Mannose binding lectin-2 gene functional polymorphisms in chronic periodontitis patients; a report from Iran

Accepted Manuscript Mannose binding lectin-2 gene functional polymorphisms in chronic periodontitis patients; a report from Iran

Somayeh Ansari Moghadam, Ali Bazi, Moghaddam, Ebrahim Miri-Moghaddam

Masoud

Miri-

PII: DOI: Reference:

S2214-5400(18)30130-0 doi:10.1016/j.mgene.2018.05.080 MGENE 449

To appear in:

Meta Gene

Received date: Revised date: Accepted date:

23 April 2018 25 May 2018 30 May 2018

Please cite this article as: Somayeh Ansari Moghadam, Ali Bazi, Masoud MiriMoghaddam, Ebrahim Miri-Moghaddam , Mannose binding lectin-2 gene functional polymorphisms in chronic periodontitis patients; a report from Iran. Mgene (2017), doi:10.1016/j.mgene.2018.05.080

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ACCEPTED MANUSCRIPT Mannose binding lectin-2 gene functional polymorphisms in chronic periodontitis patients; a report from Iran Somayeh Ansari Moghadama, Ali Bazib, Masoud Miri-Moghaddamc, Ebrahim Miri-Moghaddam d* a

Oral & Dental Disease Research Center, Department of Periodontology, Zahedan University of

Medical Sciences (ZUMS), Zahedan, Iran b

Clinical Research Development Unit, Amir-Al-Momenin Hospital, Zabol University of Medical

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Student Research Center, Faculty of Dentistry, ZUMS, Zahedan, Iran

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Sciences, Zabol, Iran

Cardiovascular Disease Research Center, Department of Molecular Medicine, Faculty of

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Medicine, Birjand University of Medical Sciences, Birjand, IR Iran *Corresponding author

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Ebrahim Miri-Moghaddam (PhD) Associated Professor of Genetics

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Address: Cardiovascular Disease Research Center, Department of Molecular Medicine,

Tel/Fax: +98-5632381230

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Faculty of Medicine, Birjand University of Medical Sciences, Birjand, IR Iran.

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E-mail: [email protected] & [email protected]

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ACCEPTED MANUSCRIPT Abstract Chronic Periodontitis (CP)1 is a bacterial inflammatory condition accompanied with destruction of tissues surrounding periodontal ligament and tooth loss. There are reports of associations between mannose binding lectin-2 (MBL2)2 gene polymorphisms and susceptibility to bacterial infections, however, a few studies have been performed on the role of MBL polymorphisms in periodontal diseases. Here, we assessed the potential impacts of two functional polymorphisms

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in MBL2 gene in CP. Forty CP patients along with 50 healthy controls were included. Two

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polymorphisms of MBL2 gene (rs1800451 and rs7096206) were assessed by Tetra-ARMS

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(Amplification Refractory Mutation System) PCR. Heterozygous (AG) and variant homozygous (AA) genotypes of rs1800451 polymorphism were detected in 17 (42.5%) and 2 (5%) of CP patients, and 17 (34%), and 1 (2%) in controls respectively (P=0.4). The variant allele frequency

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was 27.5% in CP patients and 20% in controls (P=0.2). For the rs7096206 polymorphism, heterozygous (CG) genotype was identified in 22 (55%) and 19 (38%) in healthy subjects

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respectively (P=0.1). Variant allele homozygous (GG) was only detected in one CP patient, and none of the healthy individuals. There was no significant difference in means of Clinical

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Attachment Loss 3(CAL) and Pocket Depth 4(PD) values regarding different genotypes. Although logistic regression analysis rendered odd ratio of 1.4 and 2.6, and 2 for AG and AA genotypes of

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rs1800451 polymorphism and CG genotype of rs7096206 polymorphism respectively, these were not statistically significant. Overall, there was no significant association between rs1800451 and

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rs7096206 polymorphism of MBL2 gene and CP.

