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An unusual case of identification by DNA analysis of siblings
Forensic Science International: Genetics 6 (2012) 121–123
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Case report
An unusual case of identification by DNA analysis of siblings Franz Neuhuber a,1,*, Max P. Baur b,1, Jan Cemper-Kiesslich a, Bettina Dunkelmann a, Fabio Monticelli a a b
Institute of Legal Medicine, University of Salzburg, Ignaz-Harrer-Str. 79, A-5020, Austria Institute for Medical Biometry, Informatics and Epidemiology, University of Bonn, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 October 2010 Received in revised form 14 February 2011 Accepted 21 February 2011
A badly decomposed body required identification by means of DNA analysis. A brother and sister of the deceased were available as reference subjects. Although investigation of Y-chromosomal markers established an exclusion condition, autosomal markers suggested a positive identification. In order to increase the reliability of the tests, X-chromosomal markers were also investigated. This analysis showed the body to have an XXY genotype (Klinefelter’s syndrome). A number of hypotheses were assessed using biostatistical methods, ultimately resulting in a definite identification. The special aspect of Klinefelter’s syndrome proved highly useful for biostatistical analysis. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic identification Klinefelter syndrome Biostatistics Complex pedigree analysis
1. Introduction DNA identification of unknown cadavers is usually performed by comparing the DNA of the cadaver with DNA from traces of personal possessions or by testing reference samples from relatives (parents, siblings, biological children). These methods require the tested samples to be of known origin or the relationship between the persons tested to be established beyond doubt. Otherwise, a false exclusion may be the result. The present case concerns a body found in a house scheduled for demolition. The body had been lying there for approximately one and a half years and was in an advanced state of dry decomposition and partly skeletonized. It was not possible to identify the body from dental or medical records because no comparison data was available. As no personal possessions were available as reference samples, material from two people thought to be full siblings (one brother and one sister) was used for a DNA comparison. 2. Materials and methods DNA was isolated from swabs of the body’s aorta, bladder and brain using organic extraction techniques (phenol). DNA was extracted from reference samples (buccal swabs) taken from the siblings using Chelex (Bio-Rad). 17 Y-chromosomal (Yfiler, Applied Biosystems), 28 autosomal (Powerplex 16, Promega; SEFiler Plus, Applied Biosystems; Chimera, Biotype), and 8 X-chromosomal markers (Argus X-8, Biotype) were amplified in accordance with
* Corresponding author. Tel.: +43 662 8044 3823; fax: +43 662 8044 3834. E-mail address: [email protected] (F. Neuhuber). 1 F. Neuhuber and M.P. Baur contributed equally to this manuscript. 1872-4973/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2011.02.006
the manufacturers’ instructions. The PCR products were separated on an ABI 310 Genetic Analyzer or an ABI 3100 Avant Genetic Analyzer (Applied Biosystems). Results were analyzed using the Genotyper software (Applied Biosystems). Biostatistical analysis [1,2] of the various hypotheses was carried out using the ‘‘Program for Biostatistical Paternity- and Relationship-Testing’’, version 2.1.0.24/0 (M.P. Baur, R. Fimmers, W. Spitz). 3. Results and discussion The following set of mutually exclusive hypotheses (Fig. 1) was subjected to biostatistical analysis: H1: The deceased and the two reference subjects have the same parents. H2: The sister of the deceased and the deceased have the same parents, the brother of the deceased has the same mother but a different father. H3: The brother of the deceased and the deceased have the same parents, the sister of the deceased has the same mother but a different father. H4: The brother and sister of the deceased have the same parents, the deceased has the same mother but a different father. H5: The deceased is not related to the reference subjects. Investigations of Y-chromosomal markers identified an exclusion condition in 11 (DYS456, 389I, 390, 458, 385, 439, 635, 392, 438, 448 and Y GATA H4) out of 17 tested markers between the body and the reference sample provided by the hypothetical brother. It was therefore concluded that the body and brother could not stem from the same biological father. In view of this result, further investigations of autosomal markers were carried
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F. Neuhuber et al. / Forensic Science International: Genetics 6 (2012) 121–123
Fig. 1. Relationship hypotheses for the body and the two reference subjects.
