Real-time PCR method for identification of Asian populations in forensic casework

Legal Medicine 11 (2009) S106–S108

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Legal Medicine journal homepage: www.elsevier.com/locate/legalmed

Real-time PCR method for identification of Asian populations in forensic casework Tomoharu Tokutomi *, Yuzo Takada, Takako Murayama, Masahiro Mukaida, Jun Kanetake Department of Forensic Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan

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Article history: Received 6 December 2008 Received in revised form 14 January 2009 Accepted 2 February 2009 Available online 16 March 2009 Keywords: Classifier Collagen Terrorisms Ethnic SNP Japan Self-Defense Forces

a b s t r a c t The priorities for mass disaster victim identification are rapid investigation turn-around time and low cost. We describe a DNA typing procedure to selectively identify members of Asian populations by a real-time PCR method using polymorphisms of the a2 chain of the type I collagen gene (COL1A2) and mitochondrial DNA (mtDNA). Among the 50 members of the Asian population included in the present study, 37 harbored a deleted allele in intron 33 of COL1A2 (26822–26823del) or the 10400C > T substitution mutation in mtDNA to give a probability of 0.740 for these SNPs in the Asian population. Ó 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction DNA typing provides an ideal approach for the Japan Self-Defense Forces to identify Japanese (or at least Asian) victims of mass disasters and terrorism attacks abroad. The quality of DNA from samples obtained from mass disasters can vary substantially according to the nature of the disaster and the state of degradation of the material. When only small quantities of intact DNA are available some approaches for DNA typing, including conventional short tandem repeat (STR) typing, do not always allow identification of the victim. By comparison, analysis of single nucleotide polymorphisms (SNPs) and mitochondrial DNA (mtDNA) provides a means of recovering DNA information from DNA samples of poor quality. We have previously described a deleted allele (26822– 26823del) among Asian individuals in intron 33 of the a2 chain of the type I collagen gene (COL1A2) [1]. The substitution mutation 10400C > T in mitochondrial DNA is typically associated with Asians. Here, we screened Asian populations by real-time PCR method using markers for polymorphisms in COL1A2 and mtDNA. 2. Materials and methods 2.1. Samples We analyzed DNA samples from the following individuals: 48 Japanese volunteers, J01–J48; 48 African Americans, from the Cori-

* Corresponding author. Tel.: +81 4 2995 1583; fax: +81 4 2996 5198. E-mail address: [email protected] (T. Tokutomi). 1344-6223/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2009.02.011

ell HD50AA panel, NA17101–48, (Coriel, Camden, New Jersey) and the 10th International Histocompatiblity Workshops panel; 2 Orientals, WS9066–7; 2 Africans, WS9021 and WS9058; and 40 Caucasians, WS9002, WS9004–6, WS9010, WS9013–4, WS9017–9, WS9022–3, WS9025, WS9030–3, WS9036–7, WS9040, WS9042– 3, WS9045, WS9047–8, WS9050–1, WS9054–7, WS9060, WS9062–3, WS9065, WS9069, WS9088, WS9091, and WS9104–5 [2]. 2.2. DNA extraction We extracted DNA from blood samples according to standard procedures by means of proteolytic digestion followed by phenol–chloroform purification. 2.3. COL1A2 primer design for real-time PCR Custom TaqManÒ SNP Genotyping Assays (Applied Biosystems, Foster, California) were used to design primers and probes for realtime PCR amplification of COL1A2. The primer oligonucleotide sequences were as follows: forward, COL1A2-i33F, GAATGAC AAGGTTCACTTTTGATGATACG and reverse, COL1A2-i33R, CCATGA GCACCCTAGTACCAAA. Each probe was labeled with a specific reporter. The probe specific for the wild-type fragment was VIC-labeled, while the probe specific for the deleted fragment was FAM-labeled. The same non-fluorescent quencher was used for both probes (TaqManÒ MGB Probes, Applied Biosystems). The probe oligonucleotide sequences were as follows: VIC-CATGTCTTATTAATAACACCC and FAM-ATGTCTTATTAAACACCC. Realtime PCR amplification was performed in 10 ll containing 2 Taq-

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ManÒ Fast Universal Master Mix (Applied Biosystems), 100 nM each primer, 200 nM probes, and 1 ll DNA extract. Thermal cycling conditions were 95 °C for 20 s, 40 cycles of 95 °C for 1 s and 60 °C for 20 s.

as follows: VIC-ACTATATACCAATTCGGTTCAG and FAM-ACTATATACCAATTCAGTTCAG. Real-time PCR amplication and thermal cycling conditions were performed using the same reaction conditions used for amplification of COL1A2.

