Science & Justice xxx (xxxx) xxx–xxx

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Science & Justice

journal homepage: www.elsevier.com/locate/scijus

Case review

From unknown to known: Identification of the remains at the mausoleum of

fosse Ardeatine
⁎
Elena Pillia,e, , Silvia Bocconeb, Alessandro Agostinoc, Antonino Virgilid, Giancarlo D'Erricoe,
Martina Laria, Cesare Raponee, Filippo Barnie, Jacopo Moggi Cecchib, Andrea Bertie,
David Caramellia
a
Department of Biology, University of Florence, Laboratory of Anthropology -Molecular Anthropology and Forensic Unit, Firenze, Italy
b
Department of Biology, University of Florence, Laboratory of Anthropology, Firenze, Italy
c
Diagnostic and Genomics Group. Agilent technologies, Italy
d
Istituto Superiore di Tecniche Investigative dei Carabinieri, Velletri, Roma, Italy
e
Molecular Biology and Genetics Unit, Carabinieri Scientific Investigation Department, Rome, Italy

A R T I C LE I N FO A B S T R A C T

Keywords: During the Second World War, on 24th March 1944, 335 Italians were massacred near Rome by the occupying
Forensic science forces of Nazi Germany. Four months later forensic examination led to the identification of 323 out of 335
Mass disaster victims. After approximately 60 years, the identification of the remaining unidentified twelve victims began with
Anthropological analysis anthropological and genetic analysis carried out by a team of Italian forensic experts. Anthropological analysis
Genetic identification
was performed in field in order to confirm the sex of each victim and verify the presence of only one individual in
STRs and mtDNA
each grave for a correct sampling. Selected bone fragments for each individual were then collected and trans-
ferred to the laboratory for genetic analysis. Although the anthropological ante mortem information was limited,
morphological and metrical data was collected for a possible future identification of the victims. Subsequently,
the typing of autosomal loci, Y-STR and mtDNA D-loop region of all bone and available reference samples was
conducted. LR and cumulative LRs obtained from autosomal STR and Y-STR results confirmed the alleged re-
lationship between three victims and their relatives with values over 104 (one sample) and 106 (two samples).
Therefore, the genetic analysis offered the families the possibility of replacing the number of the grave with the
name of the victim.

1. Introduction The process of identifying the victims in a mass disaster is long,

On March 23rd 1944, during the Second World War, 335 civilians
and Italian military personnel were brought by a German command to
the Fosse Ardeatine close to Rome and killed as a reprisal for the attack
suffered. For the high number of victims and tragic circumstances that
led this event, the mass disaster of Fosse Ardeatine has become the
symbol of Nazi reprisals during occupation period. On July 26th 1944,
forensic medicine investigations to identify the victims began. Bodies
did not appear distinct from each other but were piled, stacked, closely
amassed together and completely unrecognisable (Fig. 1).
Before being analysed by the medical team, all bodies were marked
with a serial number that indicated the order in which they were ex-
humed. In spite of the state of decay of the bodies due to putrefaction,
the method of investigation, mainly inductive and analytical, led to the
identification of 323 out of 335 bodies.
arduous and, of course, more difficult if bodies are unrecognisable and/
or skeletonised. However, the development of forensic science and DNA
analysis has led to resolving the main issue in mass disasters: the vic-
tims' identification. In order to identify the mass disaster victims, the
ante-mortem information obtained on missing person (such as anthro-
pological characteristics - bone disease, injury, deformation and dental
history, but also sex, age, height - and personal belongings - presence of
tattoos and/or special signs and details of victim's clothes [2–4]-) has to
be compared with corresponding results derived from post-mortem
analysis. However, in some cases, if the bodies are found long time after
death, the possibility of obtaining this type of information from wit-
nesses or relatives may often be minimal. In this case, DNA profiling can
increase the probability of identification and offer the families of the
missing persons the possibility of recovering the remains of their loved
ones, and a proper burial as required by the Geneva Convention [5].

⁎
Corresponding author at: Department of Biology, University of Florence, Laboratory of Anthropology -Molecular Anthropology and Forensic Unit, Firenze, Italy
E-mail address: elena.pilli@unifi.it (E. Pilli).

https://doi.org/10.1016/j.scijus.2018.05.007
Received 23 January 2018; Received in revised form 8 May 2018; Accepted 20 May 2018
1355-0306/ © 2018 The Chartered Society of Forensic Sciences. Published by Elsevier B.V. All rights reserved.

