japanese population were compared with those of other Asian and European population using the chi-squared test (Table 2). After Bonferroni correction (p = 0.005), the allelic frequencies of many INDELs in the Japanese population were found to be significantly different from those of the Chinese [2] and European [4–9] populations. However, the frequencies of more than 80% of the INDELs in the Japanese population were not significantly different from those of the South Korean [1] and Taiwanese [3] populations. We investigated the effectiveness of the kit for the analysis of highly degraded DNA samples. Although 7 autosomal short tandem repeat (STR) loci were detected using the AmpFLSTR IdentifilerÒ Plus PCR Amplification Kit (Applied Biosystems) with 30 PCR

cycles, the detected peaks were very low, and 5 of the 7 loci showed single peaks, which could indicate allele dropout (Fig. 1 (a)). In contrast, peaks could be detected in 27 INDELs, 9 of which indicating heterozygotes, using the DIPplexÒ kit (Fig. 1(b)). These results suggest that the DIPplexÒ Kit shows considerable potential for personal identification from degrade DNA samples due to the small amplicon size and high degree of polymorphisms. Access of data Available upon request to [email protected]. References [1] K.M. Seong, J.H. Park, Y.S. Hyun, P.W. Kang, D.H. Choi, M.S. Han, et al., Population genetics of insertion–deletion polymorphisms in South Koreans using Investigator DIPplex kit, Forensic Sci. Int. Genet. 8 (1) (2014) 80–83. [2] Y.L. Wei, C.J. Qin, H. Dong, J. Jia, C.X. Li, A validation study of a multiplex INDEL assay for forensic use in four Chinese populations, Forensic Sci. Int. Genet. 9 (2014) e22–e25. [3] W. Pepinski, M. Abreu-Glowacka, M. Koralewska-Kordel, E. Michalak, K. Kordel, A. Niemcunowicz-Janica, et al., Population genetics of 30 INDELs in populations of Poland and Taiwan, Mol. Biol. Rep. 40 (7) (2013) 4333–4338. [4] S.L. Friis, C. Børsting, E. Rockenbauer, L. Poulsen, S.F. Fredslund, C. Tomas, et al., Typing of 30 insertion/deletions in Danes using the first commercial indel kit – MentypeÒ DIPplex, Forensic Sci. Int. Genet. 6 (2) (2012) e72–e74. [5] P. Martín, O. García, B. Heinrichs, I. Yurrebaso, A. Aguirre, A. Alonso, Population genetic data of 30 autosomal indels in Central Spain and the Basque Country population, Forensic Sci. Int. Genet. 7 (2) (2013) e27–e30. [6] S. Turrina, G. Filippini, D. DeLeo, Forensic evaluation of the Investigator DIPplex typing system, Forensic Sci. Int. Genet. (Suppl. 3) (2011) e331–e332. [7] A. Zidkova, A. Horinek, V. Kebrdlova, M. Korabecna, Application of the new insertion–deletion polymorphisms kit for forensic identification and parentage testing on the Czech population, Int. J. Legal Med. 127 (1) (2013) 7–10. [8] Qiagen supplementary material: population data for Investigator DIPplex. Available at http://www.qiagen.com/literature/render.aspx?id=200253. [9] A.M. Neuvonen, J.U. Palo, M. Hedman, A. Sajantila, Discrimination power of Investigator DIPplex loci in Finnish and Somali populations, Forensic Sci. Int. Genet. 6 (4) (2012) e99–e102. [10] B.L. LaRue, J. Ge, J.L. King, B. Budowle, A validation study of the Qiagen Investigator DIPplexÒ kit; an INDEL-based assay for human identification, Int. J. Legal Med. 126 (4) (2012) 533–540. [11] K. Tanaka, Jomon skeleton from Shimekake site, Nagano prefecture, Anthropol. Sci. (Jpn. Series) 111 (1) (2003) 69–85 (in Japanese). [12] T. Kitayama, Y. Ogawa, K. Fujii, H. Nakahara, N. Mizuno, K. Sekiguchi, et al., Evaluation of a new experimental kit for the extraction of DNA from bones and teeth using a non-powder method, Legal Med. 12 (2) (2010) 84–89. [13] P.M. Schneider, Scientific standards for studies in forensic genetics, Forensic Sci. Int. 165 (2007) 238–243.