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Allele frequencies and sequence characteristics of D2S1242 STR locus in Chinese population
Legal Medicine 5 (2003) 97–99 www.elsevier.com/locate/legalmed
Brief communication
Allele frequencies and sequence characteristics of D2S1242 STR locus in Chinese population Jian Tie*, Yuka Suzuki, Shigemi Oshida Department of Legal Medicine, Nihon University School of Medicine, 30-1 Oyaguchi-Kamimachi, Itabashi-Ku, Tokyo 173-8610, Japan Received 28 February 2003; received in revised form 4 March 2003; accepted 10 March 2003
Abstract Sequence characteristics and allele frequencies of D2S1242 short tandem repeat (STR) locus were determined in a Chinese population using polymerase chain reaction (PCR) and sequence analysis. In 213 individuals studied, 32 genotypes representing the products of ten D2S1242 alleles were differentiated. The allele frequencies ranged from 0.002 to 0.234, and all the five most common alleles detected had frequencies over 10%. The observed and expected genotype values provided a complete fit to the Hardy – Weinberg equilibrium. The sequence structure of each D2S1242 allele showed a tetranucleotide repeat, and alleles 9 – 14 had different sequence structures compared to alleles 15 – 19. The power of discrimination was 0.94, showing a high degree of polymorphism as in other population data. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Polymorphism; D2S1242; Short tandem repeat; Chinese population
1. Introduction Several DNA polymorphic markers such as single nucleotide polymorphism, minisatellite DNA polymorphism and microsatellite DNA polymorphism have been used in forensic identification. However, the polymorphism of short tandem repeat (STR) still occupies an important role in forensic medicine [1 – 3]. A number of STR loci have become the main methods for paternity testing and individual identification. D2S1242 STR locus is a tetranucleotide tandem repeat polymorphism, and a high degree of polymorphism has been shown in some populations [4,5]. * Corresponding author. Tel.: þ81-3-3972-8111; fax: þ 81-33958-7776. E-mail address: [email protected] (J. Tie).
In this paper, we describe the characteristics of allele sequence structures and the allele frequency distribution of the D2S1242 STR locus in a Chinese population sample and compared the data with other populations. 2. Materials and methods 2.1. DNA extraction Genomic DNA was isolated from the peripheral blood samples of 213 unrelated Chinese individuals. DNA extraction was performed using phenol –chloroform method and precipitated with ethanol. 2.2. Amplification and DNA typing Polymerase chain reaction (PCR) for the D2S1242
1344-6223/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S1344-6223(03)00044-0
98
J. Tie et al. / Legal Medicine 5 (2003) 97–99
locus consisted of 30 cycles of denaturation at 948C for 60 s, annealing at 608C for 60 s, and extension at 728C for 90 s. Ten nanograms of each DNA sample was used for PCR amplification. The primer sequences are described in the Genomic Data Base (GDB) entry ID G00-309-429. The amplified PCR products were analyzed on 6.5% polyacrylamide gels with 7 M urea. Electrophoresis was performed at 800 V for 1.5 h. The bands of allele fragments were visualized by silver staining. DNA typing was performed using an allelic ladder constructed from ten allele fragments identified in this study.