Please cite this article as: Pilli, E., Science & Justice (2018), https://doi.org/10.1016/j.scijus.2018.05.007
E. Pilli et al.
Fig. 1. The bodies of the victims mixed with soil. The bodies were closely
amassed and completely unrecognisable when in 1944 work of identification
began [1].
The use of DNA analysis in disaster victim identification, as re-
commended by Interpol guidelines [6], is one of the primary methods
that should be used along with dental analysis and fingerprinting.
Currently, Disaster (both natural and man-made) Victim Identification
(DVI) is based mainly on molecular methodology which is considered
an effective approach in victim identification, as demonstrated by
several publications of different research groups [7–21].
In 2009 the Ministry of Defence requested the cooperation of the
Carabinieri to identify the twelve victims including two unknown
bodies and 10 known but not identified. Previously, in 2006, at the
request of the brother of one of the Fosse Ardeatine victims, Pietrangeli
et al. [22] exhumed the body from grave number 329 in order to de-
termine his identity. The mitochondrial analysis, performed by Italian
researchers, excluded a biological relationship between two living
maternal relatives and the victim. Here, we present the anthropological
examination and DNA analysis of the twelve victims present at the
Mausoleum of Fosse Ardeatine (GPS coordinates: latitude
41.857050000, longitude 12.510510000) (Fig. 2).
Our work was done at two different times and in two different
places: fieldwork and laboratory work. The first involved 1.
Photographic documentation of all phases of the investigation; 2.
Exhumation of the bodies; 3. Gathering and cataloguing everything
inside each grave and 4. Anthropometric data collection; the second
one consisted of analysis of the DNA extracted from victims' bones. In
order to obtain more reliable results, DNA typing was performed, as
described by Harder et al. [23], with a combined application of com-
mercially available short tandem repeat (STR)-multiplex kits (ESI17-
NGMSelect-miniFiler). The Y chromosome and mitochondrial haplo-
type were also studied. In order to collect reference samples for com-
parison purposes, previously and simultaneously to DNA analysis on
bones, a complex research of the immediate or the closest relatives of
the unidentified victims was conducted.
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Science & Justice xxx (xxxx) xxx–xxx
2. Materials and methods
Permission to access the study site, exhume remains, and collect
samples was granted by the Ministry of Defence –Commissariato
Generale per le Onoranze ai Caduti in guerra- (Letter 3782 of 4 May
2009), and a signed written informed consent was obtained from all the
relatives of the victims.
A forensic team (post-mortem team) was involved in carrying out
the exhumation of the bodies, anthropological field analysis, and ge-
netic analysis in laboratory. At the same time, another team (ante-
mortem team) worked with victims' families trying to gather informa-
tion about victims and their anthropological traits.
2.1. Post-mortem team activities
Fieldwork activities began at the Mausoleum of Fosse Ardeatine
with the removal of the stone slabs of twelve graves by a specialised
company. During the following days, the bodies were exhumed by
opening the graves one by one and collecting the material inside each
grave. As requested, only graves 3–52–98-122-155-264-272-273-276-
283-284 and 329 labelled “Unidentified” were opened. Then an an-
thropological examination was conducted and the skeletal remains of
each individual were collected from each grave in order to attempt a
DNA analysis.
2.1.1. Anthropological examination
An anthropological examination was performed in order to gather
as much information as possible, which could also be useful for future
identification of the victims, so that sampling could be done correctly,
and to verify that in each grave there was only one individual. The
anthropological study of skeletal remains took place close to the graves
in an area specifically prepared for the analysis. After recovery of the
skeletal remains of each individual, the post-mortem team filled a
purpose built anthropological data form in which the information such
as presence/absence of different skeletal elements and teeth (recording
ante-mortem and post-mortem losses), presence of non-metric traits of
the dentition such as the tubercle of Carabelli [24], and the morpho-
logical features for estimation of age at death and stature (Table 1) was
recorded.
All skeletal remains recovered were extensively photographed. A
skeletal element was selected from each individual, and transferred to
the laboratory for sampling. In most individuals (sample 3–4, 52–1,
98–1, 122–1, 155–1, 272–1, 273–1, 276–1 and 284–1) we chose the
femur as the bone for genetic analysis. If the femur was absent or in
poor condition, we used the humerus (264–1 and 329–1) and tibia
(283–2). Genetic analysis was also performed on two different skeletal
elements of the same portion of the right temporal bone (272–3 and
272–4).
2.1.2. Contamination prevention
In order to avoid cross-contamination during activities and obtain
reliable results, field activities were carried out by qualified personnel
wearing proper protection equipment such as mask, gloves and crime
scene coats, and the crime scene protocol proposed by US Technical
Working Group on Crime Scene Investigation [31] was adopted. The
genetic analysis was conducted according to the most stringent criteria
proposed for ancient DNA studies [32–34], and all steps of the DNA
analysis were performed in a dedicated laboratory physically separated
from that of the routine caseworks. Different precautions were taken to
avoid contamination of samples with extraneous DNA: 1. All DNA ex-
tractions and PCR involving the samples were carried out in a labora-
tory physically separated from the laboratory in which PCR cycling and
post-PCR analyses were performed; 2. Disposable masks, gloves and
laboratory coats were worn throughout and were changed frequently;
3. All DNA extractions and PCR reactions included negative controls; 4.
All surfaces were decontaminated with beach and UV-light before and
E. Pilli et al. Science & Justice xxx (xxxx) xxx–xxx

Fig. 2. Location of the Mausoleum of Fosse Ardeatine. Map of Italy showing Rome's location; map of Rome showing the location of the pozzolana quarries and
Mausoleum of Fosse Ardeatine.

