1

Atanasovska-Stojanovska A, Trajkov D, Popovska M, Spiroski M. IL10 -1082,

IL10 -819 and IL10 -592 polymorphisms are associated with chronic

periodontitis in a Macedonian population. Hum Immunol. 2012 Jul;73(7):753-

8. Epub 2012 Apr 23. PubMed PMID: 22537751.

IL10 -1082, IL10 -819 and IL10 -592 polymorphisms are associated with

chronic periodontitis in a Macedonian population

Aneta Atanasovska-Stojanovska1, Dejan Trajkov2, Mirjana Popovska1, Mirko

Spiroski2*

1Dental Clinical Center, Department of Oral Pathology and Periodontology,

Faculty of Stomatology, University "Ss. Cyril and Methodius", Skopje, Republic of

Macedonia; 2Institute of Immunobiology and Human Genetics, Faculty of

Medicine, University "Ss. Cyril and Methodius", Skopje, Republic of Macedonia

Running title: IL10 gene polymorphisms and chronic periodontitis

*Corresponding Author:

Mirko Spiroski, MD, PhD. Institute of Immunobiology and Human Genetics, Faculty of Medicine, University "Ss. Cyril and Methodius", 1109 Skopje, PO Box 60, Republic of Macedonia. Tel.: +389-2-3110556. Fax: +389-2-3110558. URL: http://www.iibhg.ukim.edu.mk. E-mail: mspiroski@yahoo.com

Conflict of interest and source of funding statement

2

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 declare we have no conflict of interest.

3

Abstract

Genetic polymorphisms in the interleukin 10 (IL10) 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 IL10 gene and its relation with periodontal disease in a Macedonian

population. The study population consisted of 111 unrelated subjects with chronic

periodontitis and 299 healthy controls. DNA was isolated and IL10 genotyping

performed by PCR-SSP (Heidelberg kit) for the alleles and genotypes of IL10 -

1082, IL10 -819 and IL10 -592. Frequencies of IL10 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 chronic periodontitis and IL10

genotypes (IL10 -1082/A:G was negative or protective and IL10 -1082/G:G was

positive or susceptible). Cytokine polymorphism on the IL10 gene appears to be

associated with susceptibility to chronic periodontitis in Macedonians.

Key words: chronic periodontitis; IL10 gene; gene polymorphism; genetics.

4

1. Introduction

Chronic periodontitis like many other common diseases (e.g.,

cardiovascular diseases, diabetes, Crohn’s disease) is considered to be a

complex multifactorial disease.. These diseases are of relative late of onset (i.e.,

postjuvenile or adult onset) and are relatively common. The phenotype of the

complex diseases is determined by both genetic and the environmental factors

that affect the individual. Although pathogenic bacteria and various other

environmental factors (e.g., smoking and stress) [1] are involved in pathogenesis

of chronic periodontitis, also genetic factors are evidenced in the aetiology of

chronic periodontitis [2, 3].

There are a large number of scientific papers searching for the role of

genes and their variants (polymorphisms) in host responses in chronic

periodontitis, and in the progression of the disease [4-12]. The genetic

polymorphisms may in some situations cause a change in the protein or its

expression possibly resulting in alterations in innate and adaptive immunity and

may thus be deterministic in disease outcome [11]. Genetic polymorphisms may

also be protective for a disease [12].

Interleukine-10 (IL-10) is considered an antiinflammatory cytokine,

downregulating the proinflammatory immune response of the monocytes and

macrophages. IL-4 and IL-10 display additive effects and induce a strong

increase in the number of viable cells. Moreover, IL-10 induces activated B cells

to secrete large amounts of IgG, IgA, and IgM, and the combination of IL-10 and

IL-4 results in the secretion of the four immunoglobulin isotypes. Thus, IL-10 may

5

play an important role in the amplification of humoral responses. [13]. Auto-

antibodies may also play a role in chronic periodontitis [14–16]. IL-10 is produced

by monocytes, macrophages, and T cells and plays a role in the regulation of

proinflammatory cytokines such as IL-1 and TNF-α.

