A novel p.S34N mutation of CAMP gene in patients with periodontal disease

A novel p.S34N mutation of CAMP gene in patients withperiodontal disease

Oya Turkoglu a,*, Afig Berdeli b, Gulnur Emingil a, Gul Atilla a

aDepartment of Periodontology, School of Dentistry, Ege University, Bornova 35100, Izmir, TurkeybMolecular Medicine Laboratory, Department of Pediatrics, School of Medicine, Ege University, Izmir, Turkey

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 5 7 3 – 5 7 9

a r t i c l e i n f o

Article history:

Accepted 15 November 2010

Keywords:

Cathelicidin antimicrobial peptide

Mutation

Periodontitis

a b s t r a c t

Objective: Recent studies have showed that genetic factors involved in the host responses

might determine the severity of periodontitis. hCAP-18/LL-37 is a part of the innate immune

response in the oral cavity. The aim of the present study was to investigate the mutation of

CAMP gene encoding hCAP-18/LL-37 in the patients with different periodontal diseases.

Design: Seventy-eight chronic periodontitis, 72 generalized aggressive periodontitis, and 149

controls were analysed for mutation of CAMP gene using direct DNA sequencing method.

Frequencies of p.S34N mutation were compared by Pearson chi-square test. Logistic regres-

sion analysis was used to analyse the association between periodontitis and p.S34N muta-

tion adjusting for bleeding on probing, age and gender.

Results: Twenty-five subjects had a novel missense mutation of CAMP gene. Single base

substitution (c.101G>A) in exon 1 led to p.S34N mutation. All amino acid substitutions were

heterozygous mutation. The patients with generalized aggressive periodontitis had signifi-

cantly higher p.S34N mutation prevalence compared to the others, whilst there was no

significant difference in prevalence of p.S34N mutation between the patients with chronic

periodontitis and the control subjects. Logistic regression analysis adjusted for BOP, age and

gender revealed that the patients with generalized aggressive periodontitis were 5.32 times

more likely to have p.S34N mutation compared to the controls (OR = 5.32, 95% CI: 1.3–22.1).

Conclusion: We report a novel missense mutation of CAMP gene. p.S34N mutation in CAMP

gene seems to be contributing factor for generalized aggressive periodontitis, but not for

chronic periodontitis.

# 2010 Elsevier Ltd. All rights reserved.

avai lab le at www.sc iencedi rec t .com

journal homepage: http://www.elsevier.com/locate/aob

1. Introduction

Periodontitis is a multifactorial inflammatory disease that

involves microbial dental plaque, genetic and environmen-

tal factors, and affects the supporting tissues of teeth.1

Tissue destruction in periodontitis depends on the balance

between both host protective and destructive mechanisms.2

The innate immune response has a primary role in the

defense against plaque-associated bacteria.3,4 In addition,

inflammatory and immune processes protecting the gingi-

* Corresponding author. Tel.: +90 232 3881105; fax: +90 232 3880325.E-mail address: [email protected] (O. Turkoglu).

0003–9969/$ – see front matter # 2010 Elsevier Ltd. All rights reservedoi:10.1016/j.archoralbio.2010.11.016

val tissues against microbial attack are harmful to the host

in that inflammation can contribute to the tissue injury

observed in periodontitis.5 Chronic periodontitis is the

common form of periodontitis and has a slow on-going

course.6,7 Aggressive type of periodontitis is the severe form

of the periodontal diseases affecting mainly young subjects,

and have a rapid attachment loss and bone resorption.8 In

addition, aggressive periodontitis subjects present polymor-

phonuclear leucocyte defects that can produce high levels

inflammatory mediators.9

d.