out using both reference samples. Of the 28 autosomal markers genotyped, five loci (D21S11, D8S1179, D2S1338, SE33, D8S1132) were inconsistent with full sibling relationships between the body and the two reference samples. Additional investigation of Xchromosomal markers revealed that the body had an XXY genotype (Klinefelter’s syndrome) which was compatible both with the XY genotype of the hypothetical brother and with the XX genotype of the hypothetical sister. The posterior probabilities W1–W5 for hypotheses H1–H5 were calculated for all autosomal, X- and Y-chromosomal markers based on their frequency in the Euro-Caucasian population (for detailed results see Electronic Supplement). The overall results (see Fig. 2) confirmed that hypotheses H1 and H3 could be excluded (W1 = 0 both autosomal and Y-chromosomal; W3 = 0 Y-chromosomal only). The autosomal and X-chromosomal information indicates that hypotheses H4 and H5 have minimal posterior probabilities W4 = 0.00035%, W5 < 0.0000001% (the Y chromosome is unable to
distinguish between these hypotheses and hypothesis H2). Hypothesis H2, with a posterior probability of W2 = 99.999651%, is almost certainly correct. For additional information it is pointed out that in the Austrian and German legal system there are no thresholds set by a laboratory but all decisions are taken by the judge. Nonetheless posterior probabilities of W > 99.99% (based on equal priors) corresponding to a likelihood ratio of LR > 10,000:1 are interpreted as convincing evidence. Klinefelter Syndrome is the most common form of sexchromosomal aneuploidy in humans. The estimated prevalence is one in 500 to one in 1000 males. Due to variable and often unspecific findings, only one in four cases is recognized [3]. The most specific morphological findings at adult age are small testes, gynecomastia, female distribution of fat and body hair, slightly increased body length due to an increased leg length and azoospermia [3,4]. In case of suspected Klinefelter syndrome a histological examination of the testes could reveal clumped Leydig
Fig. 2. Overall results W1–W5 for hypotheses H1–H5.
F. Neuhuber et al. / Forensic Science International: Genetics 6 (2012) 121–123
cells as well as sclerotic and hyalined tubules [4]. Due to advanced decomposition of the body in the present case, the diagnosis of Klinefelter syndome was impossible. 4. Conclusion The unusual XXY genotype of the body made the exclusion of hypotheses H1 and H3 by way of its Y component possible. In case of a single X-chromosome of a normal male individual the differentiation between hypotheses H1–H4 would not have been possible, because all these hypotheses differ only with regard to the paternal line of inheritance. Only by way of the two X-components in individual 1 was it possible to differentiate between H2, H4 and H5 and thus increase W2 = 99.5% (autosomal systems – LR = 200:1) to W2 = 99.9997% (all markers – LR = 333,333:1). Based on all the available genetic information, the body can with confidence be identified as a full sibling of the female reference subject and as a maternal half-brother of the male reference subject.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.fsigen.2011.02.006. References [1] M.P. Baur, C. Rittner, Program for the computation of plausibilities of paternity. I. Description of program, Z. Rechtsmed. 78 (3) (1976) 227–242. [2] D.W. Gjertson, C.H. Brenner, M.P. Baur, A. Carracedo, F. Guidet, J.A. Luque, R. Lessig, W.R. Mayr, V.L. Pascali, M. Prinz, P.M. Schneider, N. Morling, ISFG: recommendations on biostatistics in paternity testing, Forensic Sci. Int. Genet. 1 (3–4) (2007) 223–231 [Epub 2007 August 6; Review]. [3] J.C. Giltay, M.C. Maiburg, Klinefelter syndrome: clinical and molecular aspects, Expert Rev. Mol. Diagn. 10 (2010) 765–776. [4] K. Hayashi, Y. Hanaoka, S. Matsumura, T. Takagi, M. Kajiwara, N. Tamaki, K. minaguchi, Y. Sato, An autopsy case of Klinefelter’s syndrome suspected and its DNA analysis, Forensic Sci. Int. Genet. 113 (2000) 119–125.
