EVALUATION OF INTERLEUKIN-6 ...€¦ · EVALUATION OF INTERLEUKIN-6, LYMPHOTOXIN-α AND TNF-α GENE...

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868 http://www.journal-imab-bg.org / J of IMAB. 2015, vol. 21, issue 3/ EVALUATION OF INTERLEUKIN-6, LYMPHOTOXIN- α AND TNF- α GENE POLYMORPHISMS IN CHRONIC PERIODONTITIS. Velitchka Dosseva-Panova 1 , Antoaneta Mlachkova 1 , Christina Popova 1 , Maya Kicheva 2 1) Department of Periodontology, Faculty of Dental Medicine, Medical University of Sofia, Bulgaria. 2) PhD, biochemist, Progene Ltd., Sofia, Bulgaria. Journal of IMAB - Annual Proceeding (Scientific Papers) 2015, vol. 21, issue 3 Journal of IMAB ISSN: 1312-773X http://www.journal-imab-bg.org SUMMARY Background: Periodontitis is a chronic inflammatory disruption of the periodontal supportive tissues. There are the numerous evidences for his bacterial etiology. Though the occurrence of periodontal bacteria is considered to be the main cause of periodontitis, certain characteristics of the in- dividual immune response may also have inûuence on the dis- ease development and progression, and on the treatment out- comes. There are some reports that attempt to identify ge- netic factors associated with periodontitis including polymorphisms of interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) genes. We were interested from the distribution of several genotypes of the cytokines: interleukin-6 - (G-174C) and (G-597A), lymphotoxin-α (A+252G), and tumour necrosis factor-al- pha (G-308A) in patients with chronic periodontitis. Aim: To investigate the association of chronic perio- dontitis with certain gene polymorphisms of interleukin-6 (IL-6), Lymphotoxin- α, and tumor necrosis factor-alpha (TNF-α). Material and methods: The study included 30 pa- tients with moderate or severe chronic periodontitis, and 10 persons with healthy periodontium. Total genomic DNA was extracted from the buccal epithelial cells. TNF-A (G-308A), IL-6 (G-174C), IL-6 (G-597A) and LT-A (A+252G) genes polymorphisms were analyzed by Polymerase chain reaction (PCR). Results: Outcomes showed a large variety in geno- type’s distribution in the investigated groups. No important difference was observed in the distribution of IL-6, TNF-α and LT-α genotypes between chronic periodontitis patients and controls in this study be reason of the small studied group. However, a signiûcant difference in the LT-α was ob- served – a prevalence of the genotype GG in patients with severe periodontitis. In relation with IL-6 (G-597A) and IL- 6 (G-174C) genotyping - in both of them in patients with se- vere periodontitis was occurred most frequently the genotype GG. In patients with periodontitis the frequency of genotype GG of TNF-α (G-308A) was significantly increased. Conclusion: The assessment IL-6 (G-597A) and IL- 6 (G-174C), and TNF-α (G-308A) revealed that genotype GG was moderate associated with chronic periodontitis in Bul- garian individuals. As a result of these findings we may sup- pose that the G allele may play an important role in the de- velopment and progression of periodontal disease in this population. The frequency of LT-A (A252G) was significantly greater in severe periodontitis patients in this study. Key words: gene polymorphism, interleukin-6, lymphotoxin- α, tumor necrosis factor-alpha, susceptibility, periodontitis. INTRODUCTION: At present it is known that pathogenic bacteria are the key factors for initiation of periodontal disease, but the host response and the severity of clinical expression are largely determined by genetic susceptibility and environmental fac- tors. The first study of cytokine gene polymorphism was re- ported by Kornman [1] who found a significant association between severe adult periodontitis and composite genotype, namely, allele 2 of a single nucleotide polymorphism (SNP) of IL-1A+4845 and IL-1B+3954 located on chromosome 2q13. Following this, several studies have been conducted exploring the role of IL-1 gene polymorphism as a severity factor in periodontitis in various population and ethnic groups [2-4]. There are some reports that attempt to identify genetic factors associated with periodontitis including polymorphisms of interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) genes [2, 4-9]. In our study we were interested from the distribution of several genotypes of the cytokines: interleukin-6 - (G- 174C) and (G-597A), lymphotoxin- α (A+252G), and tumor necrosis factor-alpha (G-308A) in patients with chronic peri- odontitis. AIM: To investigate the association of chronic periodontitis with certain gene polymorphisms of interleukin-6 (IL-6), Lymphotoxin-α, and tumor necrosis factor-alpha (TNF-α). MATERIALS AND METHODS: Forty patients - 30 patients with chronic periodonti- tis, and 10 - healthy subjects were included in the present study. Following the conventional periodontal examination clinical parameters - hygiene index, BoP (gingival bleeding index - bleeding on probing), PD (probing depth), CAL (clini- cal attachment loss) were recorded for the diagnosis. The sub- jects with periodontitis were devised into two groups of dif- http://dx.doi.org/10.5272/jimab.2015213.868

