IL4 gene polymorphisms and their relation to periodontal disease in a Macedonian population

Human Immunology 72 (2011) 446 – 450

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IL4 gene polymorphisms and their relation to periodontal disease in a Macedonian population Aneta Atanasovska-Stojanovska a, Dejan Trajkov b, Salvador Nares c, Nikola Angelov d, Mirko Spiroski b,* a

Dental Clinical Center, Department of Oral Pathology and Periodontology, Faculty of Stomatology, University ”Ss. Kiril and Metodij”, Skopje, Republic of Macedonia Institute of Immunobiology and Human Genetics, Faculty of Medicine, University ”Ss. Kiril and Metodij”, Skopje, Republic of Macedonia c Department of Periodontology, School of Dentistry, University of North Carolina, Chapel Hill, North Carolina, USA d Department of Periodontics, School of Dentistry, Loma Linda University, Loma Linda, California, USA b

A R T I C L E

I N F O

Article history: Received 16 April 2010 Accepted 22 February 2011 Available online 25 February 2011

Keywords: Periodontitis IL4 gene Gene polymorphism Genetics

A B S T R A C T

Genetic polymorphisms in the interleukin-4 (IL4) gene have been reported to influence the host response to microbial challenge by altering levels of cytokine expression. We analyzed nucleotide polymorphisms in the promoter region of the IL4 gene and its relation with periodontal disease in a Macedonian population. The study population consisted of 92 unrelated subjects with chronic periodontitis and 286 healthy controls. DNA was isolated and IL4 genotyping performed by polymerase chain reaction–single-strand polymorphism (Heidelberg kit) for the alleles and genotypes of IL4 ⫺1098, IL4 ⫺590, and IL4 ⫺33. Frequencies of IL4 haplotypes and the haplotype zygotes were also examined. Comparisons between groups were tested using the Pearson’s p value. After Bonferroni adjustment, significant associations were detected between subjects with periodontitis and the following: (1) cytokine alleles IL4 ⫺1098 and IL4 ⫺33; (2) cytokine genotypes IL4 ⫺1098/G:T; IL4 ⫺1098/T:T, and IL4 ⫺33/T:T, (3) cytokine haplotypes IL4/GCC, IL4/TCC, and IL4/TTC; and (4) cytokine haplotype zygotes IL4/TTC: TCC, IL4/TCT:TTT, and IL4/GCC:TTC. Cytokine polymorphism on the IL4 gene appears to be associated with susceptibility to chronic periodontitis in Macedonians. 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

1. Introduction Periodontal diseases are infectious, inflammatory disorders strongly influenced by behavioral, environmental, and genetic variables. These patient factors individualize the host response to invading periodontal pathogens and significantly contribute to the clinical manifestation of disease. In this context, an increasing body of evidence points to genetic determinants as a subject variable with considerable impact on susceptibility, severity, as well as treatment responsiveness [1–5]. Because the host is primarily responsible for the tissue destruction in response to periodontal pathogens [6,7], the study of genetic factors with an impact on immune function has garnered considerable interest. Studies indicate that cytokine gene expression is influenced by genetic polymorphisms and these variations appear to be linked with progression of disease [5,8,9]. In particular, polymorphisms in the interleukin-4 (IL4) gene may have a significant impact on the host response. In human beings, the IL4 gene maps to the long arm of chromosome 5 (q31–33) within a cluster of other cytokine genes, including including IL5, IL12, and IL13 [10]. IL-4 has been shown to stimulate B-cell proliferation, to regulate immunoglobulin class switching (IgG1 and IgE), and to promote T-cell development [11].

* Corresponding author. E-mail address: [email protected] (M. Spiroski).

