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deletion polymorphisms
Forensic Science International: Genetics Supplement Series 3 (2011) e9–e10
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
When the alleged father is a close relative of the real father: The utility of insertion/deletion polymorphisms M. Magalhaes a,b,*, N. Pinto a,b,c, C. Gomes a,b, R. Pereira a,d, A. Amorim a,b, C. Alves a, L. Gusma˜o a a
IPATIMUP, Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal Faculdade de Cieˆncias da Universidade do Porto, Porto, Portugal c Centro de Matema´tica da Universidade do Porto, Porto, Portugal d Instituto de Medicina Legal da Universidade de Santiago de Compostela, Santiago de Compostela, Spain b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 July 2011 Accepted 2 August 2011
The occurrence of Mendelian incompatibilities between true father and child is not an uncommon situation. Thus, in paternity investigations, when only a few are observed, it is possible that the alleged father is either the real father or his close relative. In this study, we intended to test how a set of autosomal indels can complement the results obtained with a routine battery of STRs in paternity investigations where the alleged father is, in fact, a close relative of the real one. We analyzed 100 uncle/ nephew and grandfather/grandchild unrelated duos, through the amplification of 15 STR markers and a set of 38 non-coding bi-allelic indels. For each duo, we determined the number of observed incompatibilities and calculated the LR assuming a father/son relationship, for both STRs and indels. As expected, in a significant proportion of cases, no incompatibilities were found. In these instances, indel’s LR contribution was always in favour of the ‘‘wrong’’ hypothesis, strengthening a false paternity. Therefore, in dubious cases, an extended battery of indels can be useful to exclude false fathers but should be taken with caution when no incompatibilities are detected. We estimated that to obtain a satisfactory probability (less than 1 in 500) of finding zero incompatibilities between 2nd degree relatives, approximately 100 indels with maximum diversity would be necessary. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Paternity testing STR Insertion/deletion polymorphism Indel
1. Introduction In paternity investigations, the occurrence of Mendelian incompatibilities in the transmission of STRs (Short Tandem Repeats) between true father and child is not an uncommon situation. In some cases, ambiguous results are obtained where the claimed relationship cannot be supported either because the Likelihood Ratio (LR) is too low or because few incompatibilities are observed associated with high LR values for the remaining loci [1]. Many of such cases arise from unknowingly testing, as alleged father, a close relative of the real father (his brother, father or son). Thus, in paternity testing, when only a few incompatibilities are observed, there is the possibility that the alleged father is either the real father (having occurred mutation or presence of silent alleles) or his close relative. This problem can be overcome by the use of a large number of additional markers with low mutation rates, such as indels (insertion/deletion polymorphisms) [2]. In this study, we sought to test how a set of autosomal indels can complement the
* Corresponding author at: IPATIMUP, Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal. Tel.: +351 225 570 700; fax: +351 225 570 799. E-mail address: [email protected] (M. Magalhaes). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.08.004
results obtained with a routine battery of STRs in paternity investigations where the alleged father is, in fact, a close relative of the real one.
2. Materials and methods We analyzed 100 uncle/nephew and grandfather/grandchild unrelated duos. STR analysis was done with the commercial multiplex kit Identifiler1 (Applied Biosystems) and indel analysis with on the 38 indel-plex described by Pereira et al. [2]. Both STR and indel genotypes were compared for each analyzed duo; we determined the number of observed incompatibilities and calculated the LR assuming a father/son relationship against unrelated, using Familias pedigree analysis software (http:// familias.name). For the STR markers, we used the allele frequencies described by Amorim et al. [3] and the mutation rates from the AABB Report [4]. For the indel markers, we used the allele frequencies described by Pereira et al. [2] for the European population and a mutation rate of m = 2.3 10 9 [5]. Based on the allele frequencies for both sets, we also estimated the theoretical number of incompatibilities expected to be found in uncle/nephew and grandfather/grandchild duos.
M. Magalhaes et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e9–e10
e10
Fig. 1. Distribution of the likelihood ratios calculated for the 15 STRs (A) and for the 38 indels (B). The blue area highlights the cases where the LR values were inconclusive (below 10,000 in favour of any of the alternative hypothesis).