Table 2 Comparison of the allele frequencies of the D2S1242 locus between Chinese and Japanese populations Allele
Chinese (n ¼ 213)
Japanese (n ¼ 273)
Length (bp)
9 10 11 12 13 14 15 16 17 18 19
0.007 0.005 0.054 0.209 0.234 0.148 0.157 0.146 0.038
0.002
139 143 147 151 155 159 163 167 171 175 179
0.002
0.070 0.245 0.192 0.152 0.165 0.130 0.029 0.013 0.002
2.3. Allele sequence For sequence analysis of the each D2S1242 allele, the target bands for each allele (a total of five for each allele) were eluted from the gels, and purified using Microcon YM-100 (Millipore, USA). Sequencing was performed by the dye terminator method using ABI PRISMe 310 Genetic Analyzer (Perkin – Elmer, Applied Biosystems). 3. Results and discussion The results of genotype frequencies are shown in Table 1 Genotype frequencies of D2S1242 locus in a Chinese population Genotype Observed Expected Genotype Observed Expected No. No. No. No. 12-9 12-11 12-12 13-9 13-10 13-11 13-12 13-13 14-11 14-12 14-13 14-14 15-9 15-11 15-12 15-13 Total
1 5 9 1 2 5 26 8 3 15 18 4 1 1 10 17
0.6 4.8 9.3 0.7 0.5 5.4 24.5 11.8 3.4 13.2 14.8 4.7 0.5 3.6 13.9 15.7
15-14 15-15 16-11 16-12 16-13 16-14 16-15 16-16 17-11 17-12 17-13 17-14 17-15 17-16 17-17 19-17 32
10 6 8 13 11 8 14 2 1 1 4 1 2 4 1 1 213
9.9 5.3 3.4 13.0 14.6 9.2 9.8 4.6 0.9 3.4 3.8 2.4 2.5 2.4 0.3 0.1 213
Table 1. A total of 32 genotypes and ten alleles were observed in the present study. No statistical differences in genotype frequencies were found between the observed and expected values, and both values showed complete agreement assuming Hardy – Weinberg equilibrium (x2 ¼ 24, d.f. ¼ 22, P . 0:5). The five most common alleles were identified to be 12, 13, 14, 15 and 16, each with a frequency over 10%. Alleles 9, 10 and 19 were rare among the subjects (Table 2). Comparing the data to Japanese, both Chinese and Japanese showed the same ten alleles for D2S1242, but allele 18 was not found in the Chinese samples, and allele 10 was not reported in Japanese samples. No statistical differences were observed between the two populations (x2 ¼ 25:44, d.f. ¼ 22, P . 0:5). The allele fragment lengths ranged from 139 to 179 base pairs. All the alleles identified increased in size regularly by 4-bp increments. The major polymorphic repeat of the D2S1242 locus had a base sequence of GAAA from alleles 9 to 14. However, another polymorphic sequence was found from alleles 15 to 19 (Table 3). After the GAAA repeat, instead of (GAAAAA)2, a sequence structure of GAAG – (GAAA)3 was observed in Chinese samples. In other words, the allele length of repeat is composed of (GAAA)n – GAAG – (GAAA)3. These allele sequence characteristics are the same as those reported in Japanese and Austrian populations [4,5]. But in Austrians, a sequence structure of (GAAA) 13 – (GAAG)2 – (GAAA)3 has been described in allele 18, which is
J. Tie et al. / Legal Medicine 5 (2003) 97–99
99
Table 3 Comparison of the allele sequence structure of the D2S1242 locus between Chinese and Japanese populations Allele
Chinese (n ¼ 213)
Japanese (n ¼ 273)
9 10 11 12 13 14 15 16 17 18 19
50 -(GAAA)9 –(GAAAAA)2-30 50 -(GAAA)10 –(GAAAAA)2-30 50 -(GAAA)11 –(GAAAAA)2-30 50 -(GAAA)12 –(GAAAAA)2-30 50 -(GAAA)13 –(GAAAAA)2-30 50 -(GAAA)14 –(GAAAAA)2-30 50 -(GAAA)11 –GAAG– (GAAA)3 – 50 -(GAAA)12 –GAAG– (GAAA)3 – 50 -(GAAA)13 –GAAG– (GAAA)3 –