Furthermore, IL-4 is considered an anti-inflammatory cytokine that can modulate macrophage function [12] and induce apoptosis in monocytes [13]. Thus, genetic polymorphisms in the IL4 gene may increase periodontal disease severity by altering IL-4 levels. Indeed, polymorphisms in the IL4 gene have been implicated in the regulation of IL-4 production, and studies suggest that the ⫺590 C/T*T allele, which is in tight linkage disequilibrium with the ⫺33 C/T*T allele, is associated with increased IL-4 expression [14,15]. Considering these findings and the established ethnic differences in the distribution of genetic variants, we analyzed three single-nucleotide polymorphisms [(⫺1098, ⫺590 in the promoter (regulatory) region, and ⫺33 in the 5=UTR region of the IL4 gene (on location 5q 31.1) and its relation with periodontal disease in a Macedonian population. 2. Subjects and methods Healthy subjects were recruited from the patient pool at the Clinic for Oral Diseases and Periodontology, University Clinical Centre of Stomatology, Skopje, Macedonia [16 –18]. All subjects were at least 20 years of age, had at least 20 teeth present, were from Macedonian ethnic background to the third generation, and were unrelated residents from different geographic regions of the Republic of Macedonia. Subjects were excluded if they had any systemic disease, were pregnant, current smokers, or taking medications known to affect host immunity (e.g., steroids, immunosup-

0198-8859/11/$32.00 - see front matter 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.02.005

A. Atanasovska-Stojanovska et al. / Human Immunology 72 (2011) 446 – 450

pressants). The periodontitis group consisted of 92 subjects, aged 38.97 ⫾ 10.12 years, previously diagnosed with moderate or severe chronic periodontal disease according to established criteria [19 – 21], whereas the healthy control group consisted of 286 subjects, aged 35.20 ⫾ 9.90 years, displaying no sites with a probing depth ⬎3 mm, no clinical attachment loss (CAL) ⬎2 mm, gingival LoeSilnes Index (GI) score of 1, and bleeding on probing (BOP) 6.8% ⫾ 6.4%. The study was approved by the Committee of the Ministry of Education and Science from the Republic of Macedonia, as well as the Ethical Committee of the Medical Faculty in Skopje [22] and was part of the Cytokine Polymorphisms and Expression, 15th International Histocompatibility and Immunogenetics Workshop (IHIWS). 2.1. Testing polymorphism on IL4 gene Genomic DNA was isolated by the phenol-chloroform [23] method from peripheral leukocytes and samples stored in the Macedonian bank for human DNA as previously described [24]. IL4 gene polymorphisms were determined using the polymerase chain reaction–single-strand polymorphism (PCR-SSP; Heidelberg kit, Cytokine genotyping Tray, Invitrogen, GmbH, Karlsruhe, Germany) at the Institute for Immunobiology and Human Genetics at the Faculty of Medicine in Skopje [16,25]. Fourteen cytokine genes with 22 single nucleotide polymorphisms were typed: IL1alpha ⫺889, IL1beta ⫺511, IL1beta ⫹3962, IL1R psti1970, IL1RA mspa11100, IL4Ralpha ⫹1902, IL12 ⫺1188, IFNgammautr5644, TGFbeta1 cdn10, TGFbeta1 cdn25, TNFalpha ⫺308, TNFalpha ⫺238, IL2 ⫺330, IL2 ⫹166, IL4 ⫺1098, IL4 ⫺590, IL4 ⫺33, IL6 ⫺174, IL6 565, IL10 ⫺1082, IL10 ⫺819, and IL10 ⫺592. Briefly, the PCR-SSP typing Heidelberg kit consists of 48 PCR primer mixes aliquoted in 96-well PCR trays (two typings per tray). Master mix, which was supplied along with the reagents and consisted of MgCl2, buffer, dNTPs, and glycerol, was mixed with 1.2 to 3.0 ␮g of DNA and 20 U of Taq polymerase (GE Healthcare, Vienna, Austria) and dispensed in the 48 wells. The amplified products were separated by 2% agarose gel electrophoresis, stained with 0.5 ␮g/ml ethidium bromide and visualized by ultraviolet exposure. 2.2. Statistical analysis The population genetics analysis package PyPop, developed by the Biostatistics Core for the Workshop [26 –28], was used for analysis of the IL4 data for this report. Allele frequencies and expected Hardy–Weinberg proportions (HWP) for each IL4 allele were determined [29]. The exact test for genotype frequency deviation from HWP was calculated using the Arlequin implementation accessed via PyPop [30]. Alleles that did not fit HWP were evaluated to determine whether there was an excess of homozygotes or heterozygotes, or if any particular genotypes were significantly different from expected frequencies by the ␹2 test. The Ewens– Watterson homozygozity test of neutrality (EWN) [31] with Slatkin’s exact p values [32,33] was used to indicate any deviations from the hypothesis of neutral selection for each locus. Pearson’s p values, crude odds ratios (ORs), and Wald’s 95% confidence intervals (CIs) were calculated for associations’ analysis between IL4 (alleles, genotypes, haplotypes, and diplotypes) and periodontal disease with GraphPad QuickCalc free statistical calculators (http:// www.graphpad.com/quickcalcs/) with Bonferroni-corrected p value [34]. Values of p ⬍ 0.05 were taken as significant. 3. Results Clinical and demographic data for our Macedonian study populations are shown in Table 1. As predicted, the values of the clinical parameters GI, CAL, and BOP were higher in the periodontitis group than in the healthy controls. Observed versus expected cytokine genotypes for each SNP, HWP, and Guo and Thompson Hardy–Weinberg output (GTHWO) in healthy subjects and subjects with periodontal disease is given in

