U hrvatskom stručnom časopisu Croatian medical journal, 48 (4), Zagreb, 2007., str. ovih se dana pojavio članak Marije Definis Gojanović i Davorke Sutlović »Skeletal remains from World war II mass grave: from discover

Zagreb, 10. kolovoza 2007. (MIRIAM) – U hrvatskom stručnom časopisu Croatian medical journal, 48 (4), Zagreb, 2007., str. ovih se dana pojavio članak Marije Definis Gojanović i Davorke Sutlović »Skeletal remains from World war II mass grave: from discovery to identification. Govori o iskapanju masovne grobnice u Zagvozdu i o dosadašnjoj identifikaciji trojice ubijene naše braće, na čemu su autorice predano radile pod vodstvom pročelnika Povjerenstva za pripremu kauze mučenika fra Ante Marića. 

Članak se inače može naći na sljedećoj Mrežnoj stranici:

Forensic Science

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


Marija Definis Gojanović, Davorka Sutlović

Department of Forensic
Medicine, Split University
Hospital and School of
Medicine, Split, Croatia

> Correspondence to:

Marija Definis-Gojanović

Department of Forensic Medicine
Split University Hospital and School
of Medicine

Spinčićeva 1

21000 Split, Croatia

> Received: June 12, 2007

> Accepted: July 10, 2007

> Croat Med J. 2007;48

Aim To present the process of identification of skeletal remains from a mass
grave found on a Dalmatian mountain-range in 2005, which allegedly contained
the remains of civilians from Herzegovina killed in the World War II, including
a group of 8 Franciscan monks.

Methods Excavation of a site in Dalmatian hinterland, near the village of Zagvozd,
was accomplished according to archeological procedures. Anthropological
analysis was performed to estimate sex, age at death, and height of the individuals,
as well as pathological and traumatic changes of the bones. Due to the
lack of ante-mortem data, DNA typing using Y-chromosome was performed.
DNA was isolated from bones and teeth samples using standard phenol/chloroform/
isoamyl alcohol extraction. Two Y-chromosome short tandem repeats
(STR) systems were used for DNA quantification and amplification. Typing of
polymerase chain reaction (PCR) products was performed on an ABI Prism 310
Genetic Analyzer. PCR typing results were matched with results from DNA
analysis of samples collected from the relatives of supposed victims – blood samples
from the living relatives and bone samples collected during further exhumation
of died parents or relatives of the supposed victims.

Results The remains contained 18 almost complete skeletons, with considerable
post-mortal damage. All remains were men, mainly middle-aged, with gunshot
wounds to the head. DNA analysis and cross-matching of the results with
relatives’ data resulted in three positive identifications using the Y-chromosomal
short tandem repeat (Y-STR) systems. All of the positively identified remains
belonged to the Franciscan friars allegedly killed in Herzegovina and buried at
the analyzed site.

Conclusion Our analysis of remains from a mass grave from the World War II
confirmed the value of patrilineal lineage based on Y-STRs, even when missing
persons had left no offspring, as is the case with Franciscan monks. Although this
report is primarily focused on the identification of remains from a mass grave,
it also emphasizes the role of forensic approach in documenting human right

Croat Med J 2007;48

In investigation of human rights violations, forensic
specialists are often faced with identification
of skeletal remains from mass graves. This
is always a difficult task, especially when a lot of
time has passed since the burial. The primary focus
of such investigations is to determine the
identity of the victims and return the remains
to their families, in accordance with the Geneva
conventions (1). As a rule, a positive identification
is achieved by comparing pre-mortem data
on missing persons with corresponding findings
on the skeletons. These data include general anthropological
variables, unique bone pathological
and traumatological traits such as bone diseases,
deformations, and injuries, as well as medical
and dental histories. Determination of sex, age,
height, and individuals’ characteristics is critical
for achieving identification. Life histories and descriptions
by witnesses may also prove useful in
the process of individualization, especially data
on victims’ clothing and personal belongings,
state at the time of death, and manner of death
(2-4). The possibilities to obtain such information
for war victims are diminished due to long
period between the death and recovery of the
skeleton, negative effect on the memory of witnesses,
the quality of preserved distinctive data,
and the fact that mass graves contain skeletons
from homogenous groups, such as young men in
military clothes (5,6). The identification process
is further aggravated by the fact that lists of possible
decedents do not exist or are uncertain (5,6).
In such cases, the available pre-mortem data are
usually not sufficient, so identification and DNA
typing techniques may be the only solution.
DNA technology, including both short tandem
repeat (STR) analysis and mitochondrial DNA
(mtDNA) analysis, was already confirmed as a
method of choice in the identification of missing
person in 1991-1995 war in Croatia (2,3).