The gene encoding for IL-10 is mapped on chromosome 1q31-q32, in a cluster

with closely related interleukin genes, including IL-19, IL-20, and IL-24. Several

promoter polymorphisms have been described in the IL10 gene: -1087 (-1082), -

819 (-824), -627, -592 (-597), and -590. The IL10 -1082, -819, and -592

polymorphisms show strong linkage disequilibrium and form two common

haplotypes. The G to A nucleotide polymorphism occurs within a putative Ets

transcription factor–binding site. The C to T nucleotide polymorphism lies within a

putative positive regulatory region, and the C to A polymorphism lies within a

putative STAT 3 binding site and a negative regulatory region [17]. Initial in vitro

work on peripheral blood mononuclear cell (PBMC) cultures has suggested that

the GCC/GCC genotype is associated with higher IL-10 production than other

genotypes [18, 19]. IL-10 has a protective role towards periodontal tissue

destruction, inhibiting both matrix metalloproteinases (MMP) and receptor

activator for nuclear factor-κB (RANK) systems [20, 21].

The aim of this study was to analyze three single-nucleotide

polymorphisms (SNPs) (-1082, -819, -590) in the promoter region of IL10 gene

(on location 1q31-q32) and its relation with periodontal disease in a Macedonian

population.

6

2. Material 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 [22-26]. 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 geographical regions of the Republic

of Macedonia.

The chronic periodontitis group consisted of 111 subjects previously

diagnosed with moderate or severe chronic periodontal disease. Diagnosis

chronic (adult) periodontitis (CP), was established clinically and by x-ray

verification, in accordance to criteria of American Academy of Periodontal

Disease (AAP, 1999) [27, 28] presence of chronic gingivorrhagia, bleeding on

probing, clinical attachment loss (CAL), and horizontal or vertical loss of alveolar

bone. Degree of clinical attachment loss, in all patients was defined with

confirmed periodontal probe. For each tooth, maximal CAL (distance between

the cement-enamel junction and the bottom of the pocket) were derived by

measuring six sites around each tooth and recording the maximum values.

Patients were selected if they showed attachment loss in ≥ 30% of the teeth, with

a minimum CAL of 4 mm. Degree of gingival inflammation was fortified pursuant

to Loe-Silness’ index [29]. 0 – normal gingival (pale pink color, with hard and

paltry granular consistency); 1 – mild inflammation (marginal gingiva is mildly red,

with small oedema and does not bleed on mild provocation); 2 – modest

inflammation (gingiva with red color, with emphasized oedema on the free

7

gingival, there is bleeding on mild pressure with plug); 3 – strong inflammation

(gingiva with clearly red color, with lot of oedemas, with tendency for

spontaneous bleedings).The measures were evaluated on bucal, lingual, mesial

and distal side of each tooth, of each quadrant. Bleeding on probing BOP was

graded 1, if it occurred within 30 sec. If not it was graded 0. Excluding criteria

were: 1) diseases of oral soft and hard tissues in oral cavity, excluding caries and

periodontitis; 2) presence of orthodontic apparatuses (prosthesis) in mouth; 3)

usage of systemic antibiotics in period of three months before engagement in this

study; 4) pregnancy and lactation; 5) diabetes; 6) appliance of immune-

suppressive therapy; 7) history of any general disease which violates functions of

immune system; and 8) current or former smokers.

The healthy control group consisted of 299 subjects displaying no sites

with a probing depth >3 mm, no clinical attachment loss > 2 mm, gingival index of

1, and 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 [23] and was part of the

Cytokine Polymorphisms and Expression, 15th International Histocompatibility

and Immunogenetics Workshop (IHIWS).

2.1 Testing Polymorphism on IL10 gene

Genomic DNA was isolated by the phenol-chloroform [30] method from

peripheral leukocytes and samples stored in the Macedonian bank for human

DNA as previously described [31]. IL10 gene polymorphisms were determined

8

using the 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 [25, 32]. Briefly, the PCR-SSP

typing Heidelberg kit consists of PCR primer mixes aliquotted in 96 well PCR

trays. Master mix, which was supplied along with the reagents and consisted of

MgCl2, buffer, dNTP's, and glycerol was mixed with 1.2 - 3.0 μg DNA and 20 U

Taq polymerase and dispensed in the wells. The amplified products were

separated by 2% agarose gel electrophoresis, stained with 0.5 μg/mL ethidium

bromide and visualized by ultraviolet exposure. For IL10 gene with multiple

polymorphisms per gene [IL10 -1082 (rs1800896), IL10 -819 (rs1800871) and

IL10 -592 (rs1800872)] the Heidelberg PCR-SSP kit was able to detect true

haplotypes.

