- Email: [email protected]
Highly discriminatory capacity of the PowerPlex® Y23 System for the study of isolated populations
Accepted Manuscript Title: Highly discriminatory capacity of the PowerPlex® Y23 System for the study of isolated populations Author: Carolina Nu˜nez Miriam Baeta Miriam Fern´andez Maite Zarrabeitia Bego˜na Martinez-Jarreta Marian M.de Pancorbo PII: DOI: Reference:
S1872-4973(15)00075-7 http://dx.doi.org/doi:10.1016/j.fsigen.2015.04.005 FSIGEN 1340
To appear in:
Forensic Science International: Genetics
Received date: Revised date: Accepted date:
25-2-2015 31-3-2015 4-4-2015
Please cite this article as: Carolina Nu˜nez, Miriam Baeta, Miriam Fern´andez, Maite Zarrabeitia, Bego˜na Martinez-Jarreta, Marian M.de Pancorbo, Highly discriminatory capacity of the PowerPlexregdY23 System for the study of isolated populations, Forensic Science International: Genetics http://dx.doi.org/10.1016/j.fsigen.2015.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highly discriminatory capacity of the PowerPlex®Y23 System for the study of isolated populations Carolina Nuñez1a, Miriam Baeta1a, Miriam Fernández1, Maite Zarrabeitia2, Begoña Martinez-Jarreta3, Marian M. de Pancorbo1*. 1
BIOMICs Research Group, Centro de Investigación “Lascaray”
Ikergunea,
Universidad del País Vasco UPV/EHU, Vitoria-Gasteiz, Spain. 2
Unit of Legal Medicine, University of Cantabria, Santander, Spain.
3
Laboratory of Forensic Genetics, Faculty of Medicine, University of Zaragoza,
Zaragoza, Spain. a
These authors contributed equally to this work.
* Correspondence Author: Prof. Marian M. de Pancorbo BIOMICs Research Group Lascaray Research Center University of the Basque Country UPV/EHU Avda. Miguel de Unamuno, 3 01006 Vitoria-Gasteiz Spain Tel +34 945 01 4528 Fax +34 945 01 4458 [email protected] Highlights • First study of 23YSTR in the autochthonous Basque Country and Cantabria populations • The PPY23 is highly discriminative in the analysis of the studied populations • The PPY23 allows differentiating between similar haplotypes in isolated populations
• The use of PPY23 facilitates the identification of population exclusive haplotypes
Abstract In order to evaluate the forensic utility of the new PowerPlex®Y23 System, two Northern Spanish populations, the autochthonous Basque Country (N=105) and Cantabria (N=98), were typed. Two of the new markers incorporated in the panel, the rapid mutating loci DYS576 and DYS570, were among the most discriminative markers in both population datasets. In terms of the analysis of 23 Y-STRs, the two populations showed high haplotype diversities, with values slightly superior in the population of Cantabria (1 ± 0.0015) than in the Basque Country (0.9987 ± 0.0016). The comparison of the discrimination capacity obtained with the analysis of 23 Y-STRs and other available markers sets of 12 Y-STRs (PowerPlex®Y System) or 17 Y-STRs (YFilerTM), clearly demonstrated an improvement in the population of the Basque Country. Nevertheless, in Cantabria this augment was only seen when the number of markers was increased from 12 to 23, since the study of 17 Y-STRs was enough to differentiate all haplotypes. Therefore, this study shows that the improvement in forensic parameters by increasing the number of Y-STR markers analyzed is much more pronounced in the case of isolated populations such as the autochthonous population of the Basque Country, as it facilitates the differentiation among similar haplotypes. Moreover, by the use of the PowerPlex®Y23 identification of population specific haplotypes increased in both populations. Ultimately, the analysis of 23Y-STRs differentiated among the two geographically close populations of Basque Country and Cantabria. Indeed it showed significant differences between the Basque Country population and all European populations included, meanwhile Cantabria did exhibit significant proximity with the Iberian and the majority of European populations considered. Keywords Y chromosome, Short tandem repeats, PowerPlex®Y23 System, Basque Country, Cantabria 1. Introduction The analysis of human short tandem repeats of the Y chromosome (Y-STRs) has widely shown to be