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An Italian Jean Jacques Rousseau: A complex kinship case
Forensic Science International: Genetics Supplement Series 3 (2011) e520–e521
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
An Italian Jean Jacques Rousseau: A complex kinship case S. Inturri a,*, C. Robino a, I. Carboni b, U. Ricci b, S. Gino a a b
Laboratory of Criminalistic Sciences, Department of Anatomy, Pharmacology and Legal Medicine, University of Turin, Turin, Italy SOD Diagnostica Genetica, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 September 2011 Accepted 8 October 2011
We report the case of four women and a man, all born in an Italian village during and immediately after WWII, that recently contacted our laboratory in order to perform kinship analysis. According to their claim, the propositi were the illegitimate offspring of a country gentleman and a peasant woman, given in adoption immediately after birth. A story that curiously reminded us of Jean Jacques Rousseau, The´re`se Levasseur and their five children. Problems connected with DNA analysis in cases where all stated relationship are questioned, and a wide range of different pedigrees could be used as hypotheses in LR calculations are discussed. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Short tandem repeats Paternity testing
1. Introduction Since the introduction of haploid markers and binary polymorphisms, forensic DNA typing has no longer been confined to conventional paternity cases. Here we report the case of four women (named S1 to S4) and a man (S5), all born in an Italian village during and immediately after WWII, that recently contacted our laboratory in order to perform kinship analysis. According to their claim, the propositi were the illegitimate offspring of a country gentleman and a peasant woman, given in adoption immediately after birth. A story that curiously reminded us of Jean Jacques Rousseau, The´re`se Levasseur and their five children. In particular, the women took for granted they were full sisters and were interested in knowing whether the man was either a full-sib or unrelated. Results of likelihood ratio (LR) calculations, based on this simple alternative and genotypic data from a conventional set of CODIS STR markers, produced a LR value largely in favour of the hypothesis of full-sibship (LR > 108). However, since the women were not able to provide documentation supporting their claim (birth certificates indicated parents unknown), we decided not to convey the results in such a simplistic form and investigate the case further. 2. Materials and methods Mitochondrial DNA (mtDNA) haplotypes of Hypervariable regions HV1 (position 16024–16365) and HV2 (position 73–340)
* Corresponding author at: Corso Galileo Galilei, 22–10126 Torino, Italy. Tel.: +39 011 6705625; fax: +39 011 6705934. E-mail address: [email protected] (S. Inturri). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.10.007
were characterized by sequence analysis as described by Steighner and Holland [1]. Sequences were aligned and compared to the revised Cambridge reference sequence (rCRS). 21 X-chromosomal STR markers were amplified by the combination of two in-house developed multiplexes [2] and of the InvestigatorTM Argus X-12 Kit (Qiagen, Hilden Germany). A total of 40 autosomal STR were amplified combining the Identifiler PCR Amplification kit (Applied Biosystems Foster City, CA) and 25 non-CODIS miniSTR [3]. All STRs were typed by capillary electrophoresis on the ABI PRISM 310 (Applied Biosystems) in comparison to allelic ladders and control DNA of known genotype. For statistical analysis of X and autosomal STR data, allele frequencies described in the Italian population were used [4,5]. 