2.1. Statistical analysis

The population genetics analysis package, PyPop, developed by the

Biostatistics Core for the Workshop [33-35] was used for analysis of the IL10

data for this report. Allele frequencies and expected Hardy Weinberg proportions

(HWP) for each IL10 allele were determined [36]. The exact test for genotype

frequency deviation from HWP was calculated using the Arlequin implementation

accessed via PyPop [37]. Those 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 chi square test. The Ewens-Watterson homozygozity test of neutrality

9

(EWN) (38) with Slatkin's exact p-values (SEPV) [39, 40] was used to indicate

any deviations from the hypothesis of neutral selection for each locus. Pearson's

p-values, crude Odds Ratio (OR) and Wald’s 95% confidence interval (CI) were

calculated for associations' analysis between IL10 (alleles, genotypes,

haplotypes, and diplotypes) and periodontal disease with Simple Interactive

Statistical Analysis (SISA) free statistical calculator

(http://www.quantitativeskills.com/sisa/statistics/twoby2.htm) with Bonferroni

corrected p-value [41]. P less than 0.05 were taken as significant.

3. Results

The clinical and demographic data of 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 chronic periodontitis group than in healthy

controls.

<<Table 1 near here>>

Observed versus expected cytokine genotypes for each IL10

polymorphism, Hardy Weinberg proportion (HWP), and Guo and Thompson

Hardy Weinberg Output (GTHWO) in healthy subjects and subjects with

periodontal disease is given in the Table 2. Observed frequencies of IL10 -1082

genotypes in the healthy population were significantly different from the

expectations (HWP P<0.001, GTHWO P<0.001). In subjects with periodontal

10

disease IL10 genotypes were in equilibrium with HWP and Guo and Thompson

Hardy Weinberg Output (GTHWO) was not significant (P> 0.05) (Table 2).

<<Table 2 near here>>

DNA from our 111 chronic periodontitis and 299 healthy controls were

analyzed for polymorphisms in the promoter (-1082, -819, -592) region of the

IL10 gene. Our results indicate non significant differences in the distribution of

alleles between the two groups at the IL10 -1082, IL10 -819, and IL10 -592 gene

polymorphism (Table 3).

Homozygosity for the A allele at position IL10 -1082 was seen in 30.6% of

subjects with chronic periodontitis compared to 23.4% of healthy subjects while

13.5% of chronic periodontitis subjects were homozygous for the G allele

compared to 5.7% of healthy subjects (Table 3). The distribution of the IL10 -

1082/A:G and IL10 -1082/G:G genotypes between groups was significantly

different after Bonferroni adjustment (P<0.004, OR=0.519, 95% CI=0.331-0.814

and P<0.009, OR=2.592, 95% CI=1.247-5.389, respectively). At the -819

position, 57.7% of chronic periodontitis subjects were homozygous for the C

allele compared to 51.8% of healthy subjects. Homozygosity for the C allele at

position -590 was noted in 55.9% of chronic periodontitis compared to 51.5% of

healthy subjects, but these differences failed to reach significance (P=0.294 and

0.433, respectively). Homozygous T allele at position -819 and A allele at

position -590 were noted in 3.6% of chronic periodontitis while 6.4% of healthy

subjects were homozygous for the T allele and 9.4% for the A allele, respectively.

11

Homozygosity for the T allele at position -819 and A allele at position -590 cannot

be statistically calculated because expected <5, χ2 test (Table 3).

<<Table 3 near here>>

Six haplotypes were found in the chronic periodontitis group and in healthy

subjects with the highest frequency for IL10/GCC haplotype (0.410 in chronic

periodontitis and 0.411 in healthy subjects). Five haplotypes (IL10/ACC,

IL10/ATA, IL10/ACA, IL10/ATC, and IL10/GCA) were with lower frequency, but

differences between investigated groups were not significant (Table 4).