a powerful tool in forensic genetics for male sex and lineage identification, and it is also widely used for kinship investigation purposes [1, 2]. Until today, forensic laboratories have been generating Y chromosome haplotypes mainly based on two commonly used Y-STRs sets: the 12 Y-STRs included in the commercial kit PowerPlex®Y System (PPY12, Promega Corporation, Madison, WI) and the 17 Y-STRs contained within the AmpFlSTR YFiler® PCR Amplification Kit (YFiler, Life Technologies, Foster City, CA). Although, these kits have proved their robustness and capacity to discriminate between two male individuals, the discrimination power remains low in comparison with autosomal markers [3, 4]. Recently, the PowerPlex® Y23 System (PPY23, Promega Corporation, Madison, WI) has been developed, which allows amplification of 23 Y-STRs, combining 17 loci commonly included in commercially available kits (DYS389I, DYS448, DYS389II, DYS19, DYS391, DYS438, DYS437, DYS635, DYS390, DYS439, DYS392, DYS393, DYS458, DYS385a/b, DYS456, and YGATAH4) and six new loci (DYS481, DYS549, DYS533, DYS643, DYS576, and DYS570) with the last two being categorized as
“rapidly mutating” Y-STRs [5]. The first studies using the new PPY23 revealed a markedly increased haplotype diversity and discriminatory capacity in comparison with other marker sets [6, 7], proving to be highly beneficial for male identification in forensic casework. However, the use of the PPY23 in kinship analysis or familial searching must be taken with caution since, considering the mutation rate of some new Y-STR markers, even close relatives may exhibit one or more mismatches [6]. In order to contribute to the evaluation of the forensic utility of the new PPY23 System, we analyzed two different populations from the North of Spain, Cantabria and autochthonous population from the Basque Country. 2. Materials and methods 2.1.
Y-chromosome genotyping
A total of 203 healthy unrelated male individuals from the Basque Country (N=105) and Cantabria (N=98) were analyzed. The population sample set of the Basque Country included autochthonous individuals from three provinces, Alava (N=20), Biscay (N=61) and Gipuzkoa (N=24). The inclusion criteria used to define autochthony was Basque origin of both surnames and birthplaces of the individuals and their ancestors going back at least three generations. Informed consent was obtained from all individuals participating in the study. The 23 Y-STRs included in the PowerPlex® Y23 (Promega Corporation, Madison, WI) were analyzed according to the manufacturer’s protocol [8] with the exception of the reduction of the volume for the PCR reaction to 8.33 µl. The analyses were carried out with 1 ng of DNA. Capillary electrophoresis was conducted in an ABI PRIMS 3130 Genetic Analyzer (Applied Biosystems, Fosters City, CA). 2.2.
Analysis of data
Allelic frequencies, different and population specific haplotypes, the gene diversity (GD) of each locus and the haplotype diversity (HD) were calculated using the software Arlequin v3.5.1.2 [9]. For all statistical analyses, the repeat length of DYS389I was subtracted from DYS389II into DYS389B, and DYS385a/b alleles were treated as haplotypes. The global discrimination capacity (DC) was calculated by dividing the number of different haplotypes by the total number of individuals in the populations [10]. To assess the forensic utility of the new 23 Y-STR panel, all haplotype analysis were repeated for the 17 Y-STRs and 12 Y-STRs, included in AmpFlSTR® YFilerTM (Life Technologies, Foster City, CA) and PowerPlex Y® System (Promega Corporation, Madison, WI), respectively. Genetic distances RST and the corresponding p values between the populations from the Basque Country and Cantabria and 18 previously published European populations were determined using the software Arlequin v3.5.1.2. For this purpose, DYS385 was not considered. Significance p values were adjusted with the sequential Bonferroni correction (α=0.05/{[(1+n)/2]*n}= 2.381 x 10-4; n=number of populations) [11]. Multidimensional scaling analysis (MDS) was carried out using SPSS Statistics v18.0.0 (http://www.spss.com.hk/statistics/). 2.3.