Transcript of EVALUATION OF INTERLEUKIN-6 ...€¦ · EVALUATION OF INTERLEUKIN-6, LYMPHOTOXIN-α AND TNF-α GENE...

Page 1: EVALUATION OF INTERLEUKIN-6 ...€¦ · EVALUATION OF INTERLEUKIN-6, LYMPHOTOXIN-α AND TNF-α GENE POLYMORPHISMS IN CHRONIC PERIODONTITIS. Velitchka Dosseva-Panova 1, Antoaneta Mlachkova,

868 http://www.journal-imab-bg.org / J of IMAB. 2015, vol. 21, issue 3/

EVALUATION OF INTERLEUKIN-6, LYMPHOTOXIN-ααααα AND TNF-ααααα GENE POLYMORPHISMS INCHRONIC PERIODONTITIS.

Velitchka Dosseva-Panova1, Antoaneta Mlachkova1, Christina Popova1, MayaKicheva2

1) Department of Periodontology, Faculty of Dental Medicine, Medical Universityof Sofia, Bulgaria.2) PhD, biochemist, Progene Ltd., Sofia, Bulgaria.

Journal of IMAB - Annual Proceeding (Scientific Papers) 2015, vol. 21, issue 3Journal of IMABISSN: 1312-773Xhttp://www.journal-imab-bg.org

SUMMARYBackground: Periodontitis is a chronic inflammatory

disruption of the periodontal supportive tissues. There are thenumerous evidences for his bacterial etiology. Though theoccurrence of periodontal bacteria is considered to be themain cause of periodontitis, certain characteristics of the in-dividual immune response may also have inûuence on the dis-ease development and progression, and on the treatment out-comes. There are some reports that attempt to identify ge-netic factors associated with periodontitis includingpolymorphisms of interleukin-1 beta (IL-1β), interleukin-6(IL-6) and tumor necrosis factor-alpha (TNF-α) genes. Wewere interested from the distribution of several genotypes ofthe cytokines: interleukin-6 - (G-174C) and (G-597A),lymphotoxin-α (A+252G), and tumour necrosis factor-al-pha (G-308A) in patients with chronic periodontitis.

Aim: To investigate the association of chronic perio-dontitis with certain gene polymorphisms of interleukin-6(IL-6), Lymphotoxin- α, and tumor necrosis factor-alpha(TNF-α).

Material and methods: The study included 30 pa-tients with moderate or severe chronic periodontitis, and 10persons with healthy periodontium. Total genomic DNA wasextracted from the buccal epithelial cells. TNF-A (G-308A),IL-6 (G-174C), IL-6 (G-597A) and LT-A (A+252G) genespolymorphisms were analyzed by Polymerase chain reaction(PCR).