<<Table 4 near here>>

Table 5 shows the distribution of diplotypes (haplo-zygotes) between

groups. Of the 13 diplotypes identified in our study, three diplotypes were

significantly different between patients with chronic periodontitis and healthy

subjects (ACC:ATA, ATA:GCC, GCC:GCC), but failed to reach statistical

significance after Bonferroni adjustment. Three other diplotypes (ACC:ACC,

ACC:GCC, ATA:ATA) were statistically calculated, but differences failed to reach

significance. For seven haplotypes χ2 test was not calculated because the

expected frequencies were smaller than 5 (Table 5).

<<Table 5 near here>>

4. Discussion

In this paper we present the allelic, genotypic, and haplotypic

polymorphisms along the promoter region of the IL10 gene in a relatively

12

homogeneous population of Macedonian subjects with and without periodontal

disease.

In our study the distribution of the IL10 -1082/A:G and IL10 -1082/G:G

genotypes between groups was significantly different after Bonferroni

adjustment. Three diplotypes (ACC:ATA, ATA:GCC, GCC:GCC) were

significantly different between Macedonian patients with chronic periodontitis and

healthy subjects, but failed to reach statistical significance after Bonferroni

adjustment.

The smallest frequency of alleles, which is G allele for IL10 -1082 (-1087),

T allele for IL10 -819 (-824), and A allele for IL10 -590, is labelled with R (rare

allele) and the other allele is labelled with N (normal). The carriage rates of the

IL10 -1087 R allele (G allele) vary between 44% and 81% in Caucasians. The –

1082 (-1087) locus has not been associated with chronic periodontitis

susceptibility in most of Caucasian populations. However, the -1082 (-1087) N-

allele (A allele) was associated with chronic periodontitis in Swedish Caucasians

[42]. We present for the first time negative or protective association of IL10 -

1082/A:G with OR smaller than 1.0 (0.519) and positive or susceptible

association of IL10 -1082/G:G with OR higher than 1.0 (2.592) in Macedonian

Caucasians. However, there was no marked difference in -1082 IL10 genotype

frequencies between the controls and generalized aggressive periodontitis in

Iranian patients [43]. It was found that the proportion of IL-10+ cells in the

peripheral area of the periodontitis lesions was significantly larger in subjects with

the -1087 IL-10 GG genotype than in subjects with the AG or AA genotype [44],

13

which can be in agreement with our results. Recently, the mRNA expression of

Sp1 and IL-10 in patients with the GG genotype was four times higher than that

in patients with the AA genotype. Proportions of Sp1-positive cells overall and

Sp1-positive B cells were larger in patients with the GG genotype than in patients

with the AA genotype [45]. We can conclude from our and other published

results, that IL10 G allele at position -1082 play important role in the association

with chronic periodontitis.

The IL10 -819 polymorphism has been correlated with chronic

periodontitis in Brazilians but not in other populations [46]. Until now all three

studies on the IL10 -592 polymorphism have found a higher R-allele carriage rate

in chronic periodontitis patients [46–48]. The IL10 -592 R-allele carriage rates

varied in different populations between 68% and 75% in chronic periodontitis

patient and between 41% and 51% in controls.

One study on Japanese chronic periodontitis patients (N = 34) and

controls (N = 52) analyzed haplotypes consisting of the IL10 -1087, -819, and -

592 gene polymorphisms [49]. Only haplotype frequencies were reported and no

separate genotype frequencies were presented. No significant differences for the

carriage rates of the haplotypes of the IL10 gene were found between patients

and controls. Striking was the complete absence of the N-allele carriage at

position -1087 among the Japanese, in contrast to Caucasians, where the -1087

N-allele is the most occurring variant [42, 49].

The combination ATA/ATA, which is known as a 'low interleukin-10

producer', was found only in patients with aggressive periodontitis (15.6 vs. 0.0%,

14

p = 0.023) [50], but the Bonferroni adjustment was not applied. Without

Bonferroni correction, our results have shown significant association between

ACC:ATA, ATA:GCC, GCC:GCC diplotypes and patients with chronic

periodontitis. There was a significant (p approximately 0.02) main effect of IL10

haplotypes, with individuals having either the ATA/ACC or the ACC/ACC

genotype experiencing around 20% fewer probing depths of >or= 4 mm

compared to individuals with other genotypes, suggesting that the IL10 genotype

contributes to the progression of periodontal disease [51]. The IL10 gene

polymorphism at position -592C>A may be associated with a lower risk for

development of chronic periodontitis. The IL10 haplotype ATA is associated with

generalized aggressive periodontitis in a Taiwanese population [52].

