RICERCA ITALIANA     

Research program

DISEASES, ENVIRONMENT AND SOCIETY AT THE GRAND DUKE COURT OF FLORENCE: AN HISTORICAL, ARCHAEOLOGICAL AND PALAEOPATHOLOGICAL STUDY OF THE MEDICI FUNERARY DEPOSITIONS (XVI-XVIII CENTURIES)

University Co-ordinator

Università degli Studi di ROMA "La Sapienza" - MEDICINA SPERIMENTALE E PATOLOGIA - ROMA(RM)

Research Unit Leader

Laura OTTINI

Description

The Research Unit of Rome will be responsible of ancient DNA studies, concerning specific infectious diseases, tumors and mitochondrial DNA genealogies of the Medici family (XVI-XVIII centuries). Moreover, in collaboration with BioRep, Milan, it will be involved in the creation of centralized human ancient materials Biorepository in order to collect, process, store and later distribute samples to support a large number research studies.
As first step of the research, analysis of the state of molecular diagenesis of the remains will be performed. All samples for molecular analyses will be obtained under sterile conditions in a dedicated laboratory, subjected to 254 nm UV irradiation prior to each procedure. Whenever possible, four distinct samples, microdissected using a dedicated binocular microscope, will be obtained for each tissue. The first will be used for the assessment of the degree of amino acid racemization, as previously described. The other two samples will be used for the extraction and amplification of DNA. The fourth sample will be used for microscopic studies, after paraffin embedding. At the same time, we will conduct recognition of the pathology archives dating from the end of the XIX and early XX centuries existing at the University of Pisa and Florence. Mummified as well as paraffin-embedded samples will first be analyzed for the degree of racemization of aspartic acid (Asp), alanine (Ala) and leucine (Leu) using high performance liquid chromatography (Poinar, 1996). This analysis allows to estimate the state of preservation of endogenous DNA and may permit to better evaluate the possibility of contaminations with modern DNA, which should be suspected whenever amino acid racemization data are not compatible with DNA amplification results. The results obtained from amino acid racemization studies will be evaluated before DNA extraction and amplification. Samples with aspartic acid enantiomer racemization ratios (D/L Asp) below 0.1, which are considered compatible with ancient DNA retrieval, will be preferentially utilized for paleogenetic studies. We will then proceed to verify the amplification of endogenous DNA, extracted using the GENECLEAN kit for ancient DNA (BIO101, La Jolla, California, USA). We will focus on mitochondrial DNA, that, compared to nuclear DNA, has the advantages of being well characterized and present in multiple copies per cell (which results in a better preservation, compared to nuclear DNA). We will examine the hypervariable mitochondrial control region HVRI, which may provide data not only on DNA preservation, but also on the maternal genetic variability of the mummies examined. The strategy for the analysis of the entire 444 bp HVRI mitochondrial DNA region is based on PCR amplifications comprised within a maximum size of 183 bp. For this purpose, we designed 3 sets of primers that amplify partially overlapping sequences. The overlapping permits to exclude immediately PCR products with nucleotide variants due to polymerase errors. The data obtained in this research phase will be useful for the design and standardization of PCR protocols, and will permit to correlate taphonomic conditions and embalming procedures with biochemical alterations and ancient DNA preservation.
With regard to infectious diseases we will focus on molecular analysis of Treponema pallidum, causative agent of venereal syphilis, and Plasmodi, causative agents of malaria. Regarding the PCR systems to detect ancient pathogens DNA, we will utilize either specific or so-called "universal" systems. The diagnosis of T. pallidum will be performed utilizing primer set designed to amplify the 5' flanking region of TPP15. The diagnosis of Plasmodi will be performed utilizing at first primer sets designed to amplify a consensus region, then by using specific primer sets designed to discriminate between the different Plasmodi species. In particular, ancient DNA samples will be processed by semi-nested PCR using primers designed to amplify species-specific sequences of the small subunit ribosomal ribonucleic acid (18S SSU rRNA) genes of P. falciparum, P. vivax and P. malariae. In the external PCR, performed using primers designed on conserved regions, a 130 bp fragment DNA of the Plasmodium sp 18S genes will be amplified. The internal PCR will be performed using sense external primer in combination with each species-specific primer to P. falciparum, P. vivax and P. malariae in individual tubes. The expected band sizes will be 100 bp for P. falciparum and P. vivax and 110–115 bp for P. malariae. According to circumstances, we will utilize "universal" PCR systems that is based on the nucleotide sequence of the bacterial gene for 16s ribosomal RNA. PCR amplified DNA is cloned into a plasmid vector then the sequence of a large number of clones is determined. The sequences are