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A historical case of amelogenesisimperfecta: Giovanna of Austria,Grand Duchess of Tuscany(1547–1578)

Giuffra V, Panetta D, Salvadori PA, Fornaciari G. A historical case of amelogenesisimperfecta: Giovanna of Austria, Grand Duchess of Tuscany (1547–1578).Eur J Oral Sci 2014; 122: 1–6. © 2013 Eur J Oral Sci

The skeletal remains of Giovanna of Austria (1547–1578), daughter of the EmperorFerdinand I of Habsburg (1503–1564) and first wife of the Grand Duke of Tuscany,Francesco I (1541–1587), exhumed from the Basilica of San Lorenzo in Florence,were submitted to paleopathological study. Examination of the dentition, which wasin a good state of preservation, showed maxillary retrognathism, together with acaries lesion, moderate periodontal disease, malposition of the upper second premo-lars and tooth wear. Furthermore, several horizontal grooves were observed in boththe buccal and the lingual crown surfaces of almost all teeth, especially the anteriorones. The orthopantomogram showed hypomineralized enamel and alveolar boneloss. Two third-molar teeth were investigated using micro-computed tomography(micro-CT) analysis, revealing highly irregular enamel caps with reduced averagethickness. The observed features suggest a diagnosis of hypoplastic amelogenesis im-perfecta, a developmental condition affecting enamel formation.

Valentina Giuffra1, DanielePanetta2, Piero A. Salvadori2, GinoFornaciari1

1Division of Paleopathology, Department ofTranslational Research on New Technologiesin Medicine and Surgery, University of Pisa,Pisa; 2CNR Institute of Clinical Physiology,Pisa, Italy

Valentina Giuffra, Division of Paleopathology,Department of Translational Research onNew Technologies in Medicine and Surgery,Via Roma 57, 56126 Pisa, Italy

E-mail: v.giuffra@med.unipi.it

Key words: enamel defects; Florence; Medici;Habsburg; Renaissance

Accepted for publication September 2013

The expression amelogenesis imperfecta (AI) refers to anumber of developmental conditions with abnormaldental enamel formation. Amelogenesis imperfectaaffects the structure and the clinical appearance of theenamel in all, or nearly all, teeth, and may be associ-ated with craniofacial characteristics, such as open bite(1, 2) and taurodontism (3). It can also be found aspart of a syndrome, for example tricho–dento–osseoussyndrome (4) or Kohlschutter syndrome (5), and associ-ated with other abnormalities, such as cone-rod dystro-phy, platyspondyly, or nephrocalcinosis (5, 6). Furtherinformation is provided in the Online Mendelian Inher-itance in Man (OMIM) website (http://www.omim.org).

The incidence of AI has been reported to vary quiteconsiderably: from 1:14,000 in the USA (7) over1:8,000 in Israel (8) and 1:4,000 in western Sweden (9),to 1:700 in northern Sweden (10).

Several classifications for the subtypes of this condi-tion have been proposed (11) but, according to themost widely accepted classification based on macro-scopic appearance (phenotype) (12, 13), there are threetypes of AI: hypoplasia, hypocalcification, and hyp-omaturation. The AI phenotypes depend on the spe-cific gene involved, the location and the type ofmutation, and the corresponding putative change atthe protein level (14). Amelogenesis imperfecta shows

autosomal-dominant, autosomal-recessive, sex-linked,and sporadic inheritance patterns, as well as sporadiccases (14).

Here we report a historical case of AI observed inthe Grand Duchess Giovanna of Austria (1547–1578).

The Medici were one of the most powerful familiesof the Italian Renaissance. Starting from the 14th cen-tury, their careful management of banking venturesand skilful political actions brought them to the fore-front of social and political power in Tuscany, andespecially in Florence, the intellectual centre of the wes-tern world. Lovers of art and science, the Medici werepatrons of Michelangelo, Leonardo da Vinci, Botticelli,Galileo, and Benvenuto Cellini.

Giovanna of Austria (1547–1578), daughter of Ferdi-nand I of Habsburg (1503–1564), was the first wife ofFrancesco I (1541–1587), 2nd Grand Duke of Tuscany.Giovanna appears in numerous portraits and was con-sidered an ugly woman and described as ‘hunchbacked’by some contemporary reports. She survived seven verydifficult deliveries, but died in childbirth at the age of30 after rupture of the uterus (15). Paleopathologicalanalysis of her skeletal remains revealed clear signs ofnumerous and difficult deliveries, such as enormousretro-pubic foveae and deep pre-auricular sulci, in thepelvic bones (16).