3. Results and discussion In complex kinship cases, where all stated relationships could be questioned, the range of different pedigrees that can be used as complementary hypotheses in LR calculations expands rapidly with the number of tested subjects. In the reported case age information allowed to exclude a priori that the propositi belong to different generations. Even so, a simulation with the software Familias v1.81 produced thousands of alternative pedigrees causing the program to ‘‘hang’’ [6]. In such circumstances further simplification can derive from the inclusion in the analysis of haploid markers. All the tested subjects displayed the same mtDNA haplotype (16069[T], 16126[C], 16224[C], 73[G], 228[A], 263[G], 295[T], 315.1[C]), previously unreported in EMPOP [7]. X-chromosomal (Table 1) and autosomal STR data were consistent with the hypothesis of full-sibship, requiring a single recombination event in linkage group I (Xp22.31) of S4. LR calculations were performed
S. Inturri et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e520–e521 Table 1 Reconstructed putative parental X-chromosomes. Locus Xp22.31 DXS10148 DXS10135 DXS8378
S1
S2
Table 2 Posterior probabilities of H0 and H1 (A-STRs = autosomal STRs). S3
S4
S5
21 X-STRs
15 A-STRs
40 A-STRs
X and A-STRs
P0
P1
P0
P1
P0
P1
P0
P1
.918 .794 .830 .454 .775 .860 .498 .538 .602 .503
.082 .206 .170 .545 .225 .140 .502 .462 .398 .497
.980 .789 .953 .635 .507 .753 .778 .921 .902 .707
.020 .211 .047 .365 .493 .247 .222 .079 .098 .293
.997 .651 .997 .792 .700 .952 .934 .949 .915 .951
.003 .349 .003 .208 .300 .048 .066 .051 .085 .049
>.999 .833 >.999 .688 .848 .988 .907 .938 .919 .932
<.001 .167 <.001 .312 .152 .012 .093 .062 .081 .068
23 22 10
24 21 10
23 22 10
24 21 10
23 22 10
24 21 10
23 22 10
24 24 10
25.1 24 10
13 17 8 16
14 19 17 19
13 17 8 16
14 19 17 19
13 17 8 16
12 21 17 19
13 17 8 16
14 19 17 19
12 21 17 19
12 33 20
11 34 20
12 33 20
11 34 20
12 33 20
10 34 21
12 33 20
11 34 20
10 34 21
DXS7424 14 DXS101 28 GATA172D05 6
18 18 10
14 28 6
18 18 11
14 28 6
15 29 10
14 28 6
18 18 11
15 29 10
Xq26
DXS10103 HPRTB DXS10101
19 13 28
21 19 12 13 30.2 28
21 19 12 13 30.2 28
21 19 12 13 30.2 28
18 21 12 12 28.2 30.2
Xq28
DXS8377 DXS10146 DXS10134 DXS7423 DXS10011
48 28 33 15 30.2
51 30 35 15 33
51 30 35 15 33
50 27 38 15 40
51 30 35 15 33
Xq12
DXS7132 DXS10079 DXS10074 DXS6800
Xq21.33 DXS6801 DXS6809 DXS6789 Xq22.1
48 28 33 15 30.2
e521
48 28 33 15 30.2
48 28 33 15 30.2
50 27 38 15 33
for each pair of tested individuals. Due to the complexity of the case, we used as alternative hypothesis to full sibship (H0), the combination of distinct hypotheses (H1a–1d), all with equal weight [8]:H1athe tested subjects are maternal half-sibs;H1bthe tested subjects are three-quarter sibs;H1cthe tested subjects are double first cousins;H1cthe tested subjects are unrelated. Posterior probabilities of full-sibship (P0) and other selected kinship configurations (P1), given an a priori probability of H0 of 0.5, were calculated with the software Mendel [9] and are shown in Table 2. Based on the obtained results, full-sibship of S1, S2 and S4 could be considered reasonably proved. By this assumption, the number of pedigrees generated by Familias was reduced to 625. Fullsibship resulted the most likely relationship, with a posterior probability of .995, followed by the constellation in which S1–S3 and S5 were full-sibs and S3 their three-quarter sib (posterior probability .002). The reported case exemplifies the difficulty to infer relatedness given individual genotypes, whenever simple assumptions regarding alternative pedigrees cannot be taken a priori, as in routine paternity testing. The problem applies both to unconventional kinship cases, as the one here described, and immigration cases
S1–S2 S1–S3 S1–S4 S1–S5 S2–S3 S2–S4 S2–S5 S3–S4 S3–S5 S4–S5