50 -(GAAA)9 –(GAAAAA)2-30 50 -(GAAA)11 –(GAAAAA)2-30 50 -(GAAA)12 –(GAAAAA)2-30 50 -(GAAA)13 –(GAAAAA)2-30 50 -(GAAA)14 –(GAAAAA)2-30 50 -(GAAA)11 –GAAG–(GAAA)3 – 50 -(GAAA)12 –GAAG–(GAAA)3 – 50 -(GAAA)13 –GAAG–(GAAA)3 – 50 -(GAAA)14 –GAAG–(GAAA)3 – 50 -(GAAA)15 –GAAG–(GAAA)3 –
50 -(GAAA)15 –GAAG– (GAAA)3 –
Table 4 Comparison of forensic efficiency values of the Chinese samples with other population data [3]
Power of discrimination Heterozygosity Polymorphic information content Mean exclusion chance
Chinese
Japanese
Austrian
0.94 0.86 0.81 0.66
0.95 0.81 0.81 0.67
0.95 0.86 – 0.67
[4]
[5]
not found in Chinese and Japanese subjects. STR loci with regular 4-bp repeat units are commonly used in forensic field, some of the STR loci show irregular repeats [6 – 8]. However, alleles with regular repeat units in one part and irregular repeat units in other part was not reported in other populations at D2S1242 locus. A number of STR loci such as vWA, TH01, FESFPS and CSF1PO are commonly used in the forensic field [9 –11]. Table 4 summarizes some forensic efficiency parameters of the D2S1242 locus obtained in the present study and other population samples. The D2S1242 STR locus thus promise to be a new DNA profiling in the forensic field.
[6]
[7]
[8]
[9]
[10]
References [1] Rubocki RJ, Duffy KJ, Shepard KL, McCue BJ, Shepherd SJ, Wisecarver JL. Loss of heterozygosity detected in a short tandem repeat (STR) locus commonly used for human DNA identification. J Forensic Sci 2000;45:1087– 9. [2] Parson W, Niederstatter H, Kochl S, Steinlechner M, Berger B.
[11]
When autosomal short tandem repeats fail: optimized primer and reaction design for Y-chromosome short tandem repeat analysis in forensic casework. Croat Med J 2001;42:285 –7. Henke J, Henke L, Chatthopadhyay P, Kayser M, Dulmer M, Cleef S, Poche H, Felske-Zech H. Application of Y-chromosomal STR haplotypes to forensic genetics. Croat Med J 2001; 42:292–7. Reichenpfader B, Zehner R, Klintschar M. Characterization of a highly variable short tandem repeat polymorphism at the D2S1242 locus. Electrophoresis 1999;20:514–7. Tie J, Suzuki Y, Tsukamoto S, Oshida S. Short tandem repeat polymorphism of the D2S1242 locus in a Japanese population. Hum Biol 2002;74:601–6. Haddad AP, Sparrow RL. The short tandem repeat locus VWF2 in Intron 40 of the von Willebrand factor gene consists of two polymorphic sub-loci. Forensic Sci Int 2001;119: 299 –304. Delghandi M, Branting BJ, Jorgensen L. Evaluation of a quadruplexs short atandem repeat system (HUMVWA31/A, HUMD11S554, HUMAPOAI1, and HUMACTBP2 loci) for forensic identity testing, confident typing of complex alleles, and population databases. Croat Med J 2001;42:33–44. Barral S, Lareu MV, Salas A, Carracedo A. Sequence variation of two hypervariable short tandem repeats at the D22S683 and D6S477 loci. Int J Legal Med 2000;113:146–9. Rubocki RJ, McCue BJ, Duffy KJ, Shepard KL, Shepherd SJ, Wisecarver JL. Natural DNA mixtures generated in fraternal twins in utero. J Forensic Sci 2001;46:120– 5. Moretti TR, Baumstark AL, Defenbaugh DA, Keys KM, Smerick JB, Budowle B. Validation of short tandem repeats (STRs) for forensic usage: performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples. J Forensic Sci 2001;46:647–60. Cerda-Flores RM, Budowle B, Jin L, Barton SA, Deka R, Chakraborty R. Maximum likelihood estimates of admixture in northeastern Mexico using 13 short tandem repeat loci. Am J Hum Biol 2002;14:429–39.