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Keywords: Periodontitis, mannose binding lectin, complement system, polymorphism

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Chronic Periodontitis Mannose binding lectin-2 3 Clinical Attachment Loss 4 Pocket Depth 2

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ACCEPTED MANUSCRIPT 1. Introduction Chronic Periodontitis (CP)5 is an inflammatory condition associated with destruction of tissues surrounding periodontal ligament and tooth loss (Schätzle et al., 2004). The disease is a complex multifactorial condition caused by bacterial infections of gingival sulcus and periodontal pocket. Clinical presentation of CP is dependent on interaction between culprit organisms, environmental factors, genetic determinants, and host defense mechanisms (Karasneh et al., 2017). Various

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mediators such as prostaglandins, cytokines, and tissue remodeling enzymes (such as matrix

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metalloproteinases) take part in the pathogenesis of CP (Trindade et al., 2014, Bastos et al.,

exaggerated localized immune reactivity (Graves, 2008).

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2017). However, one of the main factors participating in tissue degeneration in periodontitis is

The role of genetic variations in genes encoding innate immunity components in the pathogenesis

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of periodontal diseases have been under extended studies. The risk of periodontitis has been associated with polymorphisms of IL-1α (da Silva et al., 2017), IL-1β (Wang et al., 2017), IL-6, IL-

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10 (Gorgun et al., 2017, Zhu et al., 2016), TLR-4 (Jin et al., 2016, Miri-Moghaddam et al., 2017), IL-17 (Chaudhari et al., 2016), IL-4 and IL-13 (Chen et al., 2016). Regarding the central role of

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complement components in innate immunity, genetic variations influencing the activity of complement pathways are potential regulators of immune responses. In accordance, complement

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activity may also contribute to the pathogenesis and progression of periodontitis (Hajishengallis, 2010). It has been shown that therapeutic modalities based on the modulation of complement

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system can be exploited to manage periodontal diseases (Hajishengallis, 2010). This however, necessitates identifying key regulators associated with complement activity.

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Mannose-binding lectins (MBLs)6 are molecules belonged to innate immune system acting as activators of the classical pathway of complement system. MBLs are members of C-type lectin

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family with biding properties for carbohydrates. MBLs are among first receptors that target foreign antigenic patterns (Soltani et al., 2014). MBLs activate complement classical pathway through interaction with MBL-associated serine proteases. Subsequently, the binding of the complex to various bacterial surfaces leads to activation of immune reactions (Eddie Ip et al., 2009) Low levels of MBL can be associated with increased predisposition to various bacterial (Holdaway et al., 2015, Guo et al., 2017) and viral infections (Gu et al., 2016). Also, low level of serum MBL

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Chronic Periodontitis Mannose-binding lectins 3

ACCEPTED MANUSCRIPT contributes to the severity of CP (Tsutsumi et al., 2009). On the other hand, MBL plasma level is influenced by its genetic polymorphisms (Tsutsumi et al., 2009). The most studied genetic polymorphisms of MBL2 gene reside in codons 52, 54, and 57 of exon1. Variant alleles in these locations are known as D (rs5030737,), B (rs1800450), and C (rs1800451,) respectively (Soltani et al., 2014). These alleles are resulted by base substitutions at nucleotides of 223 (C >T), 230 (G >A) and 239 (G > A) respectively. These alternations result

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in coding of different amino acid residues in the translated protein (Worthley et al., 2005). The

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presence of variant alleles in these sequences is usually depicted by the word “O” while “A”

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denotes the presence of wild type allele (Garred, 2008). Heterozygote individuals for variant alleles have shown reduced MBL level, while homozygotes represent with undetectable levels of MBL (Garred, 2008). In addition to exon 1 variations that lead to functional outcomes, such

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polymorphic sites also present in 5-untraslated region (5-UTR) and promoter region of MBL2 gene. The most common studied of these are variations at nucleotide -550 (G>C; denoted by H/L

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alleles, rs11003125), and -221 (G>C; denoted by X/Y alleles, rs7096206) (Navratilova et al., 2012).

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There are reports of associations between MBL2 gene polymorphisms and susceptibility to bacterial infections (Guo et al., 2017, Yanagisawa et al., 2015). Nevertheless, the association

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studies have had controversial results (Lundbo et al., 2015, Mills et al., 2015, Atan et al., 2016). A few studies have been performed on the role of MBL polymorphisms in periodontal diseases.

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In present study, we assessed two polymorphisms of MBL2 gene, rs1800451 and rs7096206 residing in exon 1 and 5-UTR region respectively, in patients with CP in south east of Iran.

