Virchows Arch (2014) 465:501–508 DOI 10.1007/s00428-014-1611-9
ORIGINAL ARTICLE
Identification of Giovanni Battista Morgagni remains following historical, anthropological, and molecular studies Alberto Zanatta & Fabio Zampieri & Girolamo Zampieri & Alice Giuliodori & Gaetano Thiene & Luciana Caenazzo
Received: 13 May 2014 / Accepted: 16 June 2014 / Published online: 16 July 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Morgagni died on December 5, 1771, 89 years old, and was buried in Saint Maxim Church in Padua, where his wife and five of his 15 children, four daughters, and one son were already buried. In 1868 and 1900, the tomb was opened to identify Morgagni. Among the remains of several adult individuals, two skulls considered of very old persons were identified and replaced in an earthenware jar inside the sepulcher. In 2011, we opened the tomb and found the remains described during the first two identifications, but additionally, we found the skulls fragments of three very young individuals which could have been Morgagni’s children. An anthropological analysis confirmed that one of the skulls inside the earthenware jar belonged to the oldest individuals (“senilis”) between those found in the tomb. A genetic analysis proved a kinship between this skull and the fragments of young individuals (one male and two females), supporting the hypothesis that they were Morgagni and his children. In conclusion, thanks to the interaction between historical studies, anthropological research, and molecular analysis that reinforce each other, we can assume that the skull is Giovanni Battista Morgagni’s and that the series of skull fragments are from his children who were buried together with their parents.
Keywords Morgagni . Anthropological analysis . Genetic analysis . Historical analysis A. Zanatta : F. Zampieri : G. Thiene (*) Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, via A. Gabelli 61, Padua 35121, Italy e-mail: [email protected] G. Zampieri Padua Archeological Museum, Via Andreini 1, Padua 35141, Italy A. Giuliodori : L. Caenazzo Department of Molecular Medicine, University of Padua, via G. Falloppio 50, Padua 35121, Italy
Introduction Giovanni Battista Morgagni is considered the father of pathological anatomy and one of the most important innovators in the history of medicine. In his De sedibus et causis morborum per anatomen indagatis (Seats and causes of diseases investigated by anatomy, Venice 1761) [1], he established pathological anatomy as a science by correlating clinical histories with autopsy findings. He considered the lesion in the organ, revealed by autopsy, as the fundamental cause of disease, its origin and progression, as well as its clinical symptoms. Organ pathology as established by Morgagni, namely that a sickened organ causes a disease, has been recently confirmed by transplantology, in which the substitution of the affected organ is the cure of the disease. Morgagni was born in Forlì in 1682 and graduated in medicine and philosophy in Bologna in 1701. He left Bologna soon after his degree, attracted by the freedom of research and teaching at the nearby University of Padua. Here he was appointed as professor of Theoretical Medicine in 1711. From 1715 to his death (1771), he acted as professor of Anatomy. Morgagni had 15 children, among them 12 daughters. Four daughters only lived for a few days. They were baptized and buried in a mass grave at Saint Maxim Church in Padua, near Morgagni’s house, with their brother Lucio Filippo, who died 2 years later in 1718. Eight daughters became nuns. The two remaining sons were Luigi Agostino, a Jesuit, and Giovanni Maria Fabrizio, the only son who got married. In 1770, when his wife died, Morgagni bought a sepulcher in Saint Maxim Church, in which he placed her bones and the remains of the other children already buried there. He also decided to leave the tomb at the disposal of any Padua University professors who would have needed a sepulcher. When Morgagni died and was placed in this family tomb, the following inscription was engraved on the headstone: “Sepulcrum Morgagni anatomici et suorum. Item Gymnasii
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patavini professorum si quem unquam iuverit hic condi. MDC[C]LXX (Sepulcher of Morgagni and his relatives. Equally of Padua University Professors if ever one would like to be buried here. 1770)” (Fig. 1). Saint Maxim Church’s archive unfortunately doesn’t report whether any professor took advantage of this opportunity, but the fact that the tomb actually contains the remains of several adult individuals seems to confirm this hypothesis. On July 18, 1868, the mayor of Forlì, Alessandro Mazzoni, sent a letter to the mayor of Padua, Andrea Meneghini, demanding the restitution of Morgagni’s remains to his hometown. To meet this request, Meneghini organized an identification of Morgagni’s tomb in Saint Maxim Church, which was carried out on August 18th of the same year. The request of Forlì couldn’t be fulfilled, because up to 11 skulls, as well as scattered osseous elements, were discovered in the tomb. Seven skulls were found at the west wall of the tomb and two at the east wall. A tenth skull was found in the center and a last one, fragmentary, was found in the south-west corner. This last skull was retained to be the oldest one of the series. Given that Morgagni died at 89 years old, it was assumed to be Morgagni’s skull and was placed inside an earthenware jar in the south-west corner of the tomb. The others were left in their original position, and the tomb was closed the same day [2]. In 1900, another attempt at identification was made by the Rector Achille de Giovanni (1838–1916), the pathologist Augusto Bonome (1857–1922), and the anatomist Dante Bertelli (1858–1946). Among the skulls found during the first identification, which had been left in their place, one was claimed to have belonged to an old man, possibly Morgagni, and was placed inside the earthenware jar with the other skull
Fig. 1 Headstone of Morgagni’s and his relatives’ tomb in Padua’s Saint Maxim Church