This report presents the identification process
of skeletal remains from a mass grave, allegedly
from the World War II. The identification
process used Y-chromosomal short tandem re

peat (Y-STR) DNA profiling as a single method
of identification from bone specimens from the
World War II.

Material and methods


The samples for DNA analysis were taken from
18 skeletons from a mass grave in the village of Zagvozd,
near the town of Imotski in the Dalmatian
hinterland in Croatia, exhumed in April 2005.

According to the survivors’ testimonies, after
partisans had captured the Franciscan monastery
in the town of Široki Brijeg, Bosnia and Herzegovina,
in February 1945, 8 friars were killed
somewhere in the region of Dalmatian mountain
range on their way to Split in Croatia (7).

In the 1970s, there were allegations that 19
civilians were killed in the village of Zagvozd in
1945, including 8 friars from neighboring Herzegovina.
Allegedly, they were buried near the
house where they had been tortured. After a
few days, the remains were removed to the field
named Ðokina sward, about a hundred meters
from the village. Intensive investigation and
data collection at the possible site of their execution
and interment started in 1990. It was initiated
by the Franciscan Province of Herzegovina
and the families of the victims, as well as by the
people of Zagvozd who wanted that friars’ bones
be buried in dignity (7). These activities started
more than 50 years after the alleged incident,
because Yugoslavian authorities after the World
War II preferred to keep such events secret (7).
In April 2005, experimental excavation started
on Ðokina sward and, after the first bones had
been discovered, the police and district attorney
were informed.

Exhumation and anthropological examination

The pit had a surface of 4×3.10 m and depth
of 0.32 m. The soil above and around the postmortal
remains was removed, exposing a number
of articulated skeletons lying on the back or

Definis Gojanović and Sutlović: Skeletal Remains from Mass Grave

on the face in an extended position. The bones
were partly commingled because about 15 bodies
were placed in the pit next to each other in
2 lines, facing each other with their legs, while 3
bodies were in the middle, laid down above them
(Figure 1). One skeleton was found without the
head, while other heads were fractured in many
fragments. Two pairs of disarticulated long bones
of the hands were found separately from the bodies.
Almost all skeletons were bound with a wire
around their necks, arms, or legs. The arms of 2
of them were tied on the back. No clothes and
footwear were found, except few buttons and
some remains of a textile material. Artifacts discovered
included a razor, scissors, pencil, spoon,
comb, ring, coins, remains of rosaries and several
bullets, and empty cartridges for high velocity
rifles. All relevant data were recorded, mapped,
measured, photographed, and described, along
with the information on the bones and all the
artifacts found. After that, the bones were removed,
placed in containers, and transported for
further analysis to the Department of Forensic
Medicine, Split University Hospital. The associated
artifacts were brought to the Museum of
Croatian Archeological Monuments in Split for
conservatory proceedings. After all of the obvious
remains had been removed, the entire area under
the bodies was examined for possible small artifacts
and bone fragments. Samples of earth were
collected from beneath and around skeletons for
chemical analysis.

Figure 1. Human skeletons exposed in mass grave after the su-
perficial layer of soil had been removed.
In the autopsy room, the material was
cleaned with water and soft brushes, dried, and
partially reconstructed. Despite the fact that
the skeletons were considerably damaged post-
mortally, they were examined to determine sex,
age, and stature. Sex was estimated by examining
the skeletal features of the skull, pelvis, and
long bones (8,9). Age estimation was performed
by examining the changes of the pubic symphysis
using the Suchey-Brooks method (10). Antemortem
stature was determined by measuring
the long bones and the results were compared
with the formulae and tables of Trotter and
Glesser (11). The remains were then analyzed in
more detail for signs of disease, injury, or skeletal

After dentition was charted, several intact,
healthy teeth were removed from each skull and
subjected to DNA testing, together with samples
of bones (femora) that seemed to be well preserved.

At the same time, ante-mortem descriptions
of the missing friars were obtained as well as
blood samples for DNA analysis from their living
relatives (brothers, sisters, or nephews). Soon
it became clear that these samples were not sufficient.
Moreover, as it was alleged that remains of
friars executed in the wider region of Herzegovina
in 1944-1945 could be among the excavated
skeletons, further collection of blood samples of
living relatives was performed, as well as two additional
exhumations of their dead parents. The
total number of analyzed specimens from relatives
was 45, among them 8 blood specimens and
37 teeth and bone samples.