Access to data
Haplotype data are accessible through YHRD under the accession numbers YA004015 (Cantabria), YA004016 (Alava, Basque Country), YA004017 (Gipuzkoa, Basque Country), and YA004018 (Biscay, Basque Country). 3. Results Allelic frequencies and the gene diversity for each marker are shown in Supplementary Table S1. The haplotypes of all individuals analyzed from the Basque Country (N=105) and Cantabria (N=98) are listed in Supplementary Table S2. Two of the new loci incorporated in the PPY23 were among the markers with the highest gene diversity in the populations studied: DYS576 (Basque Country: 0.7253; Cantabria: 0.7878) and DYS570 (Basque Country: 0.7044; Cantabria: 0.7568). Locus DYS458 showed the highest gene diversity in Cantabria (0.7926). On the other hand, the loci that contributed with the lowest gene diversity were DYS438 and DYS392 in the population from Basque Country studied (0.1443 and 0.1452, respectively), and DYS392 and DYS393 in Cantabria (0.4824 and 0.3198, respectively). A null allele was found in the Cantabrian sample at locus DYS448, which was confirmed with the YFiler kit. A total of 99/105 and 98/98 different haplotypes based on the 23 Y-STRs were obtained in the population samples of the Basque Country and Cantabria, respectively (Table 1). Accordingly, both populations showed high haplotype diversities, with values slightly superior in the population of Cantabria (1 ± 0.0015) than in the autochthonous population of the Basque Country (0.9987 ± 0.0016). To assess the forensic utility of the new panel of 23 Y-STR, haplotype parameters were compared with those provided for the 17 Y-STRs and 12 Y-STRs included in YFiler and PPY12 kits, respectively. In both populations, the forensic values obtained in the 12 Y-STRs analysis were noticeable lower to those observed with the 23 Y-STRs. In contrast, when taking into consideration the 17 Y-STRs, these values only decreased in the Basque Country population sample. In the case of Cantabria, differences were solely observed in the capacity for identifying population specific haplotypes, which was slightly lower with the 17 Y-STRs panel.
In order to study the genetic relationship between the autochthonous population of the Basque Country, Cantabria and other European populations, pairwise genetic distances (RST) and their corresponding p-values were obtained (Supplementary Table S3). As expected, significant differences (p<2.381 x 10-4) were found for the Basque Country population studied and the rest of European populations, including the neighboring Cantabria and other geographically close populations from other regions of Spain.
Contrary, the population of Cantabria only showed significant differences with the populations of Hungary, Sweden and Wales. These results are visualized in the MDS plot (Figure 1). The population sample of the Basque Country remains isolated from the other populations at the right side of the MDS graphic. On the other hand, the population of Cantabria appears in a central cluster, which encompasses the Iberian and European populations, except for Sweden, Hungary, Denmark and Wales.
4. Discussion This study provides the first population data for 23 Y-STR loci for the Basque Country and Cantabria populations from Northern Spain, genotyped with the PPY23. As expected, an improvement in forensic parameters (i.e. population specific haplotypes, discrimination capacity and haplotype diversity) was obtained in both populations with the PPY23, compared with the 12 Y-STRs included in the PPY12. However, when taking into consideration the 17 Y-STRs included in the YFiler, the discrimination capacity of the new panel was only higher in the Basque Country population sample. In Cantabria, no differences were observed in terms of discrimination as the analysis of 17 Y-STRs was able to differentiate all the haplotypes. Hence, the increment in the number of Y-STR markers does not always imply better forensic parameters, as was observed in Cantabria. On the other hand, in more isolated populations such as the autochthonous population of the Basque Country, the study of 23 Y-STRs might be very propitious to differentiate between identical haplotypes. The use of the PPY23 has also increased the identification of population specific haplotypes, as all 23 Y-STR haplotypes in Basque Country and Cantabria populations were exclusively observed in the corresponding populations. However, when the number of Y-STRs is lower, the shared haplotypes among these populations increases. Therefore, the implementation of the PPY23 in casework may strengthen the resolution of Y-STR analysis, as the statistical significance of obtaining a Y-STR match between the evidence and a known individual may increase. Furthermore, the genetic distances calculated with the 23 Y-STRs sustain the same European population structure as that observed with other marker panels [6]. Accordingly, significant differences were found between the Basque Country population and the rest of European populations. It is remarkably that this differentiation is also observed with other geographically close populations from the rest of Spain, including the neighboring Cantabria. This distinctiveness of the Basques has been previously observed in other studies based on Y and X chromosome STRs as well as mitochondrial DNA control region [12-15], and it has been attributed to the cultural and geographical isolation. Finally, the population of Cantabria, due to its non isolated nature, showed no significant differentiation with the majority of European populations.
