G gene polymorphism and the outcome of the nonsurgical periodontal treatment

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Journal of the Formosan Medical Association (2017) xx, 1e6

Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.jfma-online.com

ORIGINAL ARTICLE

Association between monocyte chemoattractant protein-1 -2518 A/G gene polymorphism and the outcome of the nonsurgical periodontal treatment Ching-Wen Chang a, Hsiu-Hui Lin b, Shih-Yun Wu a,b, Ching-Yi Wu a,c, Yu-Lin Lai a,b, Shan-Ling Hung a,b,c,* a

Department of Stomatology, Taipei Veterans General Hospital, Taipei, Taiwan Department of Dentistry, National Yang-Ming University, Taipei, Taiwan c Institute of Oral Biology, National Yang-Ming University, Taipei, Taiwan b

Received 13 January 2017; received in revised form 21 March 2017; accepted 29 March 2017

KEYWORDS Genetic polymorphism; Monocyte chemoattractant protein-1; Nonsurgical periodontal treatment; Periodontitis

Abstract Background/purpose: Elevated monocyte chemoattractant protein-1 (MCP-1) is related to severe periodontal destruction. Furthermore, MCP-1 -2518 A/G gene polymorphism affects MCP-1 after inflammatory stimuli. This study analyzed the association between MCP-1 -2518 gene polymorphism and the outcome of nonsurgical periodontal treatment. Methods: Forty periodontal patients were recruited and MCP-1 -2518 A/G gene polymorphisms were analyzed using polymerase chain reaction-restriction fragment length polymorphism assay. The clinical periodontal parameters, including probing depth (PD), clinical attachment level (CAL), gingival index (GI), bleeding index (BI) and plaque index (PI), were recorded before and six weeks after nonsurgical periodontal therapy. Patients were divided into chronic periodontitis (CP) or aggressive periodontitis (AP). Multiple linear regression analysis was performed to investigate certain predictors of the therapy outcome. Results: The frequency of MCP-1 -2518 genotype-positive (carrying allele G) was 42.5%. Poor treatment outcome in PD, GI and BI improvement could be predicted with MCP-1 -2518 A/G genotype and aggressive periodontitis status as the predictor variables. In contrast, MCP-1 -2518 A/A genotype and aggressive periodontitis status could predict better treatment response in PD and BI improvement. However, MCP-1 -2518 genotype did not affect the treatment outcome in patients with chronic periodontitis. Conclusion: MCP-1 -2518 A/G genotype might be useful in predicting less favorable nonsurgical treatment outcome in patients with aggressive periodontitis. However, MCP-1 -2518 gene

* Corresponding author. Institute of Oral Biology, National Yang-Ming University, No. 155, Sec. 2, Li-Nong St., Pei-Tou, Taipei 11221, Taiwan. Fax: þ886 2 2826 4053. E-mail address: [email protected] (S.-L. Hung). http://dx.doi.org/10.1016/j.jfma.2017.03.013 0929-6646/Copyright ª 2017, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Chang C-W, et al., Association between monocyte chemoattractant protein-1 -2518 A/G gene polymorphism and the outcome of the nonsurgical periodontal treatment, Journal of the Formosan Medical Association (2017), http:// dx.doi.org/10.1016/j.jfma.2017.03.013

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C.-W. Chang et al. polymorphism may not play a role in patients with chronic periodontitis. This study suggests that MCP-1 -2518 genotype may influence the outcome of nonsurgical periodontal treatment in aggressive periodontitis patients. Copyright ª 2017, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/).

