Single nucleotide polymorphisms in IL-1A RS1800587, IL-1B RS1143634 and vitamin D receptor rs731236 in stage III grade B/C periodontitis

As a chronic multifactorial infectious disease, periodontitis has been defined as one of the main public health problems with its high prevalence and it has been shown to lead to disability and tooth loss, impairment on aesthetic and chewing function, a presumed negative efect on general health, social inequality, and impaired quality of life [1]. It is described as microbially originated, host mediated inflammation resulting in periodontal attachment loss and, ultimately, tooth loss [2]. The etiopathogenetic mechanisms of periodontitis have not been completely discovered. Although the primary etiology of the disease is the pathogenic anaerobic bacteria found in the subgingival dental plaque; it has been shown that host susceptibility and the genetic background of individuals play substantial roles in the onset and development of the disease, in particular, with a subset of genes predominantly believed to be part of the pathological processes [3]. Researchers have completed studies on genetic association regarding the polymorphism analysis to further clarify the role of each of these genes. Polymorphisms may play a part in the presentation and/or outcome of diseases. This can also be done through conferring a degree of risk or protection from the disease.

Gene polymorphism, namely interleukin (IL)-1, is the most pronounced gene polymorphism studied in patients with periodontal diseases [4]. IL-1A and IL-1B, as members of IL-1 family, are the proinflammatory cytokines that are expressed in response to cell damage and impact cell differentiation, proliferation, and apoptosis [5]. Particularly, single nucleotide polymorphism (SNP) in position IL-1A −889 and in position IL-1B +3953 (also known as +3954) are the most investigated SNPs. In 1997, Kornman et al. [6] reported on the simultaneous presence of both IL-1A −899 and IL-1B +3953 minor alleles, known as “composite genotype”, and an elevated severity of chronic periodontitis in a subset of non‐smoker Caucasian subjects. According to the findings of a meta-analysis, comprised of 27 studies published from 1997 to 2008, 19 of these studies found a correlation between the carriage of minor IL‐1 alleles and periodontitis [7]. Controversial findings have also been reported in studies where no association has been found between rs1143634 and rs1800587 gene polymorphisms and chronic and/or aggressive periodontitis [8, 9, 10, 11, 12]. Da Silva et al. [13] reported a meta-analysis from 21 case-control research studies, published between 1998-2015, and uncovered an association between periodontitis and rs1800587. Another meta-analysis of 54 case/control studies, containing 9376 subjects, showed that rs1143634 polymorphism was linked to a high risk of chronic periodontitis in both the overall analysis and different ethnicities, such as Asian and Caucasian [14].

The vitamin D receptor (VDR) is present in many cells of the immune system, such as in monocytes and dendritic cells, as well as in bone cells such as osteoblasts [15] and osteoclasts. Beyond its established function in bone homeostasis, vitamin D also has significant anti-inflammatory and immunomodulatory properties [16]. Genetic polymorphisms in the VDR gene have been shown to be linked to bone mineral density and also bone-associated diseases [17,18]. Results of studies examining the association between periodontitis and VDR gene polymorphisms at the ApaI, BsmI, FokI, and TaqI restriction sites or their combinations are somewhat controversial [19, 20], and it is not clear which of the VDR polymorphisms are effective in periodontal disease susceptibility. A meta-analysis involving 15 studies, reported that VDR TaqI, ApaI and BsmI gene polymorphisms were associated with chronic periodontitis in Asians, yet not in Caucasians, whereas there was no relationship between chronic periodontitis and FokI polymorphism [21]. However, another meta-analysis of 18 studies concluded that chronic periodontitis linked to only TaqI polymorphism in Asians [22]. A more recent meta-analysis concluded that no significant associations were present between TaqI polymorphism and chronic periodontitis in Caucasian, Asian, African and mixed populations [23].

Until now, the effects of the genetic factor on different forms of periodontal disease have been investigated in several studies, whereas the results obtained by different researchers vary. This study hypothesized that IL-1A (rs1800587), IL-1B (rs1143634) and VDR (rs731236) gene polymorphisms may confer susceptibility to periodontitis. Thus, the purpose of the present study was to determine the frequencies of IL-1A (rs1800587), IL-1B (rs1143634) and VDR (TaqI, rs731236) gene polymorphisms in Stage III Grade B/C periodontitis patients in the Turkish population.

Table 1 demonstrates demographic data and clinical features of the periodontitis patients and the healthy subjects. All periodontal clinical parameters in the patients with periodontitis were significantly higher comparison to the healthy individuals, as expected (p<0.000).