5. Conclusions
This study shows, as expected, that the PPY23 proved to be highly discriminatory in the analysis of the populations of the Basque Country and Cantabria. However, the improvement of the discrimination capacity, by the addition of the six new loci that distinguish PPY23 from YFiler, may not be always so straightforward. When taking into consideration isolated populations such as the Basque Country, the analysis of the 23 Y-STRs might be very useful to differentiate between identical haplotypes. In summary, this new panel reveals a reinforced usefulness in forensic investigations especially in isolated populations and for identifying population specific haplotypes between geographically close groups. Quality control This paper follows the recommendations of the ISFG on the use of Y-STRs in forensic analysis [16] and the guidelines for publication of population data requested by the journal [17]. Conflict of interest Authors declare no competing interest in the content of this manuscript. Acknowledgements Funds were provided by the Basque Government (Grupo Consolidado IT833-13). The authors are grateful to all voluntary donors who cooperated generously in the development of this study. The authors thank for technical and human support provided by SGIker of UPV/EHU and the Basque Foundation for Health Research and Innovation (BIOEF). References 1. M. A. Jobling, A. Pandya, C. Tyler-Smith, The Y chromosome in forensic analysis and paternity testing, Int. J Legal Med. 110 (1997) 118-124. 2. L. Roewer, Y chromosome STR typing in crime casework, Forensic Sci. Med. Pathol. 5 (2009) 77-84. 3. E.K. Hanson, J. Ballantyne, An ultra-high discrimination Y chromosome short tandem repeat multiplex DNA typing system, PLoS ONE 2 (2007) e688. 4. M. Vermeulen, A. Wollstein, K. van der Gaag, O. Lao, Y.L. Xue, Q.J. Wang, L. Roewer, H. Knoblauch, C. Tyler-Smith, P. de Knijff, M. Kayser. Improving global and regional resolution of male lineage differentiation by simple single-copy Ychromosomal short tandem repeat polymorphisms, Forensic Sci. Int. Genet. 3 (2009) 205-213. 5. K.N. Ballantyne, V. Keerl, A. Wollstein, Y. Choi, S.B. Zuniga, A. Ralf, M. Vermeulen, P. de Knijff, M. Kayser, A new future of forensic Y-chromosome analysis: rapidly mutating Y-STRs for differentiating male relatives and paternal lineages, Forensic Sci. Int. Genet. 6 (2012) 208-218. 6. J. Purps, S. Siegert, S. Willuweit, M. Nagy, C. Alves, et al., A global analysis of Ychromosomal haplotype diversity for 23 STR loci, Forensic Sci. Int. Genet. 12 (2014) 12-23. 7. S. Turrina, S. Caratti, M. Ferrian, D. De Leo, Haplotype data and mutation rates for the 23 Y-STR loci of PowerPlex®Y23 System in a Northeast Italian population sample, Int. J Legal Med. (2014). In press.
8. Promega Corporation, Technical Manual: PowerPlex®Y23 System - Instructions for use of products DC2305 and DC2320, 2012, http://www.promega.com/. 9. L. Excoffier, H.E.L. Lischer, Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows, Mol. Ecol. Resour. 10 (2010) 564-567. 10. S. Calderon, D. Perez-Benedico, L. Mesa, D. Guyton, D.J. Rowold, R.J. Herrera, Phylogenetic and forensic studies of the Southeast Florida Hispanic population using the next-generation forensic PowerPlex®Y23 STR marker system, Legal Med. 15 (2013) 289-292. 11. Z. Zeng, R. Garcia-Bertrand, S. Calderon, L. Li, M. Zhong, R.J. Herrera, Extreme genetic heterogeneity among the nine major tribal Taiwanese island populations detected with a new generation Y23 STR system, Forensic Sci. Int. Genet. 12 (2014) 100-106. 12. L. Valverde, M. Rosique, S. Köhnemann, S. Cardoso, A. García, et al., Y-STR variation in the Basque diaspora in the Western USA: evolutionary and forensic perspectives. Int. J Legal Med. 126 (2012) 293-298. 13. S. Cardoso, M.A. Alfonso-Sánchez, L. Valverde, A. Odriozola, A.M. PérezMiranda, et al., The Maternal Legacy of Basques in Northern Navarre: New Insights Into the Mitochondrial DNA Diversity of the Franco-Cantabrian Area, Am. J Phys. Anthropol. 145 (2011) 480-488. 14. M.A. Alfonso-Sánchez, S. Cardoso, C. Martínez-Bouzas, J.A. Peña, R.J. Herrera, et al., Mitochondrial DNA haplogroup diversity in Basques: a reassessment based on HVI and HVII polymorphisms, Am. J Hum. Biol. 20 (2008) 154-164. 15. M.J. Illescas, A. Pérez, J.M. Aznar, L. Valverde, S. Cardoso, et al., Population genetic data for 10 X-STR loci in autochthonous Basques from Navarre (Spain), Forensic Sci. Int. Genet. 6 (2012) e146-e148. 16. L. Gusmao, J.M. Butler, A. Carracedo, P. Gill, M. Kayser, et al., DNA Commission of the international Society of Forensic Genetics (ISFG): an update of the recommendations on the use of Y-STRs in forensic analysis, Forensic Sci. Int. 157 (2006) 187-197. 17. A. Carracedo, J.M. Butler, L. Gusmao, A. Linacre, W. Parson, L. Roewer, P.M. Schneider, Update of the guidelines for the publication of genetic population data, Forensic Sci. Int. Genet. 10 (2014) A1-2.