Introduction Although periodontal diseases are initiated by oral bacteria, it is also evident that the host response plays a major role in the outcome of these infections. Chemokines are small signaling proteins which are involved in the physiology and pathophysiology of acute and chronic inflammatory processes, by attracting and stimulating specific subsets of leukocytes.1 Monocyte chemoattractant protein-1 (MCP-1), also known as CC Chemokine Ligand-2 (CCL-2), is one of the important chemokine to initiate, regulate, and attract monocytes.2 MCP-1 attracts chemokine receptor CCR2positive cells.3 MCP-1 expression in bacterially induced gingival inflammation is positively related to the degree of inflammation.4 In addition, levels of MCP-1 in gingival crevicular fluid (GCF) were both higher in chronic periodontitis and aggressive periodontitis compared to the healthy control.5,6 Pradeep et al. have found that MCP-1 levels in GCF and serum are positively related to the severity of periodontitis.7,8 In the same studies, they have also investigated the effects of nonsurgical periodontal treatment on MCP-1 levels in GCF and serum. Significant reduction of MCP-1 levels has been observed in periodontal patients 6e8 weeks after treatment. Smoking is a major risk factor for periodontitis.9 Anil et al. have observed higher GCF levels of MCP-1 in smokers with periodontitis than nonsmokers with periodontitis and healthy control.10 Tymkiw et al. have compared periodontitis sites to healthy sites in patients with periodontitis.11 They have found that levels of MCP-1 in GCF are higher in diseased sites than healthy sites in nonsmokers while no difference in both sites in smokers. Haytural et al. have detected higher MCP-1 levels in serum in smokers with periodontitis than nonsmokers with periodontitis.12 Smoking might have some effects on MCP-1 levels related to pathogenesis of periodontitis. Polymorphisms in the regulatory region of MCP-1 gene, which increase the expression of MCP-1, have been demonstrated.13 A biallelic A/G polymorphism has been found in the MCP-1 distal gene regulatory region at position -2518 (numbers indicate nucleotide positions relative to the major transcription start sites) that affects the level of MCP-1 expression in response to an inflammatory stimulus.14 Monocytes from individuals carrying a G allele at -2518 produce more MCP-1 than monocytes from A/A homozygous subjects.14 The MCP-1 -2518 A/G gene polymorphism appears as a genetic risk factor for myocardial infarction and cerebral infarction in two meta-analysis studies.15,16 However, a study by Zhu et al. revealed that Chinese women with generalized aggressive periodontitis had lower frequency of MCP-1 -2518 A/G gene polymorphism than healthy female

group (71.2% vs. 85.7%).17 Their another research further studied combined effect of CCR2-V64I and MCP-1 -2518 A/G on aggressive periodontitis patients. They found the odds ratio for VV genotype (CCR2) and smoking, or MCP-1 (G genotype) and smoking were 7.4 and 4.9, respectively in male patients with periodontitis.18 Elevated MCP-1 is related to severe periodontal destruction. Significant reduction of MCP-1 levels has been observed in periodontal patients after nonsurgical periodontal treatment.7,8 Furthermore, MCP-1 -2518 A/G gene polymorphism affects MCP-1 after inflammatory stimuli. However, the possible association between the genetic polymorphisms of MCP-1 and the outcome of nonsurgical periodontal treatments has not yet been investigated. The aim of the present study was to observe the frequency of the MCP-1 -2518 A/G polymorphism in the Taiwanese patients with periodontitis. The clinical parameters before and after nonsurgical periodontal treatment were also examined. Moreover, the association between the MCP-1 genotype and the outcome of non-surgical periodontal treatment in chronic and aggressive periodontitis patients was evaluated.

Materials and methods Study population A total of 40 participants with periodontitis were recruited from the patient pool at the Division of Periodontics, Department of Stomatology, Taipei Veterans General Hospital. Ethical approval for this research was approved by the Institutional Review Board of Taipei Veterans General Hospital. Demographic and personal data, including medical history, birth date, gender, family history, smoking status were collected. All participants were Taiwanese (19 male, 21 female; mean age: 46.88  11.48) and met the following criteria: (1) no known systemic diseases or antibiotics/anti-inflammatory drug therapy in the previous three months; (2) not pregnant or breast feeding; (3) no periodontal treatment, including ultrasonic scaling, root planing, and periodontal surgery in the previous three months prior to the study, since healing following nonsurgical therapy is almost complete at three months.19 Participants were all informed about the purpose and procedures and signed informed consent prior to entry into the study. The diagnostic criteria of periodontitis were based on clinical periodontal examination and full mouth periapical and bitewing radiographs. Baseline clinical parameters, probing depth (PD), clinical attachment loss (CAL), gingival index (GI),20 bleeding index (BI),20 plaque