DNA isolation

After bone surfaces were cleaned by abrasion
with a grinding tip and sandpaper, the bone was
crushed into small fragments and stored in sterile
polypropylene tubes at -20°C until analysis. Further
bone preparation and DNA extraction were
done as described by Alonso et al (12). DNA
from blood and bloodstain reference samples of

Croat Med J 2007;48

living relatives was isolated by standard Chelex
100 protocols (13).

DNA quantification

Data was collected using the ABI Prism 7000 Sequence
Detection System (Applied Biosystems,
Foster City, CA, USA). Data analysis was performed
with ABI Prism 7000 Sequence Detector
Software (SDS), version 1.0 (Applied Biosystems)
to generate an individual standard curve for each
experiment and calculate DNA amount from
each unknown sample. Human genomic DNA
9947 at 200 ng/µL concentrations (Applied Biosystems)
was used as a DNA standard. We used
QuantifilerTM human DNA quantification kit
(Applied Biosystems). The quantification assay
was performed in a total volume of 25 µL, containing
2 µL of DNA extract, Quantifiler human
primer mix, and Quantifiler PCR reaction mix,
with thermal cycling conditions according to the
manufacturer’s protocols (14). All reactions without
templates served as negative controls.

DNA amplification and typing

PCR amplification was performed on Perkin-Elmer
Thermal Cycler 9600, using the PowerPlex®
Y System (Promega Corporation, Madison,
WI, USA) and AmpFlSTR Yfiler PCR Amplification
kit (Applied Biosystems) (15,16). The
amount of DNA used for individual samples for
both kits was from 100 pg to 1 ng.

Typing of PCR products were performed on
an ABI Prism 310 Genetic Analyzer (Applied
Biosystems) with Data Collection Software.
Electropherogram data were analyzed with GeneScan
® Software and GeneMapper® ID software,
version 3.2 (Promega). The internal standard was
Liz-500 (Applied Biosystems).

Analysis of typing results

DNA profiles from bones and teeth were analyzed
and compared with DNA profiles of living
and dead relatives. DNA genotypes from
the living relatives were obtained by analyzing

the DNA isolated from blood and bloodstains,
while those from the dead relatives were obtained
by analyzing the DNA isolated from teeth
and bone. The database was kept in the Micro-
soft Access 2000 (Microsoft, Seattle, WA, USA).
Microsoft Excel 2000 (Microsoft, Seattle, WA,
USA) was used for statistical calculation. Calculation
for statistical probability of biological relationship
was performed according to standard
protocols (17-19).


Morphological investigation revealed that the remains
belonged to 18 adult male victims, mostly

All bones, but especially small and thin ones,
as well as the edges of long bones, had considerable
post-mortal damage. Consequently, the estimation
of ante-mortem stature was made only
on the basis of one or two long bones. In a single
case stature could not be determined.

The bones revealed no pathological or degenerative
changes. Only in a single case a sign of
ante-mortem trauma was found as a healed fracture
of the humerus. Gunshot injuries were present
in 11 individuals – 10 of them had gunshot
wounds of the head (Figure 2), mainly in the occipital
region, while a single individual had an
injury of the femur. Four persons had two gunshot
injuries (head plus lower extremities). The
reasons for the fracture and fragmentation of all
other skulls and of long bones in 2 cases could
not be reliably determined. The bones of forearms
were tied with wire in 6 cases (Figure 3).

The types of analyzed samples, number of
DNA isolations, and success of DNA amplification
are presented in Tables 1 and 2. DNA was
extracted from teeth and bone samples of 18 analyzed
skeletons, as well as from blood samples of
living relatives of 7 missing friars. In each case of
teeth and bone samples, several DNA isolations
were performed (Table 1). The concentration
of isolated DNA was in the range from 14.4 pg/

Definis Gojanović and Sutlović: Skeletal Remains from Mass Grave

Figure 2. Entrance gunshot wound of the head (arrow).
Figure 3. Forearm bones tied with a wire.
µL to 2.5 ng/µL. Moreover, DNA was extracted
from one blood sample collected subsequently,
and from 19 samples of teeth and bones taken
during 2 additional exhumations of missing persons’
dead parents (Table 2).

By using AmpFlSTR Yfiler PCR Amplification
kit, 3 matches between missing persons
and their living/dead relatives were obtained and
thus 3 bodies were positively identified (Table 3).
Using allele frequencies in the general Croatian
population (our unpublished data), the probability
of parenthood was calculated for 3 cases as
follows: 8.6×108 for case 1, 1.36×1013 for case 2,
and 8.6×1011 for case 3 (Table 4).


