Analysis of association between TLR-4 Asp299Gly and Thr399Ile gene polymorphisms and chronic periodontitis in a sample of south Indian population.

BACKGROUND: To analyze the association between TLR-4 Asp299Gly and Thr399Ile gene polymorphisms and chronic periodontitis in a sample of south Indian population.
MATERIALS AND METHODS: Genomic DNA was obtained from peripheral blood of 60 patients with chronic periodontitis and 60 periodontally healthy subjects. TLR-4 Asp299Gly and Thr399Ile gene polymorphisms were genotyped by a polymerase chain reaction-restriction fragment length polymorphism method. The data were analyzed by a χ(2)-test and by relative risk estimation.
RESULTS: Thr399Ile alleles were found in 4% of chronic periodontitis patients and in 1% of periodontally healthy subjects. The prevalence of a Thr399Ile heterozygote was found to be 5% in the chronic periodontitis group and 1.67% in the periodontally healthy group, respectively. Homozygosity for TLR-4 Thr399Ile was seen in chronic periodontitis patients only, which was 1.67%. The TLR-4 Asp299Gly gene polymorphism was not detected in either chronic periodontitis or periodontally healthy groups.
CONCLUSION: There is no significant association between TLR-4 Thr399Ile polymorphism and chronic periodontitis in a sample of south Indian population.

INTRODUCTION

Periodontitis is a chronic inflammatory disease of complex multifactorial etiology. Although the presence of microbes especially Gram negative anaerobes is essential in initiating and perpetuating periodontal diseases, environmental as well as genetic factors contribute to individual variation in the susceptibility to disease.[12]

Periodontal destruction is a multistage process consisting of microbial infection, host immune response, tissue destruction, and repair.[34] Risk factors can affect any of these stages through genetic and environmental interactions. The innate immune system is involved in the recognition of conserved pathogen associated with molecular patterns present on the pathogens[5] and recognition of these molecules is facilitated by a group of receptors called Toll-Like Receptors (TLRs).[6] Among the 10 human TLRs identified so far, TLR-2 and TLR-4 are the most defined members.[7] TLR-4 has been shown to specifically recognize the lipopolysaccharide (LPS) of Gram negative bacteria which work in cooperation with several protein components such as LPS-binding protein (LBP) and CD14[8] and this leads to activation of inflammatory cells via the nuclear factor-Kappa beta pathway (NF-KB) which results in the synthesis and release of proinflammatory cytokines, there by augmenting the local inflammatory response.[9]

Hence TLR-4 provides a critical link between the factors produced by pathogens and initiation of host defense. TLR-4 gene polymorphism may be an important factor in susceptibility to bacterial infections and may there by influence the inflammatory process.

Two common gene polymorphisms in the extracellular domain of the TLR-4 are ASP299Gly and Thr399Ile polymorphisms which have been shown to be associated with functional changes that predispose people to be less responsive to LPS, attenuate the TLR signaling pathway and leading to a blunted inflammatory response thus have an increased risk of susceptibility to pathogenic bacterial infections.[10]

Several studies[11—13] have investigated the role of TLR-4 gene polymorphism in patients with periodontitis and have found association between chronic periodontitis and TLR-4 gene polymorphism. As frequency of polymorphisms and gene environment interactions differ among populations, the risk of a particular gene variant for a given genetic trait might be different in different population. Hence the aim of this study is to evaluate association between the TLR-4 Asp299Gly and Thr399Ile gene polymorphisms and chronic periodontitis in a south Indian population. The objectives of this study were to determine the gene and genotype frequencies of TLR-4 polymorphisms among chronic periodontitis patients and controls in south Indian population and to estimate the risk of developing chronic periodontitis among the different genotypes of TLR-4.

MATERIALS AND METHODS

The study employed a cross-sectional design involving individuals belonging to the Dravidian race from the state of Tamil Nadu in the southern part of India. A total of 120 individuals who reported to Department of Periodontics were included in this study. The subjects were stratified into a chronic periodontitis group (n=60) and a healthy group (n=60). The chronic periodontitis group included 29 males and 31 females ranging in age from 30 years to 55 years with a mean of 36.61 ± 5.2. They had >30% of teeth exhibiting clinical attachment loss and a probing pocket depth of ≥5 mm. The control group participants (30 males and 27 females) ranging in age from 30 years to 55 years with a mean of 37 ± 5.1 were included and did not present a history of previous periodontal disease as determined by the absence of clinical attachment loss, gingival inflammation, and no sites with a probing depth of ≥3 mm. Smokers, subjects with a history of cardiovascular disorders, diabetes, malignant disease, immunodeficiency, and subjects with a previous history of periodontal surgery were excluded. Informed consent was obtained from all subjects.