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Table 1 Demographic and periodontal characteristics of the Macedonian study population

Age (y), mean ⫾ SD Female Male Loe-Silnes Index (GI) Bleeding on probing, % Clinical attachment loss

Chronic periodontitis (n ⫽ 92)

Control group (n ⫽ 286)

p Value

38.97 ⫾ 10.12 41.90% 58.10% 2.38 ⫾ 0.67 82.52 ⫾ 8.14 5.18 ⫾ 0.716

35.20 ⫾ 9.90 54.60% 46.40% 0.5 ⫾ 0 6.8 ⫾ 6.4 1.8 ⫾ 0.3

NS NS NS ⬍0.001 ⬍0.001 ⬍0.001

GI, gingivitis index; n, number of participants; NS, nonsignificant; SD, standard deviation.

Table 2. Several observed frequencies of cytokine genotypes in the healthy population were significantly different from the expectations: IL4 ⫺1098/G:T, IL4 ⫺1098/T:T, IL4 ⫺590/C:C, IL4 ⫺590/C:T, and IL4 ⫺33/T:T. In subjects with periodontal disease only IL4 ⫺33/T:T genotype was significantly different from expectations. Several SNPs (IL4 ⫺1098, IL4 ⫺590, and IL4 ⫺33 in healthy subjects and IL4 ⫺33 in subjects with periodontal disease) were not in HWP (p ⬍ 0.05), and GTHWO was significant (p ⬍ 0.05) (Table 2). DNA samples from our 92 periodontitis subjects and 286 healthy controls were analyzed for polymorphisms in the promoter (⫺1098, ⫺590) and 5= UTR (⫺33) region of the IL4 gene. Our results (Table 3) indicate significant differences (p ⬍ 0.001) in the distribution of alleles between the two groups at the IL4-1098 gene polymorphism after Bonferroni adjustment. Exponents of the T allele were associated with the periodontitis group than were exponents of the G allele (OR ⫽ 0.229, 95% CI ⫽ 0.135– 0.389). For the IL4 ⫺33 gene polymorphism, after Bonferroni adjustment, there was a significant difference in distribution of C and T alleles (p ⬍ 0.001, OR ⫽ 0.422, 95% CI ⫽ 0.288 – 0.618) in that the T allele was present in the periodontitis group, more so than in healthy controls. No significant differences in the distribution of alleles at the IL4 ⫺590 gene polymorphism were noted (p ⫽ 0.234) (Table 3). Homozygosity for the T allele at position ⫺1098 was seen in 82.6% of subjects with periodontitis compared with 38.8% of healthy subjects; and 1.1% of periodontitis subjects were homozygous for the G allele compared with 0.4% of healthy subjects (Table 3). The distribution of the IL4 ⫺1098/G:T and IL4 ⫺1098/T:T genotypes between groups was significantly different after Bonferroni adjustment (p ⬍ 0.001, OR ⫽ 0.125, 95% CI ⫽ 0.069 – 0.229, and p ⬍ 0.001, OR ⫽ 7.489, 95% CI ⫽ 4.154 –13.499, respectively). At the ⫺590 position, 44.6% of periodontitis subjects were homozygous for the C allele compared with 33.2% of healthy subjects, whereas 3.3% and 1.4% of diseased and healthy subjects, respectively were homozygous for the T allele. The frequency of the ⫺590C:C and ⫺590C:T genotypes just failed to reach statistical significance after Bonferroni adjustment (p ⫽ 0.058 and 0.023, respectively; Table 3). Homozygosity for the C allele at position ⫺33 was noted in 63% of periodontitis subjects compared with 73.1% of healthy subjects, but this difference failed to reach significance (p ⫽ 0.06). For the T allele, 26.1% vs. 5.6% of periodontitis and healthy subjects, respectively, were homozygous for this allele, and this difference was significant after Bonferroni adjustment (p ⬍ 0.001, OR ⫽ 5.956, 95% CI ⫽ 2.999 –11.829; Table 3). The IL4/GCC, IL4/TCC, and IL4/TCT haplotypes were correlated with significant differences in distribution between groups after Bonferroni adjustment (Table 4). The IL4/GCC haplotype was more commonly present in healthy subjects (p ⬍ 0.001). Conversely, the IL4/TCC and IL4/TCT haplotypes were observed more frequently in the periodontitis group (p ⬍ 0.001, OR ⫽ 1.773, 95% CI ⫽ 1.267–2.480, and p ⬍ 0.001, OR ⫽ 22.484, 95% CI ⫽ 7.712– 65.551, respectively). Table 5 shows the distribution of haplozygotes between groups. Of the three genotypes with statistically significant differences