Results: Outcomes showed a large variety in geno-type’s distribution in the investigated groups. No importantdifference was observed in the distribution of IL-6, TNF-αand LT-α genotypes between chronic periodontitis patientsand controls in this study be reason of the small studiedgroup. However, a signiûcant difference in the LT-α was ob-served – a prevalence of the genotype GG in patients withsevere periodontitis. In relation with IL-6 (G-597A) and IL-6 (G-174C) genotyping - in both of them in patients with se-vere periodontitis was occurred most frequently the genotypeGG. In patients with periodontitis the frequency of genotypeGG of TNF-α (G-308A) was significantly increased.

Conclusion: The assessment IL-6 (G-597A) and IL-6 (G-174C), and TNF-α (G-308A) revealed that genotype GGwas moderate associated with chronic periodontitis in Bul-garian individuals. As a result of these findings we may sup-pose that the G allele may play an important role in the de-

velopment and progression of periodontal disease in thispopulation. The frequency of LT-A (A252G) was significantlygreater in severe periodontitis patients in this study.

Key words: gene polymorphism, interleukin-6,lymphotoxin- α, tumor necrosis factor-alpha, susceptibility,periodontitis.

INTRODUCTION:At present it is known that pathogenic bacteria are the

key factors for initiation of periodontal disease, but the hostresponse and the severity of clinical expression are largelydetermined by genetic susceptibility and environmental fac-tors. The first study of cytokine gene polymorphism was re-ported by Kornman [1] who found a significant associationbetween severe adult periodontitis and composite genotype,namely, allele 2 of a single nucleotide polymorphism (SNP)of IL-1A+4845 and IL-1B+3954 located on chromosome2q13. Following this, several studies have been conductedexploring the role of IL-1 gene polymorphism as a severityfactor in periodontitis in various population and ethnic groups[2-4]. There are some reports that attempt to identify geneticfactors associated with periodontitis including polymorphismsof interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumornecrosis factor-alpha (TNF-α) genes [2, 4-9].

In our study we were interested from the distributionof several genotypes of the cytokines: interleukin-6 - (G-174C) and (G-597A), lymphotoxin- α (A+252G), and tumornecrosis factor-alpha (G-308A) in patients with chronic peri-odontitis.

AIM:To investigate the association of chronic periodontitis

with certain gene polymorphisms of interleukin-6 (IL-6),Lymphotoxin-α, and tumor necrosis factor-alpha (TNF-α).

MATERIALS AND METHODS:Forty patients - 30 patients with chronic periodonti-

tis, and 10 - healthy subjects were included in the presentstudy. Following the conventional periodontal examinationclinical parameters - hygiene index, BoP (gingival bleedingindex - bleeding on probing), PD (probing depth), CAL (clini-cal attachment loss) were recorded for the diagnosis. The sub-jects with periodontitis were devised into two groups of dif-

http://dx.doi.org/10.5272/jimab.2015213.868

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ferent disease severity based on CAL. Total genomic DNAwas extracted from the buccal epithelial cells. TNF-A (G-308A), IL-6 (G-174C), IL-6 (G-597A) and LT-A (A+252G)genes polymorphisms were analyzed by Polymerase chainreaction (PCR). Then, PCR amplified products were directlydigested with respective restriction enzymes (10U for Hin1IIand 20U for BseGI and NcoI) and the size controls and re-ceived bands was determined electrophoretically using 3%agarose gel (Thermo Scientific). The results obtained wereanalyzed statistically.

DNA extractionTotal genomic DNA was extracted from the buccal

epithelial cells using GeneJet Genomic DNA Purification Kit

(Thermo Scientific) according to the manufacturer’s instruc-tions for buccal swabs and stored at -20°C in 70 µl of theElution Buffer (Thermo Scientific).

Cytokine genotypingTNF-α (G-308A), IL-6 (G-174C), IL-6 (G-597A) and

LT-α (A+252G) genes polymorphisms were analyzed byPolymerase chain reaction (PCR) performed on the extractedDNA using Thermo Scientific Maxima Hot Start PCR Mas-ter Mix (2X), with the use of respective primer sets at thefinal concentration of 0,5 µM (Metabion International AG),that spanned the SNP sites, as described previously [2, 4, 5,6] and PCR conditions as listed in Table 1.

