skeletal remains from world war ii mass grave: from discovery to identification

8/2/2019 Skeletal Remains from World War II Mass Grave: from Discovery to Identification

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> Co rrespondence to:

520 www.cmj.hr

Skeletal Remains from World War II Mass Grave: from Discovery toIdentifcation

Aim o present the process o identi cation o skeletal remains rom a massgrave ound on a Dalmatian mountain-range in 2005, which allegedly containedthe remains o civilians rom Herzegovina killed in the World War II, including a group o 8 Franciscan monks.Methods Excavation o the site in Dalmatian hinterland, near the village o Za-gvozd, was accomplished according to archeological procedures. Anthropologi-cal analysis was per ormed to estimate sex, age at death, and height o the individuals, as well as pathological and traumatic changes o the bones. Due to thelack o ante-mortem data, DNA typing using Y-chromosome was per ormed.DNA was isolated rom bones and teeth samples using standard phenol/chlo-ro orm/isoamyl alcohol extraction. wo Y-chromosome short tandem repeats(S R) systems were used or DNA quanti cation and ampli cation. yping o polymerase chain reaction (PCR) products was per ormed on an ABI Prism 310Genetic Analyzer. PCR typing results were matched with results rom DNAanalysis o samples collected rom the relatives o supposed victims blood samples rom the living relatives and bone samples collected during urther exhuma-tion o died parents or relatives o the supposed victims.Results Te remains contained 18 almost complete skeletons, with consider-able post-mortal damage. All remains were men, mainly middle-aged, with gun-shot wounds to the head. DNA analysis and cross-matching o the results with

relatives data resulted in three positive identi cations using the Y-chromosomalshort tandem repeat (Y-S R) systems. All o the positively identi ed remainsbelonged to the Franciscan riars allegedly killed in Herzegovina and buried atthe analyzed site.Conclusion Our analysis o remains rom a mass grave rom the World War IIcon rmed the value o patrilineal lineage based on Y-S Rs, even when missing persons had le no o spring, as was the case with Franciscan monks. Althoughthis report is primarily ocused on the identi cation o remains rom a massgrave, it also emphasizes the role o orensic approach in documenting humanright violations.

Department of ForensicMedicine, Split UniversityHospital and School of Medicine, Split, Croatia

Marija Defnis Gojanovi, Davorka Sutlovi

Marija Defnis Gojanovi Department of Forensic Medicine

Split University Hospital and School

of Medicine

Spinieva 1 21000 Split, Croatia [email protected]

> Received: June 12, 2007

> Accepted: July 10, 2007

Forensic Science Forensic Science

> Croat Med J. 2007;48:520-7
mailto:%[email protected]:%[email protected]


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In investigation o human rights violations, o-rensic specialists are o en aced with identi ca-tion o skeletal remains rom mass graves. Tis

is always a di cult task, especially when a lot o time has passed since the burial. Te primary o-cus o such investigations is to determine theidentity o the victims and return the remainsto their amilies, in accordance with the Genevaconventions (1). As a rule, a positive identi ca-tion is achieved by comparing pre-mortem dataon missing persons with corresponding ndingson the skeletons. Tese data include general an-thropological variables, unique bone pathologi-cal and traumatological traits such as bone diseas-es, de ormations, and injuries, as well as medicaland dental histories. Determination o sex, age,height, and individuals characteristics is critical

or achieving identi cation. Li e histories and de-scriptions by witnesses may also prove use ul inthe process o individualization, especially dataon victims clothing and personal belongings,state at the time o death, and manner o death(2-4). Te possibilities to obtain such in orma-tion or war victims are diminished due to long

period between the death and recovery o theskeleton, negative e ect on the memory o wit-nesses, the quality o preserved distinctive data,and the act that mass graves contain skeletons

rom homogenous groups, such as young men inmilitary clothes (5,6). Te identi cation processis urther aggravated by the act that lists o possi-ble decedents do not exist or are uncertain (5,6).In such cases, the available pre-mortem data areusually not su cient, so identi cation and DNAtyping techniques may be the only solution.

DNA technology, including both short tandemrepeat (S R) analysis and mitochondrial DNA(mtDNA) analysis, was already con rmed as amethod o choice in the identi cation o missing person in 1991-1995 war in Croatia (2,3).

Tis report presents the identi cation pro-cess o skeletal remains rom a mass grave, alleg-edly rom the World War II. Te identi cation process used Y-chromosomal short tandem re-

peat (Y-S R) DNA pro ling as a single methodo identi cation rom bone specimens rom the World War II.

