Molecular genetic identification of the Slovene home guard victims
Abstract
BACKGROUND This paper describes the application of molecular genetic methods for identifying the skeletal remains of the three victims of the post-war killings under the Storžič Mountain. Weused femurs and teeth and compared their genetic profiles with the genetic material ofliving relatives. METHODS We cleaned the bones and teeth, removed surface contamination and ground the bonesinto powder. Prior to isolating the DNA using the Biorobot EZ1 (Qiagen), the powder wasdecalcified. The nuclear DNA of the samples were quantified using the real time PCRmethod. We aquired autosomal genetic profiles and Y-chromosome haplotypes, as well asmtDNA haplotypes, from all the bone and teeth samples and from reference persons. Forthe purposes of traceability in the event of contamination, we prepared an eliminationdata base including genetic profiles of the nuclear and mtDNA of all persons who havebeen in touch with the skeletal remains in any way. RESULTS We extracted up to 8.6 ng DNA/per g from the bone powder and, up to 55 ng DNA/per gfrom the teeth powder. When comparing genetic profiles, we matched all the bones and teeth with the living relatives. By analysing the autosomal nuclear DNA, we were able tomatch the daughter UM with the femur of skeleton 2 to identify victim UJ. By analysing theautosomal nuclear DNA and the mtDNA, we were able to match the niece CZ with thefemur of skeleton 3 to identify victim KJ. By analysing the autosomal nuclear DNA, themtDNA and the Y-chromosome, we were able to match the son KJ and the niece MZ with thefemur of the skeleton 1 and the molars of the skeleton 2 to identify victim KF. CONCLUSIONS The research showed a high probability (from 99.9999 % to 99.999999 %) that allthree victims of the killings under the Storžič Mountain are related to the living relatives,speaking in favour of the positive identification of the victims
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References
2. Zupanič I. Uvedba preiskave DNA za prepoznavanje oseb in preverjanje sorodstvenih povezav v slovenski populaciji magistrsko delo. Ljubljana: Univerza v Ljubljani; 1999.
3. Zupanič Pajnič I, Šterlinko H, Balažic J, Komel R. Parentage testing with 14 STR loci and population data for 5 STRs in the Slovenian population. Int J Legal Med 2001; 114: 178–80.
4. Zupanič Pajnič I, Balažic J, Komel R. Sequence polymorphism of the mitochondrial DNA control region in the Slovenian population. Int J Legal Med 2004; 118: 1–4.
5. Jehaes E, Decorte R, Peneau A, Petrie JH, Boiry PA, Gilissen A, et al. Mitochondrial DNA analysis on remains of a putative son of Louis XVI king of France and Marie-Antoinette. Eur J Hum Genet 1998; 6: 383–95.
6. Jehaes E, Toprak K, Vanderheyden N, Pfeiffer H, Cassiman JJ, Brinkmann B, et al. Pitfalls in the analysis of mitochondrial DNA from ancient specimens and the consequences for forensic DNA analysis: the historical case of the putative heart of Louis XVII. Int J Legal Med 2001; 115: 135–41.
7. Anslinger K, Weichhold G, Keil W, Bayer B, Eisenmenger W. Identification of the skeletal remains of Martin Bormann by mtDNA analysis. Int J Legal Med 2001; 114: 194–6.
8. Jeffreys AJ, Allen MJ, Hagelberg E, Sonnberg A. Identification of the skeletal remains of Josef Mengele by DNA analysis. Forensic Sci Int 1992; 56: 65–76.
9. Gill P, Ivanov PL, Kimpton C, Pierey R, Benson N, Tully G, et al. Identification of the remains of the Romanov family by DNA analysis. Nat Genet 1994; 6: 130–6.
10. Ivanov PI, Wadhams MJ, Roby RK, Holland MM, Weedn VW, Parsons TJ. Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II. Nat Genet 1996; 12: 417–20.
11. Primorac D, Andelinović S, Definis-Gojanović M, Drmić I, Rezić B, Baden MM, et al. Identification of war victims from mass graves in Croatia Bosnia and Herzegovina by the use of standard forensic methods and DNA typing. J Forensic Sci 1996; 41: 891–4.
12. Davoren J, Vanek D, Konjhodzić R, Crews J, Huffine E, Parsons TJ. Highly effective DNA extraction method for nuclear short tandem repeat testing of skeletal remains from mass graves. Croat Med J 2007; 48: 478–85.
13. Miloš A, Selmanović A, Smajlović L, Huel RLM, Katzmarzyk C, Rizvić A, et al. Success rates of nuclear short tandem repeat typing from different skeletal elements. Croat Med J 2007; 48:486–93.
14. Handt O, Richards M, Trommsdorff M, Kilger C, Simanainen J, Georgiev O, et al. Molecular genetic analyses of the Tyrolean Ice Man. Science 1994; 265: 1775–8.
15. Krings M, Stone A, Schmitz RW, Krainitzki H, Stoneking M, Pääbo S. Neandertal DNA sequences and the origin of modern humans. Cell 1997; 90: 19–30.
16. Alonso A, Andelinović Š, Martin P. DNA typing from skeletal remains: evaluation of multiplex and megaplex STR systems on DNA isolated from bone and teeth samples. Croat Med J 2001; 42: 260–6.
