Int Poster J Dent Oral Med 2011, Vol 13 No 3, Poster 551
The role of the c.-374T>A SNP in the promoter region of RAGE-gene in aggressive periodontitis
Diana Stosberg, Dr Susanne Schulz, Dr. Jana Klapproth, Dr. Uta Zimmermann, Prof. Dr. Hans-Günter Schaller, PD OA Dr. Stefan Reichert,
Martin-Luther-University Halle-Wittenberg, Department of Operative Dentistry and Periodontology, University School of Dental Medicine, Halle-Wittenberg, Germany
private dentist practice, Halle, Germany
PD Dr. Jamal M. Stein,
Department of Operative Dentistry, Periodontology and Preventive Dentistry, RWTH Aachen, Germany
Dr. Christiane Gläser,
Martin-Luther-University Halle-Wittenberg, Institute of Human Genetics and Medical Biology, Halle-Wittenberg, Germany
Jahrestagung der Deutschen Gesellschaft für Parodontologie
Periodontitis is characterized as a chronic inflammatory disease induced by periodontopathogens.
The receptor of advanced glycation end products (RAGE) is mediating inflammatory processes e.g. it is involved in the occurrence of severe periodontitis via activation of transcription factor NF-kB. Its expression is influenced by the c.-374T>A-SNP, located in the promoter region of this gene.
|Fig. 1a-b: Signalling pathways mediated by RAGE
Aim of the study
In the present study possible associations were investigated between this SNP and the occurrence of aggressive periodontitis and its clinical features including smoking status, plaque (API) and bleeding indexes (BOP), pocket depth (PD), clinical attachment loss (CAL) and subgingival bacterial colonization (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema denticola).
Material and Methods
Generalized aggressive periodontitis (AP) n=81:
clinical manifestation before 35th year of life
attachment loss in at least 30% of the teeth with a minimum pocket depth of 4 mm
> 3 affected teeth had to be no first molars or incisors
severity of attachment loss was inconsistent to the amount of mineralized plaque
more vertical than horizontal approximal bone loss was visible in the radiographs
Generalized chronic periodontitis (CP) n=68:
attachment loss in at least 30% of the teeth with a minimum pocket depth of 4 mm
The amount of the attachment loss was consistent with the presence of mineralized plaque
More horizontal than vertical approximal bone loss was visible in the radiographs.
Periodontitis-free controls n=85:
probing depth ≤ 3.5 mm, no gingival recession due to periodontitis
Clinical attachment loss > 3.5mm as a consequence of traumatic tooth brushing, overhanging dental fillings, orthodontic therapy etc. was not considered as a case of periodontitis.
|Fig. 2: RFLP of c.-374T>A SNP
||Fig. 3: Bacteria specific hybridization
DNA-isolation from EDTA-blood
Preparation of genomic DNA from human venous EDTA-blood was carried out using the blood extraction kit (Quiagen).
200µl EDTA-blood and 20 µl protease were mixed in a 1,5 ml tube.
After adding of 200 µl denaturation buffer AL and pulse-vortexing for 15 sec the samples were incubated at 56°C for 10 min.
200 µl of ethanol was added to the samples, vortexed and the samples were applied to a QIAamp Spin Column were the DNA is bound.
After two washing steps (buffer AW1 and AW2) the DNA bound to the column is dried by centrifugation.
200 µl distilled water is added to the samples, incubated at room temperature for 5 min and then centrifuged. The solved DNA is now in the filtrate.
Long-term storage of DNA is possible at -20°C.
Specific PCR and RFLP of c.-374T>A SNP
The detection of genotypes c.-374A>T SNP was carried out using a Forward primer 5'-3', RAGE-5: tca gag ccc ccg atc cta ttt, and a Reverse Primer 5'-3', RAGE-6: ggg ggc agt tct ctc ctc.
For every PCR 25µl of a Mastermix containing 12,5µl Mix (Promega);0,5µl primer RAGE-5; 0,5µl primer RAGE-6; 10,5µl water and 1µl genomic DNA was added.
PCR-program (2 min 94°C; 40sec 92°C, 15 cycles: 40sec 54°C, 1 min 72°C; 1sec delay; 40sec 92°C; 25 cycles: 40sec 49°C, 1min 72°C, 1sec delay; 5min 72°C; hold 4°C)
For digestion of the PCR products, to every PCR-mix 2,5µl 10* buffer (NEB) and 4U Tsp5091 (0,4µl, NEB) were added, for digestion the mixture was incubated for 8h at 65°C.