Our study is subject to limitations. After Bonferroni correction of P-values,

we found smaller number of IL10 genotypes and diplotypes to be associated with

periodontal disease in Macedonians, but the inclusion of Bonferroni correction in

this report implies a higher likelihood of false negatives with subsequently less

significant associations with periodontal disease [41, 53]. Moreover, IL10 -1082

genotypes in healthy participants were not in HWP (P<0.001) and we should be

very careful about their associations with periodontal disease. Unfortunately, we

do not have any information about HWP in other published papers connected

with IL10 gene polymorphisms and associations with chronic periodontitis [42-52]

in order to compare connection of the results with HWP. The number of patients

in our study (111), as well as in other published papers [42-52], is very small. In

the association studies, there are possibilities that some positive results might be

15

spurious and some negative findings might be a consequence of low statistical

power. It could be due to their small sample size or methodological

shortcomings, such as the selection of an appropriate control group. It is

necessary to investigate IL10 gene polymorphisms in our population in well

defined subgroups of phenotypes with bigger number of participants in order to

have more precise conclusions for genetic background of development of chronic

periodontitis in Macedonians. Multicentric studies and/or meta-analysis of the

patients with chronic periodontitis and association with cytokine polymorphisms

should be very useful.

It can be concluded that, at least in Macedonian patients with chronic

periodontitis, some IL10 gene polymorphisms contribute to

susceptibility/protection to disease. Ethnic factors might play a role in the

variability of results in different populations. Therefore, additional studies are

needed to clarify this issue.

Acknowledgements

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).

The authors thank Elena Cvetkovska (Institute of Immunobiology and Human

Genetics, Faculty of Medicine, University “Ss Cyril and Methodius”, Skopje,

Republic of Macedonia) for isolation of genomic DNA and taking care of the

Macedonian Human DNA Bank (www.hdnamkd.org.mk).

16

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24

Table 1. Demographic and periodontal findings of the Macedonian study

population.

Chronic periodontitis

(n = 111) Control group

(n = 299) P

Female (%) 41.90 54.60 NS Male (%) 58.10 46.40 NS Age, year (mean ± SD) 38.97 ± 10.12 35.20 ± 9.90 NS Loe-Silnes Index , GI (mean ± SD) 2.38 ± 0.67 0.5 ± 0 <0.001BOP, % (mean ± SD) 82.52 ± 8.14 6.8 ± 6.4 <0.001CAL (mean ± SD) 5.18 ± 0.716 1.8 ± 0.3 <0.001

SD, standard deviation; n, number of participants; NS, non significant; BOP, bleeding on probing; CAL, Clinical attachment loss.

25

Table 2. Observed vs. expected cytokine genotypes for each IL10 -1082, IL10 -819 and IL10 -592 polymorphisms, Hardy Weinberg proportions (HWP), and Guo and Thompson Hardy Weinberg Output (GTHWO) in Macedonian population.

Polymorphism Genotype Observed number

Observed frequency

Expected number

p-value HWP p-value

GTHWO p-value

Control (n=299)

IL10 -1082 A:A A:G G:G

70 212 17

0.234 0.709 0.057

103.6 144.8

50.6

0.001* <0.001* <0.001*

<0.001* <0.001*

IL10 -819 C:C C:T T:T

155 125 19

0.518 0.418 0.064

158.2 118.6

22.2

0.798 0.555 0.495

0.348 0.391

IL10 -592 A:A A:C C:C

28 117 154

0.094 0.391 0.515

25.0 123.0 151.0

0.552 0.592 0.809

0.403 0.404

Periodontitis (n=111)

IL10 -1082 A:A A:G G:G

34 62 15

0.306 0.559 0.135

38.1 53.9 19.1

0.510 0.268 0.352

0.112 0.171

IL10 -819 C:C C:T T:T

64 43

4

0.577 0.387 0.036

65.9 39.3 5.9

0.819 0.553 0.443

0.319 0.428

IL10 -592 A:A A:C C:C

62 45

4

0.559 0.405 0.036

64.3 40.3 6.3

0.355 0.464 0.772

0.224 0.302

26

Table 3. Distribution of allele and genotype polymorphisms of the IL-10 -1082, IL10 -819 and IL10 -592 in Macedonian population patients with periodontal disease, compared to control.