Table 1 Comparison of the results obtained for 10 of the analyzed cases. STRs
Indels
STRs Indels
Incompatibilities
LR
Incompatibilities
LR
Final LR
1 1 1 1 1 2 2 2 2 2
1584 55 12 12 0.0383 11 1.43E-02 0.3079 3.50E-03 1.11E-02
1 3 1 1 0 2 0 0 0 0
3.68E-06 3.05E-24 4.84E-06 5.26E-07 149.33 1.95E-16 60.491 60.05 59.93 14.441
5.82E-03 1.67E-22 5.87E-05 6.28E-06 5.72 2.10E-15 8.62E-01 18.49 2.10E-01 1.61E-01
3. Results and discussion Results obtained with the STR set (Fig. 1A) were inconclusive in 33 cases, in which only less than 3 incompatibilities were observed and the LR values were between 10 4 and 104 (note that 104 is the LR value required by most laboratories for issuing a paternity report [6]). Five of these cases were in favour of the (false) hypothesis of paternity, since their LR values were higher than 1 (Table 1). Most of these inconclusive cases were resolved with the indel analysis. However, whenever no incompatibilities were found with the indel markers, the LR value was always in favour of the ‘‘wrong’’ hypothesis, strengthening a false paternity. Overall, this happened in 17 cases (Fig. 1B), 6 of which had shown inconclusive results with the STR set. When combining the STR and indel information, 9 cases remain inconclusive, 2 of which show LR values higher than 1, hence strengthening the hypothesis of paternity, which we know to be incorrect. Moreover, in these 2 cases, the indel analysis not only did it not allow the increase in number of observed incompatibilities, as it also inverted the LR value in favour of the hypothesis of paternity (Table 1). Finally, when the a priori exclusion chance is calculated for biallelic markers with maximum diversity (considering both alleles with a frequency of 0.5), approximately 100 indels are needed to reach a value of 99.8%. 4. Conclusions An extended battery of indels can be useful to exclude false fathers but should be taken with caution when no incompatibili-
ties are detected since, in these instances, the (false) hypothesis of paternity can be strengthened by the LR value. Role of funding IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and partially supported by the Portuguese Foundation for Science and Technology (FCT). NP is supported by FCT (PhD grant SFRH/BD/ 37261/2007). Conflict of interest None. References [1] C. Phillips, M. Fondevila, M. Garcı´a-Magarin˜os, et al., Resolving relationship tests that show ambiguous STR results using autosomal SNPs as supplementary markers, Forensic Science International: Genetics 2 (2008) 198–204. [2] R. Pereira, C. Phillips, C. Alves, et al., A new multiplex for human identification using insertion/deletion polymorphisms, Electrophoresis 30 (2009) 3682–3690. [3] A. Amorim, C. Alves, L. Gusma˜o, et al., Extended Northern Portuguese database on 21 autosomal STRs used in genetic identification, Progress in Forensic Genetics 11 (2006) 364–366. [4] American Association of Blood Banks (AABB). 2008. Annual Report Summary for Testing in 2008. Available at [http://www.aabb.org/sa/facilities/Documents/ rtannrpt08.pdf]. Accessed January 26, 2011. [5] M.W. Nachman, S.L. Crowell, Estimate of the mutation rate per nucleotide in humans, Genetics 156 (2000) 297–304. [6] C. Børsting, N. Morling, Mutations and/or close relatives? Six case work examples where 49 autosomal SNPs were used as supplementary markers, Forensic Science International: Genetics 5 (2011) 236–241.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
When the alleged father is a close relative of the real father: The utility of insertion/deletion polymorphisms M. Magalhaes a,b,*, N. Pinto a,b,c, C. Gomes a,b, R. Pereira a,d, A. Amorim a,b, C. Alves a, L. Gusma˜o a a
IPATIMUP, Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal Faculdade de Cieˆncias da Universidade do Porto, Porto, Portugal c Centro de Matema´tica da Universidade do Porto, Porto, Portugal d Instituto de Medicina Legal da Universidade de Santiago de Compostela, Santiago de Compostela, Spain b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 July 2011 Accepted 2 August 2011
The occurrence of Mendelian incompatibilities between true father and child is not an uncommon situation. Thus, in paternity investigations, when only a few are observed, it is possible that the alleged father is either the real father or his close relative. In this study, we intended to test how a set of autosomal indels can complement the results obtained with a routine battery of STRs in paternity investigations where the alleged father is, in fact, a close relative of the real one. We analyzed 100 uncle/ nephew and grandfather/grandchild unrelated duos, through the amplification of 15 STR markers and a set of 38 non-coding bi-allelic indels. For each duo, we determined the number of observed incompatibilities and calculated the LR assuming a father/son relationship, for both STRs and indels. As expected, in a significant proportion of cases, no incompatibilities were found. In these instances, indel’s LR contribution was always in favour of the ‘‘wrong’’ hypothesis, strengthening a false paternity. Therefore, in dubious cases, an extended battery of indels can be useful to exclude false fathers but should be taken with caution when no incompatibilities are detected. We estimated that to obtain a satisfactory probability (less than 1 in 500) of finding zero incompatibilities between 2nd degree relatives, approximately 100 indels with maximum diversity would be necessary. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Paternity testing STR Insertion/deletion polymorphism Indel
1. Introduction In paternity investigations, the occurrence of Mendelian incompatibilities in the transmission of STRs (Short Tandem Repeats) between true father and child is not an uncommon situation. In some cases, ambiguous results are obtained where the claimed relationship cannot be supported either because the Likelihood Ratio (LR) is too low or because few incompatibilities are observed associated with high LR values for the remaining loci [1]. Many of such cases arise from unknowingly testing, as alleged father, a close relative of the real father (his brother, father or son). Thus, in paternity testing, when only a few incompatibilities are observed, there is the possibility that the alleged father is either the real father (having occurred mutation or presence of silent alleles) or his close relative. This problem can be overcome by the use of a large number of additional markers with low mutation rates, such as indels (insertion/deletion polymorphisms) [2]. In this study, we sought to test how a set of autosomal indels can complement the
* Corresponding author at: IPATIMUP, Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal. Tel.: +351 225 570 700; fax: +351 225 570 799. E-mail address: [email protected] (M. Magalhaes). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.08.004
results obtained with a routine battery of STRs in paternity investigations where the alleged father is, in fact, a close relative of the real one.