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Case report
An unusual case of identification by DNA analysis of siblings Franz Neuhuber a,1,*, Max P. Baur b,1, Jan Cemper-Kiesslich a, Bettina Dunkelmann a, Fabio Monticelli a a b
Institute of Legal Medicine, University of Salzburg, Ignaz-Harrer-Str. 79, A-5020, Austria Institute for Medical Biometry, Informatics and Epidemiology, University of Bonn, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 October 2010 Received in revised form 14 February 2011 Accepted 21 February 2011
A badly decomposed body required identification by means of DNA analysis. A brother and sister of the deceased were available as reference subjects. Although investigation of Y-chromosomal markers established an exclusion condition, autosomal markers suggested a positive identification. In order to increase the reliability of the tests, X-chromosomal markers were also investigated. This analysis showed the body to have an XXY genotype (Klinefelter’s syndrome). A number of hypotheses were assessed using biostatistical methods, ultimately resulting in a definite identification. The special aspect of Klinefelter’s syndrome proved highly useful for biostatistical analysis. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic identification Klinefelter syndrome Biostatistics Complex pedigree analysis
1. Introduction DNA identification of unknown cadavers is usually performed by comparing the DNA of the cadaver with DNA from traces of personal possessions or by testing reference samples from relatives (parents, siblings, biological children). These methods require the tested samples to be of known origin or the relationship between the persons tested to be established beyond doubt. Otherwise, a false exclusion may be the result. The present case concerns a body found in a house scheduled for demolition. The body had been lying there for approximately one and a half years and was in an advanced state of dry decomposition and partly skeletonized. It was not possible to identify the body from dental or medical records because no comparison data was available. As no personal possessions were available as reference samples, material from two people thought to be full siblings (one brother and one sister) was used for a DNA comparison. 2. Materials and methods DNA was isolated from swabs of the body’s aorta, bladder and brain using organic extraction techniques (phenol). DNA was extracted from reference samples (buccal swabs) taken from the siblings using Chelex (Bio-Rad). 17 Y-chromosomal (Yfiler, Applied Biosystems), 28 autosomal (Powerplex 16, Promega; SEFiler Plus, Applied Biosystems; Chimera, Biotype), and 8 X-chromosomal markers (Argus X-8, Biotype) were amplified in accordance with
* Corresponding author. Tel.: +43 662 8044 3823; fax: +43 662 8044 3834. E-mail address: [email protected] (F. Neuhuber). 1 F. Neuhuber and M.P. Baur contributed equally to this manuscript. 1872-4973/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2011.02.006
the manufacturers’ instructions. The PCR products were separated on an ABI 310 Genetic Analyzer or an ABI 3100 Avant Genetic Analyzer (Applied Biosystems). Results were analyzed using the Genotyper software (Applied Biosystems). Biostatistical analysis [1,2] of the various hypotheses was carried out using the ‘‘Program for Biostatistical Paternity- and Relationship-Testing’’, version 2.1.0.24/0 (M.P. Baur, R. Fimmers, W. Spitz). 3. Results and discussion The following set of mutually exclusive hypotheses (Fig. 1) was subjected to biostatistical analysis: H1: The deceased and the two reference subjects have the same parents. H2: The sister of the deceased and the deceased have the same parents, the brother of the deceased has the same mother but a different father. H3: The brother of the deceased and the deceased have the same parents, the sister of the deceased has the same mother but a different father. H4: The brother and sister of the deceased have the same parents, the deceased has the same mother but a different father. H5: The deceased is not related to the reference subjects. Investigations of Y-chromosomal markers identified an exclusion condition in 11 (DYS456, 389I, 390, 458, 385, 439, 635, 392, 438, 448 and Y GATA H4) out of 17 tested markers between the body and the reference sample provided by the hypothetical brother. It was therefore concluded that the body and brother could not stem from the same biological father. In view of this result, further investigations of autosomal markers were carried
122
F. Neuhuber et al. / Forensic Science International: Genetics 6 (2012) 121–123
Fig. 1. Relationship hypotheses for the body and the two reference subjects.