2.4. mtDNA primer design for real-time PCR

2.5. Data analysis

Custom TaqManÒ SNP Genotyping Assays was used to design primers and probes. The primer oligonucleotide sequences were as follows: forward, C10400T-mtMF CCCTAAGTCTGGCCTATGAGTGA and reverse, C10400T-mtMR TCATAATTTAATGAGTCGAAATCATTCGTTTTGTT. Each probe was labeled with a specific reporter. The probe specific for 10400C of mtDNA was VIC-labeled, while the probe specific for 10400T of mtDNA was FAM-labeled. The same non-fluorescent quencher was used for both probes (TaqManÒ MGB Probes). The probe oligonucleotide sequences were

Analysis of the data was performed using the StepOneTM RealTime PCR System and StepOneTM Software v2.0.1 (Applied Biosystems). 3. Results Among the 50 Asians included in the study, 30 individuals harbored a deleted allele in intron 33 of COL1A2 (26822–26823del), 19 individuals had 10400T in mtDNA, and 37 individuals had either of

Table 1 Real-time PCR results of three populations (N = 140). Asian (N = 50)

African (N = 50)

Caucasian (N = 40)

No.

COL1A2

mt10400T

Ethnic

No.

COL1A2

mt10400T

Ethnic

No.

COL1A2

mt10400T

Ethnic

J01 J02 J03 J04 J05 J06 J07 J08 J09 J10 J11 J12 J13 J14 J15 J16 J17 J18 J19 J20 J21 J22 J23 J24 J25 J26 J27 J28 J29 J30 J31 J32 J33 J34 J35 J36 J37 J38 J39 J40 J41 J42 J43 J44 J45 J46 J47 J48 WS9066 WS9107

WT/WT WT/WT WT/WT del/WT del/WT WT/WT del/WT WT/WT WT/WT del/WT del/WT WT/WT WT/WT U WT/WT WT/WT del/WT del/WT del/WT del/del del/WT del/WT WT/WT WT/WT WT/WT del/WT del/WT WT/WT WT/WT del/WT del/WT del/WT del/WT del/WT del/WT del/WT del/WT WT/WT del/WT del/del del/del del/WT WT/WT WT/WT del/WT WT/WT del/WT del/WT del/del del/WT

C C T C C T T C T T T C C C C C C T T T C C T T C C T T C T C C C C C C C C T T C C C C T T C C T C

J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J J O O

NA17101 NA17102 NA17103 NA17104 NA17105 NA17106 NA17107 NA17108 NA17109 NA17110 NA17111 NA17112 NA17113 NA17114 NA17115 NA17116 NA17117 NA17118 NA17119 NA17120 NA17121 NA17122 NA17123 NA17124 NA17125 NA17126 NA17127 NA17128 NA17129 NA17130 NA17131 NA17132 NA17133 NA17134 NA17135 NA17136 NA17137 NA17138 NA17139 NA17140 NA17141 NA17142 NA17143 NA17144 NA17145 NA17146 NA17147 NA17148 WS9021 WS9058

WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT

C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C T C

AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA A A

WS9002 WS9004 WS9005 WS9006 WS9010 WS9013 WS9014 WS9017 WS9018 WS9019 WS9022 WS9023 WS9025 WS9030 WS9031 WS9032 WS9033 WS9036 WS9037 WS9040 WS9042 WS9043 WS9045 WS9047 WS9048 WS9050 WS9051 WS9054 WS9055 WS9056 WS9057 WS9060 WS9062 WS9063 WS9065 WS9069 WS9088 WS9091 WS9104 WS9105

WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT U WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT WT/WT

T C C C C C T C C C C C T C C C C C C C C C C C C C C C C C C U C C C C C C C C

C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C

N, number of samples; COL1A, deleted alleles in intron 33 of the a2 chain of the type I collagen gene; mt10400T, a substitution mutation 10400C > T of mitochondrial DNA; WT, wild -type; del/WT, heterozygous deletion; del/del, homozygous deletion; U, undetermined; J, Japanese; O, Oriental; AA, African American; A, African; C, Caucasian.

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Table 2 Asian probabilities using two markers.