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which was believed to have been Morgagni’s. During the identification, a new passage was discovered inside the tomb, probably dating back from an older sepulture. Saint Maxim Church, in fact, was situated over the site of an archaic necropolis. Up to 20 skulls were found and were arranged in two lines along the east wall of the tomb, while the remaining 9 skulls from the first identification were placed next to the north wall. The earthenware jar was left in its original position, at the south-west wall. This identification also did not lead to the result which had been hoped for, and the remains of Morgagni still were not identified for sure [2]. 2011 and 2012 were two very significant years for the history of Padua University and, in particular, for its Medical School. 2011 marked 300 years since the call to this University of Giovanni Battista Morgagni (1711–2011) and 250 years since the publication of his De sedibus et causis morborum per anatomen indagatis (1761–2011) [1]. In addition to this, 2012 marked 300 years since Morgagni’s inaugural lecture at the University, which was titled Nova institutionum medicarum idea (New idea for medical curriculum, Padua 1712) (1712–2012), and in which he proposed a new program for the medical curriculum [3]. The PhD School on “Medical, Clinical and Experimental Sciences” of Padua University organized, on the occasion of theses anniversaries, a series of lectures and events that culminated in two International Congresses, one historical and one scientific, in March 2012, at the Bo Palace of Padua University [4]. On May 3, 2011, a new identification of Morgagni’s tomb was carried out, organized by a commission directed by Prof. Gaetano Thiene, who was the principal promoter of the activities celebrating the Morgagnian anniversaries. The order of skeletal remains in the tomb corresponded to that described during the second identification (Fig. 2). We observed a series of skulls next to the east wall: those of the 1900 identification which came from an older sepulture. We found, however, only 14 skulls (classified from C1 to C14), while the record indicated that there were 20 of them; probably the remaining skulls deteriorated, since the tomb and the site of the church have flooded several times during the last century. This series of skulls, being very old, had deteriorated more than the other. There were eight skulls at the north wall (classified from C15 to C22), as indicated in 1900 identification: these skulls were those which had been found in the 1868 identification, which consequently belonged to the original disposition of Morgagni’s tomb. In this case, one skull was missing. We noted the earthenware jar in the south-west corner with two skulls inside (classified with C23 and C24), one complete (C24) and the other fragmentary (C23), and some teeth and vertebras. There were also scattered osseous elements in the center and towards the south wall (classified FS). Finally, we identified in the north-east corner the fragments of several skulls (classified C25): nine lower jaws, two teeth, seven frontal bones with orbits, three left and six right frontal bones,
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Fig. 2 Schematic of the remains found in Morgagni's tomb: the series C15–C22 are the skulls found during the first identification (1868) which composed the original Morgagni’s tomb; C23 and C24 are the two skulls attributed to Morgagni during the recognitions of 1868 and 1900; C1–C14 are the skulls found during the second recognition (1900) and belonging to an older entombment; FS are scattered osseous elements; C25, finally, holds several skulls fragments
and, finally, one sphenoid bone. Of these fragments, some seem to have belonged to very young individuals. We immediately realized that we had in our hands the remains of Morgagni’s children who died prematurely. All these elements have been classified, packed, and transported to the Pathological Anatomy Museum of Padua University for scientific analysis and photographic documentation. On May 16, 2012, after one year of research, the remains of Morgagni’s tomb were replaced in their original site, protected by sterile bags, and the sepulcher was restored and reclosed (Fig. 3). We also left in the tomb a book relating to an iconographic research of Morgagni and which summarizes the activities done for the Morgagnian anniversaries [5]. The identification of Morgagni’s remains, according to the historical sources, should be focused on the skulls C15–C24, because they surely belong to the original disposition of
Fig. 3 Replacement of Morgagni’s tomb remains with the book published for the 2011–2012 Morgagnian anniversaries
Morgagni’s tomb, while C1–C14 came from an older sepulture. Of skulls C15–C24, the skull which anthropometry will confirm to belong to the oldest individual will be most likely to be Morgagni’s, because he died at an age—89 years old— which was very rare to reach in Morgagni’s time. We found skull fragments of very young individuals (C25), which, according to historical sources, could be Morgagni’s children who died prematurely. We were in the position to perform a DNA analysis to discover whether a familial relationship could be proved between the DNA extracted from these fragments and the DNA extracted from the teeth of the oldest individual of the series. When direct reference samples, such as personal items, from missing individuals are not available, identifications can be carried out indirectly based on the ranking of likelihood ratios (LRs) constructed from a comparison of the probability of observing DNA profiles of remains of a presumed relationship with profiles from reference samples of the alleged family members versus the hypothesis that the remains are unrelated to the family [6]. The 99.9 % confidence value was advocated by the DNA Commission of the International Society for Forensic Genetics [7]. There are two ways to increase the power of identification: (i) type more markers and (ii) type more relatives. The number of markers that can be typed will be limited by the quality and quantity of DNA derived from the remains. If there is sufficient DNA, then a large battery of genetic markers is available to assist in making an identification. In many cases, the quality and quantity of DNA are poor. Increasing the number of reference relatives can increase the chances of identifying
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remains, particularly for challenged samples. In some cases, the number of relatives can be quite large, but in others, the number of available relatives is very limited. Typing all the relatives of a large pedigree can be costly and may not be necessary to reach a certain threshold for identification. Because there are information and cost factors regarding which and how many relatives are selected and typed, some selection criteria should be considered to guide identity testers. The probabilities of confirming identity with certain combinations of relatives are more powerful than with certain other combinations [6]. The purpose of DNA analysis in this work is to contribute to the results of historical and anthropological research on the confirmation of the identity of the bones belonging to Morgagni. Because direct references are not available, we used indirect relative references for the identification by kinship analysis, considering the bone fragments present in the grave which hypothetically belong to children. Furthermore, to avoid the disruption of the skeletal remains, we used two teeth recovered from the skull, taking into account the problems mentioned above.
