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513www.cmj.hr

DNA Identication of Skeletal Remains from World War II Mass Graves

Uncovered in Slovenia

Aim o present the joint eort o three institutions in the identi-cation o human remains rom the World War II ound in two massgraves in the area o koa Loka, Slovenia.

Methods Te remains o 27 individuals were ound in two smalland closely located mass graves. Te DNA was isolated rom boneand teeth samples using either standard phenol/chloroorm alcohol

extraction or optimized iagen DNA extraction procedure. Somerecovered samples required the employment o additional DNA

purication methods, such as N-buthanol treatment. QuantilerHuman DNA Quantication Kit was used or DNA quantication.PowerPlex 16 kit was used to simultaneously ampliy 15 short tan-dem repeat (SR) loci. Matching probabilities were estimated usingthe DNA View program.

ResultsOut o all processed samples, 15 remains were ully proledat all 15 SR loci. Te other 12 proles were partial. Te least suc-cessul prole included 13 loci. Also, 69 reerent samples (buccalswabs) rom potential living relatives were collected and proled.Comparison o victims prole against reerent samples database

resulted in 4 strong matches. In addition, 5 other proles werematched to certain reerent samples with lower probability.

Conclusion Our results show that more than 6 decades aer theend o the World War II, DNA analysis may signicantly contrib-ute to the identication o the remains rom that period. Additionalanalysis o Y-SRs and mitochondrial DNA (mtDNA) markers willbe perormed in the second phase o the identication project.

1Institute for Genetic Engineering and

Biotechnology, Sarajevo, Bosnia and

Herzegovina2Department of Molecular Medicine,

Forensic Genetics Group, Ruer

Bokovi Institute, Zagreb, Croatia3Forensic Laboratory and Research

Center, Ministry of Interior,

Ljubljana, Slovenia4Bode Technology Group Inc,

Springeld, Va, USA5Osijek University School of Medicine,

Osijek, Croatia6Split University School of Medicine,

Split, Croatia

Damir Marjanovi1,2, Adaleta Durmi-Pai1, Narcisa Bakal1, Sanin Haveri1,Belma Kalamuji1, Lejla Kovaevi1, Jasmin Rami1, Naris Pojski1, Vedrana

karo2, Petar Proji2, Kasim Bajrovi1, Rifat Hadiselimovi1, Katja Drobni3,Edwin Hufne4, Jon Davoren4, Dragan Primorac5,6

Damir Marjanovi

Kemalbegova 10

71000 Sarajevo, Bosnia and

Herzegovina

marjanovd@hotmail.com

> Received: May 4, 2007

> Accepted: July 2, 2007

> Correspondence to:

Forensic ScienceForensic Science

> Croat Med J. 2007;48:513-9

mailto:%20marjanovd@hotmail.commailto:%20marjanovd@hotmail.com

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o the Slovenian town o koa Loka (Figure 1)during the last week o October 2006. Te larg-er o the two mass graves contained skeletal re-

mains o 20 persons (Figure 2) and the small-er contained 7 bodies. Te graves were situatedone next to another, in a wood, close to a spring(Figure 3). According to reliable testimonies osurviving witnesses, the larger o the two graves

contains the bodies o Slovenian home guards-men shot by Partisans in the spring o 1945. Tesmaller grave most probably contains the remains

o 7 German war prisoners, who buried the Slo-venians and dug out another grave where theythemselves were buried.

Te remains were recovered and processed bylocal archeologists and anthropologists. Samplesor DNA analysis (emoral ragments and teeth)

were collected and labeled. Aer that, bone rag-ments rom all 27 bodies and teeth rom 18 othem were transported to the Laboratory o Fo-rensic Genetics, at the Institute or Genetic En-gineering and Biotechnology, University o Sa-

rajevo. Upon arrival at Institute or GeneticEngineering and Biotechnology, the samples

were assigned case numbers (Figure 4) and therelevant inormation was entered into the Chaino Custody orms. Until urther processing, thesamples were stored at -80C.

For each bone sample, the entire exterior was

sanded clean to remove potential contaminants.All bone suraces were cleaned rom remnantso tissue and soil traces using grinding stone at-tached to Dremel rotary tool (Dremel Multi-unktionswerkzeuge, Leinelden-Echterdingen,Germany). Ten, the samples were successively

washed in mild detergent, 5% bleach, sterile dis-tilled water, and 100% ethanol, and air-dried.Toroughly dried samples were pulverized us-

Figure 1. Position of kofja Loka area.