requiring DNA analysis. Addition of extra markers to conventional STR loci, including mtDNA, Y and X-STRs is often mandatory. Even so, the figures attained in LR calculations and the associated posterior probabilities of pedigrees are frequently under the standard cut-off value of 99.99%. Moreover, special care must be taken when reporting results to the court and the tested subjects. In particular, awareness of the fact that LR values depend on the choice of alternative hypotheses is essential to the understanding of the conveyed information. Conflict of interest None. References [1] R.J. Steighner, M. Holland, Amplification and sequencing of mitochondrial DNA in forensic casework, Methods Mol. Biol. 98 (1998) 213–223. [2] C. Robino, A. Giolitti, S. Gino, et al., Development of two multiplex PCR systems for the analysis of 12 X-chromosomal STR loci in a northwestern Italian population sample, Int. J. Legal Med. 120 (2006) 315–318. [3] C.R. Hill, M.C. Kline, M.D. Coble, et al., Characterization of 26 MiniSTR loci for improved analysis of degraded DNA samples, J. Forensic Sci. 53 (2008) 73–80. [4] S. Inturri, S. Menegon, A. Amoroso, et al., Linkage and linkage disequilibrium analysis of X-STRs in Italian families, Forensic Sci. Int. Genet. 5 (2011) 152–154. [5] S. Presciuttini, N. Cerri, S. Turrina, et al., Validation of a large Italian database of 15 STR loci, Forensic Sci. Int. 156 (2006) 266–268. [6] T. Egeland, P.F. Mostad, B. Mevaˆg, et al., Beyond traditional paternity and identification cases. Selecting the most probable pedigree, Forensic Sci. Int. 110 (2000) 47–59. [7] W. Parson, A. Du¨r, EMPOP – a forensic mtDNA database, Forensic Sci. Int. Genet. 1 (2007) 88–92. [8] A.O. Karlsson, G. Holmlund, T. Egeland, et al., DNA-testing for immigration cases: the risk of erroneous conclusions, Forensic Sci. Int. 172 (2007) 144–149. [9] K. Lange, R. Cantor, S. Horvath, et al., Mendel Version 4.0: a complete package for the exact genetic analysis of discrete traits in pedigree and population data sets, Am. J. Hum. Genet. 69 (Suppl.) (2001) 504 (2001).
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
An Italian Jean Jacques Rousseau: A complex kinship case S. Inturri a,*, C. Robino a, I. Carboni b, U. Ricci b, S. Gino a a b
Laboratory of Criminalistic Sciences, Department of Anatomy, Pharmacology and Legal Medicine, University of Turin, Turin, Italy SOD Diagnostica Genetica, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 September 2011 Accepted 8 October 2011
We report the case of four women and a man, all born in an Italian village during and immediately after WWII, that recently contacted our laboratory in order to perform kinship analysis. According to their claim, the propositi were the illegitimate offspring of a country gentleman and a peasant woman, given in adoption immediately after birth. A story that curiously reminded us of Jean Jacques Rousseau, The´re`se Levasseur and their five children. Problems connected with DNA analysis in cases where all stated relationship are questioned, and a wide range of different pedigrees could be used as hypotheses in LR calculations are discussed. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Short tandem repeats Paternity testing
1. Introduction Since the introduction of haploid markers and binary polymorphisms, forensic DNA typing has no longer been confined to conventional paternity cases. Here we report the case of four women (named S1 to S4) and a man (S5), all born in an Italian village during and immediately after WWII, that recently contacted our laboratory in order to perform kinship analysis. According to their claim, the propositi were the illegitimate offspring of a country gentleman and a peasant woman, given in adoption immediately after birth. A story that curiously reminded us of Jean Jacques Rousseau, The´re`se Levasseur and their five children. In particular, the