Material and methods The type of deposit found inside the tomb has forced a separate investigation of anatomical elements: bones were completely mismatched and not in anatomical connection. The anthropological research carried out on all the anatomical elements began with an initial morphology of the samples and a further verification of measures and indices using appropriate anthropometric tools. There was also a valuation of the minimum number of individuals, the age, and the sex. The occupational markers and the presence of pathologies were considered. All the skeletal elements present, for a total of 647, were classified and analyzed in order to have a correct estimate of the minimum number of individuals and for a complete analysis of markers and diseases. After their recovery from the church of Saint Maxim, all the findings were studied at the Museum of Pathological Anatomy at different stages that can be summarized as follows: (1) inventory and cleaning, (2) morphological analysis and sorting, (3) cataloguing, (4) anthropometric analysis, (5) determination of age and gender, (6) estimation of stature, (7) calculation of the minimum number of individuals, (8) analysis of the occupational markers, (9) paleopathological analysis, (10) photographic documentation, (11) data collection, and (12) electronic archiving and statistical analysis. Regarding DNA analysis, the main problem is not only when working with ancient DNA stems to some degree from the low amount of starting molecules and the presence of PCR inhibitors but also includes endogenous DNA damage and
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fragmentation. Much effort has been focused on improving the extraction and purification procedures. Although some methods have been more influential than others, none has been generally accepted. In fact, if postmortem changes are probably the most important factors in the degradation of DNA, other factors also contribute to the degradation of the DNA, such as hydrolysis, oxidative action of oxygen, nonneutral pH, temperature and humidity conditions, UV radiation, and the presence of humic acids in soil. In historical cases and ancient DNA investigations, as in this study, bone and teeth samples are often the only biological material available for DNA typing. Problems which researchers face while working with DNA extracted from bones and teeth samples recovered from mass graves are limited to DNA quantity, DNA degradation, contamination, and postmortem changes [8–10]. The presence of inhibitor(s) may also prevent amplification. Inhibition is an especially significant problem when DNA is extracted from old and ancient material [10, 11]. One potential inhibitor 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 [11–13]. In the literature, most of the protocols for DNA extraction from bone and teeth suggest an incubation of powdered material in an ethylene diamine tetraacetic acid (EDTA) solution. The EDTA both demineralizes the bone/teeth (to an extent dependent on the EDTA concentration and the volume of extraction buffer) and inactivates DNAses by chelating bivalent cations such as Mg++ or Ca++ [14]. The degradation and fragmentation of DNA involve the need to dedicate extreme attention to the problem of contamination, which is present from the recovery of the remains until completion of all the experimental phases. Contamination can be caused by soil and laboratory microorganisms, especially by the human DNA (exogenous DNA) that can come from the operators during the archeological excavation, by laboratory personnel during DNA analysis, and from instrumentation which has not been properly sterilized. For the DNA analysis, we considered two teeth which fitted perfectly with the complete skull in the jar (C24), anthropological analysis of which confirmed to belong to the oldest individual of the series (Fig. 4). For the identification of kinship, it was necessary to extract bone fragments from different craniums anthropological analysis of which established to have belonged to very young individuals. These samples were conserved in sterile containers to preserve the integrity of the material and to limit contamination from chemical/physical agents, bacteria and mold, until the analysis. To prevent and reduce DNA contamination, we followed the most stringent criteria proposed for ancient DNA studies [15].
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Fig. 4 Age groups (Iuvenis61) of the skulls belonging to the Morgagni’s tomb
All DNA extractions and amplifications were carried out in sterile hoods in laboratories physically separated from the laboratory in which post-PCR analysis was conducted. Dedicated reagents and pipettes were used, together with filterplugged tips. All tools were washed with DNA RemoverTM solution and then UV-irradiated among uses. The specimens were cleaned with an abrasive brush and then soaked in agitation in a NaClO 10 % solution for 5/10 min. Subsequently, the samples were washed twice in agitation with distilled water (ddH2O) for 10 and 5 min, respectively. After this step, each side of the samples was irradiated at 240 nm (UV light) for 20 min. After decontamination, the samples were pulverized using the TissueLyserII (Qiagen); the program used consists of 1 min at 25 Hz, and it was repeated once again if the samples were not pulverized thoroughly enough. The quantity of powder obtained was 3–8 g for the skulls and 0.6–1 g for the teeth. The powder was then incubated in agitation from 3 to 5 days (depending of the specimen) with a decalcification solution (EDTA-Tris, pH 7.5), replacing the solution every day [14]. For the DNA extraction, the following procedure was adopted: about 1 g of powder was incubated overnight in 15 ml of extraction buffer (EDTA 0.5 M, 1 % NLauroylsarcosine) and 200 μl of 20 mg/ml Proteinase K, in a rotary shaker at 56 °C. This protocol was modified, adding 50 μl of DTT 1 M for each sample. The obtained solution was treated with an equivalent volume of phenol/chlororm/ isoamyl alcohol (25:24:1) and, subsequently, was concentrated and purified using a Centricon-30 filter column (Millipore) up to a 30-μl volume [14]. Low copy number Short Tandem Repeat (STR) amplifications were conducted, amplifying autosomic STR loci using different amplification kits (all produced by Applied Biosystems, Foster City, CA): D8S1179, D21S11, D7S820, CSF1PO, D3S1358, THO1, D13S357, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA, and
amelogenin for determination of sex. For each PCR reaction, twice the recommended Taq Gold concentration and 1 μl of 10 mg/ml BSA were added to the reaction mix. Moreover, six additional PCR cycles were used (overall 36 cycles). Water instead of DNA was used as negative PCR control. PCR products were separated on an Applied Biosystems 3130 (Applied Biosystems, Foster City, CA) and analyzed using GeneMapper ID software version 3.2, using GeneScan 500 LIZ as internal size standards and allelic ladders provided by the manufacturer. Results were elaborated using GeneScan Analysis 3.7 software.