Periodontitis Control Polymorphism

Allele or Genotype N F (%) N

F (%)

OR Wald

95% CI Pearson's

P-value

A 130 0.586 352 0.589IL10 -1082

G 92 0.414 246 0.4110.987

0.722-1.350

1.000

C 171 0.770 435 0.727IL10 -819

T 51 0.230 163 0.2731.256

0.876-1.802

0.214

A 53 0.239 173 0.289IL10 -590

C 169 0.761 425 0.7110.770

0.540-1.099

0.150

A:A 34 30.6 70 23.4 1.4440.890-2.344

0.135

A:G 62 55.9 212 70.9 0.5190.331-0.814

0.004* IL10 -1082

G:G 15 13.5 17 5.7 2.5921.247-5.389

0.009*

C:C 64 57.7 155 51.8 1.2650.815-1.963

0.294

C:T 43 38.7 125 41.8 0.8800.564-1.375

0.575 IL10 -819

T:T 4 3.6 19 6.4 & & &

A:A 4 3.6 28 9.4 & & &

A:C 45 40.5 117 39.1 1.0600.680-1.654

0.795 IL10 -590

C:C 62 55.9 154 51.5 1.1910.769-1.846

0.433

N = number of alleles or genotypes; F = frequency of alleles or genotypes; OR,

Odds Ratio; CI, confidence interval; *, statistically significant after Bonferroni

adjustment (p-value x number of alleles or genotypes) < 0.05; &, cannot be

calculated because expected <5, χ2 test.

27

Table 4. Distribution of haplotype polymorphisms at the IL-10 -1082, IL10 -819 and IL10 -592 in Macedonian population patients with periodontal disease, compared to control.

N = number of haplotypes; F = frequency of haplotypes; OR, Odds Ratio; CI,

confidence interval; &, cannot be calculated because expected <5, χ2 test.

Periodontitis Control Haplotype

N F N F OR

Wald

95% CI

Pearson's

P-value

GCC 91

0.410 246

0.411

0.994 0.727-1.360

1.000

ACC 72

0.324 177

0.260

1.142 0.820-1.590

0.433

ATA 46

0.207 161

0.269

0.704 0.486-1.022

0.064

ACA 7

0.032 12

0.020

1.560 0.618-4.091

0.332

ATC 5

0.023 2

0.003

& & &

GCA 1

0.005 0 0

& & &

28

Table 5. Percentage of diplotypes (haplo-zygotes) of polymorphisms of the IL10 -1082, IL10 -819 and IL10 -592 in Macedonian population patients with periodontal disease, compared to control.

Periodontitis Control Diplotype

N F (%) N F (%)

OR Wald

95% CI Pearson's

P-value

ACC:ACC 10 9.0 21 7.0 1.311 0.597-2.879 0.499 ACC:ATA 17 15.3 21 7.0 0.394 1.212-4.730 0.010 ACC:GCC 34 30.6 114 38.1 0.716 0.450-1.142 0.160 ATA:ATA 3 2.7 19 6.4 0.409 0.119-1.411 0.144 ATA:GCC 20 18.0 93 31.1 0.487 0.283-0.837 0.008 GCC:GCC 15 13.5 17 5.7 2.592 1.247-5.389 0.008 GCC:ACA 5 4.5 3 1.0 & & & ATC:GCC 2 1.8 2 0.7 & & & ACA:ATA 1 0.9 9 3.0 & & & ATC:ACC 1 0.9 0 0 & & & ATC:ATA 1 0.9 0 0 & & &

GCA:ATA 1 0.9 0 0 & & &

ATC:ACA 1 0.9 0 0 & & &

N = number of diplotypes; F = frequency of diplotypes; OR, Odds Ratio; CI,

confidence interval; &, cannot be calculated because expected <5, χ2 test.