Table 1 sample material as they were shaken together in grinding vessels. In

Information collected during the anthropological examination and associated
references.
Type of information Anthropological features of the skeleton References
Age estimation Degree of alteration of pubic symphysis [25]
Auricular surface of the pelvis [26]
Degree of wear of the molar teeth [27]
Stature estimation Length of long bones [28–30]
after usage and 5. All consumables and instruments were decontami-
nated by UV-light before and after usage. Repeated amplifications from
the same or different extracts from the same specimens are necessary: 1.
To identify contamination of a particular extraction or amplification; 2.
To allow the DNA analysis when the extracts only sporadically contain
DNA template molecules. In order to monitor and check the presence of
possible contamination, the profiles obtained from bones were com-
pared to those of all people that handled the skeletal elements in field
and the personnel of DNA laboratory [35,36].
2.1.3. DNA extraction
A 2–3 cm fragment was collected from each bone. To remove po-
tential contamination, the outer layer of the bones was physically re-
moved using a rotary sanding tool (Dremel® 300 series). After sanding,
each sample was cut into small sections using a dental diamond disk
and irradiated by ultraviolet light for 45 min in a Biolink DNA
Crosslinker (Biometra) as suggested by [36]. A TissueLyser (QIAGEN®)
was used to grind five-six bone fragments. Disruption and homo-
genisation were done by the beating and grinding effect of beads on
order to obtain a high purification efficacy and to maximize DNA yield,
as proposed by many researcher [18,37–39], we decided to isolate the
DNA from bones using a Biorobot EZ1 (Qiagen). Two different extrac-
tions using Biorobot EZ1 for each sample were carried out. About
250 mg of bone powder were transferred to a 1.5 ml tube and incubated
in 1 ml of 0.5 M EDTA solution pH 8.3 (IBI Scientific) for about 72 h at
37 °C on a rotating tube stirrer (Steroglass srl). The EDTA was changed
twice a day after centrifugation at 2500 rpm for 5 min. After 72 h of
decalcification, the precipitate was centrifuged and the supernatant was
discarded. Then a stain extraction buffer [40] containing 10 mM
Tris–HCl pH 8.0, 100 mM NaCl, 10 mM EDTA, 2% SDS, and 39 mM DTT
and proteinase K (20 mg/ml) (Invitrogen, Carlsbad, CA) was added to
precipitate and it was incubated overnight at 56 °C. DNA was purified in
a Biorobot EZ1 (Qiagen) device using EZ1 DNA Investigator Card and
EZ1 DNA Investigator Kit (QIAGEN®) and eluted in 50 μl of TE buffer.
At the same time, an optimized lysis buffer for complex forensic sam-
ples (Bone, Teeth, Adhesive) such as BTA™ lysis buffer (just released on
the market at the time of analysis), was used in combination with the
PrepFiler™ Forensic DNA Extraction kit [41–43]. Starting with 300 mg
of pulverized bone we skipped the decalcification protocol and in-
cubated samples in PrepFiler® BTA Lysis Buffer, 1.0 M DTT and pK
(20 mg /ml), according to the user's recommendation, at 56 °C for 2 h.
DNA was purified in AutoMate Express™ Instrument and eluted in 50 μl
of PrepFiler® Elution Buffer. Multiple negative controls were included
for each extraction.
2.1.4. Nuclear DNA analysis
DNA extracts of each bone sample were quantified and the presence