2.1.

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2. Patients and methods Patients

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The patient population included 40 individuals diagnosed with CP referred to the Faculty of Dental Medicine, Zahedan University of Medical Sciences within 2015. After diagnosis of CP, periodontal chart was completed for patients, then pocket depth (PD)7, and clinical attachment loss (CAL)8 were measured for patients. In parallel, 50 healthy individuals with no signs of inflammation and no detectable calculus were enrolled as controls, their plaque index was less than 15%. Interviews were carried out to obtain demographic data for the both groups. Informed consents were obtained from the participants before filling out demographic questionnaire and after explaining

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Pocket depth Clinical attachment loss 4

ACCEPTED MANUSCRIPT aims of the study to the them. Our study was approved by the Ethical Committee of Research of Zahedan University of Medical Sciences (no: 6524). 2.2.

Exclusion criteria

Subjects with systemic diseases, severe bleeding disorders, history of corticosteroids or NSAIDs consumption, antibiotic therapy in the past six months, pregnant women and tobacco smokers

Inclusion criteria

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2.3.

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were excluded.

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After diagnosis of CP, patients were examined by Wiliams probe with 1milimeter accuracy and periodontal charts were completed for them. Data of PD, CAL were recorded, patients with CAL ≥5 in at least 2 areas were included. Subjects in control group were clinically healthy, with no

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signs of inflammation, no detectable calculus, no need to periodontal therapy and plaque index <15%. Sample collection

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2.4.

Saliva samples containing oral epithelial cells were exploited for DNA extraction. Exfoliated

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mucosal epithelial cells were expectorated into centrifuge tubes (3 ml TNE solution [17 mM Tris/HCl (pH 8.0), 50 mM NaCl and 7 mM EDTA] diluted in 66% ethanol. Then the samples were

DNA extraction and polymorphisms genotyping

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2.5.

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taken to the laboratory for DNA extraction.

DNA was obtained as previously described (Aidar and Line, 2007). We applied Tetra-ARMS (Amplification Refractory Mutation System) PCR method for identification of rs1800451 and

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rs7096206 polymorphisms of MBL2 gene. In this method, four primers (two outers and two inners) are simultaneously introduced into one PCR reaction. This method provides highly specific and

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cost-effective allele-specific amplifications. Sequences of primers and the length of PCR products have been presented in table 1. 2.6.

PCR reaction

PCR reaction was run at the net reaction volume of 25 μl. Genomic DNA (50 ng), 1 μl of each primer (0.1 pMol) and 12.5 μl Taq DNA polymerase master mix (Pishgam, Iran) were admixed in a single reaction tube, and the net volume was reached by adding distilled water. PCR was initiated with 5 min of denaturation phase at 94 of denaturation (5 min at 94

. The reaction was continued with thermal cycles

), annealing (30 seconds at 60

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for rs1800450 and 58

for

ACCEPTED MANUSCRIPT rs7096206) and extension (30 seconds at 72

) and a 5 minutes final stage at 72

was allowed.

The PCR products were visualized 2% agarose gel under UV emission. 2.7.

Statistical analyses

All analyses were performed by SPSS 21 software. Distribution of quantitative variables was checked by Kolmogorov-Smirnov test. Chi-square test was exploited for seeking a possible

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association between CP with categorical variables such as sex, position of jaw or type of the teeth

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involved. When appropriate, fisher exact test was considered for reporting p values of associations between these categorical variables and the clinical outcome. Any significant

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difference between the mean age of CP and control groups was checked using independent samples t-student test. Logistic regression was used to calculate odd ratios as a dominant model

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of logistic regression. Statistical significance threshold (P value) was considered as 0.05. 3. Results

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Forty patients diagnosed with CP were included along with 50 healthy matched subjects. Males and females comprised 13 (32.5%) and 27 (67.5%) of CP group, while 15 (30%) and 35 (70%) in

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controls respectively. The mean age and BMI for CP patients were 35 ± 10.7 years old and 24.7 ± 6.6 Kg/m2, while these values were 31 ± 4.5 years old and 22.1 ± 4.3 kg/m2 in controls. Table 1