women took for granted they were full sisters and were interested in knowing whether the man was either a full-sib or unrelated. Results of likelihood ratio (LR) calculations, based on this simple alternative and genotypic data from a conventional set of CODIS STR markers, produced a LR value largely in favour of the hypothesis of full-sibship (LR > 108). However, since the women were not able to provide documentation supporting their claim (birth certificates indicated parents unknown), we decided not to convey the results in such a simplistic form and investigate the case further. 2. Materials and methods Mitochondrial DNA (mtDNA) haplotypes of Hypervariable regions HV1 (position 16024–16365) and HV2 (position 73–340)
* Corresponding author at: Corso Galileo Galilei, 22–10126 Torino, Italy. Tel.: +39 011 6705625; fax: +39 011 6705934. E-mail address: [email protected] (S. Inturri). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.10.007
were characterized by sequence analysis as described by Steighner and Holland [1]. Sequences were aligned and compared to the revised Cambridge reference sequence (rCRS). 21 X-chromosomal STR markers were amplified by the combination of two in-house developed multiplexes [2] and of the InvestigatorTM Argus X-12 Kit (Qiagen, Hilden Germany). A total of 40 autosomal STR were amplified combining the Identifiler PCR Amplification kit (Applied Biosystems Foster City, CA) and 25 non-CODIS miniSTR [3]. All STRs were typed by capillary electrophoresis on the ABI PRISM 310 (Applied Biosystems) in comparison to allelic ladders and control DNA of known genotype. For statistical analysis of X and autosomal STR data, allele frequencies described in the Italian population were used [4,5]. 3. Results and discussion In complex kinship cases, where all stated relationships could be questioned, the range of different pedigrees that can be used as complementary hypotheses in LR calculations expands rapidly with the number of tested subjects. In the reported case age information allowed to exclude a priori that the propositi belong to different generations. Even so, a simulation with the software Familias v1.81 produced thousands of alternative pedigrees causing the program to ‘‘hang’’ [6]. In such circumstances further simplification can derive from the inclusion in the analysis of haploid markers. All the tested subjects displayed the same mtDNA haplotype (16069[T], 16126[C], 16224[C], 73[G], 228[A], 263[G], 295[T], 315.1[C]), previously unreported in EMPOP [7]. X-chromosomal (Table 1) and autosomal STR data were consistent with the hypothesis of full-sibship, requiring a single recombination event in linkage group I (Xp22.31) of S4. LR calculations were performed
S. Inturri et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e520–e521 Table 1 Reconstructed putative parental X-chromosomes. Locus Xp22.31 DXS10148 DXS10135 DXS8378
S1
S2
Table 2 Posterior probabilities of H0 and H1 (A-STRs = autosomal STRs). S3
S4
S5
21 X-STRs
15 A-STRs
40 A-STRs
X and A-STRs
P0
P1
P0
P1
P0
P1
P0
P1
.918 .794 .830 .454 .775 .860 .498 .538 .602 .503
.082 .206 .170 .545 .225 .140 .502 .462 .398 .497
.980 .789 .953 .635 .507 .753 .778 .921 .902 .707
.020 .211 .047 .365 .493 .247 .222 .079 .098 .293
.997 .651 .997 .792 .700 .952 .934 .949 .915 .951
.003 .349 .003 .208 .300 .048 .066 .051 .085 .049
>.999 .833 >.999 .688 .848 .988 .907 .938 .919 .932
<.001 .167 <.001 .312 .152 .012 .093 .062 .081 .068
23 22 10
24 21 10
23 22 10
24 21 10
23 22 10
24 21 10
23 22 10
24 24 10
25.1 24 10
13 17 8 16
14 19 17 19
13 17 8 16
14 19 17 19
13 17 8 16
12 21 17 19
13 17 8 16
14 19 17 19
12 21 17 19
12 33 20
11 34 20
12 33 20
11 34 20
12 33 20
10 34 21
12 33 20
11 34 20
10 34 21
DXS7424 14 DXS101 28 GATA172D05 6
18 18 10
14 28 6
18 18 11
14 28 6
15 29 10
14 28 6
18 18 11
15 29 10
Xq26