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E. Pilli et al.
of inhibitors was monitored using Quantifiler™ Duo DNA Quantification
Kit (Thermo Fisher Scientific, Oyster Point, CA). Real-time PCR am-
plification reactions contained 10.5 μl of Primer-Probe Mix, 12.5 μl of
Master Mix, and 2.0 μl of DNA sample per the user's manual.
For all bone samples, STR typing of autosomal DNA was performed
using different amplification kits: AmpFℓSTR® NGM SElect™ PCR
Amplification Kit (Thermo Fisher Scientific, Oyster Point, CA);
PowerPlex® ESI 17 System (Promega Corporation) and AmpFℓSTR®
MiniFiler™ PCR Amplification Kit (Thermo Fisher Scientific, Oyster
Point, CA). DNA target amount was 500 pg for each amplification. In
order to use the entire amount available, in the samples in which the
total DNA quantification value was lower, 50 μl of DNA extract were
concentrated up to approximately 30 μl with Amicon Ultra-0.5 ml
Centrifugal Filters (Merck Millipore). Simultaneously with forensic
samples we amplified positive control (AmpFlSTR Control DNA 9947A,
Applied Biosystems or Control DNA 9947A, Promega) and negative PCR
and extraction controls. STR typing of Y chromosome was performed
using AmpFlSTR® YFiler® PCR Amplification Kit (Thermo Fisher
Scientific, Oyster Point, CA). Positive control (AmpFlSTR Control DNA
007 - Thermo Fisher Scientific, Oyster Point, CA), negative PCR and
extraction controls were added to amplification reaction. All reactions
were performed on Gene Amp PCR System 9700 (Thermo Fisher
Scientific, Oyster Point, CA) according to the manufacturer's re-
commendations.
PCR products were analysed by capillary electrophoresis in an ABI
3130xl Genetic Analyzer (Thermo Fisher Scientific, Oyster Point, CA)
using POP4, 36 cm capillary arrays, and default instrument settings as
recommended by the manufacturer. Genetic profiles were determined
using Data Collection v 3.0 and GeneMapper ID v 3.2 (Thermo Fisher
Scientific, Oyster Point, CA) computer software. The analytical
threshold was set at 50 RFU according to internal validation. Each
sample was amplified in triplicate. Only the alleles that appeared twice
in different amplifications were reported to create the consensus pro-
file. Then, according to low template interpretation rule [44–47] and
ISFG Commission recommendations for DVI [11], profiles generated
from different DNA extracts of the same bone were combined.
2.1.5. Mitochondrial DNA analysis
Two microliters of DNA extracted from the bone samples were
amplified as follows: 94 °C for 10 min (Taq polymerase activation),
followed by 40 cycles of PCR (denaturation, 94 °C for 45 s, annealing,
53 °C for 1 min and extension, 72 °C for 1 min) and final step at 72 °C for
10 min. The 50 μl reaction mix contained 2 U of AmpliTaq Gold
(Thermo Fisher Scientific, Oyster Point, CA), 200 mM of each dNTP and
1 mM of each primer. Due to the high degradation of the genetic ma-
terial, the entire D-loop of about 1.100 bp length was subdivided into
ten overlapping fragments as described previously [48]. Considering
only the best extraction in term of yield, each extract was amplified in
duplicate. The extraction reagent control, a known positive control and
a PCR negative control were amplified in parallel. All reactions were
performed on Veriti Thermal Cycler 96 Well (Thermo Fisher Scientific,
Oyster Point, CA). 25 μl of each PCR product were controlled on a 1.5%
agarose gel electrophoresis with a 45 min running at 80 V, visualised
with ethidium bromide and then purified by DNA gel extraction kit
(Millipore) according to the user's manual. After purification, a volume
of 3-4 μl was cycle-sequenced following the BigDye® Terminator
v3.1 Cycle Sequencing kit™ (Thermo Fisher Scientific, Oyster Point, CA)
supplier's instructions. The primers used for sequencing the PCR pro-
ducts were the same as for the amplification. Sequencing comprises
initial denaturation at 96 °C for 1 min followed by 25 cycles at 96 °C for
10s, 50 °C for 5 s and 60 °C for 4 min. Sequencing reaction products
were purified from residual dye terminators using Agencourt®
CleanSEQ® (Beckman Coulter) according to the manufacturer's manual.
Sequencing was carried out on Applied BioSystems 3130xl Genetic
Analyzer (Thermo Fisher Scientific, Oyster Point, CA) using POP7,
36 cm capillary arrays, and default instrument settings as recommended
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Science & Justice xxx (xxxx) xxx–xxx
by the manufacturer. Each template was sequenced in both forward and
reverse directions. Each consensus sequence was aligned and compared
to the revised Cambridge Reference Sequence (rCRS) [49,50] using
SeqScape® Software version 2.5 (Thermo Fisher Scientific, Oyster Point,
CA) and default software settings as recommended by the manu-
facturer, following international guidelines for mtDNA typing [51–53].
2.1.6. Statistical analysis
STR profiles obtained from bones and reference samples were
compared, and estimation of alleged familiar relationships was per-
formed. The calculation of posterior probabilities (PP) and LRs based on
both autosomal STR and Y-STRs were performed with DNAVIEW soft-
ware v. 37.25 [54], using allele frequencies of Caucasian, population
available in the software and assuming 1/12 (the number of uni-
dentified victims) and 1/342 (335 + 7) (all of the victims included in
the official list plus the ones claimed by their relatives, but not included
in the official list) as prior probability. The prior probability describes
the chance that any sample of the available remains belongs to a par-
ticular individual, based upon the available (usually non-technical)
information. In this manuscript prior probability was inversely related
to the estimated number of victims N and was lowered as the identifi-
cation progresses and the number of missing individuals decreased
[11]. EMPOP was used to determine mtDNA haplogroups [55].
Whenever an agreement of autosomal genetic profiles and Y hap-
lotypes was noticed between the bone and a relative, to confirm the
alleged relationship, the LRs were obtained by multiplying LRs based on
autosomal STR and Y-STR analysis results assuming the independence
as stated by Walsh et al. [56–59].
2.2. Ante-mortem team activities
Thanks to historical information provided by the National
Association of Italian families martyrs (ANFIM), the ante-mortem team
was able to obtain the following information: 1. the alleged names of 10
out 12 victims; 2. the victim's gender (all victims were males); 3. al-
leged age at death and 4. stature of 10 out of 12 victims. No odontology
data was available. Historical sources [1] have also reported that the
victims were all killed by one or more gunshots at the occipital bone
fired from the bottom up and back to front after their hands were tied
behind their backs. Among the victims there were four individuals who
were lame and > 60 who were Jews.
Unfortunately, only 4 out of 10 “known” families have been traced
and their DNA was collected for identification purposes. Afterward,
finding out about our investigation, another 7 families (unknown fa-
milies) who supposed that their relative had died at Fosse Ardeatine,
donated their DNA for comparative analysis.
Buccal swabs from all the living relatives of the victims as from
forensic team personnel were collected. In Table 2 the degree of kinship
between family members and victims and the possibility of genetic
comparison in consideration of the type of relationship explored are
shown.
DNA was extracted by standard Chelex 100 protocols [60]. PCR
amplifications were performed using AmpFℓSTR® NGM SElect™ PCR
Amplification Kit (Thermo Fisher Scientific, Oyster Point, CA) and
AmpFiSTR Yfiler PCR Amplification Kit (Thermo Fisher Scientific, Oy-
ster Point, CA). Typing was performed on the ABI 3130xl Genetic
Analyzer (Thermo Fisher Scientific, Oyster Point, CA) according to the
manufacturer's recommendations. mtDNA analysis was carried out for
all the families members that were to be related through the maternal
line with the exception of the family member 3 (as reported in Genetic
analysis of Results and discussion paragraph).
E. Pilli et al. Science & Justice xxx (xxxx) xxx–xxx

Table 2 is inversely proportional to the amplicon length [61–64], was observed.