FIGURE CAPTIONS Figure 1. Multidimensional scaling (MDS) plot based on pairwise RST values between the studied populations, Basque Country and Cantabria (bold dots) and 18 previously published European populations [6]. ARA: Aragón; AST: Asturias; BAS: Basque Country; CAN: Cantabria; BCN: Barcelona; DEN: Denmark; GER: Germany; ITA: Italy; SWI: Switzerland; POR: Portugal; AUS: Austria; HUN: Hungary; SWE: Sweden; MAD: Madrid; NET: Netherlands; GAL: Galicia; WAL: Wales; BEL: Belgium; OVI: Oviedo; and ENG: England. Stress: 0.02147.
Figure 1 .
Table 1. Number of different and population specific haplotypes, discrimination capacity (DC) and haplotype diversity (HD) obtained in the study of 23 (PPY23), 17 (YFiler) and 12 Y-STRs (PPY12) in the autochthonous population of the Basque Country and Cantabria.
Number of different haplotypes
Population
Number of population specific haplotypes
DC
HD
Basque Country (N=105)
PPY23 YFiler PPY12
99 85 76
99 83 61
0.9429 0.8095 0.7238
0.9987 ± 0.0016 0.9951 ± 0.0022 0.9901 ± 0.0036
Cantabria (N=98)
PPY23 YFiler PPY12
98 98 88
98 96 79
1 1 0.8980
1 ± 0.0015 1 ± 0.0015 0.9975 ± 0.0020
S1872-4973(15)00075-7 http://dx.doi.org/doi:10.1016/j.fsigen.2015.04.005 FSIGEN 1340
To appear in:
Forensic Science International: Genetics
Received date: Revised date: Accepted date:
25-2-2015 31-3-2015 4-4-2015
Please cite this article as: Carolina Nu˜nez, Miriam Baeta, Miriam Fern´andez, Maite Zarrabeitia, Bego˜na Martinez-Jarreta, Marian M.de Pancorbo, Highly discriminatory capacity of the PowerPlexregdY23 System for the study of isolated populations, Forensic Science International: Genetics http://dx.doi.org/10.1016/j.fsigen.2015.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highly discriminatory capacity of the PowerPlex®Y23 System for the study of isolated populations Carolina Nuñez1a, Miriam Baeta1a, Miriam Fernández1, Maite Zarrabeitia2, Begoña Martinez-Jarreta3, Marian M. de Pancorbo1*. 1
BIOMICs Research Group, Centro de Investigación “Lascaray”
Ikergunea,
Universidad del País Vasco UPV/EHU, Vitoria-Gasteiz, Spain. 2
Unit of Legal Medicine, University of Cantabria, Santander, Spain.
3
Laboratory of Forensic Genetics, Faculty of Medicine, University of Zaragoza,
Zaragoza, Spain. a
These authors contributed equally to this work.
* Correspondence Author: Prof. Marian M. de Pancorbo BIOMICs Research Group Lascaray Research Center University of the Basque Country UPV/EHU Avda. Miguel de Unamuno, 3 01006 Vitoria-Gasteiz Spain Tel +34 945 01 4528 Fax +34 945 01 4458 [email protected] Highlights • First study of 23YSTR in the autochthonous Basque Country and Cantabria populations • The PPY23 is highly discriminative in the analysis of the studied populations • The PPY23 allows differentiating between similar haplotypes in isolated populations
• The use of PPY23 facilitates the identification of population exclusive haplotypes
Abstract In order to evaluate the forensic utility of the new PowerPlex®Y23 System, two Northern Spanish populations, the autochthonous Basque Country (N=105) and Cantabria (N=98), were typed. Two of the new markers incorporated in the panel, the rapid mutating loci DYS576 and DYS570, were among the most discriminative markers in both population datasets. In terms of the analysis of 23 Y-STRs, the two populations showed high haplotype diversities, with values slightly superior in the population of Cantabria (1 ± 0.0015) than in the Basque Country (0.9987 ± 0.0016). The comparison of the discrimination capacity obtained with the analysis of 23 Y-STRs and other available markers sets of 12 Y-STRs (PowerPlex®Y System) or 17 Y-STRs (YFilerTM), clearly demonstrated an improvement in the population of the Basque Country. Nevertheless, in Cantabria this augment was only seen when the number of markers was increased from 12 to 23, since the study of 17 Y-STRs was enough to differentiate all haplotypes. Therefore, this study shows that the improvement in forensic parameters by increasing the number of Y-STR markers analyzed is much more pronounced in the case of isolated populations such as the autochthonous population of the Basque Country, as it facilitates the differentiation among similar haplotypes. Moreover, by the use of the PowerPlex®Y23 identification of population specific haplotypes increased in both populations. Ultimately, the analysis of 23Y-STRs differentiated among the two geographically close populations of Basque Country and Cantabria. Indeed it showed significant differences between the Basque Country population