Table 1. Primers, RCR conditions and restriction enzymes for different gene polymorphisms.

Then, PCR amplified products were directly digestedwith respective restriction enzymes (10U for Hin1II and 20Ufor BseGI and NcoI). The size of both, the amplified DNAfragments (ND controls), and bands cleaved with restrictionenzymes, was determined electrophoretically using 3%

agarose gel (Thermo Scientific) stained with ethidium bro-mide (Sigma Aldrich) and Thermo Scientific GeneRuler LowRange DNA Ladder. Table 2 summarizes lengths of cleavedbands and respective polymorphisms.

Table 2. Different gene polymorphisms according to obtained DNA fragments.

RESULTSThe clinical parameters were analyzed among the se-

lected patients (Table 3). Significant correlation between HIand PD, CAL and bone loss was observed. Similar resultswith a significant importance between PD and CAL, PD and

PolymorphismPCR amplicon / Homozygous Homozygous

HeterozygousND control size allele 1 allele 2

TNF-A (G-308A) 117 bp GG: AA: GA:

97 bp + 20bp 117 bp 117 bp + 97 bp+ 20 bp

IL-6 (G-174C) 525 bp GG: CC: GC:

327 bp +169 bp 327 bp +122bp+47 bp 327 bp+ 169 bp+122 bp+47 bp

IL-6 (G-597A) 525 bp GG: AA: GA:

525 bp 468 bp+ 57 bp 525 bp+ 468 bp+ 57 bp

LT-A (A+252G) 368 bp AA: GG: GA:

368 bp 235 bp+133 bp 368 bp+ 235 bp+ 133 bp

Polymorphism Primers and PCR conditions Restriction Enzyme

TNF-A (G-308A) (F) 5’- AGG CAA TAG GTT TTG AGG GCC AT -3’ NcoI

(R) 5’- ACA CTC CCC ATC CTC CCG GCT-3’ (4h, 37°C)

95°C 4 min., (95°C 30 s; 60°C 30 s; 72°C 15 s) x 40, 72°C 5 min.

IL-6 (G-174C) (F) 5’- GGA GTC ACA CAC TCC ACC T -3’ Hin1II=Hsp92II

(R) 5’- CTG ATT GGA AAC CTT ATT AAG -3’ (4h, 37°C)

IL-6 (G-597A) 95°C 4 min., (95°C 30 s; 57°C 30 s; 72°C 30 s) x 35, 72°C 5 min. BseGI=FokI (4h, 55°C)

LT-A (A+252G) (F) 5’- CTC CTG CAC CTG CTG CCT GGA TC -3’ NcoI

(R) 5’- GAA GAC ACG TTC AGG TGG TGT CAT -3’ (4h, 37°C)

95°C 4 min., (95°C 30 s; 65°C 30 s; 72°C 30 s) x 35, 72°C 5 min.

Bone loss, and also between CAL and Bone loss was deter-mined in this study (Table 4 and Fig. 1).

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Table 3. Descriptive analysis.

Table 4. Correlation matrix (Rang – correlation analysis (Spearman))

! *Significant correlations

Fig.1. Relationships between different parameters.

HI% BoP PD Cal BoneLoss

Age 0,169 0,0213 0,111 0,148 -0,0257

0,368 0,91 0,556 0,433 0,892

30 30 30 30 30

HI% -0,262 -0,467* -0,432* -0,505*

0,16 0,00953 0,0174 0,00464

30 30 30 30

BoP 0,208 0,124 0,197

0,268 0,512 0,294

30 30 30

PD 0,815* 0,807*

0 0

30 30

Cal 0,778*

0

30

Description Average Standartvalues Median deviation Variety Minimum Maximum Number

Age Av 43,08 43,50 14,29 52,00 18,00 70,00 43,08

HI% Av 4,38 0,00 9,07 30,00 0,00 30,00 40

BoP Av 73,63 100,00 43,36 100,00 0,00 100,00 40

PD Av 3,82 3,65 0,76 3,30 2,80 6,10 30

CAL Av 4,45 4,20 1,13 4,30 3,10 7,40 30

Bone loss 3,12 2,65 1,40 5,50 1,50 7,00 30

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Results showed a large variety in genotype’s distri-bution in the investigated groups and are presented on Table2 and Fig. 1-4.