Genetic markers panel useful for comparative purposes based on the degree of Subsequently, all samples with exception of 273–1 were typed using
kinship between victims and relatives. AmpFlSTR® Yfiler™ (Table 4), although paternal male relatives were
The immediate Degree of kinship with Autosomal STR Y-STR mtDNA available for only 8 supposed victims. The typing of Y-STR loci was
relatives the victims successful in 5 out of 13 bones, which represent a 38% success rate.
Complete Y STR profiles were not obtained for any samples. 283–2,
Family Member 1 Daughter √ × ×
52–1 and 122–1 samples showed a profile with respectively 15 (283–2)
Family Member 2 Son √ √ ×
Family Member 3 Brother √ √ √ and 14 loci (52–1 and 122–1). In the other two bone samples, 272–1
Family Member 4 Sister's son √ × √ and 276–1, 12 and 10 loci were amplified respectively. Only 6 loci were
Family Members 5 Three sons from two √ √ × amplified in the 155–1 sample, while no locus was detected in 3–4,
different brothers
98–1, 264–1, 272–3, 272–4, 284–1 and 329–1 samples. The difference
Family Member 6 Sister √ × √
Family Member 7 Brother √ √ √
in success rate between autosomal and Y typing could have been in-
Family Member 8 Son √ √ × fluenced by the combined use of the 17 loci STR kit and the mini-STR
Family Member 9 Brother's son √ √ × kit that improved the number of successful autosomal genotyped loci
Family Member 10 Son √ √ × observed in most of samples. After performing the analysis on bone
Family Member 11 Son √ √ ×
samples, the ante-mortem team generated complete autosomal STR
√ = possibility of comparison and x = no possibility of comparison. profiles of all reference samples available. Comparative analysis be-
tween bone and relative profiles suggested the presence of three pos-
3. Results and discussion sible relationships. The 273–1 bone and family member 1 (alleged
daughter) shared an emi-compatible profile across all the detected loci.
3.1. Anthropological analysis The 283–2 bone and family member 3 (alleged brother) shared the
majority of the alleles. The result was strengthened by the comparative
The bodies had been individually deposited in zinc-plated wooden analysis of Y profiles that suggested that 283–2 bone sample and family
coffins, where only the metal part was preserved. Inside the coffins, member 3 (alleged brother) shared the same Y-STR profile across all
remains of the victim clothes were still visible. Skeleton preservation detected loci. Finally, the 155–1 bone sample and family member 5
was overall good, except in grave 329. The bones in grave 122 were (three sons of two different brothers) shared some autosomal alleles and
wet, presumably due to some water infiltration. All bodies were placed the partial profile of Y STR. In accordance with the ISFG Re-
in the coffins in a prone position. In most cases, all skeletal elements commendation #11 [11], the statistical evaluation of match was per-
were present and complete. The anthropological data form filled in for formed and results were shown in Table 5. As it can be observed by the
all samples is shown in Fig. 3, where skeletal elements present are dark, value reported in Table 5, we were able to match 3 victims to living
and those missing are white. relatives with high confidence of correct identification (PP ranged from
In Table 3 the estimates of age at death, stature estimation, main 99.97% to > 99.99% assuming 1/12 as prior probability or from
dental features and other characteristics are described for each in- 99.19% to 99.99% assuming 1/342 as prior probability).
dividual. Due to the poor preservation of the skeletal remains in grave As proposed by Irwin et al. [65] and Zupanič Pajnič et al. [18], the
329, it was not possible to estimate age at death. combination of different genetic markers provided extremely high LRs
that supports the hypothesis that individuals' bones are related to the
family references, rather than unrelated individuals.
3.2. Genetic analysis Subsequently, in the hope of providing additional information on
samples from which no STR result was obtained, and adding new ones,
Quantification results are shown in Fig. 4. the typing of the entire mtDNA control region of all unidentified bone
As it can be deduced by the values reported in Fig. 4, better results samples was carried out (Table 6).
in term of DNA concentration were obtained from EZ1 extraction Typing was successful in 9 out of 11 bones (approximately 82%
method. Considering that the samples were eluted in 50 μl of TET, the success rate). Complete profiles were obtained for eight bone samples,
quantification results vary in a range of approximately 180 pg-1.5 ng whereas partial profile was observed in sample 272–4 that had a gap
and 60-180 pg for EZ1 and BTA™ lysis buffer extraction respectively. between 280 and 390 bp. As can be deduced by results reported in
Nuclear DNA quantification of samples 264–1, 272–3, 272–4, 284–1 Table 5, samples 272–3 and 272–4, collected from the same grave,
and 329–1 did not yield any result with both extraction methods. The showed two different mtDNA profiles. Sample 272–3 showed the same
internal positive control (IPC) assay highlighted the absence/removal of mtDNA profile of sample 272–1 while sample 272–4 exhibited a dif-
PCR inhibitors in all the extracted DNAs. The IPC CT value ranged be- ferent profile from 272 to 1, 272–3 and all other samples. Therefore, the
tween 28.39 and 30.02 in all extracts. Despite lower yields of BTA™ mtDNA analysis confirmed, as previously hypothesized by anthro-
method compared to EZ1 extraction, the BTA extracts were also utilized pological analysis, that the two temporal bone fragments came from
for PCR amplification after concentration. According to the criteria two different individuals. Due to the presence of an apparent hetero-
previously described for generating consensus profile, the typing of plasmy in different positions in samples 3–4 and 329–1, it was im-
autosomal loci was successful in 8 out of 14 bones, which represent possible to define their haplogroup. The presence of two dissimilar
approximately a 57% success rate. Complete autosomal STR profiles bases in four different positions of mtDNA profiles of samples 3–4 and
(17/17 loci typed) were obtained from bone samples 52–1 and 272–1. 329–1 could be presumably explained with the adventitious transfer, as
Partial profiles with SE33 locus missing were obtained from bone defined by Gill et al. [66], by having excluded all persons that had been
samples 273–1 and 283–2, whereas bone samples 122–1 and 155–1 in contact with the skeletal remains at any phase of excavation, storage,
showed a profile with a number of loci greater than or equal to 13. In anthropological analysis, or molecular genetic analysis. A different
the remaining two bone samples 276–1 and 284–1, 11 to 12 loci were profile was obtained from mtDNA analysis of sample 329 by Pietrangeli
typed (Table 3). et al. [22]. However, not being able to rule out previous contamination
The other six bone samples showed negative results (no locus was also during excavation activity and based on our procedures (analyses
detected for 264–1, 272–3, 272–4 and 329–1 samples) or only 8–9 loci were carried out in a laboratory exclusively dedicated to ancient DNA
amplified in the 3–4 and 98–1 samples. As expected, when working analysis as reported in contamination prevent paragraph) and checks
with high degraded DNA samples, signal strength was lost with the done (negative controls, additional mtDNA sequencing analyses for
larger size PCR product and the “decay curve” in which the peak height heteroplasmic sequences and exclusion of all people that handled