and all European populations included, meanwhile Cantabria did exhibit significant proximity with the Iberian and the majority of European populations considered. Keywords Y chromosome, Short tandem repeats, PowerPlex®Y23 System, Basque Country, Cantabria 1. Introduction The analysis of human short tandem repeats of the Y chromosome (Y-STRs) has widely shown to be a powerful tool in forensic genetics for male sex and lineage identification, and it is also widely used for kinship investigation purposes [1, 2]. Until today, forensic laboratories have been generating Y chromosome haplotypes mainly based on two commonly used Y-STRs sets: the 12 Y-STRs included in the commercial kit PowerPlex®Y System (PPY12, Promega Corporation, Madison, WI) and the 17 Y-STRs contained within the AmpFlSTR YFiler® PCR Amplification Kit (YFiler, Life Technologies, Foster City, CA). Although, these kits have proved their robustness and capacity to discriminate between two male individuals, the discrimination power remains low in comparison with autosomal markers [3, 4]. Recently, the PowerPlex® Y23 System (PPY23, Promega Corporation, Madison, WI) has been developed, which allows amplification of 23 Y-STRs, combining 17 loci commonly included in commercially available kits (DYS389I, DYS448, DYS389II, DYS19, DYS391, DYS438, DYS437, DYS635, DYS390, DYS439, DYS392, DYS393, DYS458, DYS385a/b, DYS456, and YGATAH4) and six new loci (DYS481, DYS549, DYS533, DYS643, DYS576, and DYS570) with the last two being categorized as
“rapidly mutating” Y-STRs [5]. The first studies using the new PPY23 revealed a markedly increased haplotype diversity and discriminatory capacity in comparison with other marker sets [6, 7], proving to be highly beneficial for male identification in forensic casework. However, the use of the PPY23 in kinship analysis or familial searching must be taken with caution since, considering the mutation rate of some new Y-STR markers, even close relatives may exhibit one or more mismatches [6]. In order to contribute to the evaluation of the forensic utility of the new PPY23 System, we analyzed two different populations from the North of Spain, Cantabria and autochthonous population from the Basque Country. 2. Materials and methods 2.1.
Y-chromosome genotyping
A total of 203 healthy unrelated male individuals from the Basque Country (N=105) and Cantabria (N=98) were analyzed. The population sample set of the Basque Country included autochthonous individuals from three provinces, Alava (N=20), Biscay (N=61) and Gipuzkoa (N=24). The inclusion criteria used to define autochthony was Basque origin of both surnames and birthplaces of the individuals and their ancestors going back at least three generations. Informed consent was obtained from all individuals participating in the study. The 23 Y-STRs included in the PowerPlex® Y23 (Promega Corporation, Madison, WI) were analyzed according to the manufacturer’s protocol [8] with the exception of the reduction of the volume for the PCR reaction to 8.33 µl. The analyses were carried out with 1 ng of DNA. Capillary electrophoresis was conducted in an ABI PRIMS 3130 Genetic Analyzer (Applied Biosystems, Fosters City, CA). 2.2.
Analysis of data
Allelic frequencies, different and population specific haplotypes, the gene diversity (GD) of each locus and the haplotype diversity (HD) were calculated using the software Arlequin v3.5.1.2 [9]. For all statistical analyses, the repeat length of DYS389I was subtracted from DYS389II into DYS389B, and DYS385a/b alleles were treated as haplotypes. The global discrimination capacity (DC) was calculated by dividing the number of different haplotypes by the total number of individuals in the populations [10]. To assess the forensic utility of the new 23 Y-STR panel, all haplotype analysis were repeated for the 17 Y-STRs and 12 Y-STRs, included in AmpFlSTR® YFilerTM (Life Technologies, Foster City, CA) and PowerPlex Y® System (Promega Corporation, Madison, WI), respectively. Genetic distances RST and the corresponding p values between the populations from the Basque Country and Cantabria and 18 previously published European populations were determined using the software Arlequin v3.5.1.2. For this purpose, DYS385 was not considered. Significance p values were adjusted with the sequential Bonferroni correction (α=0.05/{[(1+n)/2]*n}= 2.381 x 10-4; n=number of populations) [11]. Multidimensional scaling analysis (MDS) was carried out using SPSS Statistics v18.0.0 (http://www.spss.com.hk/statistics/). 2.3.