Results The minimum number of individuals inside the tomb was 32 people, according to the whole orbitals which were the most recurring anatomical element between the 647 classified. After this first step of the study, sex was determined through analysis of the femurs [16, 17]: 8 males, 3 females, 3 uncertain for the right femur, 12 males, 5 females, and 2 uncertain for the left. We also calculated the femoral robustness and pilastric index and the valuation of the height [18, 19]. On average, the height of the male was about 173 cm, while the average female’s height was 157 cm. It is also important to note the sexual dimorphism evidenced by the femoral occupational markers: they were more evident and frequent in the male than female, because of the daily posture and the different kind of work [20]. Once this stage was completed, we studied all the 24 skulls found in the tomb. Among them, we focused our attention on the complete skull presumed to be that of Morgagni which had been found inside the earthenware jar (C24) (Fig. 5). The other one inside the earthenware jar (C23), placed there during the 1868 recognition, belonged to a “maturus” individual
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Fig. 6 The most complete skull fragment among those classified C25, characterized by a clearly young morphology
Fig. 5 Skulls C23 (left) and C24 (right) found inside the earthenware jar. The C24 belongs to the oldest individual of the whole series of skulls examined (“senilis”), while C23 appeared to be a “maturus”
(Fig. 4), based on the analysis of cranial sutures [21], so it could not belong to Morgagni. Skull C24 was in a good condition, although the lower jaw appeared without the right arch. Both jaws were toothless, and the only tooth still present is the third molar on the right maxillary arch, but it is included and had not fully erupted. We also found one incisor and one premolar in the earthenware jar which fitted perfectly with the left superior jaw that was used for the molecular analysis. Many alveolar processes of the maxilla are completely reabsorbed in correspondence of dental elements lost in life: PM2, M1, and M2 of the right arch; PM1, PM2, M1, M2, and M3 of the left one. The other teeth were lost post-mortem. The biological profile of the remains corresponding to the elderly individual is male (degree of sexualization: 0.2 skull, mandible 0.5). The skull, according to the method of obliteration of the cranial sutures of Meindl and Lovejoy [21], turned out to have belonged to a “senilis” elderly man (over 61 years of age); indeed, it was the oldest skull of them all, as demonstrated by the complete obliteration of all the cranial sutures. Figure 4 shows the results according to the age groups of the 10 skulls which are surely related to Morgagni’s burial, thanks to historical documentations. The skull which is hypothetically Morgagni’s is the only one which could be considered to have belonged to a “senilis” individual. This fact was sound proof that this skull could have belonged to Morgagni, and it confirmed the supposition made during the second identification (1900). The skull had no special epigenetic characters, except for an occipital bun evident in the nuchal portion. There were also no signs of pathological disorders, even if there was some evidence of an abscess on the left maxilla, next to the nasal
cavity. In general, it is a skull of average length and is not very wide; the face is of average width, the orbits are of a medium– small size, the nasal cavity is of medium depth, and finally, the palate is long and narrow. Concerning the skull fragments classified as C25, we found that they belonged to three different young individuals, demonstrated by their complete orbital arch. The results from the morphological analysis clearly revealed that they belonged to a range of age between birth and the first few years of life. The most complete skull fragment belonged to a young individual of about 2 years old, thanks to the presence of a part of metopic suture and to the calculation of the frontal transverse index, typical of a convex forehead [22] (Fig. 6). We decided to call them C25, C25 A, and C25 B, and they have been used to test the relation of kinship with C24. Table 1 Genetic profiles obtained from the bone fragments analyzed Polymorphism
C24
C25
C25 A
C25 B
D8S1179
10–?
10–13
10–12
10–?
D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 VWA TPOX D18S51
31–32 10–11 – – – 11–12 – – 18–? 19–? 11–12 13–16
31–? 10–12 11–13 15–17 6–7 12–13 9–11 20–? – 17–19 9–11
31–? – 11–? 18–19 7–? 12–13 – 20–23 9–14 17–18 –
25.2–31 11–? 9–12 – – 8–11 – – – – –
AME D5S818 FGA
XY 10–12 31–32
16–17 XY 9–12 30–?
– XX 9–10 31–?
– XX – –
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As reported in literature [6], to evaluate whether an unidentified person belongs to a family pedigree (P), usually one or more family reference persons from the putative pedigree are typed. In a schematic explanation, the identification is assessed by comparing two alternative hypotheses: the subject is the specific member of the putative pedigree, and (the other hypothesis) the same subject is unrelated to the known reference members of the putative pedigree. The results of the statistical probability were calculated based on a ratio of the probabilities of the DNA evidence under each hypothesis (for the detailed explanation, see Ge et al. [6]). For the probability calculation, we used The Familias program (it is freely available at the website: familias.name). The Familias program may be used to compute probabilities and likelihoods in cases where DNA profiles of some people are known, but their family relationship is in doubt. The program may compute which pedigree is most likely and how much more likely it is than others. In this case, also if we obtained partial genetic profile with STR autosomal polymorphisms and no data for Y chromosome haplotype, the statistical elaboration has given three values expressed by a percentage ranging from 90.8805 to 99.8291 % that the C24 skull is the father of the three children C25, C25 A, and C25 B (1 male and 2 females), demonstrating the existence of the relationship (Table 1).
Conclusion Historical and archive documents tell us that in the tomb investigated in Saint Maxim Church of Padua, Giovanni Battista Morgagni, his children, his wife, and other unidentified individuals were buried, some of whom might have been professors at the University of Padua. We also know that Morgagni died at 89 years old, an age which was very rare to reach at the end of the eighteenth century. Historical documents tell us that two different identifications were made on this tomb, in 1868 and 1900. In the first, up to 11 individuals were found, and the skull (fragmentary) which belonged to the oldest of the series, deduced to be that of Morgagni, was placed in an earthenware jar. During the second identification, another (complete) skull from this series was claimed to have belonged to a very old individual and so was placed in the earthenware jar with the previous one. Moreover, a new passage was discovered inside the tomb, dating back from an older sepulture, where up to 20 skulls were found. During our identification in 2011, we observed the same order described in the 1900 identification. Anthropometrical analysis confirmed that the complete skull inside the earthenware jar, called by us as C24, belonged to the oldest individual of the original disposition of Morgagni’s tomb, because it was the only one that could be defined “senilis” (over 61 years of
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age). Moreover, we found fragments of skulls, called C25, which were not detected during the previous identifications and which surely belonged to very young individuals. Genetic analysis performed on skull C24 and on the C25 series of skull fragments tell us that the C24 was a male and that he was the father of the C25 series of fragments. In conclusion, thanks to the interaction between historical studies, anthropological research, and molecular analysis that reinforce each other, we can assume that the skull called C24 is that of Giovanni Battista Morgagni and that the C25 series of skull fragments are from his children who were buried together with their parents. Acknowledgements The authors express their gratitude to Antonio Mattiazzo, Archbishop of Padua, and to Giovanni Brusegan, Rector of the Saint Maxim Church, for allowing the opening of the tomb to identify the Morgagni’s remains. Conflict of interest We declare that we have no conflict of interest.