Material and methods

Location

Te samples or DNA analysis were taken rom18 skeletons rom a mass grave in the village o Za-gvozd, near the town o Imotski in the Dalmatianhinterland in Croatia, exhumed in April 2005.

According to the survivors testimonies, a er partisans had captured the Franciscan monasteryin the town o iroki Brijeg, Bosnia and Herze-govina, in February 1945, 8 riars were killedsomewhere in the region o Dalmatian mountainrange on their way to Split in Croatia (7).

In the 1970s, there were allegations that 19civilians were killed in the village o Zagvozd in1945, including 8 riars rom neighboring Her-zegovina. Allegedly, they were buried near thehouse where they had been tortured. A er a

ew days, the remains were removed to the eldnamed okina sward, about a hundred me-

ters rom the village. Intensive investigation anddata collection at the possible site o their execu-tion and interment started in 1990. It was initi-ated by the Franciscan Province o Herzegovinaand the amilies o the victims, as well as by the people o Zagvozd who wanted that riars bonesbe buried in dignity (7). Tese activities start-ed more than 50 years a er the alleged incident,because Yugoslavian authorities a er the World War II pre erred to keep such events secret (7).In April 2005, experimental excavation started

on okina sward and, a er the rst bones hadbeen discovered, the police and district attorney were in ormed.

Exhumation and anthropological examination

Te pit had a sur ace o 4 3.10 m and deptho 0.32 m. Te soil above and around the post-mortal remains was removed, exposing a num-ber o articulated skeletons lying on the back or


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on the ace in an extended position. Te bones were partly commingled because about 15 bod-ies were placed in the pit next to each other in

2 lines, acing each other with their legs, while 3bodies were in the middle, laid down above them(Figure 1). One skeleton was ound without thehead, while other heads were ractured in many

ragments. wo pairs o disarticulated long boneso the hands were ound separately rom the bod-ies. Almost all skeletons were bound with a wirearound their necks, arms, or legs. Te arms o 2o them were tied on the back. No clothes and

ootwear were ound, except ew buttons andsome remains o a textile material. Arti acts dis-covered included a razor, scissors, pencil, spoon,comb, ring, coins, remains o rosaries and sever-al bullets, and empty cartridges or high velocityrifes. All relevant data were recorded, mapped,measured, photographed, and described, along with the in ormation on the bones and all thearti acts ound. A er that, the bones were re-moved, placed in containers, and transported or

urther analysis to the Department o ForensicMedicine, Split University Hospital. Te asso-ciated arti acts were brought to the Museum o Croatian Archeological Monuments in Split orconservatory proceedings. A er all o the obviousremains had been removed, the entire area underthe bodies was examined or possible small arti-

acts and bone ragments. Samples o earth werecollected rom beneath and around skeletons orchemical analysis.

In the autopsy room, the material wascleaned with water and so brushes, dried, and partially reconstructed. Despite the act that

the skeletons were considerably damaged post-mortally, they were examined to determine sex,age, and stature. Sex was estimated by examin-ing the skeletal eatures o the skull, pelvis, andlong bones (8,9). Age estimation was per ormedby examining the changes o the pubic symphy-sis using the Suchey-Brooks method (10). Ante-mortem stature was determined by measuring the long bones and the results were compared with the ormulae and tables o rotter andGlesser (11). Te remains were then analyzed inmore detail or signs o disease, injury, or skel-etal anomalies.

A er dentition was charted, several intact,healthy teeth were removed rom each skull andsubjected to DNA testing, together with sampleso bones ( emora) that seemed to be well pre-served.

At the same time, ante-mortem descriptionso the missing riars were obtained as well asblood samples or DNA analysis rom their liv-ing relatives (brothers, sisters, or nephews). Soonit became clear that these samples were not su -cient. Moreover, as it was alleged that remains o

riars executed in the wider region o Herzegov-ina in 1944-1945 could be among the excavatedskeletons, urther collection o blood samples o living relatives was per ormed, as well as two ad-ditional exhumations o their dead parents. Tetotal number o analyzed specimens rom rela-tives was 45, among them 8 blood specimens and37 teeth and bone samples.