Access to data
Haplotype data are accessible through YHRD under the accession numbers YA004015 (Cantabria), YA004016 (Alava, Basque Country), YA004017 (Gipuzkoa, Basque Country), and YA004018 (Biscay, Basque Country). 3. Results Allelic frequencies and the gene diversity for each marker are shown in Supplementary Table S1. The haplotypes of all individuals analyzed from the Basque Country (N=105) and Cantabria (N=98) are listed in Supplementary Table S2. Two of the new loci incorporated in the PPY23 were among the markers with the highest gene diversity in the populations studied: DYS576 (Basque Country: 0.7253; Cantabria: 0.7878) and DYS570 (Basque Country: 0.7044; Cantabria: 0.7568). Locus DYS458 showed the highest gene diversity in Cantabria (0.7926). On the other hand, the loci that contributed with the lowest gene diversity were DYS438 and DYS392 in the population from Basque Country studied (0.1443 and 0.1452, respectively), and DYS392 and DYS393 in Cantabria (0.4824 and 0.3198, respectively). A null allele was found in the Cantabrian sample at locus DYS448, which was confirmed with the YFiler kit. A total of 99/105 and 98/98 different haplotypes based on the 23 Y-STRs were obtained in the population samples of the Basque Country and Cantabria, respectively (Table 1). Accordingly, both populations showed high haplotype diversities, with values slightly superior in the population of Cantabria (1 ± 0.0015) than in the autochthonous population of the Basque Country (0.9987 ± 0.0016). To assess the forensic utility of the new panel of 23 Y-STR, haplotype parameters were compared with those provided for the 17 Y-STRs and 12 Y-STRs included in YFiler and PPY12 kits, respectively. In both populations, the forensic values obtained in the 12 Y-STRs analysis were noticeable lower to those observed with the 23 Y-STRs. In contrast, when taking into consideration the 17 Y-STRs, these values only decreased in the Basque Country population sample. In the case of Cantabria, differences were solely observed in the capacity for identifying population specific haplotypes, which was slightly lower with the 17 Y-STRs panel.
In order to study the genetic relationship between the autochthonous population of the Basque Country, Cantabria and other European populations, pairwise genetic distances (RST) and their corresponding p-values were obtained (Supplementary Table S3). As expected, significant differences (p<2.381 x 10-4) were found for the Basque Country population studied and the rest of European populations, including the neighboring Cantabria and other geographically close populations from other regions of Spain.
Contrary, the population of Cantabria only showed significant differences with the populations of Hungary, Sweden and Wales. These results are visualized in the MDS plot (Figure 1). The population sample of the Basque Country remains isolated from the other populations at the right side of the MDS graphic. On the other hand, the population of Cantabria appears in a central cluster, which encompasses the Iberian and European populations, except for Sweden, Hungary, Denmark and Wales.
4. Discussion This study provides the first population data for 23 Y-STR loci for the Basque Country and Cantabria populations from Northern Spain, genotyped with the PPY23. As expected, an improvement in forensic parameters (i.e. population specific haplotypes, discrimination capacity and haplotype diversity) was obtained in both populations with the PPY23, compared with the 12 Y-STRs included in the PPY12. However, when taking into consideration the 17 Y-STRs included in the YFiler, the discrimination capacity of the new panel was only higher in the Basque Country population sample. In Cantabria, no differences were observed in terms of discrimination as the analysis of 17 Y-STRs was able to differentiate all the haplotypes. Hence, the increment in the number of Y-STR markers does not always imply better forensic parameters, as was observed in Cantabria. On the other hand, in more isolated populations such as the autochthonous population of the Basque Country, the study of 23 Y-STRs might be very propitious to differentiate between identical haplotypes. The use of the PPY23 has also increased the identification of population specific haplotypes, as all 23 Y-STR haplotypes in Basque Country and Cantabria populations were exclusively observed in the corresponding populations. However, when the number of Y-STRs is lower, the shared haplotypes among these populations increases. Therefore, the implementation of the PPY23 in casework may strengthen the resolution of Y-STR analysis, as the statistical significance of obtaining a Y-STR match between the evidence and a known individual may increase. Furthermore, the genetic distances calculated with the 23 Y-STRs sustain the same European population structure as that observed with other marker panels [6]. Accordingly, significant differences were found between the Basque Country population and the rest of European populations. It is remarkably that this differentiation is also observed with other geographically close populations from the rest of Spain, including the neighboring Cantabria. This distinctiveness of the Basques has been previously observed in other studies based on Y and X chromosome STRs as well as mitochondrial DNA control region [12-15], and it has been attributed to the cultural and geographical isolation. Finally, the population of Cantabria, due to its non isolated nature, showed no significant differentiation with the majority of European populations.