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 5 7 3 – 5 7 9574

It has been suggested that genetic factors are important

determinants of susceptibility to periodontitis since they

determine and modify host responses to the microbial

challenge.10 Inappropriate or exaggerated immune responses

against bacterial stimulus may cause variations in the

susceptibility for periodontitis.10 Genetic factors involved in

host responses might determine the severity of disease in both

chronic and aggressive periodontitis.2,11–13 Although it is

known that microbial dental plaque is the major factor

causing periodontitis, there is accumulating evidence that

genetic factors play a crucial role in the susceptibility for

especially aggressive periodontitis.14 Aggressive periodontitis

has a tendency towards familial aggregation.8,15 In family

studies, it has been suggested that the pattern of disease

transmission is consistent with Mendelian inheritance of a

gene of major effect.15–17

Human cathelicidin antimicrobial peptide of 18 kDa (hCAP-

18) is the only known cathelicidin for humans.18 These

antimicrobial peptides are products of individual genes encod-

ing the corresponding pre-pro-peptides which are cleaved to

release the mature peptide LL-37.19 It has been suggested that

hCAP18 is expressed in the squamous epithelia,20 epididymis21

and lungs.22 Expression of hCAP18 in epithelial cells is inducible

by infection and inflammation.20,22 hCAP18 binds the microbial

cell membrane and kills the bacteria by disrupting the

metabolism.23 Cathelicidin antimicrobial peptides are impor-

tant defense molecules in the oral cavity because oral

epithelium is constantly exposed to microbial pathogens.24

hCAP-18/LL-37 proteinand mRNAhavebeendetectedinhuman

tongue, buccal mucosa and saliva.20,25 hCAP-18/LL-37 is

expressed from neutrophils in gingival tissues and particularly

in junctional epithelium which serves as a route for neutrophil

migration from the connective tissue into the gingival crevice.24

The production of hCAP-18/LL-37 is upregulated in inflamed

gingival tissues compared to healthy tissues.26 In our previous

study, we demonstrated that gingival tissue samples of chronic

periodontitis had significantly higher immunostainingof hCAP-

18/LL-37 on neutrophils infiltrating in both epithelium and

connective tissue compared to controls.27 We also showed that

hCAP-18/LL-37 mRNA expression levels in gingival tissue

samples of chronic periodontitis seemed to be upregulated

compared to controls.27 On the other hand, generalized

aggressive periodontitis patients showed less immunostaining

neutrophils for hCAP-18/LL-37 in tissue samples and down-

regulated mRNA levels for hCAP-18/LL-37.27 The lack of salivary

hCAP-18/LL-37 production in patients with morbus Kostman

syndrome who have increased susceptibility to periodontal

disease28 might suggest the protective role of hCAP-18/LL-37 in

host defense. The gene encoding human hCAP-18/LL-37,

defined as CAMP, is localized on chromosome 3, and consists

of 4 exons and 3 introns.19 Up to date, the biology of the

promoters of a few cathelicidin has been studied.22,29 However,

there is currently no detailed information about the gene

encoding hCAP-18/LL-37 antimicrobial peptide.

Since hCAP-18/LL-37 antimicrobial peptide regulates in-

nate immune response in periodontitis, we hypothesized that

a mutation in CAMP gene might modify the individual

susceptibility to periodontitis. To the best of our knowledge,

there is no study investigating the CAMP gene mutation in the

patients with different types of periodontitis, and also no

reported CAMP gene mutation to date. Therefore, the present

study aimed to investigate the mutation of CAMP gene in the

patients with different types of periodontitis.

2. Materials and methods

2.1. Study population

A total of 299 unrelated Caucasians residing in the Aegean

region of Turkey and belonging to a low to moderate

socioeconomic and education level were included in the

present study. All subjects were recruited from Ege University,

School of Dentistry, and Department of Periodontology

between 2005 and 2008. Exclusion criteria were systemic

diseases, immunological disorders, infectious diseases, smok-

ing, current pregnancy or lactation, oral contraceptive usage,

antibiotic regimen within the last 3 months or periodontal

treatment within the last 6 months. Study protocol was

approved by Ethics Committee of the Ege University School of

Medicine. Subjects gave written informed consent in accor-

dance with Helsinki declaration.