Figure 2. Skeletal remains from the bigger mass grave.

Figure 3. Location of both mass graves.

Figure 4. Labeled samples upon arrival to the laboratory.

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ing a sterilized Waring blender (Waring Prod-ucts, orrington, C, USA) and powder trans-erred to sterile 15-mL conical polypropylene

tubes. Te same procedure, except or sandingphase, was applied to the teeth. Double extrac-tions were perormed or each sample, ollow-ing the previously described phenol-chloroorm(9) and optimized iagen (iagen GmbH,Hilden, Germany) procedures (Internal Com-mission on Missing Persons Bone DNA extrac-tion protocol).

Centricon-100 centriugal lter units (Mil-lipore, Billerica, MA, USA) were used or DNA

purication and concentration. Te same sam-

ples required additional purication step with n-butanol (10). Te concentrates were transerredto 1.5-mL microcentriuge tubes and diluted

with DNA-ree double distilled H2O to the nalvolume o 50-100 L.

DNA concentration was determined usinguantiler Human DNA uantication Kit(Applied Biosystems Foster City, CA, USA) asdescribed previously (11). Te reaction was car-ried out in AB 7300 Real-ime PCR System(Applied Biosystems) according to the manuac-turers recommendations.

Te PowerPlex 16 kit (Promega Corp., Mad-ison, WI, USA) was used to simultaneously am-

pliy 15 SR loci as ollows: D3S1358, H01,D21S11, D18S51, Penta E, D5S818, D13S317,D7S820, D16S539, CSF1PO, Penta D, vWA,D8S1179, POX, FGA, as well as the gender de-termination locus Amelogenin. Amplication

was carried out as described previously (12).Te total volume o each reaction was 25L.

Te PCR amplication was carried out in PEGene Amp PCR System Termal Cycler (Ap-plied Biosystems), according to the manuac-turers recommendations. In samples with smallamount o DNA, the number o cycles was in-creased to 32 and elongation time extended to90 seconds.

Electrophoresis o the amplication prod-ucts was preormed on an ABI PRISM 310 Ge-

netic Analyzer (Applied Biosystems). Te rawdata were compiled and analyzed using accesso-ry soware, 310 Data Collection Soware and

GeneMapperM 3.2 (Applied Biosystems). Nu-merical allele designations o the proles wereobtained by processing with Powertyper16 Mac-ro (Promega Corp.).

Handling of referent samples

Reerent samples (69 buccal swabs) rom poten-tial living relatives were collected, recorded, and

preliminarily labeled by local DNA experts inSlovenia. Most o the donors are still living in

Slovenia, but some o the relatives sent their sam-ples rom abroad, even rom the USA. Buccalswabs were used as the DNA source. Internal In-stitute or Genetic Engineering and Biotechnol-ogy collection kit was used or collection. Driedand labeled samples were transported to the In-stitute or Genetic Engineering and Biotechnol-ogy laboratory. Upon arrival, the samples wererelabeled and the relevant inormation enteredinto the Chain o Custody orms. Te samples

were stored at -80C until DNA extraction em-

ploying iagen Dnaeasy issue Kit (13).PowerPlex 16 kit (Promega Corp.) was used

or urther analysis. Similar amounts o DNAwere used in all PCR reactions. Amplicationwas carried out as described previously (12). Tetotal reaction volume was 25 L. PCR amplica-tion was carried out in PE Gene Amp PCR Sys-tem Termal Cycler (Applied Biosystems), ac-cording to the manuacturers recommendations.Electrophoresis o the amplication products

was perormed on ABI PRISM 377 Sequencer(Applied Biosystems), in 5% bis-acrilamide gel(Long Ranger Single Packs, Cambrex Bio Sci-ence, Rockland, ME, USA). Raw data were com-

piled and analyzed using the accessory soware:ABI PRISM Data Collection Soware andGene Scan. Numerical allele designations o the

proles were obtained by processing with Power-typer16 Macro.

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Statistical analysis

Comparative analysis o DNA proles obtained

rom skeletal remains and reerent samples, esti-mation o potential amiliar relationships, calcu-lation o paternity, and sibling indexes, as wellas calculation o matching probability were per-ormed in DNA-View program, created by C.H. Brenner (14).

