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Typing of 20 Y-chromosome STRs in the Italian population
Forensic Science International 146S (2004) S135–S138 www.elsevier.com/locate/forsciint
Announcement of population data
Typing of 20 Y-chromosome STRs in the Italian population Renato Biondoa,*, Alessandra Caglia`a, Paola Asilia, Giacomo D’Agostarob, Gregorio D’Agostinob, Aldo Spinellaa a
DCPC. Servizio Polizia Scientifica Area biologica Viale dell’Aeronautica, 7-00144 Roma, Italy b ENEA, CR ENEA Casaccia, S.P. Anguillarese, 301-00060 Roma, Italy Available online 30 October 2004
Abstract The aim of the present study is to appraise the increase of the discrimination power of the Y-specific haplotype allowed by 20 STR markers in a sample of the Italian population. The set of Y STR markers analyzed includes the European ‘‘extended haplotype’’ DYS19, DYS385, DYS389I/II, DYS390, DYS391, DYS392, DYS393 and YCAII a/b and in addition the DYS437, DYS438, DYS439, DYS447, DYS448, DYS388, DYS426, DYS460 (Y-GATA-A7.1) and Y-H4 loci. # 2004 Published by Elsevier Ireland Ltd. Keywords: Y-chromosome STRs; Population data
1. Population sample The DNA from 75 unrelated male individuals from northern, central and southern Italy was typed. The set of male DNA samples was from the DNA collection maintained in the laboratories of Italian State Police, Servizio Polizia Scientifica [1].
2. DNA extraction and quantification The DNA was purified from blood following the Chelex extraction procedure [1]. The male DNA concentration was determined using the Quantifiler Y Human Male DNA Quantification Kit (Applera).
based on the report of Butler et al. [2]. The 10 PlexY includes the dinucleotide marker YCAII, the trinucleotide loci DYS388 and DYS426, the tetranucleotide loci DYS437, DYS439, DYS460 (GATA A7.1), and Y-H4, the pentanucleotide loci DYS438 and DYS 447, and the exanucleotide marker DYS448. The two assays have in common the DYS437, DYS438, and DYS439 STR markers. This overlap provides a cross-check validation of the two procedures. The PowerPlexY amplifications were performed as recommended by the manufacturer (PowerPlexY Technical Manual, TMD018, Promega). The 10 PlexY amplifications were performed as described by Butler et al [2] with minor modifications. The concentration of DYS447 and DYS 448 primers was increased 1.5-fold, the time of synthesis at 72 8C was extended to 90 s, the number of cycles was increased to 32, and the amount DNA was lowered to 1–2 ng.
3. PCR amplification The same set of male DNA samples was amplified using the PowerPlexY system (Promega) and a 10 PlexY assay
4. Data analysis
* Corresponding author. Tel.: +39 0646544009; fax: +39 0646544380. E–mail address: [email protected] (R. Biondo).
The amplification products of the multiplex assays were resolved and detected using the ABIPRISM 3100 and 310 Genetic Analyzers (Applera), the POP4 polymer, and the
0379-0738/$ – see front matter # 2004 Published by Elsevier Ireland Ltd. doi:10.1016/j.forsciint.2004.09.041
S136
R. Biondo et al. / Forensic Science International 146S (2004) S135–S138
Table 1 Relative frequencies of 20 Y-STR marker alleles in the Italian population DYS19
DYS385a
12 13 14 15 16
0.016 0.159 0.460 0.286 0.079
DYS391 9 10 11
0.063 0.635 0.302
DYS447 17 19 20 21 22 23 24 25
0.032 0.032 0.111 0.159 0.429 0.127 0.079 0.032
DYS385b
DYS388
10 11 12 13 14 15 16 17 18
0.016 0.333 0.127 0.206 0.159 0.048 0.048 0.048 0.016
2 12 13 14 15 16 17 18 19
0.016 0.016 0.063 0.413 0.238 0.079 0.079 0.063 0.032
DYS392 11 12 13 14
0.524 0.032 0.397 0.048
DYS393 11 12 13 14 15
0.016 0.238 0.587 0.127 0.032
DYS448 200 22 23 24 25 22,23
0.032 0.349 0.429 0.127 0.048 0.016
DYS460 8 9 10 11
0.063 0.333 0.524 0.079
DYS389I
11 12 13 14 140 15 16
0.651 0.095 0.048 0.063 0.079 0.032 0.032
DYS426 10 11 12
YCAIIa 11 15 16 17 18 19
DYS389II
12 13 14 15
0.159 0.619 0.190 0.032
0.667 0.254 0.079
DYS437 14 15 16
0.302 0.556 0.143
0.048 0.048 0.762 0.032 0.079 0.032
YCAIIb 16 17 18 19 20 21
0.016 0.079 0.429 0.413 0.048 0.016
DYS390
28 29 30 31 32 33
0.190 0.286 0.381 0.079 0.048 0.016
DYS438 8 9 10 11 12
0.016 0.254 0.349 0.048 0.333
YH4 9 10 11 12
0.032 0.492 0.381 0.095
21 22 23 24 25 26
0.016 0.190 0.302 0.413 0.063 0.016
DYS439 10 11 12 13
0.079 0.333 0.460 0.127
Table 2 Statistical parameters Locus
MP
Power of discrimination
b
No.