5
E. Pilli et al. Science & Justice xxx (xxxx) xxx–xxx

Fig. 3. Anthropological data form filled in on site for all samples. Bones present in each grave are showed in gray. Hand and foot bones and ribs were not found and
considered useful for skeletal reconstruction purpose and therefore erased with lines in figure.

6
 E. Pilli et al.

Table 3
Age and stature estimation and other morphological features of the 12 individuals analysed.
Grave Age at death Stature (range- AM loss of teeth Presence of Caries Wear Other features
number (range –in in cm) Carabelli's trait
years)

3 35–45 164–168 RM2, LC, LM2 On M1, left and right Minimal Right tibia arched and 22 mm longer than the left tibia. The articular surfaces of
the distal epiphysis of tibiae, calcanei and astragali showed bone remodeling
associated with some kind of arthropathy more marked on the left, possibly
associated with evident difficulties in walking (e.g. marked limping). The
entrance and/or exit hole of the bullet were present on the bones of the skull.
Entrance hole mainly localized at or near the base of the occipital bone
52 35–60 164–169 RM1, LM2 On RM1 LP3, LP4 Minimal RM3, LM3, RM3, LM3, all missing. Possible congenital absence. Alterations at the
level of the thoracic and lumbar vertebrae possibly associated with arthropathy
and presence of slipped disc. The entrance and/or exit hole of the bullet were
present on the bones of the skull. Entrance hole mainly localized at or near the
base of the occipital bone
98 35–60 165–169 RM2, LM2, LM1 LP3, LP4 Marked RM3, LM3, missing. Possible congenital absence. Mandible missing. Evident signs
of bone resorption at the level of the frontal bone, probably associated with a
healed trauma that occurred in life. Alterations at the level of the thoracic and
lumbar vertebrae as 52 skeleton. The entrance and/or exit hole of the bullet were
present on the bones of the skull. Entrance hole mainly localized at or near the
base of the occipital bone

7
122 25–55 157–159 Marked Maxilla missing. In the upper limbs of the skeleton a marked development of
muscular insertions was observed.
155 22–35 165–170 – Skull and mandible missing. Fusion of the medial epiphysis of both clavicles was
not complete yet
264 49–73 164–170 RM3, RM2, RM1, LI2, LM1, LM2, RP4, LC only the root is – Slightly asymmetric nasal bones with a greater inclination toward the right side.
LM3, RM3, RM2, RM1, RP4, LP4, preserved, RC caries at the Sacralization of the first coccygeal vertebra. The entrance and/or exit hole of the
LM1, LM2, LM3 cervix bullet were present on the bones of the skull. Entrance hole mainly localized at or
near the base of the occipital bone.
272 35–56 163–170 RM3, RM2, RP3, RC, LP4, LM1, LM2, – Marked alterations at the level of insertion of the costo-clavicular ligament. The
LM3, RM2, RM1, RP4, RP3, LP3, LP4, entrance and/or exit hole of the bullet was present on the bones of the skull.
LM1, LM2 Entrance hole mainly localized at or near the base of the occipital bone. Two
different fragments of the same portion of the right temporal bone were
recovered (272–3 and 272–4)
273 49–73 167–171 RM1, LP3, LM1 – RM3, LM3, missing. Possible congenital absence. Mandible missing. Marked
alterations at the level of insertion of the costo-clavicular ligament
276 35–56 165–170 – Maxilla and mandible missing
283 49–73 168–174 – Skull and mandible missing. Marked alterations at the level of insertion of the
costo-clavicular ligament. A mushroom-shaped bone proliferation of about 1 cm
in diameter at the level of the ramus of the right ischium
284 26–45 161–165 RM1, RP3, LM1, LM2, LP4, RP4 On RM2 Large on RC, LM2 – RM3, LM3, RM3, LM3, all missing. Possible congenital absence
329 N/A 158–162 – Skull and mandible missing

L = left; R = right; M = molar (1,2 or 3); P = premolar and C = canine. Superscript or subscript indicate respectively upper or lower teeth. N/A = not available.
Science & Justice xxx (xxxx) xxx–xxx
E. Pilli et al. Science & Justice xxx (xxxx) xxx–xxx

Fig. 4. Quantification results of bone extracts.