Brief communication
Allele frequencies and sequence characteristics of D2S1242 STR locus in Chinese population Jian Tie*, Yuka Suzuki, Shigemi Oshida Department of Legal Medicine, Nihon University School of Medicine, 30-1 Oyaguchi-Kamimachi, Itabashi-Ku, Tokyo 173-8610, Japan Received 28 February 2003; received in revised form 4 March 2003; accepted 10 March 2003
Abstract Sequence characteristics and allele frequencies of D2S1242 short tandem repeat (STR) locus were determined in a Chinese population using polymerase chain reaction (PCR) and sequence analysis. In 213 individuals studied, 32 genotypes representing the products of ten D2S1242 alleles were differentiated. The allele frequencies ranged from 0.002 to 0.234, and all the five most common alleles detected had frequencies over 10%. The observed and expected genotype values provided a complete fit to the Hardy – Weinberg equilibrium. The sequence structure of each D2S1242 allele showed a tetranucleotide repeat, and alleles 9 – 14 had different sequence structures compared to alleles 15 – 19. The power of discrimination was 0.94, showing a high degree of polymorphism as in other population data. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Polymorphism; D2S1242; Short tandem repeat; Chinese population
1. Introduction Several DNA polymorphic markers such as single nucleotide polymorphism, minisatellite DNA polymorphism and microsatellite DNA polymorphism have been used in forensic identification. However, the polymorphism of short tandem repeat (STR) still occupies an important role in forensic medicine [1 – 3]. A number of STR loci have become the main methods for paternity testing and individual identification. D2S1242 STR locus is a tetranucleotide tandem repeat polymorphism, and a high degree of polymorphism has been shown in some populations [4,5]. * Corresponding author. Tel.: þ81-3-3972-8111; fax: þ 81-33958-7776. E-mail address: [email protected] (J. Tie).
In this paper, we describe the characteristics of allele sequence structures and the allele frequency distribution of the D2S1242 STR locus in a Chinese population sample and compared the data with other populations. 2. Materials and methods 2.1. DNA extraction Genomic DNA was isolated from the peripheral blood samples of 213 unrelated Chinese individuals. DNA extraction was performed using phenol –chloroform method and precipitated with ethanol. 2.2. Amplification and DNA typing Polymerase chain reaction (PCR) for the D2S1242
1344-6223/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S1344-6223(03)00044-0
98
J. Tie et al. / Legal Medicine 5 (2003) 97–99
locus consisted of 30 cycles of denaturation at 948C for 60 s, annealing at 608C for 60 s, and extension at 728C for 90 s. Ten nanograms of each DNA sample was used for PCR amplification. The primer sequences are described in the Genomic Data Base (GDB) entry ID G00-309-429. The amplified PCR products were analyzed on 6.5% polyacrylamide gels with 7 M urea. Electrophoresis was performed at 800 V for 1.5 h. The bands of allele fragments were visualized by silver staining. DNA typing was performed using an allelic ladder constructed from ten allele fragments identified in this study.
