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Exploring the applicability of analysing X chromosome STRs in Brazilian admixed population
Science and Justice 55 (2015) 323–328
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Science and Justice journal homepage: www.elsevier.com/locate/scijus
Exploring the applicability of analysing X chromosome STRs in Brazilian admixed population Eloisa Auler-Bittencourt a, Edna Sadayo Miazato Iwamura b,⁎, Maria Jenny Mitraud Lima c, Ismael Dale Cotrim Guerreiro da Silva a, Sidney Emannuel Batista dos Santos d a
Laboratório de Ginecologia Molecular do Departamento de Ginecologia Universidade Federal de São Paulo, SP, Brazil Departamento de Patologia da Escola Paulista de Medicina da Universidade Federal de São Paulo, SP, Brazil Divisão de Laboratório do Instituto de Criminalística de Minas Gerais, MG, Brazil d Laboratório de Genética Humana e Médica do Instituto de Ciências Biológicas da Universidade Federal do Pará, PA, Brazil b c
a r t i c l e
i n f o
Article history: Received 27 November 2014 Received in revised form 19 March 2015 Accepted 25 March 2015 Keywords: STR-Chr X Genetic kinship Forensic cases Brazilian population
a b s t r a c t Kinship and parentage analyses always involve one sample being compared to another sample or a few samples with a specific relationship question in mind. In most cases, the analysis of autosomal STR markers is sufficient to determine the genetic kinship. However, when genetic profiles are reconstructed from supposed relatives, for whom the family configuration available for analysis is deficient, the examination may be inconclusive. This study reports practical examples of actual cases analysing the efficiency of the chromosome X STR (STR-ChrX) markers. Three cases with different degrees of efficiency and impact were selected as follows: the identification of two charred bodies in a traffic accident, in which the family setting available was not complete, and one filiation analysis resulting from rape. This is the first paper reporting the use of the multiplex STR 12 ChrX in actual cases using the software Familias 1.8 and Brazilian regional frequency data. Our study clarifies the complex analysis using this powerful tool for professionals in the forensic science community, for both civil and criminal justice. We also discuss state-ofthe-art ChrX STR markers and its implications and applications for legal procedures. The data presented here should be used in other studies of complex cases to improve the progress of the current justice system. © 2015 The Chartered Society of Forensic Sciences. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction The determination of human identity to elucidate criminal cases using DNA analysis has been performed by Brazilian forensic laboratories for approximately 20 years. In particular, DNA analysis contributes to the identification of corpses in an advanced state of putrefaction, mutilation or charring. However, there are many interrelated circumstances and factors that challenge laboratories during DNA analysis for forensic genetics, such as the high number of victims in massive disasters, the extent of fragmentation of the bodies, the degree of DNA degradation, the accessibility of reference samples and an unfamiliar setting [1]. Usually, when biological kinship is investigated, the analysis of autosomal markers is sufficient to determine the genetic relationship. However, when genetic profiles are reconstructed from supposed relatives, or if the family members available for analysis are not sufficient, the number of regions analysed is increased to obtain more consistent ⁎ Corresponding author at: Department of Pathology, Escola Paulista de Medicina, Federal University of São Paulo, Rua Botucatu, 740 Edifico Lemos Torres, CEP 04023-062 São Paulo, SP, Brazil. E-mail address: [email protected] (E.S.M. Iwamura).
statistical parameters [2,3]. The currently available commercial kits enable forensic laboratories to analyse a maximum of 29 autosomal STR regions, and two of these regions, D6S1043 and Penta C, are not considered for statistical calculations. Moreover, in some situations because of the family configuration available for analysis, the regions of the autosomal STR cannot exclude the victim and his alleged blood relatives [4]. Currently, there are commercial kits available with up to 12 STRChrX markers, such as the Argus X-12 kit (Qiagen/Biotype); however only three loci, DXS7132, DXS7423, and HPRTB, are common to the Brazilian regional frequency evaluated by Ribeiro-Rodrigues et al. (2011) [5]. The markers most analysed in the Brazilian population have been described by Ribeiro-Rodrigues et al. in 2011 [5] and Gusmão et al. in 2009 [6] comprising a set of 15 STR-ChrX, and among them seven STR markers are common to both system multiplex [7]. This may change rapidly after the companies that market the kits engage in partnerships to compile the frequency of their own STR-ChrX markers, such that they can be used a greater number of Brazilian laboratories. Therefore, the cases presented here, from Rondonia [8], Minas Gerais and São Paulo, were analysed with the regional frequency data 12 STRChrX published by Ribeiro-Rodrigues et al. in 2011[5]. In these cases,
http://dx.doi.org/10.1016/j.scijus.2015.03.004 1355-0306/© 2015 The Chartered Society of Forensic Sciences. Published by Elsevier Ireland Ltd. All rights reserved.
