Gender and smoking-related risk reduction of periodontal ... - Nature

Genes and Immunity (2002) 3, 102–106  2002 Nature Publishing Group All rights reserved 1466-4879/02 $25.00 www.nature.com/gene

Gender and smoking-related risk reduction of periodontal disease with variant myeloperoxidase alleles P Meisel1, T Krause1, I Cascorbi1, W Schroeder2, F Herrmann2, U John3 and Th Kocher4 1

Department of Pharmacology, Ernst Moritz Arndt University Greifswald, Greifswald, Germany; 2Department of Genetics, Ernst Moritz Arndt University Greifswald, Greifswald, Germany; 3Department of Epidemiology, Ernst Moritz Arndt University Greifswald, Greifswald, Germany; 4Department of Periodontology, Ernst Moritz Arndt University Greifswald, Greifswald, Germany

Myeloperoxidase (MPO) is an oxidative enzyme expressed in polymorphonuclear leukocytes. It is involved in the defence against periodontal bacteria, and is also able to mediate inflammatory tissue destruction in periodontal disease. A G/A polymorphism in the promoter region of the MPO gene at position −463 has been assumed to exert profound effects on the expression of the enzyme. It is the aim of this study to evaluate whether this polymorphism may influence the risk of periodontal diseases. A total of 3148 subjects were randomly selected from the general population in the SHIP study (Study of Health in Pomerania). Periodontal status, health-related and socio-economic items were assessed. All subjects aged 40–60 years (n = 1103) were included in this study, and 1083 genotyped for the MPO −463 G/A polymorphism by PCR and RFLP methods. The genotype frequencies determined were homozygous wild type G/G 65.9% (95% CI 63.5– 68.6), heterozygous A/G 31.4% (28.8–34.4), and homozygous variant A/A 2.7% (2.0–3.8). Only female subjects have a significantly reduced risk of severe periodontal disease when bearing the variant genotypes A/G or A/A. In female subjects the reduction in periodontal risk was significant for non-smokers (OR = 0.48; 95% CI 0.23–0.96); the smokerelated increase in risk was also reduced (OR = 0.50; 95% CI 0.22–1.10). When adjusted for age, smoking, and education the odds ratios were calculated as 0.52 (P = 0.01) and 0.97 (P = 0.90) for female and male subjects, respectively. The results of this study confirm the assumption that the MPO −463A allele variants are protective in the pathogenesis of periodontal diseases. This holds true only with women but not with men. The results are discussed with respect to the known influences of sexual hormones on MPO activity. Genes and Immunity (2002) 3, 102–106. DOI: 10.1038/sj/gene/6363840 Keywords: periodontal disease; myeloperoxidase; genetic polymorphism; gender

Introduction Periodontitis is an infectious inflammatory disease, accompanied by plaque and pocket formation. It leads to attachment loss and eventually results in tooth loss. It affects a majority of all adults and is consequently a severe medical challenge. Myeloperoxidase (MPO) is an enzyme catalysing the formation of microbicidal hypochlorous acid enabling the defence against bacteria involved in the pathogenesis of various diseases including periodontitis.1 The enzyme is largely expressed in polymorphonuclear neutrophils and, therefore, the highest levels of enzyme activity were found at sites with increasing gingival inflammation.2 Correspondence: P Meisel, Department of Pharmacology, F-Loeffler-Str. 23d, D-17487 Greifswald, Germany, E-mail: meiselp얀uni-greifswald.de This work is part of the Community Medicine Research net (CMR) of the University of Greifswald, Germany, which is funded by the Federal Ministry of Education and Research (grant no. ZZ9603), the Ministry of Cultural Affairs and the Social Ministry of the Federal State of Mecklenburg-West Pomerania. The CMR gathers several research projects which share data from the population-based Study of Health in Pomerania. (SHIP; http://www.medizin.uni-greifswald.de/cm). Received 23 October 2001; revised and accepted 27 November 2001