2.2. Determination of periodontal status

Subjects were evaluated clinically and radiographically at the

first visit, and all clinical measurements were performed by a

calibrated examiner (O.T.). The whole mouth periodontal

parameters including probing depth (PD), clinical attachment

level (CAL), and presence of bleeding on probing (BOP)30 were

determined at six sites per tooth excluding third molars.

Plaque index (PI)31 and papilla bleeding index (PBI)32 were also

assessed. Measurements of PD (mm) and CAL (mm) were

performed with William’s periodontal probe (Hu-Friedy,

Chicago, IL, USA). The intra-examiner reliability was high as

was revealed by intraclass correlation coefficient 0.87 and 0.85

for PD and CAL measurements, respectively.

2.3. Study groups

The diagnosis of chronic and aggressive periodontitis was

established on consensus reports of International World

Workshop.6,8 The classification of the study groups was based

on the classification system proposed by the 1999 Interna-

tional World Workshop for a Classification of Periodontal

Disease and Conditions.33 In this case control study, the

subjects were categorized into three groups as follows:

Generalized, severe chronic periodontitis group: Seventy-eight

patients were included in chronic periodontitis group. The

patients who had at least 4 non-adjacent teeth with sites

CAL � 5 mm and PD � 6 mm were defined as chronic

periodontitis patients. All chronic periodontitis patients

included in this group had generalized, severe chronic

periodontitis, i.e. they had affected sites (with CAL � 5 mm)

greater than 30% of sites involved.33 Diagnosis of chronic

periodontitis was made if the CAL was commensurate with

the amount of plaque accumulation of the patients. All

patients were older than 35 years of age. All patients had at

least 16 teeth in their mouth, and BOP was higher than 50%

for whole mouth.

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 5 7 3 – 5 7 9 575

Generalized aggressive periodontitis group: Seventy-two

patients were included in this group. These patients demon-

strated a generalized pattern of severe destruction and

CAL � 5 mm and PD � 6 mm on eight or more teeth, at least

three of these were other than central incisors of first molars.

Additionally, CAL was not consistent with the amount of

plaque accumulation or local contributing factors. Generalized

aggressive periodontitis was diagnosed in patients younger

than 35 years of age. All subjects had at least 16 teeth.

Control group: A hundred and forty-nine systemically

healthy subjects were included into the study. The periodon-

tally healthy subjects had PD � 3 mm, no gingival recession

due to periodontitis, CAL � 2 mm at more than or equal to 90%

of the measured tooth sites. Some control subjects who had

CAL > 2 mm at <10% of sites were included into the control

group, as judged by the examining clinician this CAL was not

due to periodontitis but to traumatic tooth brushing, over-

hanging fillings, orthodontic therapy, etc. None of the patients

had clinical signs of periodontitis. No radiographic evidence of

alveolar bone loss was observed in these patients (i.e., distance

between the cemento-enamel junction and bone crest �3 mm

at >95% of the proximal tooth sites). All subjects in this group

were older than 35 years so that they could not be too young to

have periodontitis.

2.4. Blood samples and DNA isolation

Peripheral venous blood collected in EDTA coated vacutainer

sterile tubes from each subject by standard venipuncture.

Genomic DNA was isolated using a commercially available

genomic DNA isolation kit (QIAamp Mini Blood DNA, Qiagen,

Ontario, Canada) according to the manufacturers’ instruc-

tions. DNA concentration was determined by spectrophotom-

etry at 280 nm and diluted as 50 ng/ml in 200 ml volume. All

genotyping processes were performed using quantified geno-

mic DNA.

2.5. Sequence analysis of the CAMP gene

All 4 exons of CAMP gene were amplified by polymerase chain

reaction (PCR) using flanking intronic primers with designed

using ExonPrimer-Primer Design programme (http://

igh.gsf.de/igh) obtained from Helmholz Center Institute of

Human Genetics (Munich, Germany). All synthetic oligonucle-

otide primers synthesized and purchased by Invitrogen Ltd.

(Paisley, UK) as the HPLC purification grade.