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additional analyses are performed using the polymorphic regions of the ChrX STR markers, to increase in the number of regions and obtain a real power of exclusion for the analysis [9–13]. The exact location of the 12 STR-ChrX investigated in our study was obtained from the website http://www.ncbi.nlm.nih.gov and is shown in Fig. 1. A woman transmits by independent Mendelian segregation one allele of each autosomal STR and one allele from each ChrX STR to her children. By contrast, the man transmits, only for daughters, one allele from each region of the autosomal STR to his children and the single allele from the ChrX STR inherited from the mother. Therefore, all sisters share the ChrX STR haplotype they receive from their father. The analysis of sister ChrX STR markers allows the identification of the paternal allele and the reconstruction of all or part of the mother's genotype. The calculation of the likelihood ratio (LR) is widely accepted by the scientific community as the best way to evaluate the genetic linkages under various hypotheses [14–17]. However, the likelihoods should be clearly described, and it is important to consider the mode of transmission of the parental ChrX STR among females and males. To associate the autosomal STR and STR-ChrX LR, it is necessary for the H1 hypothesis (tested hypotheses or from prosecution, in some cases) and H2 (alternative or hypotheses of defence, in some cases) to be identical. It is necessary to evaluate the consistency and robustness of the examination, which should not be based solely on the value obtained by the statistical calculation, but on three parameters, the LR, the number of regions analysed and the situation of the case in question. In other words, in cases involving genetic linkages, the points commonly observed are the familiar setting available for analysis, how to reconstruct the genetic profiles of the individuals involved and the ability of exclusion.
Because of the relevance of the interpretation of the statistical analyses for the complementary analysis of ChrX STR markers in forensic practice, our objectives are as follows: a) to evaluate the efficiency of polymorphic regions of the ChrX STR for statistical calculations in cases of human identification with an incomplete family configuration and investigation of paternity arising from rape with the alleged father absent, b) to analyse the real power of exclusion that the analysis of ChrX STR adds to the examinations, and c) to use the software 1.8 Familias Program to estimate the LR involving the analysis complement of chromosome X. 2. Materials and methods All sample collection procedures were performed in accordance with the technical recommendations issued by the Brazilian forensic science community to ensure the quality, integrity and security for the forensic examinations involving DNA [18]. There is no identification of the samples used in this study referring to the real data of the cases analysed by the forensic laboratories, being maintained the necessary confidentiality. The collection of biological material from the corpses, including the tissue, teeth, bones and cartilage, depending on the condition, was performed by the coroner. An individual record signed by the responsible party for the necropsy of each victim, was assigned characteristics for comparison, such as fingerprints, tattoos, scars, dental implants, clothes and accessories. The collection of biological materials from the alleged relatives of the victims was accomplished by filling out a the form containing the following information: a) name of the victim; b) name (s) of the donor (s), age, sex, nationality, number of identity or birth certificate and
DXS9895 Xp22.31
DXS7132 Xq11.1
DXS6800 Xq21.1 DXS9898 Xq21.31 DXS6789 Xq21.33 DXS7133 Xq22.3 GATA172D05 Xq23 DXS7130 Xq24 HPRTB ~Xq26 GATA31E08 ~Xq27 DXS10011 Xq28
DXS7423 Xq28
Fig. 1. Location of XChromosomal Microsatellite. 3.1 Case # 1, Identification of corpse, Paternity analysis in the absence of the mother (duo).
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address; c) clarifying the relationship to the victim; and d) voluntary signing of the collection. The reference and alleged relative DNA samples for paternity cases resulting from rape were extracted from blood onto filter paper or from oral mucosa swabs of using the Chelex [19,20] method. For samples collected from cadavers, organic extraction with phenolchloroform-isoamyl alcohol was the preferred method and was followed by quantification using real-time PCR. The samples were amplified by polymerase chain reaction (PCR), using an AmpFlSTR Identifiler system, Applied Biosystems (fifteen autosomal regions and amelogenin) and a 16 System, Promega Corporation (fifteen autosomal regions and amelogenin) PowerPlex® system. Additional amplification was also performed with the following multiplex of twelve STR markers for ChrX: DXS7132, DXS7423, DXS7133, GATA172DO5, DX7130, DXS6800, GATA31E08, HPRTB, DXS6789, DXS9898, DXS9895 and DXS10011 for all samples collected using a thermal cycler GeneAmp PCR System 9700 (Applied Biosystems). The amplification products were subjected to capillary electrophoresis in an automatic sequencer ABI PRISM 3130 and analysed using the GeneMapper software v3.1 (Applied Biosystems). Statistical analysis was performed by considering the values of the frequencies of the alleles of the ChrX STR markers obtained from the regional database of Ribeiro et al. [5], and the autosomal markers described Poiares et al. [21] and Aguiar et al. [22]. The calculations were performed using the statistical programme Familias software version 1.8 [15] and, in some situations [23], an adaptation was necessary to insert the results. The procedure is highlighted in the each case report.
the kinship relationships and the need to complement the autosomal STR analysis with the objective of higher LR values and the ability of exclusion. The cases are presented in the form of pedigree to facilitate the understanding of the kinship relationships of the individuals involved
3. Results and discussion
Based on the comparative analysis of the genetic profiles of the 12 STR-ChrX sample I.3 (mother SMIP) and the genetic profile of sample II.2 (SMIP), we can identify the alleles of paternal origin, which must necessarily be present in the genetic profile of any daughter of this alleged father (I.2), as shown in Table S2. With the reconstruction of the genetic profile of the alleged father, we analysed the existence of familial genetic kinship between the profiles obtained for the victim sample (II.1) and the alleged father (I.2), even in the absence of the mother. The statistical calculation was performed using the Familias programme. A specific database (Table S3) and the ability to insert the results of the STR-ChrX of the alleged father as a homozygous genotype in all alleles were used. The value of LR (Paternity Index in the absence of the mother) obtained by associating the 15 autosomal STRs and 12 STR-ChrXs was 5.4 × 1011.
