A MOLECULAR APPROACH TO INVESTIGATE TUBERCULOSIS CASES IN A GOTHIC POPULATION FROM GHERĂSENI NECROPOLIS, BUZĂU COUNTY

1Molecular Biology Center, Interdisciplinary Research Institute on Bio-Nano-Sciences, “Babeș-Bolyai” University, Cluj-Napoca, Romania2Faculty of Biology and Geology, Babes-Bolyai University, Cluj-Napoca, Romania

3NIRDBS, Institute of Biological Research, Cluj-Napoca, Romania

MATERIALS AND METHODS:1. Anthropological and anthropometrical analysis1. Anthropological and anthropometrical analysis for estimation of gender, age and possible diseases of individuals;2.2. FT-IRFT-IR spectroscopy analysis - JASCO FT-IR 6000 Spectrometer tuberculosis - mycolic acids markers3.3. ancient-DNA (aDNA) extractionancient-DNA (aDNA) extraction - phenol - chloroform;4.4. PCRPCR, Cloning , Cloning andand Sequencing Sequencing

6.6. Bioinformatic analysisBioinformatic analysis

INTRODUCTION: World Health Organization (WHO) ranks tuberculosis as the second most dominant infectious disease, exceeded just by HIV/AIDS. Given the incidence of the disease today and the appearance of multi-drug-resistant strains, it is important to obtain informations about the conserved conserved andand variable genomic locivariable genomic loci and about the mutation rate of the pathogen. For this reason, ancient cases of tuberculosis have to be investigated and the co-evolution of the pathogens with modern humans should be tracked. Species pathogenic to humans are included in the M. tuberculosisM. tuberculosis complex (complex (MTBCMTBC)).

ACKNOWLEDGMENT: This study was supported by funding from the project Genetic Evolution: New Evidences for the Study of Interconnected Structures (GENESIS). A Biomolecular Journey around the Carpathians from Ancient to Medieval Times

(CNCSIS-UEFISCDI _PNII_PCCA_1153/2011).

CHIRIAC Cecilia1,3, LUPAN Iulia1,2, RADU Claudia1, KELEMEN Beatrice1,2

2. FT-IR

Figure 3. Differences between FT-IR spectra of tuberculosis infected and not infected skeletal remains.

Figure 2. A. The inferior view of a healthy V lombar vertebrae comparing with (B) a tuberculosis affected one. C. Lateral view of a normal toracic vertebrae versus (D) two fused vertebrae from an individual suffering of Pott's disease.

RESULTS AND DISSCUSION:1. Anthropological and anthropometrical analysis

5. Bioinformatic analysis

Figure 5. A. A serial dilution was made to overcome aDNA contamination with soil substances that inhibit PCR; B. A fragment of pyrazinamidase gene (117 bp) was obtaind from two bones with tuberculosis injuries; C. The regulator of hydrogen peroxide-inducible genes (oxyR pseudogene in MTBC) was amplified (110 bp) in tuberculosis infected bone samples. D. TbD1 (deletion 1) fragment (112 bp) was obtained from three bone samples and the amplicons are to be sequenced.

CONCLUSIONSOsteologic signs of Pott’s disease (spinal tuberculosis) was confirmed using physical and molecular techniques: A. the presence of mycolic acids in bone samples was determined by FT-IR spectroscopy; B. the pncA fragments obtained throught sequencing formed a cluster with MTBC in the ML phylogenetic tree; C. oxyR sequences were identical with modern ones with only one sequence having a SNP

3. aDNA extraction

D

4. PCR, Cloning and Sequencing

A B

C

Figure 8. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter. The percentage of trees in which the associated taxa clustered together is shown next to the branches. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 3.0035)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 45 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 73 positions in the final dataset. Evolutionary analyses were conducted in MEGA5.

Figure 1. Geographic location of Gherăseni necropolis

Figure 7. Sequences obtained for pncA were subjected to multiple alignment using ClustalW algorithm in Mega 5. As an outgroup we used homologous sequences from Corynebacterium glutamicum.

Figure 4. aDNA extraction 1. rib, 2. vertebrae, 3. negative control. As a consequence of post – mortem degradation, the majority of genetic material has below 50 bp in lenght.

A

B

C

A B C

Figure 9. Sequencing oxyR F3-R1 PCR products clearly reveal their identity with modern M. tuberculosis strains.

Figure 6. 3D structure of pncA fragment determined with The mfold Web Server, State University of New York.

The aim of this study is to confirm through molecular methods the presence of MTBC in human remains from 4TH-5TH century necropolis.