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Aim to Report on the Use of STR, Y-Strs, and Ministrs Typing Meth 296 FORENSIC SCIENCE CH|I doi: 10.332S/cmj.2Q09.50.296 Identification of Skeletal Remains Damir Marjanovic'•^ Adaleta Durmic- of Communist Armed Forces Pasic', Lejia Kovacevic', Jasna Avdic', Mirela Victims During and After World War Dzehverovic', Sanin II: Combined Y-chromosome Short Haveric', Jasmin Ramie', Belma Kalamujic', Lada Tandem Repeat (STR) and MiniSTR Lukic Bilela',Vedrana Skaro^ Petar Projic^ Kasim Approach Bajrovic', Katja Drobnic\ Jon Davoren\ Dragan • • • • • • "lenetic Engineering Aim To report on the use of STR, Y-STRs, and miniSTRs typing meth- •I -'""iloay, Sarajevo, ods in the identification of victims of revolutionary violence and ! , ivina crimes against humanity committed by the Communist Armed •ijen' • • •.•itia Forces during and after World War II in which bodies were exhumed "'F-" !•'• /.J ..jcurity, from mass and individual graves in Slovenia. Methods Bone fragments and teeth were removed from human re- "Bode Technology, Lorton, Va, USA mains found in severai smail and closely located hidden mass graves •'Medical School, Split University in the Skofia Loi<a area (Lovrenska Grapa and 2oisce) and 2 individual Split, Croatia graves in the Ljubljana area (Podlipogiav), Slovenia. DNA was isolat- "^Medical School, Osijek University ed using the Qiagen DNA extraction procedure optimized for bone k, Croatia and teeth. Some DNA extracts required additionai purification, such as N-buthanol treatment.The Quantifiier™ iHuman DNA Quantifica- tion Kit was used for DNA quantification. Initially PowerPlex 16 kit was used to simultaneously analyze 15 short tandem repeat (STR) loci.The PowerPlex S5 miniSTR kit and P^mpfiSTR*MiniFiier PCR Am- plification Kit was used for additional analysis if preliminary analysis yielded weak partial or no profiles at all. In 2 cases, when the Pow- erPlex 16 profiles indicated possible reiatedness ofthe remains with reference samples, but there were insufficient probabilities to call the match to possible male paternai reiatives, we resorted to an ad- ditional analysis of Y-STR markers. PowerPlex* Y System was used to simultaneously amplify 12 Y-STR loci. Fragment analysis was per- formed on an ABI PRISM 310 genetic analyzer. Matching probabili- ties were estimated using the DNA-View software. Results Foliowing the Y-STR anaiysis, 1 of the "weak matches" previ- ousiy obtained based on autosomal loci, was confirmed while the other 1 was not. Combined standard STR and miniSTR approach ap- plied to bone samples from 2 individual graves resulted in positive identifications. Finally using the same approach on 11 bone samples Received: April 23, 2009 from hidden mass grave 2olo5ce, we were able to obtain 6 useful Accepted: May 4, 2009 DNAproñies. Correspondence to: Damir Marjanovic Conclusion The results of this study in combination with previously Gajev trg 4 obtained results, demonstrate that Y-chromosome testing and mini- 71000 Sarajevo, Bosnia and STR methodology can contribute to the identification of human re- Herzegovina mains of victims of revoiutionary violence from World War II. damirmarjanovicißungeb. bg www.cmj.hr Marjanovl¿ et al: Analysis of WWII Remains - Y and MiniSTR Approach CM|I 297 Y-chromosome short tandem repeat (Y-STR) markers parts of Europe (11-13). Different DNA markers were em- are highly polymorphic. In addition, Y-chromosomes are ployed for the same purpose, from autosomal STR markers passed down from generation to generation with little in Slovenia (11 ) to mtDNA markers in Finland (12) and Y- or no change. Therefore, they may be quite useful in fol- STR markers in western Herzegovina (13). The differences lowing the paternal lineages in population studies, as well in molecular approach were mostly dictated by the quan- as in forensic DNA analysis, including the identification of tity and quality of DNA isolated from the remains. Regional human skeletal remains. Y-STRs show sufficient variability differences in the level of DNA preservation are likely to among individuals in a population and a high degree of exist due to the climatic conditions and chemical proper- geographical differentiation (1). Recent identification of ties of the soil (12). numerous, informative biallelic markers in the non-recom- bining region of Y-chromosome has already significantly There is no precise official data about the number of miss- contributed to the understanding of European pre-history ing persons in Slovenia but rough estimates suggest that and history (2-4). Y-chromosome testing may provide very the approximate number could amount to tens of thou- valuable results in forensic analysis where the nature of sands. Almost 600 scaffold and hidden mass graves are ver- evidence limits the effectiveness of autosomal tests. Typi- ified