According to Szibor et al. (2003), one of the proprieties that must be evaluated in a marker, for its posterior application in forensics practice, is the Hardy–Weinberg equilibrium. Most of population studies made using Brazilian samples with STRChrX markers described by Martin et al. (2008), Gusmão et al. (2009) and Ribeiro-Rodrigues et al. (2011) are with a 15 STR-ChrX set. Among these markers, seven STR-ChrX are common to both system multiplex [7]. In the studies made using these markers with Brazilian sample, a linkage disequilibrium among them was not observed if we do not consider the linkage among them or any other factor that could modify the linkage equilibrium (selection, mutation, random drift, founder effect, miscegenation or population subdivision) [9]. Therefore, the X-STR markers used in our studies were treated as being independent. Three (03) cases were selected for the analysis of STR ChrX markers. In addition, the autosomal STR presented different degrees of efficiency and impact. (Figs. 2, 3 and 4).The selection was based on the success in obtaining the genetic profiles of the samples using the 12 STR-ChrX multiplex system. Moreover, there was the possibility of investigating
3.1. Analysis and interpretation of results from autosomal STR markers Because the sample of the alleged paternal half-sister (II.2) and her mother (I.3) was available, it was possible to partially reconstruct the genetic profile of the biological father (I.2) of victim II.2. The compatibility between the alleles of SMIP, his father and the victim's genetic profile is shown in Table S1. To perform the statistical calculations, two hypotheses (H1 and H2), were examined: - H1. genetic findings obtained are the result of the existence of a genetic link between the victim (II.1) and the alleged paternal half-sister (II.2); - H2. genetic findings obtained are the result of the existence of a genetic link between the victim (II.1) and another individual randomly selected from the population. Using the values of the frequencies of the alleles identified by 15 STR markers according to specific databases, we calculated the likelihood ratio-LR (Index of Parenting in the absence of the mother), obtaining a value of 8.9 × 104. 3.2. Analysis and interpretation of the 12 STR ChrX results
3.3. Analysis and interpretation of results from autosomal STR markers The possible genotypes of the biological parents were reconstructed after the analysis of 15 autosomal STRs of two alleged sisters (II.2 and II.3). In only two of the fifteen STR regions analysed (2/15), we
READING Sample not available Sample not available Victim FAMILIAR SETTING I.3- mother’s II.2 II.1-victim II.2- alleged paternal half-sister - SMIP Fig. 2. Heredogram, case # 1: Paternity analysis in the absence of the mother (duo)—the supposed father was reconstructed after examining one daughter's profile. 3.2 Case # 2, Identification of corpse—kinship of filiation.
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READING or
Sample not available
Victim
FAMILIAR SETTING II.1- victim II.2 - supposed sister - SI1 II.3 - supposed sister - SI2 Fig. 3. Heredogram case # 2, genetic kinship of filiation. Alleged parents genetic profiles are reconstructed by analysing the genetic profiles of the two daughters. 3.3 Case # 3, Paternity cases resulting from rape.
identified the four alleles of the parents of SI1 and SI2. In eight of the fifteen STR analysed (8/15), only two alleles of the SI1 and SI2 parents were identified; therefore, no individual selected at random in the population could be excluded as the biological brother of SI1 and SI2 (Table S4). After identifying the possible genotypes of the putative parents, we analysed the possibility of a genetic bond of filiation to the victim. For statistical calculations, two hypotheses, H1 and H2, were evaluated: - H1. genetic findings obtained are the result of the existence of kinship between the victim and individuals II.2 (supposed sister SI1) and II.3 (supposed sister SI2); - H2. genetic findings obtained are the result of the existence of kinship between the victim and randomly selected individuals of the population. Using the values of the allele frequencies identified by the 15 STR markers according to specific databases, we calculated the likelihood ratio—LR (Index Membership), and the value obtained was 2.2 × 105.
STR-ChrX they receive from the father, it was possible to detect incompatibilities between the alleles of paternal origin SI1 and SI2 in the twelve regions, as evidenced by the alleles identified in the genetic profile of the victim (Table S5). Considering the possibility of the father's genetic profile being heterozygous in the DXS 6800, the alleles 16 and 21 were inserted as heterozygous genotype. In the analysis of the 12 STR ChrX markers, there is the possibility of exclusion regarding paternity, and in 11 regions, in which it was possible to identify the two alleles of the SM, there is the possibility of simultaneously detecting the exclusion of paternity and maternity. The statistical calculation was performed using the Familias programme. The results were entered into a specific database (Table S6) indicating that the STR-ChrX of the alleged father is heterozygous in the DXS6800 genotype and homozygous in the others STR-ChrXs. The value of LR (filiation index) obtained by associating the autosomal STR – STR-ChrX was 2.7 × 1017.
3.5. Analysis and interpretation of results from autosomal STR markers 3.4. Analysis and interpretation of the 12 STR ChrX results The analysis of the 12 STR-ChrX of sisters SI1 and SI2 allowed the identification of an inherited paternal allele and reconstruction of the genetic profile of the mother. Because all sisters share the haplotype
We performed a comparative analysis of the 15 STR profiles obtained from samples of JRC (II.2) and her biological mother, MSBRC (I.2) to reconstruct the genetic profile of I.3, the biological father of the victim, JRC, as shown in Table S7.
READING Sample not available Sample not available Sample not available FAMILIAR SETTING I.2 - Mother of the victims (MSBRC) II.1 – Victim 1 (KRC) II.2 - Victim 2 (JRC) III.1-EMRC III.2-ERRC III.3-ECRC III.4-JuRC Fig. 4. Heredogram case # 3: Paternity, alleged father reconstructed by analysing one daughter (SII).