DNA isolation

A er bone sur aces were cleaned by abrasion with a grinding tip and sandpaper, the bone wascrushed into small ragments and stored in sterile polypropylene tubes at -20C until analysis. Fur-ther bone preparation and DNA extraction weredone as described by Alonso et al (12). DNA

rom blood and bloodstain re erence samples o Figure 1. Human skeletons exposed in mass grave after the superfi-cial layer of soil had been removed.


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living relatives was isolated by standard Chelex100 protocols (13).

DNA quanti cation

Data was collected using the ABI Prism 7000 Se-quence Detection System (Applied Biosystems,Foster City, CA, USA). Data analysis was per-

ormed with ABI Prism 7000 Sequence DetectorSo ware (SDS), version 1.0 (Applied Biosystems)to generate an individual standard curve or eachexperiment and calculate DNA amount romeach unknown sample. Human genomic DNA9947 at 200 ng/L concentrations (Applied Bio-systems) was used as a DNA standard. We usedQuanti ler M human DNA quanti cation kit(Applied Biosystems). Te quanti cation assay was per ormed in a total volume o 25 L, con-taining 2 L o DNA extract, Quanti ler human primer mix, and Quanti ler PCR reaction mix, with thermal cycling conditions according to themanu acturers protocols (14). All reactions with-out templates served as negative controls.

DNA ampli cation and typing

Polymerase chain reaction (PCR) ampli cation was per ormed on Perkin-Elmer Termal Cycler9600, using the PowerPlex Y System (PromegaCorporation, Madison, WI, USA) and AmpFl-S R Y ler PCR Ampli cation kit (Applied Bio-systems) (15,16). Te amount o DNA used orindividual samples or both kits was rom 100 pg to 1 ng.

yping o PCR products were per ormed onan ABI Prism 310 Genetic Analyzer (AppliedBiosystems) with Data Collection So ware.

Electropherogram data were analyzed with Gen-eScan So ware and GeneMapper ID so ware, version 3.2 (Promega). Te internal standard wasLiz-500 (Applied Biosystems).

Analysis of typing results

DNA pro les rom bones and teeth were ana-lyzed and compared with DNA pro les o liv-ing and dead relatives. DNA genotypes rom

the living relatives were obtained by analyzing the DNA isolated rom blood and bloodstains, while those rom the dead relatives were ob-

tained by analyzing the DNA isolated rom teethand bone. Te database was kept in the Micro-so Access 2000 (Microso , Seattle, WA, USA).Microso Excel 2000 (Microso , Seattle, WA,USA) was used or statistical calculation. Calcu-lation or statistical probability o biological re-lationship was per ormed according to standard protocols (17-19).

Results

Morphological investigation revealed that the re-mains belonged to 18 adult male victims, mostlymiddle-aged.

All bones, but especially small and thin ones,as well as the edges o long bones, had consider-able post-mortal damage. Consequently, the es-timation o ante-mortem stature was made onlyon the basis o one or two long bones. In a singlecase stature could not be determined.

Te bones revealed no pathological or de-

generative changes. Only in a single case a sign o ante-mortem trauma was ound as a healed rac-ture o the humerus. Gunshot injuries were pres-ent in 11 individuals 10 o them had gunshot wounds to the head (Figure 2), mainly in the oc-cipital region, while a single individual had aninjury o the emur. Four persons had two gun-shot injuries (head plus lower extremities). Tereasons or the racture and ragmentation o allother skulls and o long bones in 2 cases couldnot be reliably determined. Te bones o ore-

arms were tied with wire in 6 cases (Figure 3).Te types o analyzed samples, number o

DNA isolations, and success o DNA ampli ca-tion are presented in ables 1 and 2. DNA wasextracted rom teeth and bone samples o 18 an-alyzed skeletons, as well as rom blood samples o living relatives o 7 missing riars. In each case o teeth and bone samples, several DNA isolations were per ormed ( able 1). Te concentration


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o isolated DNA was in the range rom 14.4 pg/L to 2.5 ng/L. Moreover, DNA was extract-ed rom one blood sample collected subsequent-ly, and rom 19 samples o teeth and bones takenduring 2 additional exhumations o missing per-sons dead parents ( able 2).

By using AmpFlS R Y ler PCR Ampli -cation kit, 3 matches between missing personsand their living/dead relatives were obtained andthus 3 bodies were positively identi ed ( able

3). Using haplotypes in the general Croatian andEuropean population database the probability o ounding a man with same genotipe as Person 1,

2 and 3, was calculated ( able 4).