Table 2 Comparison of the allele frequencies of the D2S1242 locus between Chinese and Japanese populations Allele
Chinese (n ¼ 213)
Japanese (n ¼ 273)
Length (bp)
9 10 11 12 13 14 15 16 17 18 19
0.007 0.005 0.054 0.209 0.234 0.148 0.157 0.146 0.038
0.002
139 143 147 151 155 159 163 167 171 175 179
0.002
0.070 0.245 0.192 0.152 0.165 0.130 0.029 0.013 0.002
2.3. Allele sequence For sequence analysis of the each D2S1242 allele, the target bands for each allele (a total of five for each allele) were eluted from the gels, and purified using Microcon YM-100 (Millipore, USA). Sequencing was performed by the dye terminator method using ABI PRISMe 310 Genetic Analyzer (Perkin – Elmer, Applied Biosystems). 3. Results and discussion The results of genotype frequencies are shown in Table 1 Genotype frequencies of D2S1242 locus in a Chinese population Genotype Observed Expected Genotype Observed Expected No. No. No. No. 12-9 12-11 12-12 13-9 13-10 13-11 13-12 13-13 14-11 14-12 14-13 14-14 15-9 15-11 15-12 15-13 Total
1 5 9 1 2 5 26 8 3 15 18 4 1 1 10 17
0.6 4.8 9.3 0.7 0.5 5.4 24.5 11.8 3.4 13.2 14.8 4.7 0.5 3.6 13.9 15.7
15-14 15-15 16-11 16-12 16-13 16-14 16-15 16-16 17-11 17-12 17-13 17-14 17-15 17-16 17-17 19-17 32
10 6 8 13 11 8 14 2 1 1 4 1 2 4 1 1 213
9.9 5.3 3.4 13.0 14.6 9.2 9.8 4.6 0.9 3.4 3.8 2.4 2.5 2.4 0.3 0.1 213
Table 1. A total of 32 genotypes and ten alleles were observed in the present study. No statistical differences in genotype frequencies were found between the observed and expected values, and both values showed complete agreement assuming Hardy – Weinberg equilibrium (x2 ¼ 24, d.f. ¼ 22, P . 0:5). The five most common alleles were identified to be 12, 13, 14, 15 and 16, each with a frequency over 10%. Alleles 9, 10 and 19 were rare among the subjects (Table 2). Comparing the data to Japanese, both Chinese and Japanese showed the same ten alleles for D2S1242, but allele 18 was not found in the Chinese samples, and allele 10 was not reported in Japanese samples. No statistical differences were observed between the two populations (x2 ¼ 25:44, d.f. ¼ 22, P . 0:5). The allele fragment lengths ranged from 139 to 179 base pairs. All the alleles identified increased in size regularly by 4-bp increments. The major polymorphic repeat of the D2S1242 locus had a base sequence of GAAA from alleles 9 to 14. However, another polymorphic sequence was found from alleles 15 to 19 (Table 3). After the GAAA repeat, instead of (GAAAAA)2, a sequence structure of GAAG – (GAAA)3 was observed in Chinese samples. In other words, the allele length of repeat is composed of (GAAA)n – GAAG – (GAAA)3. These allele sequence characteristics are the same as those reported in Japanese and Austrian populations [4,5]. But in Austrians, a sequence structure of (GAAA) 13 – (GAAG)2 – (GAAA)3 has been described in allele 18, which is
J. Tie et al. / Legal Medicine 5 (2003) 97–99
99
Table 3 Comparison of the allele sequence structure of the D2S1242 locus between Chinese and Japanese populations Allele
Chinese (n ¼ 213)
Japanese (n ¼ 273)
9 10 11 12 13 14 15 16 17 18 19
50 -(GAAA)9 –(GAAAAA)2-30 50 -(GAAA)10 –(GAAAAA)2-30 50 -(GAAA)11 –(GAAAAA)2-30 50 -(GAAA)12 –(GAAAAA)2-30 50 -(GAAA)13 –(GAAAAA)2-30 50 -(GAAA)14 –(GAAAAA)2-30 50 -(GAAA)11 –GAAG– (GAAA)3 – 50 -(GAAA)12 –GAAG– (GAAA)3 – 50 -(GAAA)13 –GAAG– (GAAA)3 –