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According to the 15 autosomal STR profile obtained from sample II.2 and the alleged father reconstructed in I.3, and for the biological linkages of paternity, we analysed the possibility that I.3 (JMC) is the biological father of the child III.4 (JuRC). With partial reconstruction of the genetic profile of the father, JRC, incompatibilities were detected in any of the fifteen regions analysed (Table S8). In this case, the biological father, JRC, is also the alleged father of children III.1 (EMRC), III.2 (ERRC) and III.3 (ECRC). According to the 15 STR markers profiles obtained, we analysed the possibility that I.3 (JMC) is the biological father of children III.1 (EMRC), III.2 (ERRC) and III.3 (ECRC), as shown in Table S9. The calculations were performed using the statistical programme Familia, comparing the assumptions described below: a) For child JuRC - H1. genetic findings obtained are the result of the existence of a kinship between the paternity of the JuRC child and the alleged father (biological father of JRC); - H2. genetic findings obtained are the result of the existence of a kinship between the paternity and of the child JuRC another individual randomly selected from the population. b) For child EMRC Two hypotheses, H1 and H2, were evaluated: - H1. genetic findings obtained are the result of the existence of a kinship between the paternity of the EMRC child and the alleged father (biological father of JRC); - H2. genetic findings obtained are the result of the existence of kinship between the paternity of EMRC and another individual randomly selected from the population. c) For child ERRC Two hypotheses, H1 and H2, were tested: - H1. genetic findings obtained are the result of the existence of a kinship between the paternity of the ERRC child and the alleged father (biological father of JRC); - H2. genetic findings obtained are the result of the existence of kinship between the paternity of ERRC and another individual randomly selected from the population.
Using the values of allelic frequencies identified by markers from specific databases, we calculated the likelihood ratio (Paternity Index). The values of the paternity indices were, respectively, 1.5 × 104 for JuRC (victim's daughter JRC), 8.4 × 104 for EMRC (daughter of KRC), 1.9 × 106 for ERRC (daughter of KRC) and 3.9 × 104 for ECRC (daughter of KRC). However, because we have only one allele of the alleged father, the exam is not able to detect exclusion. 3.6. Analysis and interpretation of the 12 STR-ChrX results Based on the 12 STR-ChrXs, the profile of JMC (I.3), the biological father of JRC (II.2), for each locus analysed, there is only one allele (Table S10) which must necessarily be present in the genetic profile any of his daughter. After reconstruction of the genetic profile, we analysed the possibility that the alleged father (I.3) is the biological father of child III.4 (Table S11). By comparing the genetic profiles of the X chromosome STRs of children III.1 (EMRC), III.2 (ERRC), and III.3 (ECRC) with the alleged
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reconstructed father (I.3), as provided in the Table S12, no incompatibility was detected. The statistical calculation was performed using the Familias programme. The specific database (Table S13) and the results of 12 STR-ChrX of the alleged father as heterozygous genotypes in loci GATA172D05 and HPTRB and homozygous in other 10 STR-ChrX loci were used. The values of the paternity indices obtained, associating the autosomal STR and 12 STR-ChrX were, respectively, 2.05 × 1011 for JuRC, 1.28 × 1011 for EMRC (daughter of KRC), 1.73 × 1014 for (daughter of KRC) and 2.95 × 1012 for ECRC (daughter of KRC). Recently FamLinkX software [24] has been available for statistical calculations of probability data of genetic markers, linkage disequilibrium, and mutations However, our cases were analysed in 2012 and 2013. In this period Familias software 1.8 was used in forensics labs' routine to estimate the LR of the autosomal STR profiles. Currently, there are commercial kits available with up to 12 STRChrX markers, such as the Argus X-12 kit (Qiagen/biotype); however, only three loci, DXS7132, DXS7423, and HPRTB, are common to the Brazilian regional frequency evaluated by Ribeiro-Rodrigues et al. (2011) [5]. The 10 STR-ChrX markers analysed by Martins et al. (2010) only evaluated the populations of southeastern Brazil. Therefore, the cases presented here, from Rondonia, Minas Gerais and São Paulo, were compared with the regional frequency data of 12 STR-ChrX published by Ribeiro-Rodrigues et al. in 2011. This may change rapidly after the companies that market the kits engage in partnerships to compile the frequency of their own STR-ChrX markers, such that that they can be used a greater number of Brazilian laboratories.
4. Conclusion This study uses the 12 STR-ChrX standardized by the Brazilian group that evaluated the frequency of the five geographic regions of Brazil. In forensic genetics, it is important to accurately estimate the frequency of relevant profiles in reference populations. The 1.8 Familias software programme assists in calculations involving complementary analysis of STR-ChrX for estimating LR because this software is used by routine forensic laboratories to analyse autosomal STRs. The use of STR-ChrX in specific situations to determine genetic kinship, showed great efficiency, significantly increasing the value of LR, particularly by increasing the real power of exclusion of the exam. Therefore, this study presents practical examples by which STR-ChrX genotyping can be efficiently used to solve cases that would otherwise remain inconclusive. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scijus.2015.03.004.
Acknowledgements We thank the Brazilian forensic experts, especially Elzemar Ribeiro Rodrigues for technical support, to Paulo Roberto Fagundes by providing integration between official laboratories of forensic genetics and especially to the victims. This study was supported by the Federal University of Pará (UFPA) and Federal University of São Paulo—Department of Pathology, Escola Paulista de Medicina, Universidade Federal de São Paulo (EPM/UNIFESP). This work is part of the doctoral thesis of Eloisa Auler Bittencourt with the Institutional support of the Federal University of São Paulo and Federal University of Para.