H
DYS391 DYS389 I DYS439 DYS389 II DYS438 DYS437 DYS19 DYS392 DYS393 DYS390 DYS385 a DYS385 b DYS388 DYS426 DYS447 DYS448 DYS460 YCAII a YCAII b Y-H4
0.50 0.45 0.35 0.27 0.30 0.42 0.33 0.44 0.42 0.30 0.20 0.25 0.45 0.52 0.25 0.33 0.40 0.59 0.36 0.40
0.5018896447 0.7779901188 0.9233117795 0.9791517788 0.9937439578 0.9973724308 0.9991453283 0.9996281134 0.9998441805 0.9999529283 0.9999904766 0.9999976269 0.9999989387 0.9999994532 0.9999998651 0.9999999561 0.9999999826 0.9999999897 0.9999999963 0.9999999985
0.4981103553E+00 0.2220098812E+00 0.7668822049E-01 0.2084822119E-01 0.6256042186E-02 0.2627569243E-02 0.8546716786E-03 0.3718866185E-03 0.1558194625E-03 0.4707168948E-04 0.9523448387E-05 0.2373063859E-05 0.1061271945E-05 0.5468130832E-06 0.1348778051E-06 0.4387181819E-07 0.1736523718E-07 0.1030363657E-07 0.3740876872E-08 0.1486359997E-08
3 4 4 6 5 3 5 4 5 6 9 9 7 3 8 6 4 6 6 4
0.8249649452E+00 0.1014405690E+01 0.1186479613E+01 0.1453263460E+01 0.1292417876E+01 0.9660467271E+00 0.1274066295E+01 0.9599795458E+00 0.1091602306E+01 0.1349183808E+01 0.1812528271E+01 0.1700241212E+01 0.1243645772E+01 0.8194722965E+00 0.1691128023E+01 0.1312845950E+01 0.1081038096E+01 0.9172759877E+00 0.1205973904E+01 0.1050059386E+01
MP indicates the matching probability, b the estimated Type II error probability under the hypothesis of loci independence, and No the number of observed alleles. H represents the Kolmogorov–Shennon entropy.
R. Biondo et al. / Forensic Science International 146S (2004) S135–S138
S137
Fig. 1. 10PlexY multiplex assay of DNA sample 10636. The amplification products are identified by the locus name reported above and the allele number reported below the electropherograms. The estimated size in bp of the amplification products is reported below the allele designation.
G5 matrix filter. Prior to data analysis, spectral matrices were established using the FL-JOE-TMR-CXR (Promega) and DS-33 (Applera) matrix standard sets. Electrophoretic data were analyzed with Genescan 2.5.2 (Applera). Allele designations were made by comparison to an allelic ladder (PowePlexY) or based on sizing bin windows of up to 1.25 bp (10 PlexY assay), using Genotyper 2.5.2 (Applera). The allele designations of the DYS437, DYS438, and DYS439 loci shared by the two multiplex assays were consistently identical.