out using both reference samples. Of the 28 autosomal markers genotyped, five loci (D21S11, D8S1179, D2S1338, SE33, D8S1132) were inconsistent with full sibling relationships between the body and the two reference samples. Additional investigation of Xchromosomal markers revealed that the body had an XXY genotype (Klinefelter’s syndrome) which was compatible both with the XY genotype of the hypothetical brother and with the XX genotype of the hypothetical sister. The posterior probabilities W1–W5 for hypotheses H1–H5 were calculated for all autosomal, X- and Y-chromosomal markers based on their frequency in the Euro-Caucasian population (for detailed results see Electronic Supplement). The overall results (see Fig. 2) confirmed that hypotheses H1 and H3 could be excluded (W1 = 0 both autosomal and Y-chromosomal; W3 = 0 Y-chromosomal only). The autosomal and X-chromosomal information indicates that hypotheses H4 and H5 have minimal posterior probabilities W4 = 0.00035%, W5 < 0.0000001% (the Y chromosome is unable to
distinguish between these hypotheses and hypothesis H2). Hypothesis H2, with a posterior probability of W2 = 99.999651%, is almost certainly correct. For additional information it is pointed out that in the Austrian and German legal system there are no thresholds set by a laboratory but all decisions are taken by the judge. Nonetheless posterior probabilities of W > 99.99% (based on equal priors) corresponding to a likelihood ratio of LR > 10,000:1 are interpreted as convincing evidence. Klinefelter Syndrome is the most common form of sexchromosomal aneuploidy in humans. The estimated prevalence is one in 500 to one in 1000 males. Due to variable and often unspecific findings, only one in four cases is recognized [3]. The most specific morphological findings at adult age are small testes, gynecomastia, female distribution of fat and body hair, slightly increased body length due to an increased leg length and azoospermia [3,4]. In case of suspected Klinefelter syndrome a histological examination of the testes could reveal clumped Leydig
Fig. 2. Overall results W1–W5 for hypotheses H1–H5.
F. Neuhuber et al. / Forensic Science International: Genetics 6 (2012) 121–123
cells as well as sclerotic and hyalined tubules [4]. Due to advanced decomposition of the body in the present case, the diagnosis of Klinefelter syndome was impossible. 4. Conclusion The unusual XXY genotype of the body made the exclusion of hypotheses H1 and H3 by way of its Y component possible. In case of a single X-chromosome of a normal male individual the differentiation between hypotheses H1–H4 would not have been possible, because all these hypotheses differ only with regard to the paternal line of inheritance. Only by way of the two X-components in individual 1 was it possible to differentiate between H2, H4 and H5 and thus increase W2 = 99.5% (autosomal systems – LR = 200:1) to W2 = 99.9997% (all markers – LR = 333,333:1). Based on all the available genetic information, the body can with confidence be identified as a full sibling of the female reference subject and as a maternal half-brother of the male reference subject.
123
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.fsigen.2011.02.006. References [1] M.P. Baur, C. Rittner, Program for the computation of plausibilities of paternity. I. Description of program, Z. Rechtsmed. 78 (3) (1976) 227–242. [2] D.W. Gjertson, C.H. Brenner, M.P. Baur, A. Carracedo, F. Guidet, J.A. Luque, R. Lessig, W.R. Mayr, V.L. Pascali, M. Prinz, P.M. Schneider, N. Morling, ISFG: recommendations on biostatistics in paternity testing, Forensic Sci. Int. Genet. 1 (3–4) (2007) 223–231 [Epub 2007 August 6; Review]. [3] J.C. Giltay, M.C. Maiburg, Klinefelter syndrome: clinical and molecular aspects, Expert Rev. Mol. Diagn. 10 (2010) 765–776. [4] K. Hayashi, Y. Hanaoka, S. Matsumura, T. Takagi, M. Kajiwara, N. Tamaki, K. minaguchi, Y. Sato, An autopsy case of Klinefelter’s syndrome suspected and its DNA analysis, Forensic Sci. Int. Genet. 113 (2000) 119–125.