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Science and Justice journal homepage: www.elsevier.com/locate/scijus
Exploring the applicability of analysing X chromosome STRs in Brazilian admixed population Eloisa Auler-Bittencourt a, Edna Sadayo Miazato Iwamura b,⁎, Maria Jenny Mitraud Lima c, Ismael Dale Cotrim Guerreiro da Silva a, Sidney Emannuel Batista dos Santos d a
Laboratório de Ginecologia Molecular do Departamento de Ginecologia Universidade Federal de São Paulo, SP, Brazil Departamento de Patologia da Escola Paulista de Medicina da Universidade Federal de São Paulo, SP, Brazil Divisão de Laboratório do Instituto de Criminalística de Minas Gerais, MG, Brazil d Laboratório de Genética Humana e Médica do Instituto de Ciências Biológicas da Universidade Federal do Pará, PA, Brazil b c
a r t i c l e
i n f o
Article history: Received 27 November 2014 Received in revised form 19 March 2015 Accepted 25 March 2015 Keywords: STR-Chr X Genetic kinship Forensic cases Brazilian population
a b s t r a c t Kinship and parentage analyses always involve one sample being compared to another sample or a few samples with a specific relationship question in mind. In most cases, the analysis of autosomal STR markers is sufficient to determine the genetic kinship. However, when genetic profiles are reconstructed from supposed relatives, for whom the family configuration available for analysis is deficient, the examination may be inconclusive. This study reports practical examples of actual cases analysing the efficiency of the chromosome X STR (STR-ChrX) markers. Three cases with different degrees of efficiency and impact were selected as follows: the identification of two charred bodies in a traffic accident, in which the family setting available was not complete, and one filiation analysis resulting from rape. This is the first paper reporting the use of the multiplex STR 12 ChrX in actual cases using the software Familias 1.8 and Brazilian regional frequency data. Our study clarifies the complex analysis using this powerful tool for professionals in the forensic science community, for both civil and criminal justice. We also discuss state-ofthe-art ChrX STR markers and its implications and applications for legal procedures. The data presented here should be used in other studies of complex cases to improve the progress of the current justice system. © 2015 The Chartered Society of Forensic Sciences. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction The determination of human identity to elucidate criminal cases using DNA analysis has been performed by Brazilian forensic laboratories for approximately 20 years. In particular, DNA analysis contributes to the identification of corpses in an advanced state of putrefaction, mutilation or charring. However, there are many interrelated circumstances and factors that challenge laboratories during DNA analysis for forensic genetics, such as the high number of victims in massive disasters, the extent of fragmentation of the bodies, the degree of DNA degradation, the accessibility of reference samples and an unfamiliar setting [1]. Usually, when biological kinship is investigated, the analysis of autosomal markers is sufficient to determine the genetic relationship. However, when genetic profiles are reconstructed from supposed relatives, or if the family members available for analysis are not sufficient, the number of regions analysed is increased to obtain more consistent ⁎ Corresponding author at: Department of Pathology, Escola Paulista de Medicina, Federal University of São Paulo, Rua Botucatu, 740 Edifico Lemos Torres, CEP 04023-062 São Paulo, SP, Brazil. E-mail address: [email protected] (E.S.M. Iwamura).
statistical parameters [2,3]. The currently available commercial kits enable forensic laboratories to analyse a maximum of 29 autosomal STR regions, and two of these regions, D6S1043 and Penta C, are not considered for statistical calculations. Moreover, in some situations because of the family configuration available for analysis, the regions of the autosomal STR cannot exclude the victim and his alleged blood relatives [4]. Currently, there are commercial kits available with up to 12 STRChrX markers, such as the Argus X-12 kit (Qiagen/Biotype); however only three loci, DXS7132, DXS7423, and HPRTB, are common to the Brazilian regional frequency evaluated by Ribeiro-Rodrigues et al. (2011) [5]. The markers most analysed in the Brazilian population have been described by Ribeiro-Rodrigues et al. in 2011 [5] and Gusmão et al. in 2009 [6] comprising a set of 15 STR-ChrX, and among them seven STR markers are common to both system multiplex [7]. This may change rapidly after the companies that market the kits engage in partnerships to compile the frequency of their own STR-ChrX markers, such that they can be used a greater number of Brazilian laboratories. Therefore, the cases presented here, from Rondonia [8], Minas Gerais and São Paulo, were analysed with the regional frequency data 12 STRChrX published by Ribeiro-Rodrigues et al. in 2011[5]. In these cases,
http://dx.doi.org/10.1016/j.scijus.2015.03.004 1355-0306/© 2015 The Chartered Society of Forensic Sciences. Published by Elsevier Ireland Ltd. All rights reserved.