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ORIGINAL ARTICLE
Identification of Giovanni Battista Morgagni remains following historical, anthropological, and molecular studies Alberto Zanatta & Fabio Zampieri & Girolamo Zampieri & Alice Giuliodori & Gaetano Thiene & Luciana Caenazzo
Received: 13 May 2014 / Accepted: 16 June 2014 / Published online: 16 July 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Morgagni died on December 5, 1771, 89 years old, and was buried in Saint Maxim Church in Padua, where his wife and five of his 15 children, four daughters, and one son were already buried. In 1868 and 1900, the tomb was opened to identify Morgagni. Among the remains of several adult individuals, two skulls considered of very old persons were identified and replaced in an earthenware jar inside the sepulcher. In 2011, we opened the tomb and found the remains described during the first two identifications, but additionally, we found the skulls fragments of three very young individuals which could have been Morgagni’s children. An anthropological analysis confirmed that one of the skulls inside the earthenware jar belonged to the oldest individuals (“senilis”) between those found in the tomb. A genetic analysis proved a kinship between this skull and the fragments of young individuals (one male and two females), supporting the hypothesis that they were Morgagni and his children. In conclusion, thanks to the interaction between historical studies, anthropological research, and molecular analysis that reinforce each other, we can assume that the skull is Giovanni Battista Morgagni’s and that the series of skull fragments are from his children who were buried together with their parents.
Keywords Morgagni . Anthropological analysis . Genetic analysis . Historical analysis A. Zanatta : F. Zampieri : G. Thiene (*) Department of Cardiac, Thoracic and Vascular Sciences, University of Padua, via A. Gabelli 61, Padua 35121, Italy e-mail: [email protected] G. Zampieri Padua Archeological Museum, Via Andreini 1, Padua 35141, Italy A. Giuliodori : L. Caenazzo Department of Molecular Medicine, University of Padua, via G. Falloppio 50, Padua 35121, Italy
Introduction Giovanni Battista Morgagni is considered the father of pathological anatomy and one of the most important innovators in the history of medicine. In his De sedibus et causis morborum per anatomen indagatis (Seats and causes of diseases investigated by anatomy, Venice 1761) [1], he established pathological anatomy as a science by correlating clinical histories with autopsy findings. He considered the lesion in the organ, revealed by autopsy, as the fundamental cause of disease, its origin and progression, as well as its clinical symptoms. Organ pathology as established by Morgagni, namely that a sickened organ causes a disease, has been recently confirmed by transplantology, in which the substitution of the affected organ is the cure of the disease. Morgagni was born in Forlì in 1682 and graduated in medicine and philosophy in Bologna in 1701. He left Bologna soon after his degree, attracted by the freedom of research and teaching at the nearby University of Padua. Here he was appointed as professor of Theoretical Medicine in 1711. From 1715 to his death (1771), he acted as professor of Anatomy. Morgagni had 15 children, among them 12 daughters. Four daughters only lived for a few days. They were baptized and buried in a mass grave at Saint Maxim Church in Padua, near Morgagni’s house, with their brother Lucio Filippo, who died 2 years later in 1718. Eight daughters became nuns. The two remaining sons were Luigi Agostino, a Jesuit, and Giovanni Maria Fabrizio, the only son who got married. In 1770, when his wife died, Morgagni bought a sepulcher in Saint Maxim Church, in which he placed her bones and the remains of the other children already buried there. He also decided to leave the tomb at the disposal of any Padua University professors who would have needed a sepulcher. When Morgagni died and was placed in this family tomb, the following inscription was engraved on the headstone: “Sepulcrum Morgagni anatomici et suorum. Item Gymnasii
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patavini professorum si quem unquam iuverit hic condi. MDC[C]LXX (Sepulcher of Morgagni and his relatives. Equally of Padua University Professors if ever one would like to be buried here. 1770)” (Fig. 1). Saint Maxim Church’s archive unfortunately doesn’t report whether any professor took advantage of this opportunity, but the fact that the tomb actually contains the remains of several adult individuals seems to confirm this hypothesis. On July 18, 1868, the mayor of Forlì, Alessandro Mazzoni, sent a letter to the mayor of Padua, Andrea Meneghini, demanding the restitution of Morgagni’s remains to his hometown. To meet this request, Meneghini organized an identification of Morgagni’s tomb in Saint Maxim Church, which was carried out on August 18th of the same year. The request of Forlì couldn’t be fulfilled, because up to 11 skulls, as well as scattered osseous elements, were discovered in the tomb. Seven skulls were found at the west wall of the tomb and two at the east wall. A tenth skull was found in the center and a last one, fragmentary, was found in the south-west corner. This last skull was retained to be the oldest one of the series. Given that Morgagni died at 89 years old, it was assumed to be Morgagni’s skull and was placed inside an earthenware jar in the south-west corner of the tomb. The others were left in their original position, and the tomb was closed the same day [2]. In 1900, another attempt at identification was made by the Rector Achille de Giovanni (1838–1916), the pathologist Augusto Bonome (1857–1922), and the anatomist Dante Bertelli (1858–1946). Among the skulls found during the first identification, which had been left in their place, one was claimed to have belonged to an old man, possibly Morgagni, and was placed inside the earthenware jar with the other skull
Fig. 1 Headstone of Morgagni’s and his relatives’ tomb in Padua’s Saint Maxim Church