2. Materials and methods We analyzed 100 uncle/nephew and grandfather/grandchild unrelated duos. STR analysis was done with the commercial multiplex kit Identifiler1 (Applied Biosystems) and indel analysis with on the 38 indel-plex described by Pereira et al. [2]. Both STR and indel genotypes were compared for each analyzed duo; we determined the number of observed incompatibilities and calculated the LR assuming a father/son relationship against unrelated, using Familias pedigree analysis software (http:// familias.name). For the STR markers, we used the allele frequencies described by Amorim et al. [3] and the mutation rates from the AABB Report [4]. For the indel markers, we used the allele frequencies described by Pereira et al. [2] for the European population and a mutation rate of m = 2.3 10 9 [5]. Based on the allele frequencies for both sets, we also estimated the theoretical number of incompatibilities expected to be found in uncle/nephew and grandfather/grandchild duos.
M. Magalhaes et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e9–e10
e10
Fig. 1. Distribution of the likelihood ratios calculated for the 15 STRs (A) and for the 38 indels (B). The blue area highlights the cases where the LR values were inconclusive (below 10,000 in favour of any of the alternative hypothesis).
Table 1 Comparison of the results obtained for 10 of the analyzed cases. STRs
Indels
STRs Indels
Incompatibilities
LR
Incompatibilities
LR
Final LR
1 1 1 1 1 2 2 2 2 2
1584 55 12 12 0.0383 11 1.43E-02 0.3079 3.50E-03 1.11E-02
1 3 1 1 0 2 0 0 0 0
3.68E-06 3.05E-24 4.84E-06 5.26E-07 149.33 1.95E-16 60.491 60.05 59.93 14.441
5.82E-03 1.67E-22 5.87E-05 6.28E-06 5.72 2.10E-15 8.62E-01 18.49 2.10E-01 1.61E-01
3. Results and discussion Results obtained with the STR set (Fig. 1A) were inconclusive in 33 cases, in which only less than 3 incompatibilities were observed and the LR values were between 10 4 and 104 (note that 104 is the LR value required by most laboratories for issuing a paternity report [6]). Five of these cases were in favour of the (false) hypothesis of paternity, since their LR values were higher than 1 (Table 1). Most of these inconclusive cases were resolved with the indel analysis. However, whenever no incompatibilities were found with the indel markers, the LR value was always in favour of the ‘‘wrong’’ hypothesis, strengthening a false paternity. Overall, this happened in 17 cases (Fig. 1B), 6 of which had shown inconclusive results with the STR set. When combining the STR and indel information, 9 cases remain inconclusive, 2 of which show LR values higher than 1, hence strengthening the hypothesis of paternity, which we know to be incorrect. Moreover, in these 2 cases, the indel analysis not only did it not allow the increase in number of observed incompatibilities, as it also inverted the LR value in favour of the hypothesis of paternity (Table 1). Finally, when the a priori exclusion chance is calculated for biallelic markers with maximum diversity (considering both alleles with a frequency of 0.5), approximately 100 indels are needed to reach a value of 99.8%. 4. Conclusions An extended battery of indels can be useful to exclude false fathers but should be taken with caution when no incompatibili-
ties are detected since, in these instances, the (false) hypothesis of paternity can be strengthened by the LR value. Role of funding IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and partially supported by the Portuguese Foundation for Science and Technology (FCT). NP is supported by FCT (PhD grant SFRH/BD/ 37261/2007). Conflict of interest None. References [1] C. Phillips, M. Fondevila, M. Garcı´a-Magarin˜os, et al., Resolving relationship tests that show ambiguous STR results using autosomal SNPs as supplementary markers, Forensic Science International: Genetics 2 (2008) 198–204. [2] R. Pereira, C. Phillips, C. Alves, et al., A new multiplex for human identification using insertion/deletion polymorphisms, Electrophoresis 30 (2009) 3682–3690. [3] A. Amorim, C. Alves, L. Gusma˜o, et al., Extended Northern Portuguese database on 21 autosomal STRs used in genetic identification, Progress in Forensic Genetics 11 (2006) 364–366. [4] American Association of Blood Banks (AABB). 2008. Annual Report Summary for Testing in 2008. Available at [http://www.aabb.org/sa/facilities/Documents/ rtannrpt08.pdf]. Accessed January 26, 2011. [5] M.W. Nachman, S.L. Crowell, Estimate of the mutation rate per nucleotide in humans, Genetics 156 (2000) 297–304. [6] C. Børsting, N. Morling, Mutations and/or close relatives? Six case work examples where 49 autosomal SNPs were used as supplementary markers, Forensic Science International: Genetics 5 (2011) 236–241.