Genomic DNA preparation

Five milliliters of venous blood was collected by vein punctures and dispersed into a 15 ml sterile centrifuge tube containing a pinch of ethylenediaminetetraacetic acid (EDTA). It was mixed thoroughly to avoid clot formation. It was stored at 4°C until further processing.

Human genomic DNA was isolated from the leukocytes using the modified Miller's 1998 protocol. First red blood cell (RBC) lysis was done using the buffer NH4Cl (0.155 M) and Tris base (0.17 M). White blood cell (WBC) lysis was done using Tris–HCl (1 M), disodium EDTA (0.5 M), NaCl (1 M), and double distilled water. A total of 500 μl of proteinase K and 200 μl of sodium dodecyl sulfate were added to the tube. It was incubated for 16 h in a water bath at 37°C. A total of 1 ml of 6 M NaCl was added and vigorously shaken for 30 s. The tube was centrifuged at 3000 rpm for 20 min and 4 ml of supernatant was transferred to another fresh tube. Double the volume of ice-cold ethanol was added and tilted once or twice. The DNA fibers were transferred to a 1.5 ml centrifuge tube and the DNA was dissolved in 200 μl of TE buffer. It was left at room temperature for an hour and the sample was stored at –20°C.

Polymerase chain reaction and enzyme digest

Determination of the TLR-4 gene polymorphism was accomplished with PCR and restriction fragment length polymorphism by the method of Lorenz et al.[14] The primers for TLR-4 Asp299Gly were forward 5’- GATTACATACTTAGACTACTACCTCGA-3’ and reverse 5’-GATCAACTTCTGAAAAAGCATTCLACC-3’. For Thr399Ile primers were forward 5’-ATTGCTGTTCTCAAAGTGATTTTGGGACCAA-3’ and reverse 5’-CCTG AAGACT GGAGA GTGAGTTAAATGCT-3’. Amplification was carried out in a Eppendorf thermal cycler. The cycling conditions comprise initial denaturation at 95°C for 3 min, followed by 35 amplifications cycles at 95°C for 30 s, 65°C for 30 s and 72°C for 30 s, followed by one elongation step at 72°C for 4 min.

The digest reaction was set up using restriction enzyme NcoI (for Asp299Gly) and HinfI (for Thr399Ile). It is incubated at 37°C for 4 h and electrophoresed in 3% agarose gel to identify the TLR-4 alleles on the basis of the respective allele size. After digestion, wild type TLR-4 allele sizes of 249 bp for the Asp299Gly and 406 bp for the Thr399Ile will not change but fragment sizes for carriers of polymorphic allele will decrease to 23 bp for the Asp299Gly residue and 26 bp for the Thr399Ile residue [Figures 1 and 2].

Figure 1

The 3% agarose gel electrophoregram showing the restriction digestion patterns of The399Ile polymorphisms of TLR-4 gene 4 Thr using the HinfI enzyme. Lanes 1, 2, and 7: genotype CC (homozygous normal). Lane 5: genotype TT (homozygous mutant). Lanes 3 and 6: genotype CT (heterozygous mutant)

Figure 2

The 3% agarose gel electrophoregram showing the restriction digestion patterns of Asp299Gly polymorphism of TLR-4 gene using the NcoI enzyme. All lanes show genotype AA (homozygous normal)

Statistical analysis

The statistical analysis of comparison between a periodontitis group and a healthy group with regard to genotypes was done by the χ2-test. Risk estimation between the genotypes was analyzed using relative risk.

RESULTS

In this research study, the allele and genotype frequencies of TLR-4 gene polymorphisms were compared between chronic periodontitis and control groups and risk of getting chronic periodontitis among different genotypes of TLR-4 was estimated.