5. Conclusions
This study shows, as expected, that the PPY23 proved to be highly discriminatory in the analysis of the populations of the Basque Country and Cantabria. However, the improvement of the discrimination capacity, by the addition of the six new loci that distinguish PPY23 from YFiler, may not be always so straightforward. When taking into consideration isolated populations such as the Basque Country, the analysis of the 23 Y-STRs might be very useful to differentiate between identical haplotypes. In summary, this new panel reveals a reinforced usefulness in forensic investigations especially in isolated populations and for identifying population specific haplotypes between geographically close groups. Quality control This paper follows the recommendations of the ISFG on the use of Y-STRs in forensic analysis [16] and the guidelines for publication of population data requested by the journal [17]. Conflict of interest Authors declare no competing interest in the content of this manuscript. Acknowledgements Funds were provided by the Basque Government (Grupo Consolidado IT833-13). The authors are grateful to all voluntary donors who cooperated generously in the development of this study. The authors thank for technical and human support provided by SGIker of UPV/EHU and the Basque Foundation for Health Research and Innovation (BIOEF). References 1. M. A. Jobling, A. Pandya, C. Tyler-Smith, The Y chromosome in forensic analysis and paternity testing, Int. J Legal Med. 110 (1997) 118-124. 2. L. Roewer, Y chromosome STR typing in crime casework, Forensic Sci. Med. Pathol. 5 (2009) 77-84. 3. E.K. Hanson, J. Ballantyne, An ultra-high discrimination Y chromosome short tandem repeat multiplex DNA typing system, PLoS ONE 2 (2007) e688. 4. M. Vermeulen, A. Wollstein, K. van der Gaag, O. Lao, Y.L. Xue, Q.J. Wang, L. Roewer, H. Knoblauch, C. Tyler-Smith, P. de Knijff, M. Kayser. Improving global and regional resolution of male lineage differentiation by simple single-copy Ychromosomal short tandem repeat polymorphisms, Forensic Sci. Int. Genet. 3 (2009) 205-213. 5. K.N. Ballantyne, V. Keerl, A. Wollstein, Y. Choi, S.B. Zuniga, A. Ralf, M. Vermeulen, P. de Knijff, M. Kayser, A new future of forensic Y-chromosome analysis: rapidly mutating Y-STRs for differentiating male relatives and paternal lineages, Forensic Sci. Int. Genet. 6 (2012) 208-218. 6. J. Purps, S. Siegert, S. Willuweit, M. Nagy, C. Alves, et al., A global analysis of Ychromosomal haplotype diversity for 23 STR loci, Forensic Sci. Int. Genet. 12 (2014) 12-23. 7. S. Turrina, S. Caratti, M. Ferrian, D. De Leo, Haplotype data and mutation rates for the 23 Y-STR loci of PowerPlex®Y23 System in a Northeast Italian population sample, Int. J Legal Med. (2014). In press.
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FIGURE CAPTIONS Figure 1. Multidimensional scaling (MDS) plot based on pairwise RST values between the studied populations, Basque Country and Cantabria (bold dots) and 18 previously published European populations [6]. ARA: Aragón; AST: Asturias; BAS: Basque Country; CAN: Cantabria; BCN: Barcelona; DEN: Denmark; GER: Germany; ITA: Italy; SWI: Switzerland; POR: Portugal; AUS: Austria; HUN: Hungary; SWE: Sweden; MAD: Madrid; NET: Netherlands; GAL: Galicia; WAL: Wales; BEL: Belgium; OVI: Oviedo; and ENG: England. Stress: 0.02147.
Figure 1 .
Table 1. Number of different and population specific haplotypes, discrimination capacity (DC) and haplotype diversity (HD) obtained in the study of 23 (PPY23), 17 (YFiler) and 12 Y-STRs (PPY12) in the autochthonous population of the Basque Country and Cantabria.
Number of different haplotypes
Population
Number of population specific haplotypes
DC
HD
Basque Country (N=105)
PPY23 YFiler PPY12
99 85 76
99 83 61
0.9429 0.8095 0.7238
0.9987 ± 0.0016 0.9951 ± 0.0022 0.9901 ± 0.0036
Cantabria (N=98)
PPY23 YFiler PPY12
98 98 88
98 96 79
1 1 0.8980
1 ± 0.0015 1 ± 0.0015 0.9975 ± 0.0020