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Table 2 Observed versus expected cytokine genotypes for each single nucleotide polymorphism, Hardy–Weinberg proportions (HWP), and Guo and Thompson Hardy–Weinberg output (GTHWO) in a Macedonian population Polymorphism Control IL4 ⫺1098

IL4 ⫺590

IL4 ⫺33

Periodontitis IL4 ⫺1098

IL4 ⫺590

IL4 ⫺33

Genotype

Observed number

Observed frequency

Expected number

p Value

HWP p value

GTHWO p value

G:T T:T G:G C:C C:T T:T C:C C:T T:T

174 111 1 95 187 4 209 61 16

0.608 0.388 0.004 0.332 0.654 0.014 0.731 0.213 0.056

121.8 137.1 27.1 124.2 128.5 33.2 200.6 77.9 7.6

⬍0.001 ⬍0.001a

⬍0.001

⬍0.001

0.009 ⬍0.001a

⬍0.001

⬍0.001

0.551 0.558 0.002

⬍0.001

⬍0.001

G:T T:T G:G C:C C:T T:T C:C C:T T:T

15 76 1 41 48 3 58 10 24

0.163 0.826 0.011 0.446 0.522 0.032 0.630 0.109 0.261

15.4 75.8 0.8 38.2 45.9 7.9 43.1 39.7 9.1

0.913 0.980a

0.911

0.558

0.013

0.021

⬍0.001

⬍0.001

0.467 0.111 0.080 0.024 ⬍0.001 ⬍0.001

Cannot be calculated because expected to be ⬍5 (␹2 test).

a

accumulation of activated macrophages releasing copious amount of proinflammatory mediators. Indeed, human studies have linked reduced gingival and serum IL-4 levels with periodontal disease [40 – 43] and resorption of alveolar bone [44,45], and IL4 mRNA levels were also reported to be significantly repressed in diseased compared with healthy peri-implant tissues [46]. Numerous studies have investigated the relationship between periodontal disease and genetic polymorphisms within the promoter region of IL4; however these studies have yielded conflicting results likely resulting from a number of different factors, including the study population, number of subjects, and variables tested (SNPs vs haplotypes) [16,45,47–54]. Two polymorphisms within the IL4 gene, C:T polymorphism on position ⫺590 and at the IL4 70-bp repeat polymorphism in intron 2, were reported in subjects with aggressive periodontitis [45]. Contrary to these findings, our study and those of others [48,48,51,53,54] failed to demonstrate any statistical differences in the frequency of the IL4 ⫺590 allele in subjects with chronic periodontitis. However, we noted a frequency of 90% for the IL4 ⫺1098 T allele in chronic periodontitis subjects, compared with 69% for controls, whereas the IL4 ⫺33 T allele was present in 31% of subjects with periodontitis compared