Eur J Oral Sci 2014; 122: 1–6DOI: 10.1111/eos.12097Printed in Singapore. All rights reserved

� 2013 Eur J Oral Sci

European Journal ofOral Sciences

The paleopathological examination also revealed alarge number of other disorders. The skullcap wasfound to be considerably thickened, showing a markedcongenital hyperostosis of about 1 cm. An open-biteClass III malocclusion with maxillary retrognathism,usually referred to as the famous ‘Habsburg jaw’, wasevident (17, 18). Severe juvenile scoliosis had affectedthe dorso-lumbar column, resulting in an impressivedeformity of the pelvis, which would indeed explain herdifficult deliveries and death following rupture of theuterus (19). In addition, bilateral acetabular dysplasiawas observed in the hip joints (20).

Material and methods

Like the other members of the Medici family, Giovannawas buried under the crypt floor of the Basilica of SanLorenzo in Florence. Her skeletal remains were exhumedduring the ‘Medici Project’, aimed at the study and valori-zation of the burials of the Medici family, and submittedto anthropological and paleopathological analysis (16).Age at death was determined on the basis of standardanthropological methods (21).

After macroscopic examination, the skeletal remains ofGiovanna were moved to the Careggi Hospital of Florenceand underwent standard radiography, which included anorthopantomogram.

The third upper-right molar and third lower-left molarwere analysed with micro-computed tomography (micro-CT) using the Xalt micro-CT scanner (22). Both teethwere scanned at 50 kVp, 2-mm Al filtration, 960 viewsover 360°, and 1.6 mAs/view. All images were recon-structed using a modified Feldkamp algorithm, with anisotropic voxel size of 37 lm on a 480 9 480 9 700 vol-ume data set. After reconstruction, ImageJ (23) and Vol-View (http://www.kitware.com/opensource/volview.html)were used for two-dimensional (2D) and three-dimensional(3D) volume visualization, and enamel segmentation wasperformed using Seg3D (http://www.seg3d.org). The localthickness of the segmented enamel cap of these two thirdmolars was then quantified (24). Moreover, the averageenamel thickness (AET) and relative enamel thickness(RET) were measured on micro-CT-derived buccolingualvirtual planes passing through mesial cusp tips, asdescribed by SMITH et al. (25).

Results

The anthropological study of the skeleton of Giovannarevealed an age at death of 25–35 yr.

The analysis of the dentition, which was completeand in a good state of preservation, revealed a series ofpathologies and abnormalities. There was marginalalveolar bone resorption of grade 2, with root expo-sure, presumably as a result of periodontal disease (26)(Figs 1A,B and 2A,B). A deep caries lesion of grade 5(a larger cavity, which clearly penetrates the dentine;ref. 27) affected the occlusal surface of the second left-upper molar (Fig. 1C). Malposition of both the secondupper premolars was evident: the left one was rotated90°, whilst the right one was displaced lingually

(Fig. 1C). Moderate tooth wear, exposing some areasof dentine on the occlusal surfaces of all teeth, exceptfor the second and third molars, was visible (28).

The tooth crowns had a white/brown colour andreduced lucency. Several horizontal rows of grooveswere observed on both the buccal and lingual surfacesof almost all teeth, and these grooves were especiallypronounced on the anterior teeth (Figs 1 and 2). Insome limited areas the margins of the enamel seemedto be chipping off, probably as a result of weak adhe-sion to the underlying dentine. The lesions stopped atthe amelo–cemental junction without extending apicallyto involve the cementum on the root surfaces.

The orthopantomogram (Fig. 3) showed hypominer-alized enamel and marginal alveolar bone loss. Noabnormalities were evident in the roots or pulp cham-bers, except for minor intrapulpal calcifications andpossible periapical radiolucencies, the latter possiblyrelated to the presence of chronic abscesses or granulo-mas. Quantitative micro-CT analysis revealed severaldefects of the enamel caps on both the third upper-right molar and the third lower-left molar. This couldalready be observed qualitatively by the 3D volumerenderings of the micro-CT scans, as shown in Fig. 4A,B. Figure 4C shows a colour-coded thickness map ofthe enamel cap. The arrows indicate several features ofabnormally reduced thickness and irregular shapes ofthe enamel.