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which was believed to have been Morgagni’s. During the identification, a new passage was discovered inside the tomb, probably dating back from an older sepulture. Saint Maxim Church, in fact, was situated over the site of an archaic necropolis. Up to 20 skulls were found and were arranged in two lines along the east wall of the tomb, while the remaining 9 skulls from the first identification were placed next to the north wall. The earthenware jar was left in its original position, at the south-west wall. This identification also did not lead to the result which had been hoped for, and the remains of Morgagni still were not identified for sure [2]. 2011 and 2012 were two very significant years for the history of Padua University and, in particular, for its Medical School. 2011 marked 300 years since the call to this University of Giovanni Battista Morgagni (1711–2011) and 250 years since the publication of his De sedibus et causis morborum per anatomen indagatis (1761–2011) [1]. In addition to this, 2012 marked 300 years since Morgagni’s inaugural lecture at the University, which was titled Nova institutionum medicarum idea (New idea for medical curriculum, Padua 1712) (1712–2012), and in which he proposed a new program for the medical curriculum [3]. The PhD School on “Medical, Clinical and Experimental Sciences” of Padua University organized, on the occasion of theses anniversaries, a series of lectures and events that culminated in two International Congresses, one historical and one scientific, in March 2012, at the Bo Palace of Padua University [4]. On May 3, 2011, a new identification of Morgagni’s tomb was carried out, organized by a commission directed by Prof. Gaetano Thiene, who was the principal promoter of the activities celebrating the Morgagnian anniversaries. The order of skeletal remains in the tomb corresponded to that described during the second identification (Fig. 2). We observed a series of skulls next to the east wall: those of the 1900 identification which came from an older sepulture. We found, however, only 14 skulls (classified from C1 to C14), while the record indicated that there were 20 of them; probably the remaining skulls deteriorated, since the tomb and the site of the church have flooded several times during the last century. This series of skulls, being very old, had deteriorated more than the other. There were eight skulls at the north wall (classified from C15 to C22), as indicated in 1900 identification: these skulls were those which had been found in the 1868 identification, which consequently belonged to the original disposition of Morgagni’s tomb. In this case, one skull was missing. We noted the earthenware jar in the south-west corner with two skulls inside (classified with C23 and C24), one complete (C24) and the other fragmentary (C23), and some teeth and vertebras. There were also scattered osseous elements in the center and towards the south wall (classified FS). Finally, we identified in the north-east corner the fragments of several skulls (classified C25): nine lower jaws, two teeth, seven frontal bones with orbits, three left and six right frontal bones,
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Fig. 2 Schematic of the remains found in Morgagni's tomb: the series C15–C22 are the skulls found during the first identification (1868) which composed the original Morgagni’s tomb; C23 and C24 are the two skulls attributed to Morgagni during the recognitions of 1868 and 1900; C1–C14 are the skulls found during the second recognition (1900) and belonging to an older entombment; FS are scattered osseous elements; C25, finally, holds several skulls fragments
and, finally, one sphenoid bone. Of these fragments, some seem to have belonged to very young individuals. We immediately realized that we had in our hands the remains of Morgagni’s children who died prematurely. All these elements have been classified, packed, and transported to the Pathological Anatomy Museum of Padua University for scientific analysis and photographic documentation. On May 16, 2012, after one year of research, the remains of Morgagni’s tomb were replaced in their original site, protected by sterile bags, and the sepulcher was restored and reclosed (Fig. 3). We also left in the tomb a book relating to an iconographic research of Morgagni and which summarizes the activities done for the Morgagnian anniversaries [5]. The identification of Morgagni’s remains, according to the historical sources, should be focused on the skulls C15–C24, because they surely belong to the original disposition of
Fig. 3 Replacement of Morgagni’s tomb remains with the book published for the 2011–2012 Morgagnian anniversaries
Morgagni’s tomb, while C1–C14 came from an older sepulture. Of skulls C15–C24, the skull which anthropometry will confirm to belong to the oldest individual will be most likely to be Morgagni’s, because he died at an age—89 years old— which was very rare to reach in Morgagni’s time. We found skull fragments of very young individuals (C25), which, according to historical sources, could be Morgagni’s children who died prematurely. We were in the position to perform a DNA analysis to discover whether a familial relationship could be proved between the DNA extracted from these fragments and the DNA extracted from the teeth of the oldest individual of the series. When direct reference samples, such as personal items, from missing individuals are not available, identifications can be carried out indirectly based on the ranking of likelihood ratios (LRs) constructed from a comparison of the probability of observing DNA profiles of remains of a presumed relationship with profiles from reference samples of the alleged family members versus the hypothesis that the remains are unrelated to the family [6]. The 99.9 % confidence value was advocated by the DNA Commission of the International Society for Forensic Genetics [7]. There are two ways to increase the power of identification: (i) type more markers and (ii) type more relatives. The number of markers that can be typed will be limited by the quality and quantity of DNA derived from the remains. If there is sufficient DNA, then a large battery of genetic markers is available to assist in making an identification. In many cases, the quality and quantity of DNA are poor. Increasing the number of reference relatives can increase the chances of identifying
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remains, particularly for challenged samples. In some cases, the number of relatives can be quite large, but in others, the number of available relatives is very limited. Typing all the relatives of a large pedigree can be costly and may not be necessary to reach a certain threshold for identification. Because there are information and cost factors regarding which and how many relatives are selected and typed, some selection criteria should be considered to guide identity testers. The probabilities of confirming identity with certain combinations of relatives are more powerful than with certain other combinations [6]. The purpose of DNA analysis in this work is to contribute to the results of historical and anthropological research on the confirmation of the identity of the bones belonging to Morgagni. Because direct references are not available, we used indirect relative references for the identification by kinship analysis, considering the bone fragments present in the grave which hypothetically belong to children. Furthermore, to avoid the disruption of the skeletal remains, we used two teeth recovered from the skull, taking into account the problems mentioned above.