The DNA fragments were loaded onto a 2% agarosegel for electrophoresis. After electrophoresis, the ethidium bromide stained gel is photographed and interpreted.
Evaluation of periodontopathic bacteria in subgingival pockets
Paper points for collection of subgingival samples were used to bind periodontopathogens of the deepest pocket of each quadrant.
Preparation of bacterial DNA was carried out using the QIAamp DNA Mini Kit (Quiagen).
The paper points were incubated with 180 µl ATL-buffer and 20 µl proteinase K and incubated at 70°C for 10 min.
200 µl buffer Al was added and the mixture was incubated at 96°C for 5 min.
The mixture (without paper points) was applied to a QIAamp Spin Column and washed twice with buffer AW1 and AW2.
The DNA was solved in 400 µl AE-buffer and stored at -20°C.
For specific amplification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema denticola the micro-Ident® test of HAIN-Diagnostik based on alkaline phosphatase mediated staining reaction was used.
Mastermix provided in the micro-Ident® test (containing buffer, biotynilated primer, DNA for positive control), 2U Taq-polymerase (Eppendorf), and 5 µl of isolated bacgterial DNA were mixed.
PCR was performed (5 min 95°C; 10 cycles: 30 sec 95°C, 2 min 58°C; 20 cycles: 25 sec 95°C, 40 sec 53°C, 40 sec 70°C; 8 min 70°C)
The quality of PCR product was checked by agarosegelelectrophoresis.
Bacteria specific hybridization
20 µl of the PCR product were mixed with 20 µl of the denaturation solution in the well of the tray and incubated at room temperature for 5 min.
1 ml prewarmed (45°C) hybridization buffer was added to the sample and a strip (hybridized with DNA sequences of each bacteria as well as a positive control) was placed in the well of the tray.
The tray was incubated at 45°C for 30 min in a shaking water bath.
1 ml of stringent wash solution was added and incubated at 45°C for 15 min.
The strip was washed once with 1 ml rinse solution for 1 min and 1 ml of conjugate solution was added (room temperature for 30 min).
After washing 1 ml of substrate solution was added.
The occurrence of bacteria was evaluated visually by means of colored bands.
Two positive controls for amplification reaction and for conjugate were included in the test.
Clinical characterization of the patient groups
No significant differences between the patient groups and the healthy control group could be proven investigating gender, smoking status, and age. Sole exception was the age of the patients suffering from aggressive periodontitis because of the young age of onset of disease. As expected, both patient groups showed significant more severe clinical symptoms compared to the control group.
||Aggressive periodontitis (AP)
||Chronic periodontitis (CP)
|Mean age (years)
|Gender (% female)
|Approximal plaque index (%)
|Bleeding on probing (%)
|Pocket depth (mm)
|Pocket depth on microbial test site (mm)
|Clinical attachment loss in general (mm)
|Clinical attachment loss on microbial test site (mm)
|Tab. 1: Clinical characterization of the patient group – I
* p < 0.05 vs. periodontitis-free controls
As expected, both patient groups showed distinct and mostly significant increase in the occurrence of periodontopathic bacteria. Interestingly, no significant difference in the subgingival colonization with Aa could be shown for patients suffering from CP.
|Aggregatibacter actinomycetemcomitans (%)
|Porphyromonas gingivalis (%)
|Prevotella intermedia (%)
|Tannerella forsythia (%)
|Treponema denticola (%)
|Pg, Td, Tf (%)
|Tab. 2: Clinical characterization of the patient group – II: Microbiological assessment
Since the T-allele was associated with decreased promoter activity the genotypes carrying the T-allele were evaluated as "risk genotypes" (TT+AT) according to a dominant genetic model.
Significant differences in genotype as well as allele distribution could be proven between the periodontitis-free control group and the group of AP patients. In binary logistic regression analysis the T-allele could be proven as an independent risk factor for aggressive periodontitis.
The same tendency (n.s.) was proven for genotype and allele distribution in patients suffering from chronic periodontitis.
|Fig. 4a-b: Genetic evaluation
|Fig. 5: Multivariate evaluation
The results emphasize the role of the T-allele of RAGE SNP c.-372T>A as a putative risk indicator for aggressive periodontitis in this German Cohort irrespective of further periodontal risk factors.
This finding could be possibly based on an impaired immune response due to the decraesed promoter activity associated with the T-allele.
This Poster was submitted by Diana Stosberg.
University School of Dental Medicine, Department of Operative Dentistry and Periodontology