5. Statistical analysis The matching probability at k-th locus (i. e. the probability for two randomly chosen individuals to exhibit the same allele), hereafter referred to as bk, was estimated by meansPof the observed relative frequencies of its alleles: bk ¼ i¼1;Nk ðnki =NÞ2 , where nki represents the i-th allele frequency at k-th locus, N the sample dimension and Nk the number of different alleles at k-th locus. Under the hypothesis that allele distributions at different loci are independent,
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R. Biondo et al. / Forensic Science International 146S (2004) S135–S138
the probability of joint occurrence of alleles at different loci factorizes and so does b. The power of discrimination is simply: Power ¼ 1 b ¼ 1 P k¼1;nloc bK ; where bk represents the matching probability for the k-th locus. The Kolmogorov–Shennon entropy was used as an index of locus polymorphism P and estimated from the allele frequencies:Hk ¼ i¼1;Nk ðnki =NÞ logðnki =NÞ. 6. Results The relative frequencies of the observed alleles are reported in Table 1 It is worth noting that a DYS388 variant (140 ), a DYS488 variant (200 ) and a DYS448 duplication (Fig. 1) were detected with a sizeable frequency: 0.079, 0.032, and 0.016, respectively. The discrimination power of the various loci varies, as indicated by the estimated matching probabilities (Table 2). To further characterize the polymorphism of the STR markers, the entropy values and the number of observed alleles are reported in Table 2. The DYS385 a/b, DYS447, DYS389 II, and DYS448 loci appear to be the most informative markers. The overall discrimination power (Table 2) was estimated assuming that the allele distributions of the different loci were independent. As a matter of fact, two different pair of unrelated individuals which exhibit the same partial Y haplotype, deter-
mined using only one of the two multiplex assay, were resolved by the entire Y haplotype resulting from the analysis of 20 Y-STR markers.
7. Discussion The results of the present study, based on a small sample of 75 individuals, provide novel information on the allele frequencies of 12 Y-STR markers in the Italian population. The entire Y haplotype resulting from the analysis of 20 YSTR markers was capable to discriminate all of the examined Italian individuals. The data bank is being extended to a larger representative sample and will possibly include additional loci.
References [1] R. Biondo, A. Spinella, P. Montagna, P.S. Walsh, C. Holt, B. Budowle, Regional Italian allele frequencies at nine short tandem repeat loci, Forensic Sci. Int. 115 (2001) 95–98. [2] J.M. Butler, R. Schoske, P.M. Vallone, M.C. Kline, A.J. Reed, M.F. Hammer, A novel multiplex for the simultaneous amplification of 20 Y chromosome STR markers, Forensic Sci. Int. 129 (2002) 10–24.
Announcement of population data
Typing of 20 Y-chromosome STRs in the Italian population Renato Biondoa,*, Alessandra Caglia`a, Paola Asilia, Giacomo D’Agostarob, Gregorio D’Agostinob, Aldo Spinellaa a
DCPC. Servizio Polizia Scientifica Area biologica Viale dell’Aeronautica, 7-00144 Roma, Italy b ENEA, CR ENEA Casaccia, S.P. Anguillarese, 301-00060 Roma, Italy Available online 30 October 2004
Abstract The aim of the present study is to appraise the increase of the discrimination power of the Y-specific haplotype allowed by 20 STR markers in a sample of the Italian population. The set of Y STR markers analyzed includes the European ‘‘extended haplotype’’ DYS19, DYS385, DYS389I/II, DYS390, DYS391, DYS392, DYS393 and YCAII a/b and in addition the DYS437, DYS438, DYS439, DYS447, DYS448, DYS388, DYS426, DYS460 (Y-GATA-A7.1) and Y-H4 loci. # 2004 Published by Elsevier Ireland Ltd. Keywords: Y-chromosome STRs; Population data
1. Population sample The DNA from 75 unrelated male individuals from northern, central and southern Italy was typed. The set of male DNA samples was from the DNA collection maintained in the laboratories of Italian State Police, Servizio Polizia Scientifica [1].
2. DNA extraction and quantification The DNA was purified from blood following the Chelex extraction procedure [1]. The male DNA concentration was determined using the Quantifiler Y Human Male DNA Quantification Kit (Applera).
based on the report of Butler et al. [2]. The 10 PlexY includes the dinucleotide marker YCAII, the trinucleotide loci DYS388 and DYS426, the tetranucleotide loci DYS437, DYS439, DYS460 (GATA A7.1), and Y-H4, the pentanucleotide loci DYS438 and DYS 447, and the exanucleotide marker DYS448. The two assays have in common the DYS437, DYS438, and DYS439 STR markers. This overlap provides a cross-check validation of the two procedures. The PowerPlexY amplifications were performed as recommended by the manufacturer (PowerPlexY Technical Manual, TMD018, Promega). The 10 PlexY amplifications were performed as described by Butler et al [2] with minor modifications. The concentration of DYS447 and DYS 448 primers was increased 1.5-fold, the time of synthesis at 72 8C was extended to 90 s, the number of cycles was increased to 32, and the amount DNA was lowered to 1–2 ng.