Please cite this article in press as: Chang C-W, et al., Association between monocyte chemoattractant protein-1 -2518 A/G gene polymorphism and the outcome of the nonsurgical periodontal treatment, Journal of the Formosan Medical Association (2017), http:// dx.doi.org/10.1016/j.jfma.2017.03.013

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MCP-1 -2518 gene polymorphism index (PI),21 were measured at six sites of each tooth. In addition, the number of tooth extracted was recorded and the reason of tooth extraction was all due to periodontal origin. Patients were categorized into chronic periodontitis (CP) or aggressive periodontitis (AP) according to the 1999 World Workshop for the classification of periodontal disease.22 The percentage distribution of different PD or CAL in chronic or aggressive periodontitis was calculated. Furthermore, patients were classified as nonsmokers (n Z 32), former smokers (n Z 3) and current smokers (n Z 5) according to the smoking status. Former smokers reported not smoking at all, and quit for at least 3 months.23 All patients received nonsurgical periodontal treatments, including scaling and root planing, as well as oral hygiene reinforcement by two well-trained periodontists. The nonsurgical periodontal treatments were separated in 3e4 appointments, including 1e2 times oral hygiene check-up. Six weeks after treatments, the patients were re-checked for all of the clinical parameters as described above. The time period, six-weeks, was chosen, since significant reduction of MCP-1 levels in GCF and serum, has been observed in periodontal patients 6e8 weeks after nonsurgical periodontal treatment.8

Blood collection and isolation of genomic DNA A 2 ml of anti-coagulated peripheral blood sample was obtained from each patient by venipuncture. Genomic DNA was isolated from each sample using Blood and Tissue Genomic DNA Extraction Miniprep System (Viogene-Biotek Corp, Taipei, Taiwan).

3 improvement of each periodontal parameter (PD, CAL, GI, BI or PI).

Results In our study, there were 27 patients with chronic periodontitis and 13 patients with generalized aggressive periodontitis. The MCP-1 -2518 A/G genotype distribution was shown in Table 1. MCP-1 allele G frequency was 42.5% (34/ 80) (Table 1). The periodontal characteristics of study groups were shown in Table 2 and Table 3. The genotype distribution between non-smokers with chronic periodontitis and aggressive periodontitis was not statistically different (X2 Z 5.131, P Z 0.077). No distribution difference in subjects with history of the smoking habit was also observed (X2 Z 0.533, P Z 0.465). After nonsurgical periodontal treatment, patients had significant improvement in all clinical parameters examined (P < 0.001). A mean PD decrease of 0.85 mm (0.57 mm) and a mean CAL gain of 0.79 mm (0.53 mm) were observed. The average number of tooth extraction was 0.875 in each patient. Furthermore, Table 4 listed the improvement of PD, CAL, GI, BI and PI in patients with different MCP-1 genotypes and periodontitis types. A multiple linear regression analysis was used to determine the possible effects of the variables on the treatment outcomes. These independent variables included MCP-1 genotype, periodontitis type, smoking habit, tooth extraction number, age, baseline PD, CAL, GI, BI and PI (Table 5). According to these results, aggressive periodontitis and MCP-1 -2518 A/G genotype had significant negative

Genotyping Genotyping of MCP-1 -2518 polymorphism was determined by the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) techniques using a PvuII site affected by the G/A polymorphism.14,24 Amplification with the primers MCP417S: 50 -TCT CTC ACG CCA GCA CTG ACC-30 and MCP650AS: 50 -GAG TGT TCA CAT AGG CTT CTG-30 generated a 234-base pairs (bp) product. Cycling was carried out for 1 cycle at 95  C for 5 min; 35 cycles for 1 min each at 95  C, 58  C and 72  C; and 1 cycle at 72  C for 5 min. Digestion with PvuII yields 75 and 159 bp fragments (allele [G]) or a single 234 bp fragment (allele [A]).