DXS10103 HPRTB DXS10101
19 13 28
21 19 12 13 30.2 28
21 19 12 13 30.2 28
21 19 12 13 30.2 28
18 21 12 12 28.2 30.2
Xq28
DXS8377 DXS10146 DXS10134 DXS7423 DXS10011
48 28 33 15 30.2
51 30 35 15 33
51 30 35 15 33
50 27 38 15 40
51 30 35 15 33
Xq12
DXS7132 DXS10079 DXS10074 DXS6800
Xq21.33 DXS6801 DXS6809 DXS6789 Xq22.1
48 28 33 15 30.2
e521
48 28 33 15 30.2
48 28 33 15 30.2
50 27 38 15 33
for each pair of tested individuals. Due to the complexity of the case, we used as alternative hypothesis to full sibship (H0), the combination of distinct hypotheses (H1a–1d), all with equal weight [8]:H1athe tested subjects are maternal half-sibs;H1bthe tested subjects are three-quarter sibs;H1cthe tested subjects are double first cousins;H1cthe tested subjects are unrelated. Posterior probabilities of full-sibship (P0) and other selected kinship configurations (P1), given an a priori probability of H0 of 0.5, were calculated with the software Mendel [9] and are shown in Table 2. Based on the obtained results, full-sibship of S1, S2 and S4 could be considered reasonably proved. By this assumption, the number of pedigrees generated by Familias was reduced to 625. Fullsibship resulted the most likely relationship, with a posterior probability of .995, followed by the constellation in which S1–S3 and S5 were full-sibs and S3 their three-quarter sib (posterior probability .002). The reported case exemplifies the difficulty to infer relatedness given individual genotypes, whenever simple assumptions regarding alternative pedigrees cannot be taken a priori, as in routine paternity testing. The problem applies both to unconventional kinship cases, as the one here described, and immigration cases
S1–S2 S1–S3 S1–S4 S1–S5 S2–S3 S2–S4 S2–S5 S3–S4 S3–S5 S4–S5
requiring DNA analysis. Addition of extra markers to conventional STR loci, including mtDNA, Y and X-STRs is often mandatory. Even so, the figures attained in LR calculations and the associated posterior probabilities of pedigrees are frequently under the standard cut-off value of 99.99%. Moreover, special care must be taken when reporting results to the court and the tested subjects. In particular, awareness of the fact that LR values depend on the choice of alternative hypotheses is essential to the understanding of the conveyed information. Conflict of interest None. References [1] R.J. Steighner, M. Holland, Amplification and sequencing of mitochondrial DNA in forensic casework, Methods Mol. Biol. 98 (1998) 213–223. [2] C. Robino, A. Giolitti, S. Gino, et al., Development of two multiplex PCR systems for the analysis of 12 X-chromosomal STR loci in a northwestern Italian population sample, Int. J. Legal Med. 120 (2006) 315–318. [3] C.R. Hill, M.C. Kline, M.D. Coble, et al., Characterization of 26 MiniSTR loci for improved analysis of degraded DNA samples, J. Forensic Sci. 53 (2008) 73–80. [4] S. Inturri, S. Menegon, A. Amoroso, et al., Linkage and linkage disequilibrium analysis of X-STRs in Italian families, Forensic Sci. Int. Genet. 5 (2011) 152–154. [5] S. Presciuttini, N. Cerri, S. Turrina, et al., Validation of a large Italian database of 15 STR loci, Forensic Sci. Int. 156 (2006) 266–268. [6] T. Egeland, P.F. Mostad, B. Mevaˆg, et al., Beyond traditional paternity and identification cases. Selecting the most probable pedigree, Forensic Sci. Int. 110 (2000) 47–59. [7] W. Parson, A. Du¨r, EMPOP – a forensic mtDNA database, Forensic Sci. Int. Genet. 1 (2007) 88–92. [8] A.O. Karlsson, G. Holmlund, T. Egeland, et al., DNA-testing for immigration cases: the risk of erroneous conclusions, Forensic Sci. Int. 172 (2007) 144–149. [9] K. Lange, R. Cantor, S. Horvath, et al., Mendel Version 4.0: a complete package for the exact genetic analysis of discrete traits in pedigree and population data sets, Am. J. Hum. Genet. 69 (Suppl.) (2001) 504 (2001).