3. PCR amplification The same set of male DNA samples was amplified using the PowerPlexY system (Promega) and a 10 PlexY assay
4. Data analysis
* Corresponding author. Tel.: +39 0646544009; fax: +39 0646544380. E–mail address: [email protected] (R. Biondo).
The amplification products of the multiplex assays were resolved and detected using the ABIPRISM 3100 and 310 Genetic Analyzers (Applera), the POP4 polymer, and the
0379-0738/$ – see front matter # 2004 Published by Elsevier Ireland Ltd. doi:10.1016/j.forsciint.2004.09.041
S136
R. Biondo et al. / Forensic Science International 146S (2004) S135–S138
Table 1 Relative frequencies of 20 Y-STR marker alleles in the Italian population DYS19
DYS385a
12 13 14 15 16
0.016 0.159 0.460 0.286 0.079
DYS391 9 10 11
0.063 0.635 0.302
DYS447 17 19 20 21 22 23 24 25
0.032 0.032 0.111 0.159 0.429 0.127 0.079 0.032
DYS385b
DYS388
10 11 12 13 14 15 16 17 18
0.016 0.333 0.127 0.206 0.159 0.048 0.048 0.048 0.016
2 12 13 14 15 16 17 18 19
0.016 0.016 0.063 0.413 0.238 0.079 0.079 0.063 0.032
DYS392 11 12 13 14
0.524 0.032 0.397 0.048
DYS393 11 12 13 14 15
0.016 0.238 0.587 0.127 0.032
DYS448 200 22 23 24 25 22,23
0.032 0.349 0.429 0.127 0.048 0.016
DYS460 8 9 10 11
0.063 0.333 0.524 0.079
DYS389I
11 12 13 14 140 15 16
0.651 0.095 0.048 0.063 0.079 0.032 0.032
DYS426 10 11 12
YCAIIa 11 15 16 17 18 19
DYS389II
12 13 14 15
0.159 0.619 0.190 0.032
0.667 0.254 0.079
DYS437 14 15 16
0.302 0.556 0.143
0.048 0.048 0.762 0.032 0.079 0.032
YCAIIb 16 17 18 19 20 21
0.016 0.079 0.429 0.413 0.048 0.016
DYS390
28 29 30 31 32 33
0.190 0.286 0.381 0.079 0.048 0.016
DYS438 8 9 10 11 12
0.016 0.254 0.349 0.048 0.333
YH4 9 10 11 12
0.032 0.492 0.381 0.095
21 22 23 24 25 26
0.016 0.190 0.302 0.413 0.063 0.016
DYS439 10 11 12 13
0.079 0.333 0.460 0.127
Table 2 Statistical parameters Locus
MP
Power of discrimination
b
No.
H
DYS391 DYS389 I DYS439 DYS389 II DYS438 DYS437 DYS19 DYS392 DYS393 DYS390 DYS385 a DYS385 b DYS388 DYS426 DYS447 DYS448 DYS460 YCAII a YCAII b Y-H4
0.50 0.45 0.35 0.27 0.30 0.42 0.33 0.44 0.42 0.30 0.20 0.25 0.45 0.52 0.25 0.33 0.40 0.59 0.36 0.40
0.5018896447 0.7779901188 0.9233117795 0.9791517788 0.9937439578 0.9973724308 0.9991453283 0.9996281134 0.9998441805 0.9999529283 0.9999904766 0.9999976269 0.9999989387 0.9999994532 0.9999998651 0.9999999561 0.9999999826 0.9999999897 0.9999999963 0.9999999985
0.4981103553E+00 0.2220098812E+00 0.7668822049E-01 0.2084822119E-01 0.6256042186E-02 0.2627569243E-02 0.8546716786E-03 0.3718866185E-03 0.1558194625E-03 0.4707168948E-04 0.9523448387E-05 0.2373063859E-05 0.1061271945E-05 0.5468130832E-06 0.1348778051E-06 0.4387181819E-07 0.1736523718E-07 0.1030363657E-07 0.3740876872E-08 0.1486359997E-08
3 4 4 6 5 3 5 4 5 6 9 9 7 3 8 6 4 6 6 4
0.8249649452E+00 0.1014405690E+01 0.1186479613E+01 0.1453263460E+01 0.1292417876E+01 0.9660467271E+00 0.1274066295E+01 0.9599795458E+00 0.1091602306E+01 0.1349183808E+01 0.1812528271E+01 0.1700241212E+01 0.1243645772E+01 0.8194722965E+00 0.1691128023E+01 0.1312845950E+01 0.1081038096E+01 0.9172759877E+00 0.1205973904E+01 0.1050059386E+01
MP indicates the matching probability, b the estimated Type II error probability under the hypothesis of loci independence, and No the number of observed alleles. H represents the Kolmogorov–Shennon entropy.