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shows basic and clinical features in CP patients and controls. Heterozygous (AG) and variant homozygous (AA) genotypes of rs1800451 (variant C, codon 57)

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of MBL 2 were detected in 17 (42.5%) and 2 (5%) of CP patients. For the 5-UTR polymorphism (rs7096206), heterozygous (CG) and variant homozygous (GG) were identified in 22 (55%), and

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1 (2.5%) of CP patients. There was no significant difference in genotypes and allele frequencies between CP patients and normal individuals (table 2). Also, there was no significant difference in the means of CAL and PD values regarding different genotypes of either rs1800451 and

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rs7096206 polymorphisms (table 3). Although logistic regression analysis rendered the odd ratios of 1.4 and 2.6 for AG and AA genotypes of rs1800451 polymorphism respectively, and odd ratio of 2 for CG genotype of rs7096206 polymorphism, these were not statistically significant (table 4). 4. Discussion In present study, we assessed the potential impact of two polymorphisms in MBL2 gene; rs1800450 and rs7096206 residing in exon 1 and 5-UTR respectively. There was no significant association between either of these polymorphisms and CP. In line, no significant difference was

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ACCEPTED MANUSCRIPT reported in the frequencies of variant alleles of codons 52, 54, and 57 of MBL exon 1 comparing CP and normal subjects in a previous report (Ozcaka et al., 2010). In another study, Tsutsumi et al found no association between codon 54 polymorphism of MBL2 gene with CP (Tsutsumi et al., 2009). In another study in Caucasians, functional polymorphisms in MBL2 gene which resulted in either lower or higher plasma level of MBL showed no significance difference between periodontitis patients and normal individuals (Louropoulou et al., 2008). In another report,

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polymorphisms in innate immune receptors including TLR-2, TLR4, and MBL2 were associated

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with nor aggressive periodontitis or CP (Han et al., 2015). However, a relationship was found between the severity of periodontitis and codon 54 polymorphism (rs1800450) of MBL2 gene

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(Han et al., 2015). Polymorphisms of MBL2 gene has been associated with susceptibility to dental caries in adolescents, in a study in Saudi Aribia (Alyousef et al., 2017).

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Polymorphisms of MBL2 gene can be associated with lower levels of the respective protein in plasma. The most common polymorphisms accompanied with low serum levels of MBL are those

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identified in exon 1 (Liukkonen et al., 2016, Soltani et al., 2014). In addition, the level of MBL in serum may be determined by an interaction between different alleles residing in MBL2 gene

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(Soltani et al., 2014). In accordance, there is a highly broad range of serum MBL level among individuals raging form 50µg/L to more than 5000µg/L that are supposed to be the results of

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inheritance of genetic variations in MBL genetic loci (Lee et al., 2005). Although rs1800450 and rs7096206 polymorphisms are associated with depressed MBL protein,

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they have not been identified in correlation with CP susceptibility. In addition to the influence of genetic determinants on complement activity, bacterial profile of the disease also can influence

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this phenomenon, as some bacteria can themselves inhibit or activate complement components, and therefore obviate the action of inherent complement activators such as MBLs (Hajishengallis, 2010). In line with this, periodontitis patients with presence of Aggregatibacter actinomycetem

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comitans were found to represent higher destructive changes and complement activity in patients who carry the variant form of codon 54 polymorphisms of MBL-2 gene (Liukkonen et al., 2016). An interaction with bacterial populated within inflamed periodontal tissue and MBL variant allele of codon 54 was noted to be associated with higher local concentration of inflammatory cytokine IL-1β. This is while the elevated IL-1β was not the case in patients carried wild type allele of MBL2 codon 54 (Liukkonen et al., 2016). This is supported by some notions that relate the extent of inflammatory conditions in periodontitis to local complement activity (Hajishengallis, 2010). On the other hand, inflammatory cytokines are supposed to be the main culprits in tissue damage of periodontal area (Graves, 2008). In particular, the level of IL1β produced by leukocytes isolated 7

ACCEPTED MANUSCRIPT from CP patients was significantly higher than normal cases, and this was correlated with disease severity (Sakalauskiene et al., 2016). In this regard, coincidence of variant homozygote of MBL2 polymorphism along with TLR-2 gene polymorphism was correlated with lower level of IFN-γ (Poyhonen et al., 2017). A complex interaction between various genotypic signatures in multiple immune related genes may be more important than variations in individual genes in determination of CP clinical course.