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additional analyses are performed using the polymorphic regions of the ChrX STR markers, to increase in the number of regions and obtain a real power of exclusion for the analysis [9–13]. The exact location of the 12 STR-ChrX investigated in our study was obtained from the website http://www.ncbi.nlm.nih.gov and is shown in Fig. 1. A woman transmits by independent Mendelian segregation one allele of each autosomal STR and one allele from each ChrX STR to her children. By contrast, the man transmits, only for daughters, one allele from each region of the autosomal STR to his children and the single allele from the ChrX STR inherited from the mother. Therefore, all sisters share the ChrX STR haplotype they receive from their father. The analysis of sister ChrX STR markers allows the identification of the paternal allele and the reconstruction of all or part of the mother's genotype. The calculation of the likelihood ratio (LR) is widely accepted by the scientific community as the best way to evaluate the genetic linkages under various hypotheses [14–17]. However, the likelihoods should be clearly described, and it is important to consider the mode of transmission of the parental ChrX STR among females and males. To associate the autosomal STR and STR-ChrX LR, it is necessary for the H1 hypothesis (tested hypotheses or from prosecution, in some cases) and H2 (alternative or hypotheses of defence, in some cases) to be identical. It is necessary to evaluate the consistency and robustness of the examination, which should not be based solely on the value obtained by the statistical calculation, but on three parameters, the LR, the number of regions analysed and the situation of the case in question. In other words, in cases involving genetic linkages, the points commonly observed are the familiar setting available for analysis, how to reconstruct the genetic profiles of the individuals involved and the ability of exclusion.
Because of the relevance of the interpretation of the statistical analyses for the complementary analysis of ChrX STR markers in forensic practice, our objectives are as follows: a) to evaluate the efficiency of polymorphic regions of the ChrX STR for statistical calculations in cases of human identification with an incomplete family configuration and investigation of paternity arising from rape with the alleged father absent, b) to analyse the real power of exclusion that the analysis of ChrX STR adds to the examinations, and c) to use the software 1.8 Familias Program to estimate the LR involving the analysis complement of chromosome X. 2. Materials and methods All sample collection procedures were performed in accordance with the technical recommendations issued by the Brazilian forensic science community to ensure the quality, integrity and security for the forensic examinations involving DNA [18]. There is no identification of the samples used in this study referring to the real data of the cases analysed by the forensic laboratories, being maintained the necessary confidentiality. The collection of biological material from the corpses, including the tissue, teeth, bones and cartilage, depending on the condition, was performed by the coroner. An individual record signed by the responsible party for the necropsy of each victim, was assigned characteristics for comparison, such as fingerprints, tattoos, scars, dental implants, clothes and accessories. The collection of biological materials from the alleged relatives of the victims was accomplished by filling out a the form containing the following information: a) name of the victim; b) name (s) of the donor (s), age, sex, nationality, number of identity or birth certificate and
DXS9895 Xp22.31
DXS7132 Xq11.1
DXS6800 Xq21.1 DXS9898 Xq21.31 DXS6789 Xq21.33 DXS7133 Xq22.3 GATA172D05 Xq23 DXS7130 Xq24 HPRTB ~Xq26 GATA31E08 ~Xq27 DXS10011 Xq28
DXS7423 Xq28
Fig. 1. Location of XChromosomal Microsatellite. 3.1 Case # 1, Identification of corpse, Paternity analysis in the absence of the mother (duo).
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address; c) clarifying the relationship to the victim; and d) voluntary signing of the collection. The reference and alleged relative DNA samples for paternity cases resulting from rape were extracted from blood onto filter paper or from oral mucosa swabs of using the Chelex [19,20] method. For samples collected from cadavers, organic extraction with phenolchloroform-isoamyl alcohol was the preferred method and was followed by quantification using real-time PCR. The samples were amplified by polymerase chain reaction (PCR), using an AmpFlSTR Identifiler system, Applied Biosystems (fifteen autosomal regions and amelogenin) and a 16 System, Promega Corporation (fifteen autosomal regions and amelogenin) PowerPlex® system. Additional amplification was also performed with the following multiplex of twelve STR markers for ChrX: DXS7132, DXS7423, DXS7133, GATA172DO5, DX7130, DXS6800, GATA31E08, HPRTB, DXS6789, DXS9898, DXS9895 and DXS10011 for all samples collected using a thermal cycler GeneAmp PCR System 9700 (Applied Biosystems). The amplification products were subjected to capillary electrophoresis in an automatic sequencer ABI PRISM 3130 and analysed using the GeneMapper software v3.1 (Applied Biosystems). Statistical analysis was performed by considering the values of the frequencies of the alleles of the ChrX STR markers obtained from the regional database of Ribeiro et al. [5], and the autosomal markers described Poiares et al. [21] and Aguiar et al. [22]. The calculations were performed using the statistical programme Familias software version 1.8 [15] and, in some situations [23], an adaptation was necessary to insert the results. The procedure is highlighted in the each case report.
the kinship relationships and the need to complement the autosomal STR analysis with the objective of higher LR values and the ability of exclusion. The cases are presented in the form of pedigree to facilitate the understanding of the kinship relationships of the individuals involved
3. Results and discussion
Based on the comparative analysis of the genetic profiles of the 12 STR-ChrX sample I.3 (mother SMIP) and the genetic profile of sample II.2 (SMIP), we can identify the alleles of paternal origin, which must necessarily be present in the genetic profile of any daughter of this alleged father (I.2), as shown in Table S2. With the reconstruction of the genetic profile of the alleged father, we analysed the existence of familial genetic kinship between the profiles obtained for the victim sample (II.1) and the alleged father (I.2), even in the absence of the mother. The statistical calculation was performed using the Familias programme. A specific database (Table S3) and the ability to insert the results of the STR-ChrX of the alleged father as a homozygous genotype in all alleles were used. The value of LR (Paternity Index in the absence of the mother) obtained by associating the 15 autosomal STRs and 12 STR-ChrXs was 5.4 × 1011.