In this study, a total of 120 subjects who came to Department of Periodontics were selected. This comprises 60 healthy subjects and 60 patients who had chronic periodontitis. In the chronic periodontitis group, 29 (48.33%) were males and 31 (51.67%) were females whereas in the healthy group, 33 (55%) were males and 27 (45%) were females. The mean age group was 36.65 ± 5.22 in the chronic periodontitis group whereas it was 37.04 ± 5.16 in the healthy group [Table 1].

Table 1

Characteristics of the study groups

The results for TLR-4 Thr399Ile have shown that C allele frequency was 96% in the chronic periodontitis group and 99% in the healthy group whereas T allele frequency was 4% in the chronic periodontitis group and 1% in the healthy group. For CT genotype (heterozygous mutant) frequency was found to be 5% in the chronic periodontitis group and 1% in the healthy group whereas TT genotype (homozygous mutant) frequency was found only in 1.67% the chronic periodontitis group [Table 2]. The results for TLR-4 Asp299Gly did not show any polymorphic mutants, so AA genotype (homozygous normal) frequency was found to be 100%.

Table 2

Genotype distributions and allele frequencies of the Thr399Ile polymorphism in the TLR- 4gene of chronic periodontitis and healthy groups

In this study, there was no significant difference among chronic periodontitis groups and healthy groups in TLR-4 Thr399Ile genotype frequencies (χ2= 2.08, P=0.35). Also, allele frequencies did not show any significant difference among chronic periodontitis and healthy groups for TLR-4 Thr399Ile gene polymorphism (χ2 = 27, P=0.2) [Tables 3 and 4]. When the genotype CC was compared to genotype CT, there was no statistically significant difference between chronic periodontitis groups and healthy groups (P=0.3). There was also no statistical significant difference between chronic periodontitis groups and healthy groups when the genotypes CC compared with TT and CT compared with TT (with P=0.5 and 1).

Table 3

Comparing chronic periodontitis and healthy groups with genotypes distribution for Thr399Ile polymorphism using χ2 tests

Table 4

Comparing chronic periodontitis and healthy groups with alleles for Thr399Ile polymorphism using χ2 tests

Estimation of relative risk of getting periodontitis between the genotypes showed that the risk of getting disease is 1.54 times more for CT when compared to CC, with 95% confidence interval {0.85–2.80} which is statistically insignificant. Whereas risk getting disease is 2.05 times more for TT when compared to CC genotype and 1.3 times more for TT when compared to CT which is also statistically insignificant [Table 5].

Table 5

Estimation of relative risk (RR) of getting chronic periodontitis between the genotypes

DISCUSSION

Periodontitis is a chronic multifactorial disease with strong genetic components. Gene polymorphisms that modulate host response to the microbial challenge have been associated with different clinical forms of periodontitis.[15] Several studies have shown that allelic variation in genes encoding molecules of the host defense system such as cytokines could affect the susceptibility and progression of periodontal disease.[16] Because TLR-4 provide a critical link between factors produced by pathogens and initiation of host defense, TLR-4 gene polymorphism may be important factors in susceptibility to bacterial infection and there by influence the inflammatory process. Two common gene polymorphisms in the extracellular domain of the TLR-4 are Asp299Gly and Thr399Ile polymorphisms which have been shown to be associated with functional changes that predispose people to be less responsive to LPS, attenuate the TLR signaling pathway and leading to a blunted inflammatory response and thus have an increased risk of susceptibility to pathogenic bacterial infections.[10]

Several studies have investigated the role of TLR-4 gene polymorphism in patients with periodontitis. In these, studies by Schröder et al.[11] in German population, Brett et al.[12] in Great Britain and by Fukusaki et al.[13] in Japanese population found association between chronic periodontitis and TLR-4 gene polymorphism. As frequency of polymorphisms and gene environment interactions differ among populations, the risk of a particular gene variant for a given genetic trait might be different in different population. Hence, the aim of this study was to evaluate association between the TLR-4 Asp299Gly and Thr399Ile gene polymorphisms and chronic periodontitis in south Indian population. Objectives were to determine the gene and genotype frequencies of TLR-4 polymorphisms among chronic periodontitis patients and controls in south Indian population and to estimate the risk of getting chronic periodontitis among the different genotypes of TLR-4.