50 -(GAAA)9 –(GAAAAA)2-30 50 -(GAAA)11 –(GAAAAA)2-30 50 -(GAAA)12 –(GAAAAA)2-30 50 -(GAAA)13 –(GAAAAA)2-30 50 -(GAAA)14 –(GAAAAA)2-30 50 -(GAAA)11 –GAAG–(GAAA)3 – 50 -(GAAA)12 –GAAG–(GAAA)3 – 50 -(GAAA)13 –GAAG–(GAAA)3 – 50 -(GAAA)14 –GAAG–(GAAA)3 – 50 -(GAAA)15 –GAAG–(GAAA)3 –
50 -(GAAA)15 –GAAG– (GAAA)3 –
Table 4 Comparison of forensic efficiency values of the Chinese samples with other population data [3]
Power of discrimination Heterozygosity Polymorphic information content Mean exclusion chance
Chinese
Japanese
Austrian
0.94 0.86 0.81 0.66
0.95 0.81 0.81 0.67
0.95 0.86 – 0.67
[4]
[5]
not found in Chinese and Japanese subjects. STR loci with regular 4-bp repeat units are commonly used in forensic field, some of the STR loci show irregular repeats [6 – 8]. However, alleles with regular repeat units in one part and irregular repeat units in other part was not reported in other populations at D2S1242 locus. A number of STR loci such as vWA, TH01, FESFPS and CSF1PO are commonly used in the forensic field [9 –11]. Table 4 summarizes some forensic efficiency parameters of the D2S1242 locus obtained in the present study and other population samples. The D2S1242 STR locus thus promise to be a new DNA profiling in the forensic field.
[6]
[7]
[8]
[9]
[10]
References [1] Rubocki RJ, Duffy KJ, Shepard KL, McCue BJ, Shepherd SJ, Wisecarver JL. Loss of heterozygosity detected in a short tandem repeat (STR) locus commonly used for human DNA identification. J Forensic Sci 2000;45:1087– 9. [2] Parson W, Niederstatter H, Kochl S, Steinlechner M, Berger B.
[11]
When autosomal short tandem repeats fail: optimized primer and reaction design for Y-chromosome short tandem repeat analysis in forensic casework. Croat Med J 2001;42:285 –7. Henke J, Henke L, Chatthopadhyay P, Kayser M, Dulmer M, Cleef S, Poche H, Felske-Zech H. Application of Y-chromosomal STR haplotypes to forensic genetics. Croat Med J 2001; 42:292–7. Reichenpfader B, Zehner R, Klintschar M. Characterization of a highly variable short tandem repeat polymorphism at the D2S1242 locus. Electrophoresis 1999;20:514–7. Tie J, Suzuki Y, Tsukamoto S, Oshida S. Short tandem repeat polymorphism of the D2S1242 locus in a Japanese population. Hum Biol 2002;74:601–6. Haddad AP, Sparrow RL. The short tandem repeat locus VWF2 in Intron 40 of the von Willebrand factor gene consists of two polymorphic sub-loci. Forensic Sci Int 2001;119: 299 –304. Delghandi M, Branting BJ, Jorgensen L. Evaluation of a quadruplexs short atandem repeat system (HUMVWA31/A, HUMD11S554, HUMAPOAI1, and HUMACTBP2 loci) for forensic identity testing, confident typing of complex alleles, and population databases. Croat Med J 2001;42:33–44. Barral S, Lareu MV, Salas A, Carracedo A. Sequence variation of two hypervariable short tandem repeats at the D22S683 and D6S477 loci. Int J Legal Med 2000;113:146–9. Rubocki RJ, McCue BJ, Duffy KJ, Shepard KL, Shepherd SJ, Wisecarver JL. Natural DNA mixtures generated in fraternal twins in utero. J Forensic Sci 2001;46:120– 5. Moretti TR, Baumstark AL, Defenbaugh DA, Keys KM, Smerick JB, Budowle B. Validation of short tandem repeats (STRs) for forensic usage: performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples. J Forensic Sci 2001;46:647–60. Cerda-Flores RM, Budowle B, Jin L, Barton SA, Deka R, Chakraborty R. Maximum likelihood estimates of admixture in northeastern Mexico using 13 short tandem repeat loci. Am J Hum Biol 2002;14:429–39.