In Table 2, the distributions of polymorphisms in all study populations agreed to the Hardy–Weinberg equilibrium (p > 0.05), except IL-1B (rs1143634) in the healthy group (X²=7.75; p < 0.05). Comparisons of genotype frequencies between the periodontitis patients and healthy individuals are presented in Table 2. IL-1A (rs1800587) and IL-1B (rs1143634) polymorphisms did not show any association with periodontitis (OR=1.142, p=0.793, OR=1.726, p=0.371, respectively). Considering the VDR TaqI gene polymorphism, the frequencies of TT genotype in the periodontitis and healthy subjects were 36% and 49%, respectively, and CC genotype 19% and 10%, respectively. The results showed that the genotype CC was associated with a 2.586 times higher risk of periodontitis with a statistically significant risk coefficient (p=0.031).

The allelic distributions of each gene polymorphisms are presented in Table 3. Allele frequency regarding IL-1A (rs1800587) demonstrated no significant differences between the healthy individuals and the periodontitis patients. The C allele in IL-1B (rs1143634) was detected more frequently in healthy individuals than patients (84.5% versus 76.5% OR=1.167, p=0.045). In VDR TaqI gene polymorphism, the C allele frequency in the periodontitis patients (83%) was higher than in the healthy subjects (61%). The C allele was statistically significant in the periodontitis patients with an OR of 1.61 (95% CI= 1.071–2.441, p=0.022).

Model wise genotypic distributions regarding co-dominant, dominant, recessive and overdominant models are presented in Table 4. Model wise genotypic distribution of IL-1A (rs1800587) did not demonstrate any association with periodontitis (Table 4). Regarding the IL-1B (rs1143634) polymorphic region, the frequency of CC genotype was higher in healthy subjects and associated with a decreased risk of periodontitis under dominant model [OR (95% CI) =0.527 (0.289-0.960), p=0.035]. Likewise, VDR TaqI gene polymorphism showed alliance with periodontitis, on the other hand, the dominant, recessive and overdominant models did not demonstrate any relation with periodontitis (Table 4).

Associations between different grades of periodontitis and gene polymorphisms in IL-1A (rs1800587), IL-1B (rs1143634) and VDR TaqI are demonstrated in Table 5. Both allele and genotype frequency of IL-1A (rs1800587) gene polymorphism did not show any association with any grade of periodontitis (p>0.05). The C and T alleles frequencies of IL-1B (rs1143634) polymorphism were 76 (74.5%) and 26 (25.5%) in the Grade B periodontitis, 77 (78.6%) and 21 (21.4%) in the Grade C periodontitis and 169 (84.5%) and 31 (15.5%) in the healthy subjects, respectively. The difference was found to be statistically significant only between the Grade B periodontitis and healthy subjects [OR=1.865; 95% CI (1.037-3.355), p=0.036]. A comparison of the VDR TaqI genotypes and alleles between the Grade B periodontitis and healthy subjects revealed that the TT genotype, and T allele were more frequently observed in the healthy subjects (TT vs. CC, TT vs. CT, T vs. C, p˂0.05). In the evaluation of TaqI polymorphism in Grade B and C periodontitis, individuals with the CT genotype have a 3.207 times higher risk of Grade B periodontitis than individuals with the TT genotype. [CT vs. TT, OR (95% CI) =3.207 (1.286-7.997), p=0.011].

Recent evidence has shown that, with the continuous advancement of genetic engineering technology, gene polymorphism can be an important basis for the individual diferences in the development and progression of periodontitis. In this study, the frequencies of IL-1A (rs1800587), IL-1B (rs1143634) and VDR (rs731236) gene polymorphisms were evaluated for the first time in Stage III Grade B/C periodontitis patients in comparison with periodontally healthy individuals. Among different genotyping methods, Real-time PCR that could rapidly and simultaneously detect SNPs was preferred in this study.

SNPs from the promoter region, which play an essential role the transcriptional regulation in the coding region, can afect the expression of a gene. The genes in IL-1 family possess allele polymorphisms in which SNP -889C/T (rs1800587) in IL-1A gene and SNP +3654 (rs1143634) in IL-1B gene are researched extensively. Our findings revealed that the genotypic distributions of IL-1A (rs1800587) and IL-1B (rs1143634) gene polymorphisms are not associated with Stage III periodontitis. These results are consistent with the previous studies reporting no relationship between SNPs rs1800587 or rs 1143634 and neither chronic nor aggressive periodontitis [8,27, 28, 29, 30, 31, 32, 33]. On the other hand, contradictory findings were published declaring significant associations between either of the two SNPs and periodontal diseases [29, 34, 35, 36, 37, 38, 39]. Majumder et al. [40] and Wagner et al. [41] concluded that IL-1A (rs1800587) polymorphism was related to the susceptibility of chronic periodontitis. Recently, a meta-analysis of ethnicity assessment revealed that IL-1A (rs1800587) gene polymorphism is associated with an increased risk of chronic periodontitis in Europeans, US Americans and Africans, but not in Algerians and Mexicans [42]. The results of the present study displayed T allele for IL-1B (rs1143634) to be associated with the periodontitis patients, while the C allele was associated with the healthy individuals. Similarly, T allele in IL-1B (+3954C/T) was linked to high risk of periodontal disease in Asians, Caucasians, and in mixed populations, however not in Africans [14]. Considering that these differences may be related to the population, the literature contains no data on the Turkish population regarding IL-1A (-889C/T) and only 2 research studies have been published about IL-1B (+3954C/T). Yücel et al. [43] stated that the distribution of allele and genotype frequencies for IL-1B (+3954C/T) were similar among chronic periodontitis, aggressive periodontitis and healthy control groups. On the other hand, our result was contrary to the results of Güzeldemir et al. [37] who show that susceptibility to localized aggressive periodontitis is increased by homozygosity for allele 1 of rs1143634 which referred to (C→T) at position +3954 in the fifth exon of the IL-1B gene. In the new classification, localized aggressive periodontitis may refer to the Stage III or IV and Grade C. It is thought that different outcomes can be obtained depending on the size (31 healthy subjects and 31 localized aggressive periodontitis patients) and the different status of the patients based on the new classification of periodontal diseases.