Table 1 shows the quantitative results of averageenamel thickness (AET) and relative enamel thickness(RET) measurements on the two-third molars, togetherwith the normal values for adult women found in theliterature (25). Even though the RET appeared normalin both teeth, it is worth noting that the AET of thelower third molar was considerably lower than normal,thus supporting our observation of hypomineralization

A B

C

Fig. 1. (A) The skull of Giovanna showing reduced lucencyof tooth crowns, white/brown tooth colour, and horizontalgrooves on the buccal surfaces of teeth. (B) Left lateral viewof maxilla and mandible showing the horizontal grooves, peri-odontal disease, and chipped-off enamel in some limited areas.Note maxillary retrognathism. (C) Crowns of the upper teethwith horizontal grooves on the lingual surfaces of teeth. Notedental wear, caries on second left molar (M2), rotation ofsecond left premolar (PM2), and shift of second rightpremolar (PM2).

2 Giuffra et al.

of the teeth. On the other hand, the reduced values ofthe dentin area and enamel dentin junction lengthreflect an overall reduced crown size.

Discussion

The dental crowns showed a, presumably, congenitalmalformation involving the enamel and mainly com-prising horizontal grooves on both the lingual and thebuccal surfaces of the teeth. The dentition of the fatherand the mother of Giovanna has been examined byCzech researchers (29); however, neither Ferdinand Inor Anna Jagellone revealed signs of any dental enamelanomalies.

Several conditions may affect the quality and mor-phology of the enamel and need to be taken into con-sideration for a differential diagnosis to explain thelesions observed in Giovanna of Austria.

Osteogenesis imperfecta (OI) is a genetic disorder ofconnective tissues caused by a collagen type I mutationand may be characterized by different degrees of bone

fragility, growth retardation, joint laxity, hearing loss,blue sclerae, and dentinogenesis imperfecta (DI) type 1.The teeth appear opalescent greyish-blue to brown, andthe deciduous dentition is generally more severelyaffected than the permanent dentition; the pulp cham-bers and root canals are completely or partially obliter-ated by abnormal dentin; and the enamel has a normalstructure and mineral content (30). In Giovanna’s teeththere was a brownish coloration, but the pulp chamberand root canals were normal. The main featureobserved in the Duchess was grooves in the dentalenamel, which is not a feature of OI, thus ruling outthis diagnosis.

Dentinogenesis imperfecta belongs to a group ofgenetic disorders affecting the development of dentine.Three types of DI have been described in the literature,all of which share an opalescent blue/grey or yellow/brown discoloration of the tooth enamel. Radiographi-cally, the teeth may show structural defects, such asbulbous crowns and small or obliterated pulp chambers(31). Apart from a white/brown colour, no discolor-ation, bulbous crowns, or pulp chambers were observedin Giovanna, which made it possible to rule out DI.

Dental fluorosis, also referred to as mottling of toothenamel, is a developmental disturbance of dentalenamel caused by excessive exposure to high concentra-tions of fluoride during tooth development. The toothcrowns appear white opaque owing to the presence of ahypomineralized enamel subsurface. With more severedental fluorosis, pitting and loss of the enamel surfacemay occur, leading to a brownish staining. Severely flu-orosed enamel can easily chip off during normalmechanical use (32). Although pits may appear on theenamel surfaces in dental fluorosis, there are no pro-nounced horizontal grooves such as those seen in Giov-anna’s teeth. Some countries have areas with highnatural fluoride levels in the water; these include India,China, and parts of Africa. However, people living inAustria, as Giovanna did in her first 18 years of life,would not be expected to have ingested any significantamounts of fluoride.

Tricho–dento–osseous syndrome is a rare, autoso-mal-dominant disorder characterized by defects in hair,teeth and bones, and also by other defects (4). Severalfeatures of tricho–dento–osseous syndrome wereobserved in Giovanna, including enamel hypoplasia,maxillary retrusion, and cranial hyperostosis. Neverthe-less, an essential diagnostic trait of tricho–dento–osse-ous syndrome (taurodontism) (33), was not found inGiovanna, and therefore the diagnosis of tricho–dento–osseous syndrome seems unlikely.

Therefore, the most probable diagnosis for the dentalanomalies observed in Giovanna is AI. Hypoplastic AIis caused by some ectodermal disturbance related toalterations in the production of a normal organicenamel matrix, resulting in defects ranging from whiteflecks, narrow horizontal bands, lines of pits, grooves,and even massive loss of enamel volume. Discolorationof the teeth, varying from yellow to dark brown, is themost common feature. In hypocalcified AI the primaryenamel matrix is laid down appropriately, but the min-

A

B

Fig. 2. Detail of the anterior maxillary teeth (A) and of theright lateral view of the maxilla with horizontal grooves evi-dent on the teeth (B).