The function to combat pathogenic oral bacteria may protect from infection, on the other hand activated neutrophils promote cell destruction and MPO activity measurements have been used as predictors of periodontal disease severity.3,4 Besides this dual role in the pathogenesis of periodontitis, MPO-generated oxidants are capable of oxidising a wide variety of compounds, among these are also products of tobacco smoke. Leukocytes are recruited in immune response and therefore, reactive intermediates from xenobiotics generated by leukocyte metabolism may play a role in idiosyncratic drug reactions.5,6 Various drugs, but also arylamines and benz[a]pyrene from tobacco smoke are converted to cytotoxic products. The degranulation of these cells and also their hyperactive state in the presence of chronic antigenic stimulation may transform environmental pre-carcinogens to highly reactive intermediates7 as was shown for heterocyclic amine activation by MPO in fibroblasts and epithelial cells.8 Moreover, MPO provides one pathway for mutagenesis and cytotoxicity at sites of inflammation. Thus, MPO activity may contribute to the smokerelated risk of periodontitis. Most of the increased MPO activity in periodontally diseased patients can be attributed to the increased number of neutrophils.9

Gender and smoking-related risk reduction P Miesel et al

As myeloperoxidase activity expressed in neutrophils recruited to the gingiva after chemical or immunological insults contributes to tissue destruction, it seems promising to hypothesise that genetic variants of MPO might influence the extent and/or the severity of periodontal diseases. A polymorphism in the promoter region of the MPO gene at position −463 with a G/A exchange was detected by Austin et al.10 It results in the loss of a transcription factor binding site and strongly reduced mRNA expression.11 It is the aim of this study to assess the distribution of the MPO polymorphism in a randomly selected population which has been examined for their periodontal state. We hypothesise that this polymorphism could influence the risk of periodontal disease.

only, odds ratio (OR) = 0.50, 95% CI 0.22–1.10, P = 0.06 (Table 1, smoking GG genotype as reference). The same relationships are obtained when restricting the analysis to subjects still smoking (excluding all ex-smokers) as compared with non-smokers. In females smoking still at present the risk associated with smoking results in an OR of 3.51 and 1.85 for GG homozygotes and AG/AA variants, respectively. In their non-smoking counterparts the risk reduction with the A allele variant gives OR = 0.42 (95% CI 0.21–0.81, P = 0.005). Assuming a multiplicative model for gene–environmental interactions, results for female subjects only in relationships as expected, are namely

Results

In Table 1 for females the figure is 1.25/0.48 × 2.51 = 1.04, for men it is 1.48. As expected, the number of former (quitted) smokers is the highest in the group with minor periodontal signs (‘controls’). In this respect sex-related differences are also obvious as there are fewer smokers who quitted among the female subjects than among their male counterparts. Adjusting for the factors influencing the periodontal outcome the most by logistic regression analysis confirmed that females carrying the variant MPO genotypes −463 A/G or A/A had a significantly reduced risk of periodontal disease. This is not the case with male individuals (Table 2). In both sexes, increasing age is associated with higher risk, the educational factor (high school 12th grade or higher), however, reduces the risk of more severe forms of periodontal disease. Diabetes, also known as a risk factor, was without significant influence.

The MPO −463 genotype distribution determined in this study comprising a north-east German population were: homozygous wild type −463 G/G 65.9% (95% confidence interval (CI) 63.5–68.6%), heterozygous A/G 31.4% (95% CI 28.8–34.4%), and homozygous variant A/A 2.7% (95% CI 2.0–3.8%). The frequencies fitted the Hardy-Weinberg equilibrium. There are marginal sex-differences in allele frequencies as yet unexplained as this is a populationbased, randomly selected study group. Nevertheless, in each of the gender subgroups the genotype distribution obeys the Hardy-Weinberg equilibrium. Smoking is one of the most important risk factors of periodontal diseases. In Table 1, the risks are given associated with the genetic factor alone, the environmental factor smoking alone, and that for both factors combined. The relationship is also given for those who ever smoked, ie for subjects who formerly smoked (quitted smokers) together with those who are active smokers. For male as well as female subjects smoking is a significant risk factor for more severe forms of periodontitis, increasing the risk by a factor of nearly three. Possessing the MPO variant genotype −463 A/G or A/A reduces the risk of advanced periodontitis by nearly a half in female subjects, but not so in males (Table 1). In females, even the increased risk imposed by smoking is reduced by the variant genotypes: female smokers

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ORgene–smoking = 1. ORgene × ORsmoking

Discussion It is well known that MPO activity is increased in inflammatory gingival tissue.2 The polymorphism of the MPO gene promoter affects the risk of periodontal diseases. As revealed in this study, female subjects bearing the A/G or A/A allele have a reduced risk of suffering from advanced periodontal disease. This observation appears sex-specific as this association could not be observed among male subjects. It is most intriguing that