17. Tully G, Bär W, Brinkmann B, Carracedo A, Gill P, Morling N. Considerations by the European DNA profiling (EDNAP) group on the working practices nomenclature and interpretation of mitochondrial DNA profiles. Forensic Sci Int 2001; 124:83–91.
18. Bär W, Brinkmann B, Budowle B, Carracedo A, Gill P, Holland M. DNA commission of the International society for forensic genetics: guidelines for mitochondrial DNA typing. Int J Legal Med 2000; 113: 193–6.
19. Carracedo A, Bär W, Lincoln P, Mary W. DNA commission of the International society for forensic genetics: guidelines for mitochondrial DNA typing. Forensic Sci Int 2000; 110: 79–85.
20. Kemp BM, Smith DG. Use of bleach to eliminate contaminating DNA from the surface of bones and teeth. Forensic Sci Int 2005;154: 53–61.
21. Wilson MR, DiZinno JA, Polanskey D, Replogle J, Budowle B. Validation of mitochondrial DNA sequencing for forensic casework analysis. Int J Legal Med 1995; 108: 68–74.
22. Kalmar T, Bachrati CZ, Marcsik A, Rasko I. A simple and efficient method for PCR amplifiable DNA extraction from ancient bones. Nucleic Acids Res 2000; 28: 67.
23. Tamariz J, Voynarovska K, Prinz M, Caragine T. The application of ultraviolet irradiation to exogenous sources of DNA in plasticware and water for the amplification of low copy number DNA. J Forensic Sci 2006; 51: 790–4.
24. Shaw K, Sesardić I, Bristol N, Ames C, Dagnall K, Ellis C, et al. Comparison of the effects of sterilisation techniques on subsequent DNA profiling. Int J Legal Med 2008; 122: 29–33.
25. Zupanič Pajnič I. Molekularno genetska identifikacija neznanih trupel iz skeletnih ostankov in zob. In: Luzar B, Poljak M, Glavač D, Balažic J, eds. Molekularna diagnostika v medicini – 15. spominsko srečanje akademika Janeza Milčinskega in 36. memorialni sestanek profesorja Janeza Plečnika in 1. srečanje slovenskega društva za humano genetiko z mednarodno udeležbo; 2005 Nov 30-Dec 2; Ljubljana, Slovenija. Ljubljana: Medicinska fakulteta; 2005.
26. Loreille OM, Diegoli TM, Irwin JA, Coble MD, Parsons TJ. High efficiency DNA extraction from bone by total demineralization. Forensic Sci Int Genet 2007; 1: 191–5.
27. Nagy M, Otremba P, Krüger C, Bergner-Greiner S, Anders P, Henske B, et al. Optimization and validation of a fully automated silica-coated magnetic beads technology in forensics. Forensic Sci Int 2005; 152: 13–22.
28. Montpetit SA, Fitch IT, O‘Donnell PT. A simple automated instrument for DNA extraction in forensic casework. J Forensic Sci 2005; 50: 1–9.
29. Kishore R, Hardy WR, Anderson VJ, Sanchez NA, Buoncristiani MR. Optimization of DNA extraction from low-yield and degraded samples using the biorobot EZ1 and biorobot M48. J Forensic Sci 2006; 51: 1055–61.
30. Vanderheyden N, Mai A, Gilissen A, Cassiman JJ, Decorte R. Identification and sequence analysis of discordant phenotypes between AmpFlSTR SGM Plus and PowerPlex 16. Int J Legal Med 2007; 121: 297–301.
31. Hill CR, Kline MC, Mulero JJ, Lagace RE, Chang CW, Hennessy LK, et al. Concordance study between the AmpFlSTR MiniFiler PCR Amplification Kit and conventional STR typing kits. J Forensic Sci 2007; 52: 870–3.
32. Gross AM, Berdos P, Ballantyne J. Y-STR concordance study between Y-Plex 5, Y-Plex 6, Y-Plex 12, Powerplex Y, Y-Filer, MPI, and MPII. J Forensic Sci 2006; 51: 1423–8.
33. Gross AM, Liberty AA, Ulland MM, Kuriger JK. Internal validation of the AmpFlSTR Yfiler amplification kit for use in forensic casework. J Forensic Sci 2008; 53: 1–10.
34. Mayntz-Press K, Ballantyne J. Performance characteristics of commercial Y-STR multiplex systems. J Forensic Sci 2007; 52:1025–34.
35. Gill P, Whitaker J, Flaxman C, Brown N, Buckleton J. An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA. Forensic Sci Int 2000; 112: 17–40.
36. Zupanič Pajnič I. Identifikacija oseb iz starih in slabo ohranjenih bioloških materialov s polimorfizmi mitohondrijske DNA (doktorsko delo). Ljubljana: Univerza v Ljubljani; 2007.
37. Gusmao L, Butler JM, Carracedo A, Gill P, Kayser M, Mayr WR, et al. DNA commission of the international society of forensic genetics (ISFG): an update of the recommendations on the use of Y STRs in the forensic analysis. Int J Legal Med 2006; 120:191–200.
38. Willuweit S, Roewer L. Y Chromosome Haplotype Reference Database. Dosegljivo na: www.yhrd.org
39. Parson W. European Mitochondrial DNA Control Region Database. Dosegljivo na: www.empop.org
40. Walsh B, Redd AJ, Hammer MF. Joint match probabilities for Y chromosomal and autosomal markers. Forensic Sci Int 2008;174: 234–8.
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