subsequently matched to the reference sequences in a database. Cloning of PCR products is a necessary step in the analysis of ancient DNA, because nucleic acid degradation increases the frequency of occasional nucleotide substitutions in PCR products. Cloning will be performed using the TOPO-TA DUAL PROMOTER kit (Invitrogen Corporation, CA, USA), based on the plasmid TPO10F' as a vector. For cloning verification, plasmid DNA will be amplified with the same primers used in the original DNA amplifications and then sequenced. Cloned sequences will be compared to discriminate between shared and occasional nucleotide variants. Sequences will be obtained with an ABI Prism 310 Sequencing Analyzer (Applied Biosystems, Perkin Elmer Corporation, Norwalk, CT, USA) and the ABI Prism RR Big-Dye Terminator Cycle Sequencing Kit. Sequence analyses will be conducted in both sense and antisense strands (H and L chain for mitochondriat DNA control sequences). Variant sequences of pathogens and mitochondrial DNA haplotypes will be analyzed using computerized programs for sequence studies (MacPairwise, Arlequine, Clustal, MacVector). In particular, sharing between parasites sequences obtained from ancient remains and modern global world data sequences avalaible on GenBank will be obtained using Arlequin 2000. The cladistic tree will be constructed through neighbor-joining algorithm using PHYLIP 3.6 Inference Package. Distance matrixes will be based on the F84 model with a gamma-distribution for substitution rate heterogeneity (Felsenstein, Evolution 38: 16, 1984). The gamma-shape and alfa-transition/transversion parameters will be set as estimated using TREE-PUZZLE 5.0. Significance will be determined from bootstrap percentages obtained after replication of 1000 trees. Furthermore, to obtain phylogenetic networks the DnaSP v.4.2 software (available at http://www.ub.es/dnasp/) will be applied to sequence data to identify segregating variants. Tables of variants will be used in the median-joining algorithm option of NETWORK 4.108 (Fluxus Tecnology Ltd). The rho statistics for time estimates of parasites clades that includes sequences and their standard deviations will be calculated according to Foster et al. (1996) and Saillard et al. (2000), as implemented in NETWORK 4.108. Molecular data will be considered valid only when independenlty replicated in distinct laboratories.
Tumors will be subjected to mutational analysis for the identification of specific tumor-associated mutations in p53 (exons 5 to 8), K-ras (codons 12-13 and 61) Mutational analyses will be conducted on nested PCR products. Sense and antisense primers will be designed in order to amplify partially overlapping sequences under 180-200 bp in size. Negative controls, based on PCR reactions with no template, will always be performed to exclude contaminants in the reagents used, and/or the introduction of contaminants during analytical procedures.
To obtain information on genetic relationships between mummies, ancient DNA from the Medici family will be investigated through the study of the hypervariable mitochondrial DNA control region including D-loop (I e II) and COII/tRNAlys. Moreover, taking advantage of previous mitochondrial DNA genealogies analyses conducted in collaboration with Dr. Giovanna Masala (Centro Studi per la Prevenzione Oncologica, CSPO, Florence) on DNA from a population-based series living at present in the area of Florence we will be able to investigate the genetic contribute of the Medici family to the genetic pool of the population in Florence.
Finally, the research unit will be involved in the creation of a centralized ancient human remains biorepository to address the need of permanent preservation of ancient biological samples of historical and cultural relevance. This objective provides a basic tool for implementing the upstream objectives, and represents an important goal in itself, as it will entail development of an advanced International facility that will be extremely valuable as a resource for furthering studies also beyond the present scopes. The centralized Biorepository will be established, in collaboration with Prof. Pasquale De Blasio, at BioRep located in Milan, Italy, where expertise and infrastructures are available. The BioRep facility (www.biorep.it) is one of the largest research facilities of this kind in Europe offering cryopreservation cell biology and high throughput services (HTS). BioRep with its partner, Coriell Institute for Medical Research USA (www.coriell.org), one of the oldest and largest Biorepositories in the world, has established a State-of-the-Art Biorepository designed to give the utmost efficiency and security in the management of bio-specimens and related resources. Here, we plan to create a centralized ancient human remains biorepository in order to collect, preserve and analyze biological samples from the Medici family. New and dedicated protocols for extraction and manipulation of ancient DNA will be also tested. Each sample will be characterized with accompanying provenance information including historical data. Moreover, ethical and legal issues related to collection, storage and distribution of biomaterials from human remains will be carefully considered.