after Bonferroni adjustment, two (TTC: TCC, TCT: TTT) were observed more frequently in periodontitis subjects (p ⬍ 0.001, OR ⫽ 5.413, 95% CI ⫽ 2.033–14.413, and p ⬍ 0.001, OR ⫽ 19.583, 95% CI ⫽ 6.491–59.085, respectively). Conversely, the GCC:TTC genotype was more likely to be prevalent in healthy subjects (p ⬍ 0.001, OR ⫽ 0.169, 95% CI ⫽ 0.079 – 0.363). Of note, the IL4/TTC:TCC and IL4/TCT: TTT genotypes were linked with a 5.41 and 19.58 times greater risk for periodontal disease occurrence, respectively. For nine haplozygotes the ␹2 test was not calculated because the expected frequency was less than 5 (Table 5). 4. Discussion We examined the allelic, genotypic, and haplotypic polymorphisms along the promoter region of the IL4 gene in a relatively homogeneous population of Macedonian subjects with and without periodontal disease. IL-4 is a Th2-derived, pleomorphic cytokine with anti-inflammatory properties [12] recognized as a modulator of macrophage function that downregulates CD14 levels [35] and inhibits secretion of PGE2, IL-1␤, IL-6, and tumor necrosis factor (TNF)–␣ (36 –38). IL-4 also induces apoptosis in macrophages and monocytes [13,39]. Thus, genetic variations within the promoter region may affect transcriptional regulation of IL-4, resulting in an

Table 3 Distribution of allele and genotype polymorphism of the IL4 gene in a Macedonian population of patients with periodontal disease, compared with control subjects Polymorphism

IL4 ⫺1098 IL4 ⫺590 IL4 ⫺33 IL4 ⫺1098

IL4 ⫺590

IL4 ⫺33

Allele or genotype

Periodontitis n

F (%)

Control n

F (%)

G T C T C T G:T T:T G:G C:C C:T T:T C:C C:T T:T

17 167 130 54 126 58 15 76 1 41 48 3 58 10 24

9.2 90.8 70.7 29.3 68.5 31.5 16.3 82.6 1.1 44.6 52.2 3.3 63.0 10.9 26.1

176 396 377 195 479 93 174 111 1 95 187 4 209 61 16

30.8 69.2 65.9 34.1 83.7 16.3 60.8 38.8 0.4 33.2 65.4 1.4 73.1 21.3 5.6

CI, confidence interval; F, frequency of alleles or genotypes; n, number of alleles or genotypes; OR, odds ratio. a Statistically significant after Bonferroni adjustment (p value ⫻ number of alleles or genotypes) ⬍0.05.

OR

Wald 95% CI

Pearson’s p value

0.229

0.135–0.389

⬍0.001a

1.245

0.868–1.787

0.234

0.422

0.288–0.618

⬍0.001a

0.125 7.489 3.132 1.585 0.577 2.376 0.628 0.450 5.956

0.069–0.229 4.154–13.499 0.194–50.578 0.983–2.556 0.359–0.930 0.522–10.820 0.382–1.034 0.220–0.919 2.999–11.829

⬍0.001a ⬍0.001a 0.396 0.058 0.023 0.249 0.066 0.024 ⬍0.001a

A. Atanasovska-Stojanovska et al. / Human Immunology 72 (2011) 446 – 450

Table 4 Distribution of haplotype polymorphism at the IL4 gene in Macedonian population patients with periodontal disease, compared with control Haplotype