Fig. 3. 3% agarose gel electrophoresis of RFLP with ethidium bromide staining of IL-6 (G-597A): Lane 1 – NDcontrol; Lanes 7 and 11 – ladder; Lanes 2, 3, 4, 6, 10, 14 – heterozygous GA genotype – two fragments (525+468); Lanes9, 12, 13 – homozygous GG for allele 1 with one fragment (525); Lanes 5 and 8 -homozygous AA for allele 2 with twofragments (468+57).

Fig. 2. 3% agarose gel electrophoresis of RFLP with ethidium bromide staining of LT-A (A252G): Lanes 1, 6 and16 - 100 bp ladder (L); Lanes 2, 5, 7, 11, 12 – heterozygous AG genotype with 3 fragments after restriction (368 +235+133)length of the PCR product; ND – lane 10 – non digested control; Lanes 3, 8, 13, 14 – homozygous AA for allele 1 with onefragment (368); Lanes 4, 9, 15 – homozygous GG for allele 2 with two fragments (235+133).

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Fig. 4. 3% agarose gel electrophoresis of RFLP with ethidium bromide staining of IL-6 (G-174C): Lanes 2 and 7 –ND control; Lanes 1 and 13 – ladder; Lanes 5, 7, 8, 10, 11, 15 – heterozygous GC genotype – 4 fragments (327+169+122+47);Lanes 3, 4, 6, 10 – homozygous GG for allele 1 with 2 fragments (327+169); Lane 14 - homozygous CC for allele 2 with 3fragments (327+122+47).

Fig. 5. 3% agarose gel electrophoresis of RFLP with ethidium bromide staining of TNF-A (G-308A): Lane 1– NDcontrol; Lane 8– ladder; Lanes 3-7, 9 and 13 – heterozygous GA genotype – 3 fragments (117+97+20); Lanes 10 and 12 –homozygous GG for allele 1 with 2 fragments (97+20); Lane 11 - homozygous AA for allele 2 with one fragment (117).

Genotyping results:IL-6 (G-174C) – in the control group was lack of

genotype GG. In patients with severe periodontitis this geno-type occurred most frequently.

IL-6 (G-597A) – in the control group was lack ofgenotype GG. In patients with severe periodontitis this geno-type occurred most frequently.

LT-A (A252G) – in the control group was detectedonly genotype AG. In patients with chronic periodontitis were

detected both AA and GG genotypes (in patients with severeperiodontitis most frequently - genotype GG).

TNF-A (G-308A) – in the control group most fre-quently was observed genotype GA (70% of cases). In pa-tients with periodontitis the frequency of genotype GG wassignificantly increased. Only in one patient with severe peri-odontitis was observed genotype AA.

All results are presented on Table 5.

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Assesment of the predictive value:The regression analysis - Backward Elimination for

prognosis verification was applied. The results obtained inthis study showed that the periodontitis severity may be pre-

The combination of this genotype with the others of(IL-6 (G-174C), IL-6 (G-597A) and TNF-A (G-308A)) hasn’texceeded the total precision of the model.

Following model has showed that the severe periodon-titis have more frequently LT-A (A252G) AG genotype,whereas AA genotype was related with the moderate peri-odontitis.