Material and methods The type of deposit found inside the tomb has forced a separate investigation of anatomical elements: bones were completely mismatched and not in anatomical connection. The anthropological research carried out on all the anatomical elements began with an initial morphology of the samples and a further verification of measures and indices using appropriate anthropometric tools. There was also a valuation of the minimum number of individuals, the age, and the sex. The occupational markers and the presence of pathologies were considered. All the skeletal elements present, for a total of 647, were classified and analyzed in order to have a correct estimate of the minimum number of individuals and for a complete analysis of markers and diseases. After their recovery from the church of Saint Maxim, all the findings were studied at the Museum of Pathological Anatomy at different stages that can be summarized as follows: (1) inventory and cleaning, (2) morphological analysis and sorting, (3) cataloguing, (4) anthropometric analysis, (5) determination of age and gender, (6) estimation of stature, (7) calculation of the minimum number of individuals, (8) analysis of the occupational markers, (9) paleopathological analysis, (10) photographic documentation, (11) data collection, and (12) electronic archiving and statistical analysis. Regarding DNA analysis, the main problem is not only when working with ancient DNA stems to some degree from the low amount of starting molecules and the presence of PCR inhibitors but also includes endogenous DNA damage and
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fragmentation. Much effort has been focused on improving the extraction and purification procedures. Although some methods have been more influential than others, none has been generally accepted. In fact, if postmortem changes are probably the most important factors in the degradation of DNA, other factors also contribute to the degradation of the DNA, such as hydrolysis, oxidative action of oxygen, nonneutral pH, temperature and humidity conditions, UV radiation, and the presence of humic acids in soil. In historical cases and ancient DNA investigations, as in this study, bone and teeth samples are often the only biological material available for DNA typing. Problems which researchers face while working with DNA extracted from bones and teeth samples recovered from mass graves are limited to DNA quantity, DNA degradation, contamination, and postmortem changes [8–10]. The presence of inhibitor(s) may also prevent amplification. Inhibition is an especially significant problem when DNA is extracted from old and ancient material [10, 11]. One potential inhibitor 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 [11–13]. In the literature, most of the protocols for DNA extraction from bone and teeth suggest an incubation of powdered material in an ethylene diamine tetraacetic acid (EDTA) solution. The EDTA both demineralizes the bone/teeth (to an extent dependent on the EDTA concentration and the volume of extraction buffer) and inactivates DNAses by chelating bivalent cations such as Mg++ or Ca++ [14]. The degradation and fragmentation of DNA involve the need to dedicate extreme attention to the problem of contamination, which is present from the recovery of the remains until completion of all the experimental phases. Contamination can be caused by soil and laboratory microorganisms, especially by the human DNA (exogenous DNA) that can come from the operators during the archeological excavation, by laboratory personnel during DNA analysis, and from instrumentation which has not been properly sterilized. For the DNA analysis, we considered two teeth which fitted perfectly with the complete skull in the jar (C24), anthropological analysis of which confirmed to belong to the oldest individual of the series (Fig. 4). For the identification of kinship, it was necessary to extract bone fragments from different craniums anthropological analysis of which established to have belonged to very young individuals. These samples were conserved in sterile containers to preserve the integrity of the material and to limit contamination from chemical/physical agents, bacteria and mold, until the analysis. To prevent and reduce DNA contamination, we followed the most stringent criteria proposed for ancient DNA studies [15].
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Fig. 4 Age groups (Iuvenis61) of the skulls belonging to the Morgagni’s tomb
All DNA extractions and amplifications were carried out in sterile hoods in laboratories physically separated from the laboratory in which post-PCR analysis was conducted. Dedicated reagents and pipettes were used, together with filterplugged tips. All tools were washed with DNA RemoverTM solution and then UV-irradiated among uses. The specimens were cleaned with an abrasive brush and then soaked in agitation in a NaClO 10 % solution for 5/10 min. Subsequently, the samples were washed twice in agitation with distilled water (ddH2O) for 10 and 5 min, respectively. After this step, each side of the samples was irradiated at 240 nm (UV light) for 20 min. After decontamination, the samples were pulverized using the TissueLyserII (Qiagen); the program used consists of 1 min at 25 Hz, and it was repeated once again if the samples were not pulverized thoroughly enough. The quantity of powder obtained was 3–8 g for the skulls and 0.6–1 g for the teeth. The powder was then incubated in agitation from 3 to 5 days (depending of the specimen) with a decalcification solution (EDTA-Tris, pH 7.5), replacing the solution every day [14]. For the DNA extraction, the following procedure was adopted: about 1 g of powder was incubated overnight in 15 ml of extraction buffer (EDTA 0.5 M, 1 % NLauroylsarcosine) and 200 μl of 20 mg/ml Proteinase K, in a rotary shaker at 56 °C. This protocol was modified, adding 50 μl of DTT 1 M for each sample. The obtained solution was treated with an equivalent volume of phenol/chlororm/ isoamyl alcohol (25:24:1) and, subsequently, was concentrated and purified using a Centricon-30 filter column (Millipore) up to a 30-μl volume [14]. Low copy number Short Tandem Repeat (STR) amplifications were conducted, amplifying autosomic STR loci using different amplification kits (all produced by Applied Biosystems, Foster City, CA): D8S1179, D21S11, D7S820, CSF1PO, D3S1358, THO1, D13S357, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA, and
amelogenin for determination of sex. For each PCR reaction, twice the recommended Taq Gold concentration and 1 μl of 10 mg/ml BSA were added to the reaction mix. Moreover, six additional PCR cycles were used (overall 36 cycles). Water instead of DNA was used as negative PCR control. PCR products were separated on an Applied Biosystems 3130 (Applied Biosystems, Foster City, CA) and analyzed using GeneMapper ID software version 3.2, using GeneScan 500 LIZ as internal size standards and allelic ladders provided by the manufacturer. Results were elaborated using GeneScan Analysis 3.7 software.