Table 1 Genetic and smoking-associated risk for attachment loss in men and women who ever smoked. Subjects were considered as cases, ie periodontally diseased if their extent of attachment loss ⬎4 mm exceeded 52.5% (4th quartile), and as controls having less than 25.0% attachment loss ⬎4 mm (the median). Reference set at an odds ratio = 1, to which each row has been compared Smoking Male no no yes yes

MPO

Cases

Controls

Odds ratio (95% CI)

P

GG AG or AA GG AG or AA

21 7 75 36

61 27 90 39

1.00 0.75 (0.25–2.16) 2.42 (1.30–4.53) 2.68 (1.30–5.56)

reference NS 0.003 0.004

43 (39%)

80 (62%)

42 14 44 14

127 89 53 34

1.00 0.48 (0.23–0.96) 2.51 (1.43–4.42) 1.25 (0.57–2.68)

reference 0.025 0.0006 0.547

13 (22%)

41 (47%)

Ex-smokers Female no no yes yes Ex-smokers

GG AG or AA GG AG or AA

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Table 2 Logistic regression analysis for the risk of attachment loss ⬎4 mm (4th quartile vs 1.–2. quartiles as dependent variable). Smoking (yes: 1/no: 0), education (school 12th grade yes: 1/no: 0), and MPO genotype (wild type GG: 0 vs variant AG/AA: 1) as dichotomous and age as continuous independent variables Male Variable

Odds ratio (95% CI)

Age (years) Smoking (ever) Education MPO genotype

1.084 3.008 0.343 0.968

a

(1.041–1.128)a (1.785–5.067) (0.184–0.639) (0.591–1.585)

Female P

Odds ratio (95% CI)

⬍0.0001 ⬍0.0001 0.0007 0.898

1.086 3.185 0.393 0.518

(1.042–1.132)a (1.963–5.167) (0.176–0.880) (0.311–0.861)

P ⬍0.0001 ⬍0.0001 0.023 0.011

For intervals of 10 years these figures are OR = 2.23 and 2.29 for males and females, respectively.

these results are corresponding to the associations reported for different diseases such as lung cancer,12–14 esophageal cancer,15 coronary artery disease,16 Helicobacter pylori infections17 and neuro-degenerative disorders, ie Alzheimer’s disease18 and multiple sclerosis.19 In all these studies, the allelic variant MPO −463A elicited a protective effect with nearly identical odds ratios in the range of 0.3–0.7. In some of these studies gender-specific results were presented showing the protective effect of the MPO A-allele either only in females18,19 or only in males.17 There must be a common link leading to this correspondence in the different clinical settings. Inflammationrelated recruitment of neutrophil leukocytes into the affected tissue is most likely responsible for the different protective effects seen in the various studies mentioned. Exchange of an adenosine for a guanosine at position −463 in the 5⬘-untranslated region of the MPO gene leads to the loss of a transcription factor binding site. Reduced binding of transcription factor SP1 results in diminished expression of MPO.11 The observed sex-specific differences suggest that binding of sexual hormones may be affected by the loss of the MPO promoter binding motif. Possible interactions are known between the SP1 binding site and binding of the estrogen receptor to regulatory gene sequences.20 In vitro experiments demonstrated that the −463 G/A results in estrogen receptor ␣ binding to the MPO A promoter, but not the MPO G promoter.21 These results suggest that the −463 G/A base exchange affects the MPO expression differently in men and women. Potential estrogen responsive elements were also detected within the introns 7 and 9 of the MPO gene.22 As a potential cause of MPO-induced tissue destruction in inflammatory diseases, it was reported that estrogen enhances MPO activity in human polymorphonuclear leukocytes.23 Reports on variations in MPO activities depending on estradiol levels during the menstrual cycle support this view24 as well as the observation that hormone replacement restores the reduced neutrophil myeloperoxidase release and activity in menopausal women.25 Subjects with high estradiol levels also showed an increase in MPO protein in the plasma as a sign of the ability of estradiol to induce MPO.26 Clinical experience confirms the influence of sexual steroids on the periodontium under several clinical conditions. Especially, the function of polymorphonuclear leukocytes invading the gingiva may be affected by estrogens.27 Known background risk factors associated with periodontal disease include gender (with males being at Genes and Immunity

a higher risk) and osteopenia associated with estrogen deficiency, among others.28 The results shown here support these observations and show that the MPO promoter polymorphism is a sex-specific risk factor (as far as for the GG genotype) or protective factor (AG or AA variants) in periodontal diseases.