DISCUSSION:The current data show there are also investigations that

attempt to identify genetic factors associated with periodon-titis including polymorphisms of interleukin-1 beta (IL-1β),interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) genes. [10-16]

In this study our aim was to determine the distribu-tion of TNF-A (G-308A), IL-6 (G-174C), IL-6 (G-597A) and

LT-A (A+252G) genes polymorphisms in patients withchronic periodontitis. Interestingly the literature data havepublished mainly results obtained in several small and con-flicting studies. [4, 5, 12, 14, 15, 17] Yoshie et al. reviewedthe role of IL-6 polymorphism in periodontitis [12] andfounded a correlation between IL-6 (G-174C) and chronicperiodontitis susceptibility in Brazilian Caucasian populationbut not in Czech Caucasians. The studies of Brett et al., Babeland al. and Tervonen et al. showed correlation of IL-6 (G-174C) with susceptibility to chronic periodontitis [13, 14, 18].Still more the investigation of Tervonen et al. was focusedon polymorphism in the gene encoding IL-6, especially theIL-6-174 GG genotype, which is associated with prevalenceof periodontal disease in patients with moderate and severeperiodontitis. [18] This is in agreement with the results ob-tained in our study and this genotype was not presented in

dicted base on LT-A (A252G) genotype. The total accuracyof conducted predictive model based on logistic regressionwas 70%.

Table 6. Classification table:

Predicted cases Correctly predicted

cases in %

Cases Severity 1 Severity 2

Severity 1 7 5 58.3%

Severity 2 4 14 77.8%

Total accuracy 70%

Table 5. Distribution of genotypes in different patients groups

Control group Patients with Patients with P -(healthy) moderate periodontitis severe periodontitis coefficient

n % n % n %

Genotypes 10 12 18

IL-6 (G-174C)

CC 2 20.0% 1 5.6%

GC 8 80.0% 8 66.7% 8 44.4%

GG 4 33.3% 9 50.0% 0.05

IL-6 (G-597A)

AA 2 20.0% 1 5.6%

GA 8 80.0% 8 66.7% 8 44.4%

GG 4 33.3% 9 50.0% 0.05

LT-A (A252G)

AA 7 58.3% 4 22.2%

AG 10 100.0% 4 33.3% 12 66.7%

GG 1 8.3% 2 11.1% 0.02

TNF-A (G-308A)

AA 1 5.6%

GA 7 70.0% 4 33.3% 5 27.8%

GG 3 30.0% 8 66.7% 12 66.7% 0.20

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1. Kornman KS, Crane A, Wang HY,di Giovine FS, Newman MG, Pirk FW,et al. The interleukin-1 genotype as aseverity factor in adult periodontal dis-ease. J Clin Periodontol. 1997 Jan;24(1):72–7. [PubMed] [CrossRef]

2. Papapanou PN, Neiderud AM,Sandros J, Dahlen G. Interleukin-1 genepolymorphism and periodontal status.A

case-control study. J Clin Periodontol.2001 May;28(5):389–96. [PubMed][CrossRef]

3. McDevitt MJ, Wang HY, Knobel-man C, Newman MG, di Giovine FS,Timms J, et al. Interleukin-1 genetic as-sociation with periodontitis in clinicalpractice. J Periodontol. 2000 Feb;71(2):156–63. [PubMed] [CrossRef]

4. Archana PM, Salman AA, KumarTS, Saraswathi PK, Panishankar KH,Kumarasamy P. Association betweeninterleukin-1 gene polymorphism andseverity of chronic periodontitis in asouth Indian population group. J IndianSoc Periodontol. 2012 Apr-Jun;16(2):174-178. [PubMed] [CrossRef]

5. Costa AM, Guimaraes MC, de

control group. In the present study was found an increasedpresence of IL-6 (G-174C) G/G genotype similarly to the re-sults of Costa et al. In accordance to the other investigatorswe may conclude that G homozygous subjects (GG) were sig-nificantly more affected by periodontal disease than individu-als who carry the C allele that was a great distribution inhealthy individuals [5].

On the other hand, the Czech investigation suggestedthat the gene polymorphism IL-6 -572 may be regarded as aprotective factor for the disease [17].

Regarding gene polymorphism of IL-6 -597 our re-sults showed a prevalence of IL-6 (G-597A) GA genotypein healthy group and in moderate periodontitis group andthence may be related with the protective factors associatedwith lower susceptibility to chronic periodontitis.