According to Szibor et al. (2003), one of the proprieties that must be evaluated in a marker, for its posterior application in forensics practice, is the Hardy–Weinberg equilibrium. Most of population studies made using Brazilian samples with STRChrX markers described by Martin et al. (2008), Gusmão et al. (2009) and Ribeiro-Rodrigues et al. (2011) are with a 15 STR-ChrX set. Among these markers, seven STR-ChrX are common to both system multiplex [7]. In the studies made using these markers with Brazilian sample, a linkage disequilibrium among them was not observed if we do not consider the linkage among them or any other factor that could modify the linkage equilibrium (selection, mutation, random drift, founder effect, miscegenation or population subdivision) [9]. Therefore, the X-STR markers used in our studies were treated as being independent. Three (03) cases were selected for the analysis of STR ChrX markers. In addition, the autosomal STR presented different degrees of efficiency and impact. (Figs. 2, 3 and 4).The selection was based on the success in obtaining the genetic profiles of the samples using the 12 STR-ChrX multiplex system. Moreover, there was the possibility of investigating
3.1. Analysis and interpretation of results from autosomal STR markers Because the sample of the alleged paternal half-sister (II.2) and her mother (I.3) was available, it was possible to partially reconstruct the genetic profile of the biological father (I.2) of victim II.2. The compatibility between the alleles of SMIP, his father and the victim's genetic profile is shown in Table S1. To perform the statistical calculations, two hypotheses (H1 and H2), were examined: - H1. genetic findings obtained are the result of the existence of a genetic link between the victim (II.1) and the alleged paternal half-sister (II.2); - H2. genetic findings obtained are the result of the existence of a genetic link between the victim (II.1) and another individual randomly selected from the population. Using the values of the frequencies of the alleles identified by 15 STR markers according to specific databases, we calculated the likelihood ratio-LR (Index of Parenting in the absence of the mother), obtaining a value of 8.9 × 104. 3.2. Analysis and interpretation of the 12 STR ChrX results
3.3. Analysis and interpretation of results from autosomal STR markers The possible genotypes of the biological parents were reconstructed after the analysis of 15 autosomal STRs of two alleged sisters (II.2 and II.3). In only two of the fifteen STR regions analysed (2/15), we
READING Sample not available Sample not available Victim FAMILIAR SETTING I.3- mother’s II.2 II.1-victim II.2- alleged paternal half-sister - SMIP Fig. 2. Heredogram, case # 1: Paternity analysis in the absence of the mother (duo)—the supposed father was reconstructed after examining one daughter's profile. 3.2 Case # 2, Identification of corpse—kinship of filiation.
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READING or
Sample not available
Victim
FAMILIAR SETTING II.1- victim II.2 - supposed sister - SI1 II.3 - supposed sister - SI2 Fig. 3. Heredogram case # 2, genetic kinship of filiation. Alleged parents genetic profiles are reconstructed by analysing the genetic profiles of the two daughters. 3.3 Case # 3, Paternity cases resulting from rape.
identified the four alleles of the parents of SI1 and SI2. In eight of the fifteen STR analysed (8/15), only two alleles of the SI1 and SI2 parents were identified; therefore, no individual selected at random in the population could be excluded as the biological brother of SI1 and SI2 (Table S4). After identifying the possible genotypes of the putative parents, we analysed the possibility of a genetic bond of filiation to the victim. For statistical calculations, two hypotheses, H1 and H2, were evaluated: - H1. genetic findings obtained are the result of the existence of kinship between the victim and individuals II.2 (supposed sister SI1) and II.3 (supposed sister SI2); - H2. genetic findings obtained are the result of the existence of kinship between the victim and randomly selected individuals of the population. Using the values of the allele frequencies identified by the 15 STR markers according to specific databases, we calculated the likelihood ratio—LR (Index Membership), and the value obtained was 2.2 × 105.
STR-ChrX they receive from the father, it was possible to detect incompatibilities between the alleles of paternal origin SI1 and SI2 in the twelve regions, as evidenced by the alleles identified in the genetic profile of the victim (Table S5). Considering the possibility of the father's genetic profile being heterozygous in the DXS 6800, the alleles 16 and 21 were inserted as heterozygous genotype. In the analysis of the 12 STR ChrX markers, there is the possibility of exclusion regarding paternity, and in 11 regions, in which it was possible to identify the two alleles of the SM, there is the possibility of simultaneously detecting the exclusion of paternity and maternity. The statistical calculation was performed using the Familias programme. The results were entered into a specific database (Table S6) indicating that the STR-ChrX of the alleged father is heterozygous in the DXS6800 genotype and homozygous in the others STR-ChrXs. The value of LR (filiation index) obtained by associating the autosomal STR – STR-ChrX was 2.7 × 1017.
3.5. Analysis and interpretation of results from autosomal STR markers 3.4. Analysis and interpretation of the 12 STR ChrX results The analysis of the 12 STR-ChrX of sisters SI1 and SI2 allowed the identification of an inherited paternal allele and reconstruction of the genetic profile of the mother. Because all sisters share the haplotype
We performed a comparative analysis of the 15 STR profiles obtained from samples of JRC (II.2) and her biological mother, MSBRC (I.2) to reconstruct the genetic profile of I.3, the biological father of the victim, JRC, as shown in Table S7.
READING Sample not available Sample not available Sample not available FAMILIAR SETTING I.2 - Mother of the victims (MSBRC) II.1 – Victim 1 (KRC) II.2 - Victim 2 (JRC) III.1-EMRC III.2-ERRC III.3-ECRC III.4-JuRC Fig. 4. Heredogram case # 3: Paternity, alleged father reconstructed by analysing one daughter (SII).