The main goal of mass grave exhumation and examination
of skeletal remains was the identifi-

Table 1. Types of analyzed samples, number of DNA isolations

and success of DNA amplification with PowerPlex Y System and
AmpFlSTR Yfiler PCR Amplification kit

Types of Success of DNA amplification

Case references No. of DNA PowerPlex® Y AmpFlSTR
No. samples isolations System (12 loci) Yfiler (17 loci)

1 teeth 3 –* 16/17
2 teeth 2 – 12/17
3 teeth 2 – 17/17
4 teeth 3 12/12 17/17
5 femur and teeth 5 10/12 12/17
6 teeth 3– 17/17
7 teeth 4– 12/17
8 femur and teeth 6 12/12 17/17
9 teeth 5 12/12 17/17
10 teeth 3 10/12 17/17
11 teeth 2 12/12 17/17
12 teeth 3 11/12 11/17
13 teeth 5 12/12 12/17
14 teeth 4– 17/17
15 teeth 3– 17/17
16 teeth 3– 17/17
17 teeth 4– 17/17
18 teeth 3 5/12 –
19 blood 1 12/12 17/17
20 blood 1 12/12 17/17
21 blood 1 12/12 17/17
22 blood 1 12/12 17/17
23 blood 1 12/12 17/17
24 blood 1 12/12 17/17
25 blood 1 12/12 17/17

*unsuccessful DNA amplification

Table 2. Types of analyzed samples, number of DNA isolations

and success of DNA amplification with AmpFlSTR Yfiler PCR
Amplification kit

Types of
Sample references Number of Success of DNA
No. samples DNA isolations amplification (17 loci)

1 femur and teeth 5 3/17
2 femur and teeth 9 17/17
3 femur and teeth 3 –*
4 femur and teeth 4 17/17
5 femur and teeth 3 16/17
6 femur and teeth 4 8/17
7 femur and teeth 3 15/17
8 femur and teeth 3 15/17
9 femur 2 17/17
10 femur and teeth 2 –
11 blood 1 17/17
12 femur 3 17/17
13 femur 4 17/17
14 femur and teeth 3 17/17
15 femur and teeth 3–
16 femur and teeth 3 17/17
17 femur 2 17/17
18 femur 4 –
19 femur 4 –
20 femur 4 –

*unsuccessful DNA amplification.

cation and determination of cause and manner
of death. Allegedly, a group of 19 civilians was
killed in 1945 and (re)buried after several days in
a common grave. Although the region of their al


Croat Med J 2007;48

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

Case 1 Case 2 Case 3

nephew’s 1
missing person
NN 1
nephew’s 2
missing person
NN 2
skeletal remains
missing person
NN 3
skeletal remains
DYS391 11 11 11 10 10 11 11
DYS389 I 13 13 13 13 13 13 13
DYS439 13 13 13 11 11 13 13
DYS389 II 31 31 31 29 29 31 31
DYS438 10 10 10 11 11 10 10
DYS437 15 15 15 14 14 15 15
DYS19 15 15 15 16 16 14 14
DYS392 11 11 11 11 11 11 11
DYS393 13 13 13 13 13 13 13
DYS390 24 24 24 25 25 24 24
DYS385 14;15 14;15 14;15 11;14 11;14 14;15 14;15

Table 4. Example of statistical calculations of biological relationship between missing person and his presumptive relatives

Allele frequency*
Locus Obligate genotype pq RMNE†

DYS 391 11 0.624 0.39172224
DYS 389I 13 0.697 0.48792091
DYS 439 13 0.321 0.10522059
DYS 389II 31 0.413 0.17299331
DYS 438 10 0.697 0.48792091
DYS 437 15 0.615 0.38059275
DYS 19 14 0.018 0.00050076
DYS 392 11 0.908 0.82529936
DYS 393 13 0.908 0.82529936
DYS 390 24 0.642 0.41446236
DYS 385 14;15 0.009 0.018 0.000324
combined RMNE 1.15912×10-11
Probability‡ = 1: 8.6×108

*Allele frequencies in general Croatian population.

†random man not excluded (RMNE) the proportion of the population that could
contribute all of the obligate alleles and therefore could not be excluded.
‡Chance probability of founding a person with same genotype in population.
leged first burial ground was investigated, no archeological
evidence was found, suggesting that
the bodies had been removed to the grave analyzed
in this study. Using morphological and anthropological
analyses, remains were shown to
contain bones of 18 adult male victims. Considerable
post-mortal damage and changes of the
bones compromised the possibility to estimate
the precise age and stature of the individuals.