There exist several investigation that support carriageof TNF-A (G-308A)*2 polymorphism with the severity ofperiodontitis in Caucasian population [10, 11]. Controversy,Fassman et al. demonstrated that TNF-A (G-308A) polymor-phism have not association with chronic periodontitis inCzech population, whereas the LT-A (A252G) genotypeshave statistically significant differences between periodonti-tis cases and healthy controls similarly to our study results.Therefore, according to the authors the LT-α gene may beconsidered an informative marker and it may be one of theprotective genetic factors versus chronic periodontitis in ourpopulation [15].

As regard with the possible link between genetic vari-ants and their expresion, the polymorphisms in TNF-α wereconsidered to be risk factors in periodontitis. There are sev-eral clinical studies that address to this suggestion. However,the results obtained from these studies are controversial [5,10, 11, 14-16, 19-22].

As regards to TNF-A (G-308A) gene polymorphismin the present study G homozygous subjects were signifi-cantly more affected by periodontal disease than individualswho carry the A allele. These results also are in accordancewith the results obtained in the study of Costa et al. [5].

From the review of Laine et al. it become clear thatno gene polymorphism has, as yet, been definitely to be arisk factor for chronic periodontitis susceptibility. The expla-nation of this may be that small sample size studies, as inour study, are greatly underpowered since most associations

have small ods ratio’s (1,1-1,5) and greatly contribute to therisk for false positive or negative outcomes [23].

In our study we investigated the IL-6 polymorphisms,at positions -597 and -174, TNF-α polymorphism at posi-tion -308, and LT-α polymorphism at position -252. No im-portant difference was observed in the distribution of IL-6,TNF-α and LT-α genotypes between chronic periodontitispatients and controls in this study be reason of the smallstudied group. However, a signiûcant difference in the LT-α was observed - a prevalence of the genotype GG in pa-tients with severe periodontitis. In relation with IL-6 (G-597A) and IL-6 (G-174C) genotyping - in both of them inpatients with severe periodontitis was occurred most fre-quently the genotype GG. In patients with periodontitis thefrequency of genotype GG of TNF-α (G-308A) was signifi-cantly increased. In addition, based on the results of thisstudy and on the available literature, there are limited datato support associations between TNF-α gene polymorphismat position -308, IL-6 polymorphisms at positions -597 and-174, and LT-α polymorphism at position -252 and chronicperiodontitis. On the other hand, the assessment IL-6 (G-597A) and IL-6 (G-174C), and TNF-α (G-308A) revealedthat genotype GG was moderate associated with chronicperiodontitis in Bulgarian individuals. As a result of theseûndings we may suppose that the G allele may play an im-portant role in the development and progression of periodon-tal disease in this population. The obtained results are inagreement with these of received in other investigations andauthors and published in the specialized literature [5, 13,14, 18, 22].

CONCLUSION:The frequency of LT-α (A252G) was significantly

greater in severe periodontitis patients in this study. How-ever our investigation was limited on the number of sub-jects included. For that reason we may present our resultsonly as a tendency or prevalence in determined genotypesof investigated genes without an important significance. Atpresent, the results obtained in this study suggest that fur-ther studies are needed on gene polymorphisms of the ma-jor cytokines that are known to be involved in the protec-tive-destructive immune response in periodontal diseases fordefinite conclusion.

Acknowledgements:This work was supported by the Medical University of Sofia (grant No. 16/2014).

REFERENCES:

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Address for correspondence:Velitchka Dosseva-Panova,Department of Periodontology, Faculty of Dental Medicine, Medical University– Sofia,1, George Sofiiski Str., 1431 Sofia, Bulgaria.E-mail: [email protected]

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Please cite this article as: Dosseva-Panova V, Antoaneta Mlachkova A, Popova Ch, Kicheva M. Evaluation of interleukin-6, Lymphotoxin-α and TNF-α gene polymorphisms in chronic periodontitis. J of IMAB. 2015 Jul-Sep;21(3):868-875.DOI: http://dx.doi.org/10.5272/jimab.2015213.868

Received: 21/06/2015; Published online: 15/09/2015