Results The minimum number of individuals inside the tomb was 32 people, according to the whole orbitals which were the most recurring anatomical element between the 647 classified. After this first step of the study, sex was determined through analysis of the femurs [16, 17]: 8 males, 3 females, 3 uncertain for the right femur, 12 males, 5 females, and 2 uncertain for the left. We also calculated the femoral robustness and pilastric index and the valuation of the height [18, 19]. On average, the height of the male was about 173 cm, while the average female’s height was 157 cm. It is also important to note the sexual dimorphism evidenced by the femoral occupational markers: they were more evident and frequent in the male than female, because of the daily posture and the different kind of work [20]. Once this stage was completed, we studied all the 24 skulls found in the tomb. Among them, we focused our attention on the complete skull presumed to be that of Morgagni which had been found inside the earthenware jar (C24) (Fig. 5). The other one inside the earthenware jar (C23), placed there during the 1868 recognition, belonged to a “maturus” individual
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Fig. 6 The most complete skull fragment among those classified C25, characterized by a clearly young morphology
Fig. 5 Skulls C23 (left) and C24 (right) found inside the earthenware jar. The C24 belongs to the oldest individual of the whole series of skulls examined (“senilis”), while C23 appeared to be a “maturus”
(Fig. 4), based on the analysis of cranial sutures [21], so it could not belong to Morgagni. Skull C24 was in a good condition, although the lower jaw appeared without the right arch. Both jaws were toothless, and the only tooth still present is the third molar on the right maxillary arch, but it is included and had not fully erupted. We also found one incisor and one premolar in the earthenware jar which fitted perfectly with the left superior jaw that was used for the molecular analysis. Many alveolar processes of the maxilla are completely reabsorbed in correspondence of dental elements lost in life: PM2, M1, and M2 of the right arch; PM1, PM2, M1, M2, and M3 of the left one. The other teeth were lost post-mortem. The biological profile of the remains corresponding to the elderly individual is male (degree of sexualization: 0.2 skull, mandible 0.5). The skull, according to the method of obliteration of the cranial sutures of Meindl and Lovejoy [21], turned out to have belonged to a “senilis” elderly man (over 61 years of age); indeed, it was the oldest skull of them all, as demonstrated by the complete obliteration of all the cranial sutures. Figure 4 shows the results according to the age groups of the 10 skulls which are surely related to Morgagni’s burial, thanks to historical documentations. The skull which is hypothetically Morgagni’s is the only one which could be considered to have belonged to a “senilis” individual. This fact was sound proof that this skull could have belonged to Morgagni, and it confirmed the supposition made during the second identification (1900). The skull had no special epigenetic characters, except for an occipital bun evident in the nuchal portion. There were also no signs of pathological disorders, even if there was some evidence of an abscess on the left maxilla, next to the nasal
cavity. In general, it is a skull of average length and is not very wide; the face is of average width, the orbits are of a medium– small size, the nasal cavity is of medium depth, and finally, the palate is long and narrow. Concerning the skull fragments classified as C25, we found that they belonged to three different young individuals, demonstrated by their complete orbital arch. The results from the morphological analysis clearly revealed that they belonged to a range of age between birth and the first few years of life. The most complete skull fragment belonged to a young individual of about 2 years old, thanks to the presence of a part of metopic suture and to the calculation of the frontal transverse index, typical of a convex forehead [22] (Fig. 6). We decided to call them C25, C25 A, and C25 B, and they have been used to test the relation of kinship with C24. Table 1 Genetic profiles obtained from the bone fragments analyzed Polymorphism
C24
C25
C25 A
C25 B
D8S1179
10–?
10–13
10–12
10–?
D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 VWA TPOX D18S51
31–32 10–11 – – – 11–12 – – 18–? 19–? 11–12 13–16
31–? 10–12 11–13 15–17 6–7 12–13 9–11 20–? – 17–19 9–11
31–? – 11–? 18–19 7–? 12–13 – 20–23 9–14 17–18 –
25.2–31 11–? 9–12 – – 8–11 – – – – –
AME D5S818 FGA
XY 10–12 31–32
16–17 XY 9–12 30–?
– XX 9–10 31–?
– XX – –
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As reported in literature [6], to evaluate whether an unidentified person belongs to a family pedigree (P), usually one or more family reference persons from the putative pedigree are typed. In a schematic explanation, the identification is assessed by comparing two alternative hypotheses: the subject is the specific member of the putative pedigree, and (the other hypothesis) the same subject is unrelated to the known reference members of the putative pedigree. The results of the statistical probability were calculated based on a ratio of the probabilities of the DNA evidence under each hypothesis (for the detailed explanation, see Ge et al. [6]). For the probability calculation, we used The Familias program (it is freely available at the website: familias.name). The Familias program may be used to compute probabilities and likelihoods in cases where DNA profiles of some people are known, but their family relationship is in doubt. The program may compute which pedigree is most likely and how much more likely it is than others. In this case, also if we obtained partial genetic profile with STR autosomal polymorphisms and no data for Y chromosome haplotype, the statistical elaboration has given three values expressed by a percentage ranging from 90.8805 to 99.8291 % that the C24 skull is the father of the three children C25, C25 A, and C25 B (1 male and 2 females), demonstrating the existence of the relationship (Table 1).
Conclusion Historical and archive documents tell us that in the tomb investigated in Saint Maxim Church of Padua, Giovanni Battista Morgagni, his children, his wife, and other unidentified individuals were buried, some of whom might have been professors at the University of Padua. We also know that Morgagni died at 89 years old, an age which was very rare to reach at the end of the eighteenth century. Historical documents tell us that two different identifications were made on this tomb, in 1868 and 1900. In the first, up to 11 individuals were found, and the skull (fragmentary) which belonged to the oldest of the series, deduced to be that of Morgagni, was placed in an earthenware jar. During the second identification, another (complete) skull from this series was claimed to have belonged to a very old individual and so was placed in the earthenware jar with the previous one. Moreover, a new passage was discovered inside the tomb, dating back from an older sepulture, where up to 20 skulls were found. During our identification in 2011, we observed the same order described in the 1900 identification. Anthropometrical analysis confirmed that the complete skull inside the earthenware jar, called by us as C24, belonged to the oldest individual of the original disposition of Morgagni’s tomb, because it was the only one that could be defined “senilis” (over 61 years of
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age). Moreover, we found fragments of skulls, called C25, which were not detected during the previous identifications and which surely belonged to very young individuals. Genetic analysis performed on skull C24 and on the C25 series of skull fragments tell us that the C24 was a male and that he was the father of the C25 series of fragments. In conclusion, thanks to the interaction between historical studies, anthropological research, and molecular analysis that reinforce each other, we can assume that the skull called C24 is that of Giovanni Battista Morgagni and that the C25 series of skull fragments are from his children who were buried together with their parents. Acknowledgements The authors express their gratitude to Antonio Mattiazzo, Archbishop of Padua, and to Giovanni Brusegan, Rector of the Saint Maxim Church, for allowing the opening of the tomb to identify the Morgagni’s remains. Conflict of interest We declare that we have no conflict of interest.
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