Table 1 Genotype and allele frequencies for MCP-1 -2518A/G. Periodontitis (n Z 40) Genotype A/A A/G G/G Allele A G

11 (27.5%) 24 (60.0%) 5 (12.5%) 46 (57.5%) 34 (42.5%)

MCP-1 Z monocyte chemoattractant protein-1.

Statistical analysis Genotype and allele frequencies for MCP-1 -2518 A/G were analyzed on a percentage basis. The distribution and significance of baseline PD, CAL, GI, BI, and PI between CP and AP patients were analyzed using the independent T test, while the distribution and significance of the genotypes of MCP-1 and smoking habits between CP and AP patients were analyzed by the chi-square test. Furthermore, multiple linear regression analysis and stepwise procedure were used to select parameters which could best “predict” the clinical outcomes. P-value <0.05 was considered statistically significant. Post hoc power calculations were performed, setting type I error as 0.05. It was shown that the linear regression analyses had at least 99% power to predict

Table 2 Baseline clinical parameters in patients with chronic and aggressive periodontitis.

PD (mm) CAL (mm) GI (%) BI (%) PI (%)

Chronic periodontitis (n Z 27)

Aggressive periodontitis (n Z 13)

P value

3.07  0.49 4.00  0.74 59.15  20.70 56.33  19.87 66.59  27.74

3.78  1.10 5.06  1.81 62.31  17.85 64.08  21.30 72.62  24.72

0.044 0.062 0.640 0.266 0.510

BI Z bleeding index; CAL Z clinical attachment level; GI Z gingival index; PD Z probing depth; PI Z plaque index.

Please cite this article in press as: Chang C-W, et al., Association between monocyte chemoattractant protein-1 -2518 A/G gene polymorphism and the outcome of the nonsurgical periodontal treatment, Journal of the Formosan Medical Association (2017), http:// dx.doi.org/10.1016/j.jfma.2017.03.013

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C.-W. Chang et al. Table 3 The percentage distribution of different PD and CAL in chronic or aggressive periodontitis.

PD 1e3 mm (%) PD 4e6 mm (%) PD  7 mm (%) CAL 1e2 mm (%) CAL 3e4 mm (%) CAL  5 mm (%)

Chronic periodontitis (n Z 27)

Aggressive periodontitis (n Z 13)

73.83  13.99 22.74  12.26 3.45  3.12 20.35  12.95 46.39  11.13 33.26  17.18

59.70 27.59 12.73 15.56 39.06 45.38

     

22.67 12.51 12.46 10.99 19.49 28.47

P value

0.020 0.252 0.020 0.258 0.225 0.176

CAL Z clinical attachment level; PD Z probing depth.

influence on the treatment outcome in PD, GI and BI improvement (P < 0.05). Smoking habits were also related to less improvement in PD, CAL, and GI, while patients with older age had less GI and BI improvement. On the other hand, combined aggressive periodontitis and MCP-1 -2518 A/A genotype factors resulted in better treatment outcome in PD and BI parameters (P < 0.05). Moreover, more severe baseline measurements and more extracted teeth number were related to better treatment outcome. However, MCP1 gene polymorphism may not play a role in patients with chronic periodontitis.

Discussion Interleukin-1 (IL-1) gene polymorphism showed diverse frequency across multiple ethnic populations.14,25 The G allele frequency of MCP-1 -2518 A/G in our study was similar to other studies in Asian groups (42.5e66%),17,26,27 but it was different with Caucasian, AfricaneAmerican and Hispanic groups (22e30%).14,28,29 Our study reported that 42.5% of Taiwanese had allele G at MCP-1 -2518 and revealed there are different distributions of MCP-1 gene polymorphism among various ethnic groups which was consistent with the findings of IL-1 gene polymorphism. To the best of our knowledge, this study is the first research to investigate the effect of MCP-1 genotype on the outcomes of nonsurgical periodontal treatment. Although Pradeep et al. have found nonsurgical periodontal treatment significantly reduced MCP-1 levels in GCF and serum in periodontal patients,8 the association between MCP-1

