A MOLECULAR APPROACH TO INVESTIGATE TUBERCULOSIS CASES IN A GOTHIC POPULATION FROM GHERASENI NECROPOLIS, BUZAU COUNTY

1
A MOLECULAR APPROACH TO INVESTIGATE TUBERCULOSIS
CASES IN A GOTHIC POPULATION FROM GHERASENI
NECROPOLIS, BUZAU COUNTY
CHIRIAC Cecilia1,3, LUPAN Iulia1,2, RADU
Claudia1, KELEMEN Beatrice1,2
1Molecular Biology Center, Interdisciplinary
Research Institute on Bio-Nano-Sciences,
Babe?-Bolyai University, Cluj-Napoca,
Romania 2Faculty 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 analysis
  for estimation of gender, age and possible
  diseases of individuals
• 2. FT-IR spectroscopy analysis - JASCO FT-IR 6000
  Spectrometer
• tuberculosis - mycolic acids
  markers
• 3. ancient-DNA (aDNA) extraction - phenol -
  chloroform
• 4. PCR, Cloning and Sequencing
• 6. Bioinformatic 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 and variable 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. tuberculosis complex (MTBC).
The aim of this study is to confirm through
molecular methods the presence of MTBC in human
remains from 4TH-5TH century necropolis.
Figure 1. Geographic location of Gheraseni
necropolis
RESULTS AND DISSCUSION
1. Anthropological and anthropometrical analysis
2. FT-IR
Figure 3. Differences between FT-IR spectra of
tuberculosis infected and not infected skeletal
remains.
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.
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.
5. Bioinformatic analysis
A
3. aDNA extraction
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.
B
Figure 6. 3D structure of pncA fragment
determined with The mfold Web Server, State
University of New York.
C
B
A
C
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.
CONCLUSIONS Osteologic signs of Potts 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
Figure 9. Sequencing oxyR F3-R1 PCR products
clearly reveal their identity with modern M.
tuberculosis strains.
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).