Discussion

Te main goal o mass grave exhumation and ex-amination o skeletal remains was the identi -

cation and determination o cause and mannero death. Allegedly, a group o 19 civilians waskilled in 1945 and (re)buried a er several days ina common grave. Although the region o their al-

Figure 2. Entrance gunshot wound of the head.

Figure 3. Forearm bones tied with a wire.

Table 1. Types of analyzed samples, number of DNA isolationsand success of DNA ampli cation with PowerPlex Y System andAmpFlSTR Y ler PCR Ampli cation kit

Types of Success of DNA ampli cation

CaseNo.

referencessamples

No. of DNAisolations

PowerPlex YSystem (12 loci)

AmpFlSTRY ler (17 loci)

1 teeth 3 * 16/172 teeth 2 12/173 teeth 2 17/174 teeth 3 12/12 17/175 femur and teeth 5 10/12 12/176 teeth 3 17/177 teeth 4 12/178 femur and teeth 6 12/12 17/179 teeth 5 12/12 17/1710 teeth 3 10/12 17/1711 teeth 2 12/12 17/1712 teeth 3 11/12 11/1713 teeth 5 12/12 12/1714 teeth 4 17/1715 teeth 3 17/1716 teeth 3 17/1717 teeth 4 17/1718 teeth 3 5/12 19 blood 1 12/12 17/1720 blood 1 12/12 17/1721 blood 1 12/12 17/1722 blood 1 12/12 17/1723 blood 1 12/12 17/1724 blood 1 12/12 17/1725 blood 1 12/12 17/17*nsuccessful DNA amplification.

Table 2. Types of analyzed samples, number of DNA isolationsand success of DNA ampli cation with AmpFlSTR Y ler PCRAmpli cation kit

Sample No.

Types of

referencessamples

Number of DNA isolations

Success of DNAampli cation (17 loci)1 femur and teeth 5 3/172 femur and teeth 9 17/173 femur and teeth 3 *4 femur and teeth 4 17/175 femur and teeth 3 16/176 femur and teeth 4 8/177 femur and teeth 3 15/178 femur and teeth 3 15/179 femur 2 17/1710 femur and teeth 2 11 blood 1 17/1712 femur 3 17/1713 femur 4 17/1714 femur and teeth 3 17/1715 femur and teeth 3

16 femur and teeth 3 17/1717 femur 2 17/1718 femur 4 19 femur 4 20 femur 4 *Unsuccessful DNA amplification.


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leged rst burial ground was investigated, no ar-cheological evidence was ound, suggesting thatthe bodies had been removed to the grave ana-lyzed in this study. Using morphological and an-thropological analyses, remains were shown to

contain bones o 18 adult male victims. Consid-erable post-mortal damage and changes o thebones compromised the possibility to estimatethe precise age and stature o the individuals.

Gunshot injuries, likely caused by assault ri-fes, were identi ed in 11 out o 18 cases. Wecould not exclude the possibility that racturesand ragmentations o bones in uncertain cases were the result o gunshot injuries. Althoughit is not possible to make conclusions wheth-er these injuries were inficted be ore or a er

death, the anatomical distribution o the injuries, bullet trajectories through the skulls, as well as the act that no clothes were ound andthat all victims were bound with a wire, strong-ly suggest that the victims were prisoners rath-er than soldiers killed in con rontation betweentwo armed groups (20-22).

Tere was no ante-mortal in ormation tomatch the post-mortal data obtained by standard

orensic identi cation techniques, and the application o molecular methods in identi cation process seemed to be an imperative. We used Y-S R systems or several reasons as ollows: allskeletal remains were male, some o them alleg-edly did not have any o spring, the presump-tive parents were dead, and the living relatives brothers, sisters, or nephews were not availablein the su cient number. S R systems located inthe non-recombining region o the Y chromo-some are widely used in orensic science or theidenti cation o male individuals (18). Te Y

chromosome is passed down through generationrom ather to son, and does not change a lot be-tween generations. Tus, it can be used to track parental lineage to see i the men in question arerelated through their athers. Tis report con-

rmed the value o patrilineal lineage based on Y chromosome S Rs, which is the only applicablemethod o DNA analysis in some circumstanc-es. Some other studies have already shown thatY chromosome could provide important in or-mation i there are di culties in identi ying lin-

eages rom a speci c male (23,24). On the otherhand, our work clearly showed the importanceo other DNA identi cation methods and theirapplication in case work, such as mitochondrialDNA. However, the possibilities or the use o these methods or identi cation are o en limit-ed, especially in situations like the case presented, where ante-mortem data are completely absent.Moreover, in cases similar to this one, the sam-