gene polymorphism and periodontal treatment outcomes remains unclear. Our study revealed that in aggressive periodontitis patients, MCP-1 -2518 A/G genotype had significant negative impact on the treatment outcome in PD, GI and BI, while MCP-1 -2518 A/A genotype was associated with better treatment outcome in PD and BI. There was a study investigating the relationship between IL-1 gene polymorphism and nonsurgical periodontal treatment.30 IL1 gene positive is defined as composite genotype with allele 2 of both the genes IL-1A and IL-1B. Ehmke et al. have revealed no association between IL-1 gene polymorphism and clinical attachment loss after scaling and root planing.31 In contrast, Lang et al. have investigated the effects of supportive periodontal therapy and demonstrated that nonsmokers with IL-1 genotype positive had higher percentage of bleeding on probing.32 McGuire and Nunn have found that IL-1 genotype positive elevated 2.7 times of tooth loss while combined IL-1 genotype positive and heavy smoking elevated 7.7 times of tooth loss in patients under supportive periodontal therapy.33 In our study, we found that MCP-1 -2518 A/G genotype had significant negative impact in treatment response in the patients with aggressive periodontitis. Moreover, combined aggressive periodontitis and MCP-1 -2518 A/A genotype factors resulted in better treatment outcome in PD and BI parameters. Monocytes from individuals carrying a G allele at -2518 produce more MCP-1 than monocytes from A/ A homozygous subjects.14 The possible mechanisms involved may be related to dose-dependent effect of MCP-1. Besides MCP-1 -2518 gene polymorphism, many scientists analyzed the association of different gene polymorphism with aggressive periodontitis. Gene of interleukin-1 receptor antagonist was found with a variable number tandem repeat polymorphism and may be associated with a decreased risk on aggressive periodontitis.34 IL-6 -174 G allele and -572 C/G polymorphism may increase the risk of aggressive periodontitis.35 Furthermore, FcgRIIIB NA2 allele might elevate the risk for aggressive periodontitis.36 TLR4 polymorphisms may also play roles in the pathogenesis of aggressive periodontitis.37 In the present study, smoking habits and older age both contributed to worse treatment outcome which are consistent with previous reports.38,39 Higher GCF levels of MCP-1 in smokers with periodontitis than nonsmokers with periodontitis have been observed.10 Higher levels of MCP-1 in serum of smokers with periodontitis than nonsmokers

Table 4 Changes of clinical parameters after nonsurgical periodontal treatment according to MCP-1 -2518A/G genotype and periodontitis type. MCP-1 -2518 genotype

Periodontitis type

n

PD reduction (mm)

A/A

CP AP CP AP CP AP

10 1 13 11 4 1

0.67 1.17 0.74 0.87 0.93 0.95

A/G G/G

 0.27  0.26  0.90  0.60

CAL gain (mm) 0.65 0.73 0.66 0.71 0.73 0.93

 0.50  0.39  0.64  0.36

GI reduction (%) 35.80 49 42.46 33.45 48.00 33

 15.08  17.98  16.32  11.34

BI reduction (%) 31.80 50 38.69 36.64 39.50 31

 12.24  19.09  20.25  18.48

PI reduction (%) 38.2  20.64 74 40.62  24.76 40.27  14.64 58.75  18.08 58

AP Z aggressive periodontitis; BI Z bleeding index; CAL Z clinical attachment level; CP Z chronic periodontitis; GI Z gingival index; MCP-1 Z monocyte chemoattractant protein-1; PD Z probing depth; PI Z plaque index.

Please cite this article in press as: Chang C-W, et al., Association between monocyte chemoattractant protein-1 -2518 A/G gene polymorphism and the outcome of the nonsurgical periodontal treatment, Journal of the Formosan Medical Association (2017), http:// dx.doi.org/10.1016/j.jfma.2017.03.013

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MCP-1 -2518 gene polymorphism Table 5

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Multiple linear regression of predictors for improvement of PD, CAL, GI, BI and PI.