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According to the 15 autosomal STR profile obtained from sample II.2 and the alleged father reconstructed in I.3, and for the biological linkages of paternity, we analysed the possibility that I.3 (JMC) is the biological father of the child III.4 (JuRC). With partial reconstruction of the genetic profile of the father, JRC, incompatibilities were detected in any of the fifteen regions analysed (Table S8). In this case, the biological father, JRC, is also the alleged father of children III.1 (EMRC), III.2 (ERRC) and III.3 (ECRC). According to the 15 STR markers profiles obtained, we analysed the possibility that I.3 (JMC) is the biological father of children III.1 (EMRC), III.2 (ERRC) and III.3 (ECRC), as shown in Table S9. The calculations were performed using the statistical programme Familia, comparing the assumptions described below: a) For child JuRC - H1. genetic findings obtained are the result of the existence of a kinship between the paternity of the JuRC child and the alleged father (biological father of JRC); - H2. genetic findings obtained are the result of the existence of a kinship between the paternity and of the child JuRC another individual randomly selected from the population. b) For child EMRC Two hypotheses, H1 and H2, were evaluated: - H1. genetic findings obtained are the result of the existence of a kinship between the paternity of the EMRC child and the alleged father (biological father of JRC); - H2. genetic findings obtained are the result of the existence of kinship between the paternity of EMRC and another individual randomly selected from the population. c) For child ERRC Two hypotheses, H1 and H2, were tested: - H1. genetic findings obtained are the result of the existence of a kinship between the paternity of the ERRC child and the alleged father (biological father of JRC); - H2. genetic findings obtained are the result of the existence of kinship between the paternity of ERRC and another individual randomly selected from the population.
Using the values of allelic frequencies identified by markers from specific databases, we calculated the likelihood ratio (Paternity Index). The values of the paternity indices were, respectively, 1.5 × 104 for JuRC (victim's daughter JRC), 8.4 × 104 for EMRC (daughter of KRC), 1.9 × 106 for ERRC (daughter of KRC) and 3.9 × 104 for ECRC (daughter of KRC). However, because we have only one allele of the alleged father, the exam is not able to detect exclusion. 3.6. Analysis and interpretation of the 12 STR-ChrX results Based on the 12 STR-ChrXs, the profile of JMC (I.3), the biological father of JRC (II.2), for each locus analysed, there is only one allele (Table S10) which must necessarily be present in the genetic profile any of his daughter. After reconstruction of the genetic profile, we analysed the possibility that the alleged father (I.3) is the biological father of child III.4 (Table S11). By comparing the genetic profiles of the X chromosome STRs of children III.1 (EMRC), III.2 (ERRC), and III.3 (ECRC) with the alleged
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reconstructed father (I.3), as provided in the Table S12, no incompatibility was detected. The statistical calculation was performed using the Familias programme. The specific database (Table S13) and the results of 12 STR-ChrX of the alleged father as heterozygous genotypes in loci GATA172D05 and HPTRB and homozygous in other 10 STR-ChrX loci were used. The values of the paternity indices obtained, associating the autosomal STR and 12 STR-ChrX were, respectively, 2.05 × 1011 for JuRC, 1.28 × 1011 for EMRC (daughter of KRC), 1.73 × 1014 for (daughter of KRC) and 2.95 × 1012 for ECRC (daughter of KRC). Recently FamLinkX software [24] has been available for statistical calculations of probability data of genetic markers, linkage disequilibrium, and mutations However, our cases were analysed in 2012 and 2013. In this period Familias software 1.8 was used in forensics labs' routine to estimate the LR of the autosomal STR profiles. Currently, there are commercial kits available with up to 12 STRChrX markers, such as the Argus X-12 kit (Qiagen/biotype); however, only three loci, DXS7132, DXS7423, and HPRTB, are common to the Brazilian regional frequency evaluated by Ribeiro-Rodrigues et al. (2011) [5]. The 10 STR-ChrX markers analysed by Martins et al. (2010) only evaluated the populations of southeastern Brazil. Therefore, the cases presented here, from Rondonia, Minas Gerais and São Paulo, were compared with the regional frequency data of 12 STR-ChrX published by Ribeiro-Rodrigues et al. in 2011. This may change rapidly after the companies that market the kits engage in partnerships to compile the frequency of their own STR-ChrX markers, such that that they can be used a greater number of Brazilian laboratories.
4. Conclusion This study uses the 12 STR-ChrX standardized by the Brazilian group that evaluated the frequency of the five geographic regions of Brazil. In forensic genetics, it is important to accurately estimate the frequency of relevant profiles in reference populations. The 1.8 Familias software programme assists in calculations involving complementary analysis of STR-ChrX for estimating LR because this software is used by routine forensic laboratories to analyse autosomal STRs. The use of STR-ChrX in specific situations to determine genetic kinship, showed great efficiency, significantly increasing the value of LR, particularly by increasing the real power of exclusion of the exam. Therefore, this study presents practical examples by which STR-ChrX genotyping can be efficiently used to solve cases that would otherwise remain inconclusive. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scijus.2015.03.004.
Acknowledgements We thank the Brazilian forensic experts, especially Elzemar Ribeiro Rodrigues for technical support, to Paulo Roberto Fagundes by providing integration between official laboratories of forensic genetics and especially to the victims. This study was supported by the Federal University of Pará (UFPA) and Federal University of São Paulo—Department of Pathology, Escola Paulista de Medicina, Universidade Federal de São Paulo (EPM/UNIFESP). This work is part of the doctoral thesis of Eloisa Auler Bittencourt with the Institutional support of the Federal University of São Paulo and Federal University of Para.
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