Table 4 Table 6
Consensus profiles obtained from bone samples. Mitochondrial DNA analysis results.
Samples name Consensus Autosomal STR Profile Consensus Y STR Profile Sample Motif of entire control region (16024–576) Haplogroup
Name
3–4 8/17 =
52–1 17/17 14/16 3–4 16519Y 263G 477Y 489Y 513R Not available
98–1 9/17 = 52–1 16442C and T 16519C 059C 263G 315.1C H2a2a
122–1 14/17 14/16 98–1 16126C 16519C 073G 152C 194 T 195C 263G JT
155–1 13/17 6/16 122–1 16519C 263G 309.1C 309.2C 315.1C H2a2a
264–1 = = 264–1 16519C 263G 309.1C 315.1C H2a2a
272–1 17/17 12/16 272–1 16224C 16311C 16519C 073G 146C 152C 263G K2a2a1
272–3 = = 315.1C 512C
272–4 = = 272–3 16224C 16311C 16519C 073G 146C 152C 263G K2a2a1
273–1 16/17 = 315.1C 512C
276–1 11/17 10/16 272–4* 16265C 16311C 152C 263G H2a
283–2 16/17 15/16 276–1 16129A 16183C 16189C 16,223 T 16249C M1a1
284–1 12/17 = 16289G 16311C 16359C 16519C 073G 195C
329–1 = = 263G 315.1C 375G 489C
284–1 16,069 T 16126C 16,193 T 16294 T 16519C 073G J1d
Consensus autosomal STR (AmpFℓSTR® NGM SElect™ PCR Amplification Kit 146C 152C 263G 295T 309.1C 315.1C 462 T 489C
-Thermo Fisher Scientific, Oyster Point, CA- and Power Plex ESI 17 System - 329–1* 16224Y 16519C 073G 150Y 195Y 198Y 263G Not available
Promega Corporation-) profiles expressed as the number of loci positively typed 295T 315.1C
for each sample; consensus Y-STR (AmpFlSTR® YFiler® PCR Amplification Kit
Name, mitochondrial profiles and haplogroup of each sample analysed. *partial
-Thermo Fisher Scientific, Oyster Point, CA-) profile expressed as the number of
profile: 272–4 had a gap between 280 and 390 bp while 329–1 D-loop sequence
loci positively typed for each sample. =failure in STRs typing. The underlined
terminated at approximately 460 bp instead of 580 bp as for the others.
samples showed a number of positively typed loci below 10 considered as
threshold value for a genetic identification according to the Italian National
Database Law. of 276–1 showed European or Middle Eastern haplogroup. Instead,
sample 276–1 exhibited M1a1 haplogroup. In their manuscript Gon-
Table 5 zales et al. [67] provided evidence that M1, or its ancestor, had an
Statistical results. Asiatic origin and highlighted that a relevant fraction of M1a hap-
logroups present today in the European Continent supposedly had a
Bone/ Family LR(n-STR) LR(Y-STR) LR(n-STR × PP(n-STR × Y-STR)
samples member
Jewish maternal ascendance. As sample 276–1, all European Ashkenazi
Y-STR)
name Jews have M1a lineage characterized by a transition in the 16,289
position that has not been detected in other Jew or non-Jew popula-
155–1 5 –three 23.61 1578 41,990.58 99.97%* tions. Since only 4 of 11 families members were related through ma-
sons of two 99.19%**
different
ternal line with the victims and one of them was identified by auto-
brothers- somal and Y STR analysis (LR value provided an extremely strong
273–1 1 –daughter- 2.38 × 106 > 99.99%* support for the hypothesis that individuals' bones are related to the
99.98%** family reference, rather than unrelated individual [68]), the ante-
283–2 3 –brother- 21,580 11,850 2.56 × 10 8
> 99.99%*
mortem team generated complete mtDNA profile for three family
99.99%**
members −4, 6 and 7-. Comparative analysis between bones and re-
Autosomal LR, Y LR, combined LR and posterior probability (PP), assuming *1/ ference mtDNA profiles suggested that there was not any genetic
12 and ** 1/342 as prior probability, for identified victims at the Mausoleum of compatibility between victims and reference mtDNA profiles. There-
Fosse Ardeatine. In family member column, kinship with the victim is indicated. fore, this incompatibility led us to exclude the presence of the victims
related to the family members 4, 6 and 7 among the 12 unknowns.
samples), the previous data was not used for comparative analysis with All our anthropological and genetic activities were carried between
our reference samples. Haplogroup of bone samples is reported in 2010 and 2012, therefore, the use of most suitable kits for old DNA
Table 6. As can be deduced from the Table 6, all samples with exception samples quantification, such as e.g. Quantifiler® Trio (Thermo Fisher