Independent variables MCP-1 A/G  AP MCP-1 A/A  APb Smoker Former smoker Nonsmoker Tooth extraction no. Age Baseline PD Baseline CAL Baseline GI GI1.s75.675  GI1c GI1.be75.675d Baseline BI Baseline PI a

PD (r2 Z 0.91) Estimate

P value

0.27 0.44 0.74

0.002 0.047 <0.001

0.08

<0.001

0.69

<0.001

CAL (r2 Z 0.60) Estimate

0.78

P value

<0.001

0.09

0.038

0.27

<0.001

GI (r2 Z 0.76)

BI (r2 Z 0.78)

Estimate

P value

Estimate

P value

17.56

<0.001

11.69 21.08

0.006 0.049

9.22 15.95

0.041 0.006

3.64 0.29

0.002 0.032

28.93 12.52 2.79 0.39

<0.001 0.016 0.016 0.006

0.79 51.48

<0.001 <0.001 0.61

<0.001

PI (r2 Z 0.74) Estimate

P value

0.56376

<0.001

AP Z aggressive periodontitis; BI Z bleeding index; CAL Z clinical attachment level; GI Z gingival index; MCP-1 Z monocyte chemoattractant protein-1; PD Z probing depth; PI Z plaque index. a MCP-1 A/G  AP: patients with MCP-1 -2518 A/G and aggressive periodontitis. b MCP-1 A/A  AP: patients with MCP-1 -2518 A/A and aggressive periodontitis. c When baseline GI < 75.675%, it had positive linear relationship with GI improvement. d When baseline GI  75.675%, GI improvement remained the constant level.

with periodontitis have also been observed.12 However, smoking status did not affect the association between MCP1 polymorphism and chronic/aggressive periodontitis in this study. On the other hand, we included teeth extraction number as an independent variable and observed that the more teeth extracted during treatment, the better therapy results obtained. Maybe it is because the responses of the remaining teeth were not masked by these severe and poorly responded extracted teeth. In contrast to the study by Preshaw et al., our research showed that more severe baseline parameters were statistically associated with better response to treatment.40 The possible explanation might be that we performed high quality of nonsurgical periodontal treatment by two well-trained periodontists. In Preshaw’s study, the procedure was performed by ten dental hygienists in training.40 More experienced and welltrained operators may decrease the chance of residual calculus remaining on root surfaces which may result in better improvement in more severe periodontitis teeth in our study. The frequency of MCP-1 -2518 G genotype has been shown to be significantly lower in female patients with aggressive periodontitis compared to the female healthy group.17 On the other hand, they found aggressive periodontitis female patients with MCP-1 -2518 A/A genotype had deeper probing depth than patients with MCP-1 -2518 A/G and G/G genotype. Moreover, confounding factors such as smoking habits, age, baseline parameters in the periodontal treatment can be important considerations for dentists to predict the outcomes of periodontal treatments. MCP-1 -2518 A/G genotype might be a useful predictor for less favorable outcome of nonsurgical periodontal treatment in subjects with aggressive periodontitis, whereas MCP-1 -2518 A/A genotype could predict better treatment results. However, the results demonstrated that

MCP-1 -2518 gene polymorphism might not play a role in chronic periodontitis patients. With a limited number of patients, this study suggests that MCP-1 -2518 genotype may influence the outcome of nonsurgical periodontal treatment in patients with aggressive periodontitis. Post hoc power calculations were performed and the linear regression analyses had at least 99% power to predict improvement of each periodontal parameter (PD, CAL, GI, BI or PI). Nevertheless, additional large sample-size studies in other ethnic populations may be necessary to confirm the findings. Further studies to evaluate the predictability of MCP-1 gene polymorphism on periodontal treatment response are also needed.

Conflicts of interest The authors have no conflicts of interest relevant to this article.

Acknowledgments No external funding, apart from the support of the authors’ institutions, was used for this study. The authors declare that there are no conflicts of interest in this study.

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Please cite this article in press as: Chang C-W, et al., Association between monocyte chemoattractant protein-1 -2518 A/G gene polymorphism and the outcome of the nonsurgical periodontal treatment, Journal of the Formosan Medical Association (2017), http:// dx.doi.org/10.1016/j.jfma.2017.03.013