AI (N = 50) A (N = 50) C (N = 40)

COL1A2del

mt10400T

COL1A2del and mt10400T

COL1A2del or mt10400T

0.600 0.000 0.000

0.380 0.020 0.075

0.240 0.000 0.000

0.740 0.020 0.075

N, number of samples; COL1Adel, a deleted allele in intron 33 of the a2 chain of the type I collagen gene; mt10400T, a substitution mutation 10400C > T mitochondrial DNA; AI, Asian; A, African; C, Caucasian.

the above markers (Table 1). The probability of an individual in the Asian population having a deleted allele in intron 33 of COL1A2 or 10400T of mtDNA was 0.740 (Table 2). 4. Discussion Gill (2001) estimated that 50 SNPs gave match probabilities equivalent to 12 STRs [3]. Analysis of 50–60 SNP loci instead of 13 STR loci is preferable as most SNPs can be amplified from PCR products less than 100 bp in length. By comparison, PCR products need to be 200 bp or longer for amplification of STR loci. In highly degraded DNA samples, the average length of DNA fragments is less than 150 bp. Thus, many STR loci are not amplified from samples in which the DNA is degraded due to exposure to high temperature or high humidity or other environmental factors associated with samples from victims of mass disasters and terrorism attacks. Frudakis et al. (2003) developed a classifier for the SNP-based inference of ancestry using 56 markers and demonstrated an accuracy of 99%, 98%, and 100% for identifying individuals of European, African, Asian descent, respectively [4]. In the event of mass disasters and terrorism attacks abroad, the efficient identification of individuals requires investigators from each country to cooperate and place victims or samples in order of priority for identification. To achieve the efficient identification of individuals screening methods need to be rapid, simple, transportable, and be comprised of a limited number of markers. COL1A2 (MIM  120160) is a type I collagen genes located at 7q21.3–q22.1 and mutations in this gene appear to cause osteogenesis imperfecta. Polymorphisms in human COL1A2 have natural variation across an ethnically diverse population [5,6]. Haplogroups of mtDNA correspond to control region polymorphisms as well as entire mtGenome variation. Haplogroups A, B, C, D, F, F, G, and M are typically associated with Asians, while most Native Americans fall into haplogroups A, B, C, and D [7]. Haplogroups L1, L2, and L3 are associated with the African population, and haplogroups H, I, J, K, T, U, V, W, X are typically associated with European populations [7]. Haplogroup M is a coding region polymorphism for the substitution mutation 10400C > T in mitochondrial DNA. Recent advances in rapid-cycle and real-time PCR instrumentation, for example the SmartCyclerÒ (Cepheid, Sunnyvale, Califor-

nia) and StepOneTM (Applied Biosystems), provide a high level of transportability allowing analysis of samples on site. The use of these instruments, together with the method described in the present report, provides an efficient system for placing samples in order of priority for the identification of Japanese (Asian) individuals in the event of mass disasters and terrorism attacks abroad. In addition to assisting in the identification process, this method will save time and money. Several SNP panels have been proposed for forensic casework providing the basis for the selection of SNPs in the mtDNA coding region for identification of Asian populations. Conflict of interest We have no conflicting financial interests to declare. Acknowledgment We thank Dr. Ken Kobayashi, Nihon Pharmaceutical University, for suggestions and comments on this paper. References [1] Masuda T, Takada Y, Murayama T, Tokutomi T, Kumaki F, Mukaida M. Genetic polymorphism in collagen type 1 alpha 2 intron 33 can be used for the initial screening of East Asians quickly and easily by a heteroduplex analysis. Legal Med 2008;10:88–91. [2] Yang SY, Milford E, Hämmerling U, Dupont B. In: Dupont B, editor. Immunobiology of HLA volume I histocompatibility testing, 1987: description of the reference panel of B-lymphoblastoid cell lines for factors of the HLA system: the B-cell line panel designed for the tenth international histocompatibility workshop. New York: Springer-Verlag; 1989. p. 11–9. [3] Gill P. An assessment of the utility of single nucleotide polymorphisms (SNPs) for forensic purpose. Int J Legal Med 2001;114:204–10. [4] Frudakis T, Venkateswarlu K, Thomas MJ, Gaskin Z, Ginjupalli S, Gunturi S, et al. A classifier for the SNP-based inference of ancestry. J Forensic Sci 2003;48:771–82. [5] Mitchell RJ, Howlett S, White NG, Federle L, Papiha SS, Briceno I, et al. Deletion polymorphism in the human COL1A2 gene: genetic evidence of a non-African population whose descendants spread to all continents. Hum Biol 1999;71:901–14. [6] Chan TF, Poon A, Basu A, Addleman NR, Chen J, Phong A, et al. Natural variation in four human collagen genes across an ethnically diverse population. Genomics 2008;91:307–14. [7] Wallace DC, Brown MD, Lott MT. Mitochondrial DNA variation in human evolution and disease. Gene 1999;238:211–30.