Gunshot injuries, likely caused by assault rifles,
were identified in 11 out of 18 cases. We
could not exclude the possibility that fractures
and fragmentations of bones in uncertain cases
were the result of gunshot injuries. Although
it is not possible to make conclusions whether
these injuries were inflicted before or after death,
the anatomical distribution of the injuries, bullet

trajectories through the skulls, as well as the fact
that no clothes were found and that all victims
were bound with a wire, strongly suggest that the
victims were prisoners rather than soldiers killed
in confrontation between two armed groups (2022).

There was no ante-mortal information to
match the post-mortal data obtained by standard
forensic identification techniques, and the application
of molecular methods in identification
process seemed to be an imperative. We used YSTR
systems for several reasons as follows: all
skeletal remains were male, some of them allegedly
did not have any offspring, the presumptive
parents were dead, and the living relatives –
brothers, sisters, or nephews were not available
in the sufficient number. STR systems located in
the non-recombining region of the Y chromosome
are widely used in forensic science for the
identification of male individuals (18). The Y
chromosome is passed down through generation
from father to son, and does not change a lot between
generations. Thus, it can be used to track
parental lineage to see if the men in question are
related through their fathers. This report confirmed
the value of patrilineal lineage based on Y
chromosome STRs, which is the only applicable
method of DNA analysis in some circumstances.
Some other studies have already shown that
Y chromosome could provide important information
if there are difficulties in identifying lineages
from a specific male (23,24). On the other

Definis Gojanović and Sutlović: Skeletal Remains from Mass Grave

hand, our work clearly showed the importance
of other DNA identification methods and their
application in case work, such as mitochondrial
DNA. However, the possibilities for the use of
these methods for identification are often limited,
especially in situations like the case presented,
where ante-mortem data are completely absent.
Moreover, in cases similar to this one, the samples
are often of poor quality, which hinders the
DNA extraction, subsequent amplification, and
final identification (22).

In our study, DNA amplification was performed
by PowerPlex® Y System first (12 loci)
and then repeated by new, more informative
AmpFlSTR Yfiler PCR Amplification kit (17
loci). In both systems, the results were not completely
sufficient, and in some cases no profiles
or partial profiles only were obtained. DNA
degradation or DNA polymerase inhibitions
was the most likely explanation for unsuccessful
amplification of some of the loci. Problems that
forensic scientists most often face while working
with DNA extracted from bones and teeth
samples recovered from mass graves are limited
DNA quantity, DNA degradation, contamination,
and postmortem changes (3,12,25). The
presence of inhibitor(s) may also prevent amplification.
Inhibition is an especially significant
problem when DNA is extracted from old and
ancient material (25,26). One of the potential
inhibitors is humic acid. DNA extraction from
soil always results in co-extraction of other soil
components, mainly humic acid or other humic
substances, which negatively interfere with
DNA detecting processes (26-28). The soil from
the site described in this study was collected; its
composition and possible PCR inhibition is still
under analysis.

We successfully identified 3 persons out of 18
in the grave. One of them was a friar captured in
a hydro-electric power station in Široki Brijeg in
1945. He sustained a gunshot wound of the parietal
region of the head. Two other persons were
also friars, allegedly captured in 1945 and killed

somewhere near Ljubuški, Bosnia and Herzegovina.
One had a gunshot wound of the occipital
region of the head, while no signs of trauma
were found in the other. These results confirm
the presumption that there were more than 8 friars
in the mass grave. Also, they confirm that our
decision to collect the samples for DNA analysis
from more relatives, living and dead, requiring 2
additional exhumations, was correct. We hope
that our experience and data will be of value in
further identifications of skeletal remains from
other alleged graves in this part of Croatia and
Bosnia and Herzegovina.

The ultimate goal of recoveries is to positively
identify the remains and return them to their
families. However, personal identification of war
victims has many aspects, from ethical to humanitarian
and medico-legal. In this context, the
excavation of graves, examination of their contents,
and analysis the post-mortal remains has
another additional purpose – to collect forensic
evidence that would permit prosecution of those
responsible for the creation of the mass grave,
crimes against humanity, and international human
rights abuses (4,21,29). Proper and systematic
exhumation of mass graves and post-mortem
examination could demonstrate what happened
and when, as well as help discover those who are
accountable. These answers are essential for promoting
reconciliation and justice, give the rights
to the dead, and provide moral and emotional
satisfaction to the living.


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