Fig. 3. Orthopantomogram of Giovanna showing hypominer-alized enamel and alveolar bone loss. Slight intrapulpal calcifi-cations and possible periapical radiolucencies are visible.

Amelogenesis imperfecta of Giovanna of Austria 3

eralization process is defective. Therefore, the soft andfriable coronal enamel is removed after some years offunction. In hypomaturation AI the final stage of themineralization process is abnormal. Therefore, theenamel is hard but has a mottled opaque white to yel-low/brown or red/brown discoloration (12, 13).

All macroscopic, radiological and micro-CT scandata obtained from Giovanna of Austria suggest thediagnosis of AI of the hypoplastic type. A morphologi-cally very similar condition has been described byM�ARDH et al. (see Fig. 1 in ref. 34), which, in that case,was caused by a mutation in the enamelin gene.

Genetic studies have demonstrated that hypoplasticAI may be caused by mutations in several differentgenes. The human enamelin gene (ENAM) is impliedthrough autosomal-dominant inheritance patterns (34).Depending on the specific ENAM mutation, the hypo-plastic enamel may result in pits or horizontal ridges,or a more generalized effect may be observed with amarked reduction in thickness of the entire enamel. Inaddition, several patients with ENAM gene mutationshave been reported to display an anterior open-bitemalocclusion (35). Mutations in the amelogenin, X iso-form (AMELX) gene cause hypoplastic (and/or hyp-

A B

C

Fig. 4. Micro-computed tomography (micro-CT) cross-sections and three-dimensional (3D) volume rendering of (A) the thirdlower-left molar and (B) the third upper-right molar of Giovanna of Austria. An irregular shape of the enamel grooves is evident,especially in (A). (C) Colour-coded enamel thickness map obtained following quantitative analysis of the micro-CT images. Thearrows indicate abnormal regions of reduced enamel thickness (coded by darker colours) in both teeth of Giovanna.

Table 1

Micro-computed tomography (micro-CT)-derived morphometric parameters of the two-third molars of Giovanna, in comparison withnormal values for adult women (25)

M3 (mandibular) M3 (maxillary)

Joanna

Normal values*

Joanna

Normal values†

Mean Minimum Maximum SD Mean Minimum Maximum SD

Dentin area (mm2) 20.71 35.2 27.35 40.37 4.35 18.34 41.71 28.57 54.64 7.08Area of enamel cap (mm2) 15.24 24.57 19.26 29.42 3.43 18.90 28.32 22.15 38.39 3.42Length of enamel–dentinjunction (mm)

15.08 18.61 15.9 21.21 1.32 13.59 19.88 16.25 23.15 1.69

AET (mm) 1.01 1.32 1.08 1.67 0.19 1.39 1.43 1.21 1.95 0.15RET 22.20 22.44 18.67 31.84 3.77 32.47 22.35 18.04 30.01 3.15BCD (mm) 8.50 8.42 7.13 9.99 0.71 7.55 10.44 8.78 11.65 0.72

AET, average enamel thickness; BCD, bi-cervical diameter; M3, third molar; RET, relative enamel thickness.*n = 14.†n = 33.

4 Giuffra et al.

omaturation) AI with an X-linked inheritance pattern(36). Recently, a mutation in the FAM20A gene hasbeen identified as a cause of hypoplastic AI with gingi-val hyperplasia in a large consanguineous family (37),and the identification of a frameshift mutation inLAMB3, which encodes the beta 3 subunit of laminin,as a cause of dominant hypoplastic AI has beenreported (38). Besides the above-mentioned examples,other mutations have also been reported in the litera-ture.

Amelogenesis imperfecta may be classified accordingto both the appearance of the enamel and the patternof genetic inheritance. Following recent informationgained about the genetic causes of AI, a gene-basedclassification system would be preferred to the pheno-type-based one (39). However, for osteoarchaeologicalfindings, which are scarce (40, 41) despite a relativelyhigh incidence of AI in living populations, a diagnosishas to be based on the phenotype. For the future, it ishoped that the improvements in sequencing technologyin ancient DNA research will allow the identification ofspecific genetic defects in ancient human remains.

Acknowledgements – We would like to thank Professor Natale Vil-lari for the orthopantomogram; and Dr Monica Bietti, Directorof the Medici Chapels in Florence, and her staff for their supportduring the explorations of the Medici burials. This work was sup-ported, in part, by a grant from the ARPA Foundation (www.fondazionearpa.it).

Conflicts of interest – All authors declare no conflicts of interest.

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