Materials and methods Subjects A total of 3148 subjects were randomly selected from a population of 210 000 inhabitants of the German part of Pomerania designated as the SHIP study. The design of the study, recruiting of participants and the scope of this population-based cross-sectional health survey were outlined by John et al.29 In this group, the most profound smoking-related differences in periodontal parameters were recognised between the ages of 40–60 years. Therefore, all subjects in the age range of 40–60 years (n = 1103) were included in this study. Characteristics of these subjects, relevant to the objective of the study, are displayed in Table 3. Clinical status The smoking status and habits of all subjects were assessed by an extensive questionnaire comprising 31 items concerning present and past quality and quantity of smoking. The items included questions as to whether the subjects had ever used tobacco products, the age at which they started smoking (and if applicable ceased smoking), and the frequency and duration of smoking. In an independent study (n = 335), we proved by cotinine determinations that not more than 2–3% of the subjects gave answers not consistent with their true smoking behaviour. Investigations in representative population samples provided evidence that self-reported smoking status is accurate.30 Periodontal state was assessed by trained dentists including probing depth, attachment loss, bleeding and plaque index. The periodontal examination was carried out either on the left or right quadrants, the examination side was changed from subject to subject. All fully erupted teeth were assessed excluding third molars. A maximum of 14 teeth per subject was examined. Attachment loss and probing depth were assessed with a periodontal probe (PCP 11, HuFriedy) at mesiobuccal, distobuccal, midbuccal and midlingual aspect on each selected tooth. The measurements were made in whole millimeters.


Table 3 Subjects enrolled in the Study of Health in Pomerania (SHIP) which were assessed for their periodontal state and genotyped for the MPO polymorphism −463 G/A. Shown are numbers of subjects, ratios, or the median and range of parameters where appropriate. Significance tests were performed by ANOVA or contingency tables (␹2)

Number of subjects Age, median (range 40–60) MPO allele frequency −463A Number of teeth, mediana % Attachment loss ⬎3 mm, median % Attachment loss ⬎4 mm, median % Probing depth ⬎4 mm, median Probing depth, mean ± s.d. Smoker (ever/quitted) Packyears, mean ± s.d. (smokers) Diabetes School, 12th grade a

Female

Male

P

578 50 0.198 21 50.0 19.2 6.8 2.52 ± 0.73 201/72 8.7 ± 6.3 27 86 (= 15%)

505 51 0.167 22 61.8 30.0 11.5 2.75 ± 0.80 335/161 17.5 ± 13.1 39 95 (= 19%)

– NS 0.053 0.014 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.036 0.083

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Excluding the third molars.

Genotyping and statistics Of the 1103 subjects enrolled, 1083 were successfully genotyped for MPO polymorphism, the remaining refused DNA analyses, or genotyping failed. Genotyping for MPO G −463A was performed by PCR/RFLP according to the method described by Cascorbi et al.12 For PCR we used the primers forward 5⬘-CGG TAT AGG CAC ACA ATG GTG AG, reverse 5⬘-GCA ATG GTT CAA GCG ATT CTT C; the amplified PCR fragment was digested with endonuclease AciI in order to distinguish the genotypes wild type −463 G/G, heterozygous G/A, and homozygous A/A. Periodontal diseases impose themselves as a continuous range from minor signs to severe disease, complicated by the variability between the teeth of a particular individual. Thus, in order to avoid any arbitrarily chosen disease criteria or cut-offs, we used strictly statistical methods to distinguish periodontally ‘healthy’ from ‘diseased’ subjects. To evaluate the extent of attachment loss (percent of sites exceeding 4 mm) quartiles were calculated and subjects in the upper quartile of distribution (‘cases’) were compared with those in the lower quartiles with no or minor attachment loss (‘controls’). Gene–environmental interactions were calculated from 2 × 4 tables as outlined by Botto and Khoury.31 Subjects were compared having the G/G genotype wild-type vs those bearing at least one copy of −463A. Adjusting for sex, age, education level, and smoking was carried out by logistic regression analyses.

Acknowledgements We wish to thank Mrs Ingrid Geissler for her skilful technical assistance.

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