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5. Conclusion

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We identified no significant association between rs1800451 and rs7096206 polymorphisms of

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MBL2 gene and CP. One limitation of our study was low number of our patients which may be a potential reason precluding achievement of a statistically significant association. We recommend

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assessing these polymorphisms in larger populations.

Conflict of Interest: The authors declare that they have no conflict of interest.

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Informed consent: Informed consents were obtained from the participant before filling out demographic questionnaire and after explaining aims of the study to the participant.

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Acknowledgment: The authors would like to thank the patients that participated in the research. The current research was funded by Research Deputy of Zahedan University of Medical Sciences

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under the grant number 6524.

Funding: This research did not receive any specific grant from funding agencies in the public,

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commercial, or not-for-profit sectors.

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Tables Table 1. Demographic and clinical parameters in 40 patients with chronic periodontitis and 50 healthy counterparts.

N=40

N=50

n (%)

n (%)

Male

13 (32.5%)

Female

27 (67.5%)

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Healthy controls

15 (30%)

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Sex

Chronic periodontitis

35 (70%)

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Parameters

35 ± 10.7

Body mass index (kg/m2)

24.7 ± 6.6

Pocket depth

2.9 ± 0.8

Clinical attachment loss

3.2 ± 0.5

P index

68.8 ± 22.7

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Age (years, mean ± SD)

31 ± 4.5 22.1 ± 4.3 -

Generalized

31 (77.5%)

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localization

Localized

9 (22.5%)

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Disease

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Table 2. Frequencies of genotypes and alleles of two polymorphisms in mannose-biding lectin gene; Rs1800451 and Rs7096206 in chronic periodontitis and healthy controls. Genotypes

Chronic

Healthy controls

periodontitis

N=50

N=40

n (%)

P value

32 (64)

AG

17 (42.5)

17 (34)

AA

2 (5)

1 (2)

G allele

29 (72.5)

40 (80)

A allele

11 (27.5)

10 (20)

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17 (42.5) 22 (55)

GG

1 (2.5)

C allele

28 (70)

G allele

12 (30)

0.1

0 (0) 40 (80)

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PT

ED

*; Fisher's exact test

0.2*

19 (38)

AN

CG

31 (62)

M

Rs7096206 CC

0.4

IP

21 (52.5)

CR

Rs1800451 GG

T

n (%)

13

10 (20)

0.1*

ACCEPTED MANUSCRIPT

Table 3. A comparison of clinical attachment loss and pocket depth of 50 patients with chronic periodontist regarding different genotype of rs1800451 and rs7096206 of mannose binding lectin 2 gene Genotypes

Clinical attachment Pocket depth loss

Mean ± SD

2.6±0.7

AG

4±1.4

3.1±0.8

AA

3.3±0.2

2.5±1

P value

0.1

0.1

Rs7096206 CC

3.2±0.5

2.6±0.7

CG

3.9±1.3

2.9±0.8

GG

NA a

US

P value

CR

3.3±0.7

IP

Rs1800451 GG

T

Mean ± SD

NA a 0.5

AN

0.1

a; not applicable, only one patient was detected with GG genotype in

AC

CE

PT

ED

M

chronic periodontitis patients

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ACCEPTED MANUSCRIPT

Table 4. Odd ratios and 95% confidence interval for genotypes and alleles of the two polymorphisms of MBL2 gene for chronic periodontitis Genotypes

Reference group

AG

1.4

0.5-3.5

0.4

AA

2.6

0. 2-32.7

0.4

G

Reference group

A

1.4 Reference group

CG

2

C

Reference group

G

1.6

IP

0.5-3.9

T

GG

Rs7096206a CC

Alleles

P value

CR

Alleles

95% CI

0.8-4.9

US

Rs1800451

Odd ratio

0.6-4.4

0.4

0.09

0.2

AN

a; Odd ratio for GG genotype was not retrieved because inadequate participants with

AC

CE

PT

ED

M

this genotype (only one individual in control group was identified with this combination)

15