GCC GCT GTC GTT TCC TCT TTC TTT

Periodontitis

Control

n

F

n

F

16 0 0 1 90 25 20 32

0.087 0 0 0.005 0.489 0.136 0.109 0.174

163 8 4 1 202 4 110 80

0.285 0.014 0.007 0.002 0.353 0.007 0.192 0.140

OR

Wald 95% CI

Pearson’s p value

0.139–0.412

⬍0.001a

b

b

b

b

b

b

b

b

b

1.773 22.484 0.517 1.305

1.267–2.480 7.712–65.551 0.311–0.859 0.834–2.044

⬍0.001a ⬍0.001a 0.010 0.243

0.239

CI, confidence interval; F, frequency of haplotypes; n, number of haplotypes; OR, odds ratio. a Statistically significant after Bonferroni adjustment (p value ⫻ number of haplotypes) ⬍0.05. b Cannot be calculated because expected ⬍5 (␹2 test).

with 16% of controls. These alleles may be associated with the occurrence of chronic periodontal disease in Macedonians. We identify an associative finding of one other polymorphism of the IL4 cytokine gene (SNPs ⫺1098) which have not been previously reported in periodontitis subjects. Our study revealed a significantly higher frequency of the G:T IL4-1098 genotype in healthy subjects, indicating a possible protective role. Interestingly, although carriage of the C:T IL4 ⫺33 genotype was associated with healthy subjects, this finding is in contrast to the highly significant relation of allele T with the occurrence of periodontitis (p ⬍ 0.001). Furthermore, we demonstrate that exponents of the homozygote T:T genotype was associated with a 5.956 times greater risk of periodontitis, emphasizing an associative role of IL4 ⫺33/T allele with periodontitis in our cohort of Macedonian subjects. However, our findings relating the C:T IL4 ⫺590 genotype in healthy subjects (protective in relation to periodontitis) is in contrast to those reported for Brazilian and Iranian populations [49,55]. Considering that populations of different ethnicities may have different allele/ genotype frequencies, and also considering that many studies that enrolled ethnically different populations point to a similar association of a polymorphism with a disease, these facts can be interpreted as growing evidence of the role of this polymorphism as a genetic marker of the disease [56]. Gonzales et al. [50] failed to demonstrate any differences based on racial background when comparing the frequency of the IL4 ⫺590C-T polymorphism in Japanese and Caucasian subjects with aggressive periodontitis. Similarly, Pontes et al. [51] did not find any racial differences after investigating the association of the IL4 ⫺590C-T polymorphism with chronic periodontitis in Brazilians of Afro-American and non– Afro-American descent. Haplotype GCC was correlated with healthy subjects and may have a protective role in relation to periodontal disease. By contrast, bearers of the TCC and TCT haplotypes were more likely to be in the periodontitis group (OR ⫽ 1.77 and 22.48, respectively). This is in concordance with the finding for the allele distribution of IL4 polymorphism, where exponents of T allele on position ⫺1098 and ⫺33 were linked with a significant association with periodontitis in our study population (p ⬍ 0.001). The distribution of genotypes in our study showed the strongest association with periodontitis within the TCT:TTT (OR ⫽ 19.58, 95% CI ⫽ 6.49 –59.08) and TTC:TCC (OR ⫽ 5.41, 95% CI ⫽ 2.03–14.41) genotypes. These data are also in accordance with our findings demonstrating a significant association of the TCT and TCC haplotypes with periodontal disease, and point to a possible role of these haplotypes in periodontal pathogenesis. In contrast, the GCC: TTC genotype demonstrated an apparent protective role for periodontitis in our Macedonian population (Table 5). In agreement, Holla et al. [57] recently examined differences in IL4 alleles, genotypes, and haplotypes in a Czech popula-