Results

Samples (13 bones and 14 teeth) rom 27 skele-tal remains were processed. Preliminary quanti-

cation o the extracted DNA dened the samplesthat required additional purication by n-butha-nol procedure, and those were quantied again.Te quantication results showed that the meanconcentration varied between 2.13 103ng/L and 5.20 101ng/L, or samples or whichDNA proles were obtained. Based on the quan-tication results, extended PCR procedure (32cycles with elongation time extended to 90 sec-onds) was recommended or 12 samples.

Te characteristics and procedures employedor all the obtained remains SR proles are

presented in able 1. Te number o detected

loci varied rom 13 to 16 per prole. PP16 SRproling was least successul or bone samplesBS0016 and BS0025. Partial proles o thesesamples consisted o alleles detected across 13SR loci. Finally, 15 samples were proled atall 15 SR loci. Other 12 proles were partiallygenerated. Also, 69 reerent buccal swabs collect-ed rom relatives o persons missing in that area

were successully proled.Comparison o victims prole against reer-

ent samples database resulted in 4 strong match-

es. In addition, 5 other proles were matched tocertain reerent samples with lower probability.

Discussion

Tis study demonstrated that the experi-ence gathered over the last almost een yearsthrough the identication projects o missing

persons in Bosnia and Herzegovina and Croa-tia could be successully used to identiy skeletal

Table 1. Summary of DNA analysis of skeletal remains samples for submitted DNA proles

Body code

Samplecode

Sampletype

DNA extractionprocedure

Additionaln-butanol precipitation

Meanqantity

PCRprotocol

Number of lociwith detected alleles

Positiveidentication

LG-06/D1 BS0001 tooth Qiagen + 6.65 102 Standard 16/16 LG-06/D2 BS0002 tooth phenol-chloroform 2.35 102 Extended 16/16 LG-06/D3 BS0003 tooth phenol-chloroform 2.48 102 Standard 15/16 LG-06/D4 BS0004 tooth Qiagen + 2.75 102 Standard 15/16 LG-06/D5 BS0005 tooth phenol-chloroform 4.15 102 Standard 15/16 LG-06/D6 BS0006 tooth Qiagen + 4.21 102 Standard 16/16 LG-06/D7 BS0007 tooth Qiagen 7.11 102 Standard 16/16 LG-06/D8 BS0008 tooth Qiagen 2.97 102 Standard 16/16 LG-06/D9 BS0009 tooth phenol-chloroform 7.95 103 Extended 16/16 LG-06/D10 BS0010 tooth phenol-chloroform 6.06 101 Standard 16/16 +LG-06/D11 BS0011 bone Qiagen + 1.03 102 Extended 16/16 +LG-06/D12 BS0012 tooth Qiagen 1.02 101 Standard 16/16 LG-06/D13 BS0013 tooth phenol-chloroform 5.09 102 Standard 16/16

LG-06/D14 BS0014 tooth Qiagen + 5.29 102 Standard 15/16 LG-06/D15 BS0015 bone Qiagen + 1.76 102 Extended 14/16 LG-06/D16 BS0016 bone Qiagen 2.51 102 Extended 13/16 LG-06/D17 BS0017 bone phenol-chloroform + 3.11 103 Extended 15/16 LG-06/D18 BS0018 tooth Qiagen 2.26 102 Extended 16/16 +LG-06/D19 BS0019 bone Qiagen + 1.32 102 Extended 14/16 +LG-06/D20 BS0020 bone Qiagen 2.97 102 Standard 16/16 LG-06/1 BS0021 bone phenol-chloroform 7.46 103 Extended 16/16 LG-06/2 BS0022 bone phenol-chloroform 1.55 102 Extended 16/16 LG-06/3 BS0023 bone phenol-chloroform + 2.13 103 Extended 14/16 LG-06/4 BS0024 bone phenol-chloroform 7.96 102 Standard 16/16 LG-06/5 BS0025 bone phenol-chloroform + 5.57 103 Extended 13/16 LG-06/6 BS0026 bone Qiagen 4.27 102 Standard 14/16 LG-2005/0 BS0027 bone Qiagen 5.20 101 Standard 14/16

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http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=1398379&dopt=Abstrachttp://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=8426158&dopt=Abstrachttp://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=8426158&dopt=Abstrachttp://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=15317190&dopt=Abstrachttp://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=16343834&dopt=Abstrachttp://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=16343834&dopt=Abstrachttp://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=15317190&dopt=Abstrachttp://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=8426158&dopt=Abstrachttp://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=1398379&dopt=Abstrac