8
E. Pilli et al.
Scientific, Oyster Point, CA) –optimized compared to Quantifiler® Duo
to improve detection sensitivity and evaluate DNA degradation level- or
PowerQuant® System (Promega Corporation) [69] and the new gen-
eration STR kits like GlobalFiler (Thermo Fisher Scientific) or Power-
Plex® Fusion 6C System (Promega Corporation) and Yfiler Plus (Thermo
Fisher Scientific, Oyster Point, CA) or PowerPlex® Y23 System (Pro-
mega Corporation), was not possible. Also the choice of the better
skeletal element, in terms of DNA amount and preservation, than femur
for genetic analysis such as petrous bone [70] could have increased the
possibility of analytical success. Mundorff et al. [71,72] proposed the
use of the small elements of the hands and feet for DNA analysis due to
the fact that they were very similar in DNA yield as femur and tibia
however, in our graves, these small skeletal elements were not found/
collected in most cases. To date, the identification activity of the re-
mains at the Mausoleum of Fosse Ardeatine is completed and, therefore,
we do not have access to the skeletons but, if the competent authority
decided to resume identification activities, it could be very interesting
to test all the new technologies present in the forensic market including
the NGS (Next Generation Sequencing) approach.
4. Conclusions
During the Second World War, the Fosse Ardeatine massacre was a
mass execution in which 335 Italians were killed. At the end of the war,
323 out of 335 bodies were identified. Approximately 60 years after, in
2009, at the request of the Ministry of Defence, the identification of the
remaining twelve victims began via a multidisciplinary approach that
involved anthropologists and forensic genetic experts. Despite the
scarcity of AM anthropological information, careful inspection of the
skeletal elements of all the individuals led us to confirm the male sex of
each victim and verify the presence of only one individual in each grave
with the exception of 272 in which two different skeletal elements of
the same portion of the right temporal bone were collected. Moreover,
data gathered on morphological features of each individual showed the
presence between victims of an individual with a clear arthropathy,
possibly associated with evident difficulties in walking. This anthro-
pological result confirmed the ante mortem information collected
(among the victims there were four lame), but unfortunately it did not
help to identify any victim, presumably due to the fact that only 4 out of
10 “known” families of the victims (2 out of 12 victims unidentified in
1944 were unknown i.e. their name was not known to the National
Association of Italian families martyrs), were available for identifica-
tion purpose. However, despite the lack of reference samples, 3 in-
dividuals were successfully identified via genetic analysis using an ef-
fective DNA extraction method and a combination of different
amplification systems. LR and cumulative LRs obtained from autosomal
STR and Y-STR results confirmed the alleged relationship between
victims and their relatives with high probabilities. Excluding any pos-
sible misidentifications during the DVI activities in 1944, the genetic
analysis results enabled us to rule out the presence of relatives of the 7
“unknown” families among Fosse Ardeatine victims. Moreover, al-
though the mtDNA analysis suggested that there was not any genetic
compatibility between the victims and reference mtDNA profiles, a
Jewish maternal ascendance (among the victims there were > 60 Jews)
of one of the victims, useful for any future identification purpose, was
supposed. The effort lavished by all experts involved in the realization
of this project together with the good genetic results successfully al-
lowed us to replace the numbers on the graves with the names of the
victims. To date, the Ministry of Defence efforts are focusing on the
research of additional victims' families in order to try to identify re-
maining unknown victims. Due to the time spent and the absence of a
complete list of people who died at the Fosse Ardeatine, this research
could be time consuming. If some relatives were found, we could
compare their genetic profiles with the ones obtained in the present
work or the competent authority could evaluate whether to re-analyze
the unknown bone samples with more advanced and new technologies
9
Science & Justice xxx (xxxx) xxx–xxx
and/or reopen all of the graves to assess possible mis-identifications
during the forensic examination in 1944.
Acknowledgements
We are very grateful to Jonathan Kyte and Claudia Agostino for
providing language support and invaluable advices in proof reading the
article.
All operations were performed in the presence of the ANFIM's
(National Association of Italian Families Martyrs) representatives (Mrs.
Nicoletta Leoni -ANFIM Associate Director- and Mr. Aladino Lombardi
-ANFIM Secretary-General-) and the Religious Authorities of the
Catholic and Jewish Communities. We are very grateful to them for all
their encouragement and support during our fieldwork. We would like
to thank ANFIM for the photos from their historical archive. Moreover,
we would like to show our gratitude to all the Biologists of Forensic
Science Department of Rome who helped us during the challenging
activities of exhumation. In particular, we would like to thank
Francesco Cominetti that he was able to capture every moment of our
fieldwork through his beautiful pictures. Finally, a truly heartfelt
thanks to the Families of the victims: only their suffered desire for truth
made this work possible.
Author contributions
Exhumation of the bodies: EP, SB, JMC, ML, DC, AV, GD and AB.
Fieldwork activities and anthropological analysis: SB and JMC.
DNA analysis on bone samples: EP and AB. AA participated in the
extraction with PrepFiler® BTA Lysis Buffer.
Sample collection and DNA analysis on relatives: GD, AV and CR.
Data analysis: EP and AB.
Statistical analysis: FB.
Writing – original draft: EP.
Writing – review: EP, FB, DC, JMC, SB, CR, AA and AB.
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