The VDR, a ligand-controlled transcription factor, intervenes the actions of the vitamin D hormone 1,25-di-hydroxyvitamin D3 to alter bone mineral homeostasis. VDR TaqI gene polymorphism is positioned at exon 9. The current study demonstrated that increased TT genotype and T allele of the VDR TaqI polymorphism in healthy subjects were associated with the decreased risk of periodontitis. Similar to the present study, De Brito et al. [19] reported that the patients with any form of C (formerly t) allele (Tt or tt genotypes) were 2.4 times more susceptible to chronic periodontitis than the patients who lacked C allele. However, Gunes et al. [44] contradicted this finding and suggested that TaqI polymorphisms of the VDR gene were not associated with severe generalized chronic periodontitis in the Turkish population studied. In two meta-analyses published in 2011 [21] and 2012 [22], no association was observed in the Caucasian population, while chronic, but not aggressive, periodontitis cases, in patients among the Asian population were reported to have a weak, significantly higher frequency of the TT genotype and T allele of TaqI. The findings of a recently published meta-analysis, in contrast with the above meta-analyses, reveal a link between periodontitis susceptibility and the VDR TaqI polymorphism in Caucasian patients under the dominant model without any apparent correlation in Asian participants [45]. The frequency TaqI alleles may vary among different populations. It has been shown that the T allele frequency of the VDR TaqI polymorphism is lowest among Asians, about of the 8% population, and highest in Caucasians, at 43% [46]. The genetic susceptibility of a subject to periodontitis due to polymorphism might change on account of the ethnicity of the population.

Genetic factors are of great importance in identifying the host’s immune response to infection and could account for significant variation in the severity, distribution, and extension of the disease [47, 48, 49]. The grade of periodontitis is predicted by direct or indirect evidence of the progression rate of in three levels: Grade A (slow progression), Grade B (moderate progression) and Grade C (rapid progression) [2]. With the given facts, the present study investigated whether SNPs rs1800587, rs1143634 and rs731236 might be related with the progression of periodontal disease in a group of the Turkish population. In our study, healthy individuals and Grade B and Grade C Stage III periodontitis patients were evaluated and compared. T allele of the rs1143634 was linked to patients having Grade B Stage III periodontitis. Our finding was in accordance with da Silva et al. [14], who revealed that T allele of the rs1143634 was related with an increased risk of periodontal disease in Asians, Caucasians and in a mixed population. The outcome of the present study demonstrated that rs731236 (TaqI) polymorphism is significantly associated with Grade B Stage III periodontitis, but not Grade C, in comparison with healthy individuals. In the assessment of associations between the grading of periodontitis and gene polymorphism, TaqI polymorphism CT genotype is higher in Grade B periodontitis patients. In a research study conducted by Chantarangsu et al. [50], with a homogeneous Thai population, the VDR gene polymorphism was examined according to the severity of chronic periodontitis (no/mild, moderate and severe). The study revealed that there is no relationship between TaqI and periodontitis. To the authors’ knowledge, this is the first study evaluating susceptibility to periodontitis according to Grade and Stage approach in the classification of periodontitis. A limitation of our study may be the absence of other stages or grades of periodontitis.

In conclusion, CC genotype and C allele of TaqI polymorphism were associated with enhanced susceptibility to Stage III periodontitis in a Turkish study group. Furthermore, VDR TaqI polymorphism may be useful to discriminate between Grade B and Grade C in Stage III periodontitis. Identifying genetic risk factors according to the stage and grade of periodontitis will improve our knowledge about the pathophysiology and can potentially be used in defining individuals at risk of developing disease and predicting its progression rate.