Table 3. Y-chromosome short tandem repeat (STR) genotypes of the skeletal remains of three missing persons and their presumptiverelatives (nephews blood and skeletal remains of dead brothers)

Case 1 Case 2 Case 3

Locusnephews 1

bloodmissing person

NN 1nephews 2

bloodmissing person

NN 2brothers

skeletal remainsmissing person

NN 3brothers

skeletal remainsDYS391 11 11 11 10 10 11 11DYS389 I 13 13 13 13 13 13 13DYS439 13 13 13 11 11 13 13DYS389 II 31 31 31 29 29 31 31DYS438 10 10 10 11 11 10 10DYS437 15 15 15 14 14 15 15DYS19 15 15 15 16 16 14 14DYS392 11 11 11 11 11 11 11DYS393 13 13 13 13 13 13 13DYS390 24 24 24 25 25 24 24DYS385 14;15 14;15 14;15 11;14 11;14 14;15 14;15

Table 4. Statistical calculations of probability of nding a manwith the same genotype as Person 1, 2, and 3 in populationProbability Person 1 Person 2 Person 3P1* 1.76 10-2 1.76 10-2 1.76 10-2P2 2.09 10-3 3.80 10-3 2.09 10-3*Statistical calculations of probability according to the Croatian unpublished popula-tion data (n=167 haplotypes).Statistical calculations according to the eight loci from European database onhttp://www.yhrd.org (n=51 253 haplotypes).
http://www.yhrd.org/http://www.yhrd.org/


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ples are o en o poor quality, which hinders theDNA extraction, subsequent ampli cation, and

nal identi cation (22).

In our study, DNA ampli cation was per-ormed by PowerPlex Y System rst (12 loci)and then repeated by new, more in ormativeAmpFlS R Y ler PCR Ampli cation kit (17loci). In both systems, the results were not completely su cient, and in some cases no pro lesor partial pro les only were obtained. DNAdegradation or DNA polymerase inhibitions was the most likely explanation or unsuccess ulampli cation o some o the loci. Problems that

orensic scientists most o en ace while work-ing with DNA extracted rom bones and teethsamples recovered rom mass graves are limitedDNA quantity, DNA degradation, contamina-tion, and postmortem changes (3,12,25). Te presence o inhibitor(s) may also prevent ampli cation. Inhibition is an especially signi cant problem when DNA is extracted rom old andancient material (25,26). One o the potentialinhibitors is humic acid. DNA extraction romsoil always results in co-extraction o other soilcomponents, mainly humic acid or other hu-mic substances, which negatively inter ere withDNA detecting processes (26-28). Te soil romthe site described in this study was collected; itscomposition and possible PCR inhibition is stillunder analysis.

We success ully identi ed 3 persons out o 18 in the grave. One o them was a riar cap-tured in a hydro-electric power station in irokiBrijeg in 1945. He sustained a gunshot woundo the parietal region o the head. wo oth-

er persons were also riars, allegedly capturedin 1945 and killed somewhere near Ljubuki,Bosnia and Herzegovina. One had a gunshot wound o the occipital region o the head, while no signs o trauma were ound in the oth-er. Tese results con rm the presumption thatthere were more than 8 riars in the mass grave.Also, they con rm that our decision to collectthe samples or DNA analysis rom more rela-

tives, living and dead, requiring 2 additionalexhumations, was correct. We hope that ourexperience and data will be o value in urther

identi cations o skeletal remains rom otheralleged graves in this part o Croatia and Bosniaand Herzegovina.

Te ultimate goal o recoveries is to positive-ly identi y the remains and return them to their

amilies. However, personal identi cation o war victims has many aspects, rom ethical to hu-manitarian and medico-legal. In this context, theexcavation o graves, examination o their con-tents, and analysis the post-mortal remains hasanother additional purpose to collect orensicevidence that would permit prosecution o thoseresponsible or the creation o the mass grave,crimes against humanity, and international hu-man rights abuses (4,21,29). Proper and system-atic exhumation o mass graves and post-mortemexamination could demonstrate what happenedand when, as well as help discover those who areaccountable. Tese answers are essential or pro-moting reconciliation and justice, give the rightsto the dead, and provide moral and emotionalsatis action to the living.References

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skeletal remains from world war ii mass grave: from discovery to identification

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