R. Biondo et al. / Forensic Science International 146S (2004) S135–S138
S137
Fig. 1. 10PlexY multiplex assay of DNA sample 10636. The amplification products are identified by the locus name reported above and the allele number reported below the electropherograms. The estimated size in bp of the amplification products is reported below the allele designation.
G5 matrix filter. Prior to data analysis, spectral matrices were established using the FL-JOE-TMR-CXR (Promega) and DS-33 (Applera) matrix standard sets. Electrophoretic data were analyzed with Genescan 2.5.2 (Applera). Allele designations were made by comparison to an allelic ladder (PowePlexY) or based on sizing bin windows of up to 1.25 bp (10 PlexY assay), using Genotyper 2.5.2 (Applera). The allele designations of the DYS437, DYS438, and DYS439 loci shared by the two multiplex assays were consistently identical.
5. Statistical analysis The matching probability at k-th locus (i. e. the probability for two randomly chosen individuals to exhibit the same allele), hereafter referred to as bk, was estimated by meansPof the observed relative frequencies of its alleles: bk ¼ i¼1;Nk ðnki =NÞ2 , where nki represents the i-th allele frequency at k-th locus, N the sample dimension and Nk the number of different alleles at k-th locus. Under the hypothesis that allele distributions at different loci are independent,
S138
R. Biondo et al. / Forensic Science International 146S (2004) S135–S138
the probability of joint occurrence of alleles at different loci factorizes and so does b. The power of discrimination is simply: Power ¼ 1 b ¼ 1 P k¼1;nloc bK ; where bk represents the matching probability for the k-th locus. The Kolmogorov–Shennon entropy was used as an index of locus polymorphism P and estimated from the allele frequencies:Hk ¼ i¼1;Nk ðnki =NÞ logðnki =NÞ. 6. Results The relative frequencies of the observed alleles are reported in Table 1 It is worth noting that a DYS388 variant (140 ), a DYS488 variant (200 ) and a DYS448 duplication (Fig. 1) were detected with a sizeable frequency: 0.079, 0.032, and 0.016, respectively. The discrimination power of the various loci varies, as indicated by the estimated matching probabilities (Table 2). To further characterize the polymorphism of the STR markers, the entropy values and the number of observed alleles are reported in Table 2. The DYS385 a/b, DYS447, DYS389 II, and DYS448 loci appear to be the most informative markers. The overall discrimination power (Table 2) was estimated assuming that the allele distributions of the different loci were independent. As a matter of fact, two different pair of unrelated individuals which exhibit the same partial Y haplotype, deter-
mined using only one of the two multiplex assay, were resolved by the entire Y haplotype resulting from the analysis of 20 Y-STR markers.
7. Discussion The results of the present study, based on a small sample of 75 individuals, provide novel information on the allele frequencies of 12 Y-STR markers in the Italian population. The entire Y haplotype resulting from the analysis of 20 YSTR markers was capable to discriminate all of the examined Italian individuals. The data bank is being extended to a larger representative sample and will possibly include additional loci.
References [1] R. Biondo, A. Spinella, P. Montagna, P.S. Walsh, C. Holt, B. Budowle, Regional Italian allele frequencies at nine short tandem repeat loci, Forensic Sci. Int. 115 (2001) 95–98. [2] J.M. Butler, R. Schoske, P.M. Vallone, M.C. Kline, A.J. Reed, M.F. Hammer, A novel multiplex for the simultaneous amplification of 20 Y chromosome STR markers, Forensic Sci. Int. 129 (2002) 10–24.