In this study for TLR-4 Thr399Ile the gene polymorphism shows that the distribution of allele C was more compared to allele T (mutant variety). The frequency of occurrence of allele T (mutant variety) in chronic periodontitis is 4% and that of the healthy group is 1%. The results of this study are comparable to studies by Folwaczny et al.[17] and Laine et al.[18] who also reported that T allele frequency was 4.5% and 5%, respectively, in chronic periodontitis groups. In contrast, a study by Izakovicova et al.[19] in Czech population reported that T allele frequency was 93% in chronic periodontitis groups and this higher frequencies of T allele compared with other population are considered to reflect some ethnic difference in the relative frequencies of the TLR-4genotype in Czech population.

In this study, most of the polymorphic mutants are heterozygous. Even though the occurrence of heterozygous mutant variety (CT genotype) was detected in both the groups, it is less compared to homozygous normal (CC genotype). The homozygous normal (CC genotype) frequency was found to be 93% in the chronic periodontitis group and that of the healthy group is 98% whereas heterozygous mutant (CT genotype) frequency was found to be 5% in the chronic periodontitis group and 1.67% in the healthy group. These results are in agreement with the studies by Berdeli et al.[20] and Brett et al.[12] who also reported that heterozygous mutant (CT genotype) was 3% and 7% in chronic periodontitis groups. Another important finding in this study is less occurrence of homozygous mutants (TT genotype) with 1% in the study population group correlating with the study by Laine et al.[18] but in contrast to the study by Izakovicova[19] reported that homozygous mutant (genotype) frequency was 88% in Czech population.

In this study, genotype and allele frequencies of the Thr399Ile polymorphisms in both groups of healthy and chronic periodontitis subjects compared using χ2 showed no significant difference among chronic periodontitis groups and healthy groups in TLR-4 Thr399Ile genotype frequencies (χ2 = 2.08, P=0.35). Also, allele frequencies did not show any significant difference among chronic periodontitis and healthy groups for TLR-4 Thr399Ile polymorphisms (χ2 = 2.7, P=0.2). This result was in accordance with studies by Folwaczny et al.,[17] Izakovicova et al.[19] and Laine et al.[18] who also found that there is no significant difference among chronic periodontitis and healthy groups in TLR-4 Thr399Ile genotype and allele frequencies. In contrast to this, study by Schröder et al.[11] reported significant association between chronic periodontitis groups and TLR-4 Thr399Ile gene polymorphisms conducted in German population (P=0.002). Here the frequency of TLR-4 Thr399Ile gene polymorphism was found to be higher in patients with chronic periodontitis (18.97%) than that of healthy subjects (5.17). These contradictory results may be due to difference in allele and genotype frequencies in various ethnic populations.

In this study, when the genotype CC was compared with genotype CT, there was no statistically significant difference between chronic periodontitis groups and control groups (P>0.05). There was also no statistical significant difference between chronic periodontitis groups and control groups when the genotypes CC compared with TT and CT was compared with TT.

When calculating relative risk, we found that risk of getting disease is 1.54 times more for CT compared to CC. Whereas risk of getting disease is 2.05 times more for TT compared to CC and 1.33 times more for TT when compared to CT.

Another important finding in this study is that the results for TLR-4 Asp299Gly gene polymorphisms did not show any polymorphic mutants. These results were in accordance with the study by Okayama et al.[21] and Hang et al.[22] who reported that Asp299 Gly polymorphism in TLR-4 is not present in Japanese and Chinese population. Thus, TLR-4 Asp299Gly gene polymorphism seemed to be rather low in the south Indian population, which does not permit any conclusion regarding its effect on periodontitis.

The biological mechanism behind the association between TLR-4 gene polymorphisms and periodontal disease lies in the hypothesis that gene polymorphism of TLR-4 has been shown to be associated with functional changes that predispose people to be less responsive to LPS, with decreased NF-κB activity and proinflammatory cytokine production and leading to blunted inflammatory response, thus increasing the risk of susceptibility to pathogenic bacterial infections.[102324]

Thus, the results of this study have clearly shown that there is no significant association between Thr399Ile gene polymorphisms and chronic periodontitis as far as south Indian population is concerned. Thus, the findings from this study question the usefulness of using TLR-4 Thr399Ile gene polymorphisms as a screening test for chronic periodontitis. However, extensive studies in large groups of patients and also in other ethnic populations should be undertaken in order to analyze the putative relevance of the TLR-4 gene polymorphism in the pathogenesis of periodontitis.