449

tion, and more recently Anovazzi et al. [58] in Brazilian population, concluding that there was an association between IL4 haplotypes and chronic periodontitis. Thus, it is likely that the interaction of multiple genetic markers within a haplotype appears to be a major determinant of disease susceptibility [57,59]. Moreover, the expression of cytokines within tissues, including IL4 expression, will be influenced by host–microbe interactions and gene-gene interactions that may increase or decrease the chance of disease onset [57]. Our study is subject to limitations. After Bonferroni correction of p values, we found smaller number of alleles, genotypes, haplotypes, and diplotypes to be associated with periodontal disease in Macedonians. Several types of multiple testing corrections are used: Bonferroni; Bonferroni step-down (Holm); Westfall and Young Permutation; and Benjamini and Hochberg false discovery rate (FDR) [34,60]. The methods are listed in order of their stringency, with the Bonferroni being the most stringent, and the Benjamini and Hochberg FDR being the least stringent. The more stringent a multiple testing correction, the less false-positive genes are allowed. The trade-off of a stringent multiple testing corrections is that the rate of false negatives (alleles, genotypes, haplotypes, and diplotypes that are called nonsignificant when they are) is very high. Thus, the inclusion of Bonferroni correction of p values in this report implies a higher likelihood of false negatives with subsequently less significant associations with periodontal disease. Moreover, all SNPs in healthy participants and IL4 ⫺33 in patients with periodontal disease were not in HWP (p ⬍ 0.001), and we should be very careful about their associations with periodontal disease. Our sample size (N ⫽ 92) is similar to, or larger than, that in other published reports [3,41,54] investigating cytokine polymorphisms and will certainly benefit by including a larger study population. Furthermore, we did not directly measure GCF or serum IL-4 levels and their association with periodontitis and genetic differences. Nevertheless, we report evidence of an association between the IL4 ⫺1098, IL4 ⫺33, and IL4 ⫺590 haplotype and haplotype zygosity and chronic periodontitis in Macedonians. At this point, there is insufficient literature to support a definitive claim regarding this association, but it can clearly be designated as a goal for further research.

Table 5 Percentage of haplozygotes (diplotypes) of polymorphism of the IL4 gene in Macedonian population patients with periodontal disease, compared with control subjects Genotype

GCC:TCC TCC:TCC TTT:TCC TTC:TCC GCC:TTT TCT:TTT GCC:GCC GCT:TTT GCC:TTC TTT:TTT GTC:TCC GTT:TCC TCT:TCT TTC:TCT GTT:GCC

Periodontitis Control

OR

n

F (%)

n

F (%)

7 32 8 11 0 20 0 0 8 2 0 0 2 1 1

7.6 34.8 8.7 12.0 0.0 21.7 0.0 0.0 8.7 2.2 0.0 0.0 2.2 1.1 1.1

26 68 28 7 32 4 1 8 103 4 4 1 0 0 0

9.1 23.8 9.8 2.4 11.2 1.4 0.3 2.8 36 1.4 1.4 0.4 0 0 0

0.823 1.710 0.878 5.413

Wald 95% CI

Pearson’s p value

0.345–1.965 1.029–2.842 0.385–1.999 2.033–14.413

0.661 0.037 0.756 ⬍0.001a

b

b

b

19.583

6.491–59.085

⬍0.001a

b

b

b

b

b

b

0.079–0.363

⬍0.001a

b

b

b

b

b

b

b

b

b

b

b

b

b

b

b

b

b

b

0.169

CI, confidence interval; F, frequency of haplotypes; n, number of haplotypes; OR, odds ratio. a Statistically significant after Bonferroni adjustment (p value ⫻ number of diplotypes) ⬍0.05. b Cannot be calculated because expected to be ⬍5 (␹2 test).

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Acknowledgments This research is part of the project “Molecular analysis of cytokine gene polymorphisms in the Republic of Macedonia” supported by the Ministry of Education and Science from Republic of Macedonia (Project No. 13-874/3-05). We would like to gratefully acknowledge Prof. G. Opelz and Dr J. Mytilineos from the Institute of Immunology, Department of Transplantation Immunology, University of Heidelberg, Heidelberg, Germany for kindly supplying the Heidelberg PCR-SSP kit reagents in this project. For sample collection, technical support, and laboratory direction, we thank Elena Cvetkovska. References [1] Michalowich BS, Diehl SR, Gunsolley JC, et al. Evidence of a substantial genetic basis for risk of adult periodontitis. J Periodontol 2000;71:1699 –707. [2] De Santis M, Zuccelli G. Interleukin 1 gene polymorphisms and long term stability following guided tissue regeneration therapy. J Periodontol 2000;71: 606 –13. [3] Cattabriga M, Rotundo R, Muzzi L, et al. 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