in Slovenia (14) and the latest mass grave was discov- cal examples are sexual assault cases with the evidence ered in Barbarin rov (Huda jama, Lasko) in March 2009 by in the form of mixtures with predominant female fraction Slovenian Government Commission Regarding the Ques- (5). Also, Y-linked markers usually significantly increase the tion of Hidden Mass Graves. In this grave, skeletal remains possibility of identification of male missing persons when of 300 victims killed by Communist Armed Forces after the only available reference is a male paternal relative. WWII were found (15). In 2006, the Commission for Re- cording and Managing Hidden Graves of the Major of the MiniSTR assays can help recover information from de- Municipality of 5kofia Loka (a city located around 40 km graded DNA samples that typically result in partial profiles to the northwest from the Slovenian capital Ljubljana) put and total loss of information from regular STR amplicons in a significant effort to identify victims of the Communist (6). This approach has already been used in the analysis Armed Forces discovered in 2 hidden mass graves uncov- of highly degraded samples like those processed within ered at the location Lovrenska Grapa (Figure 1 ). Due to the the identification of victims from the World Trade Center advanced state of the remains decomposition, application terrorist attacks (7). Also, miniSTR primer sets for all CODIS of conventional methods of human identification was un- STR loci have already been developed (6), but many other feasible and DNA identification was requested. We have re- loci were taken in consideration (8). Finally, several mini- ported the first results of these analyses earlier (11). In our STR commercial kits have been released in the last 2 years. previous study (11), comparison of victims'profile against a Considering all relevant information, the most recent con- reference sample database resulted in 4 statistically signifi- cept of miniSTR kits siiould certainly upgrade the analysis cant matches. In addition, another 5 profiles were associ- of DNA from old bones and teeth, including those origi- nating from World War II (WWII) skeletal remains. Figure 1. Identification of human remains relies on various proce- dures, such as identification by direct facial recognition, fingerprint and dental analysis, identification of special features, recognition of clothing and belongings, autopsy findings, forensic anthropologist's findings, reconstruction of facial features from skulls, hair comparisons, and DNA analysis (9). Unfortunately, with significant number of re- mains buried in one mass grave or reburied in secondary graves in order to hide the location, identification of such remains is much more difficult (10). Considering a tempo- ral gap of 60 years, DNA analysis seems to be the only vi- able approach to the identifying the remains dating from the period during and immediately after the WWII (11). Similar studies have already been performed in different Mass grave at Lovrenska Grapa site. www.cm|.hf 298 FORENSICSCIENCE Croat Med J. 2009; BO: 29B-304 ated to certain reference samples with insufficiently high cedures leading from the process of skeletal remains re- probability. Two of those victims were associated with pos- covery to DNA amplification were described in detail in sible male paternal relatives, thus the samples were further our previous article (11), PowerPlex Y kit (Promega Corp,, subjected to Y-5TR typing. At this point, we present the re- Madison, WI, USA) was used to simultaneously amplify 12 sults of additional analysis based on Y-STR markers. Y-STR loci. Amplification reactions were prepared accord- ing to the Promega technical manual (16), The polymerase Encouraged by these results, the same authorities request- chain reaction (PCR} amplification was carried out in the ed DNA analysis for the victims found in newly uncovered PE GeneAmp PCR System Thermal Cycler [Applied Biosys- hidden mass grave situated near íkof¡a Loka, location tems, Foster City, CA, USA) according to the manufactur- 2olosca (Figure 2), Interestingly, after the public announce- er's recommendations with 32 amplification cycles. Frag- ments of previously achieved results in the local media, ment analysis was preformed on an ABI PRISM 310 Genetic numerous persons requested DNA analysis for the skele- Analyzer (Applied Biosystems),The raw data was compiled tal remains found in several individual graves, presumed and analyzed using accessory software; 310 Data Collec- to belong to their beloved. These samples turned to be tion Software and GeneMapper™ 3,2 (Applied Biosystems). extremely challenging for processing and at this point we Numerical alíele designations of the profiles were obtained present some of the obtained results. by processing
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