Primer sequences were 50-CAT AAA GGA GGC TCC TGT

GGG-30 sense and 50R-AGGC AGT GCA GCA GGA GCA GA-30

nonsense for exon 1, 50-GAGGTCATGG ACACAAATCA-30 sense

and 50-GAG TAG TTA ATT GAC CCA TT-30 nonsense for exon 2,

50-AACCTGTT TCTTCCTGTA CACAACCCC-30 sense and 50-

CAG AGC CCA GAA GCC TGA GCC-30 nonsense for exons 3 and

4. PCR amplification was carried out on a BioNeerMyGenieTM 96

Gradient Thermal Block the peltier-based systems (BioNeer

Corp, Daejeon, Korea) in a 25 ml reaction mixture in 0.2 ml

thin-wall PCR strip tubes (Axygen Scientific, Inc., CA, USA)

containing 1 ml genomic DNA solution, Platinium TAQ with

Enhancer Buffer (Invitrogen Ltd., Paisley, UK) 50 mmol/l each of

the dGTP, dATp, dTTP and dCTP (Promega, Madison, WI, USA),

5 pmol each forward and reverse primers and 1.0 U Platinium

TAQ Polymerase (Invitrogen Ltd., Paisley, UK). The cycling

conditions comprised a hot start at 95 8C for 10 min, followed

by 35 amplification cycles at gradient programme. Yielded PCR

products were purified using Exo-SAP PCR purification kit

(Amersham Life Science, Buckinghamshire HP7 9NA, UK).

Yielded purified PCR products were done cycle sequencing PCR

using the BigDye Terminator Cycle Sequencing Ready Reac-

tion Kit v.3.1 (Applied Biosystems, Forster City, CA, USA)

according to manufacturers’ instructions. Before sequencing

the PCR products were purified using BigDye XT Purification

Kit (Applied Biosystems, Forster City, CA, USA). DNA direct

sequence analysis in both forward and reverse direction was

performed by ABI3130xl Genetic Analyzer (Applied Biosys-

tems, Foster City, CA, USA). For sequence evaluation, the

SeqScape 2.0 sequencing analysis software (Applied Biosys-

tems, Foster City, CA, USA) was used. The obtained nucleotide

sequences were compared to publish Genbank (NM_004345)

and protein reference sequence (NP_004336) obtained from

NCBI (http://www.ncbi.nlm.nih.gov).

2.6. Statistical analysis

Minimal sample size was determined to detect 7% difference

in intergroup comparisons for allele frequency with 80%

power and p = 0.05 significance level. Statistical analysis was

performed a statistician who is blinded to the clinical

diagnosis of the study subjects. One-way ANOVA test was

used for comparisons of demographic and periodontal

parameters amongst the study groups. When there were

significant differences (p < 0.05), post hoc two group compar-

isons were performed with Dunnett T3 or Bonferroni tests.

Chi-square tests were used to evaluate the concordance of

genotype frequencies with Hardy–Weinberg equilibrium in

each group. The N34 allele and carrier frequencies for p.S34N

mutation were compared by Pearson chi-square test amongst

study groups. The risk of genotypes with periodontitis was

estimated by computing the odds ratio (OR) and 95% CI from

logistic regression analysis, adjusting for BOP, age and gender.

All data analysis was performed using a statistical package

(SPSS Inc., ver. 17.0, Chicago, IL, USA), and p-values < 0.05

were considered to be statistically significant.

3. Results

The genotype frequencies of the whole study population as

well as the frequencies of the three study groups were in

Hardy–Weinberg equilibrium (p > 0.05, x2 < 5.99). Minimum

required sample size was calculated as 70 and 145 subjects for

patient and control groups, respectively.

3.1. Demographic and clinical periodontal findings

The demographics and periodontal parameters of the study

groups are presented in Table 1. Generalized aggressive

periodontitis patients were younger than the other subjects

(p < 0.05). There was no significant difference in gender

amongst the study groups (p > 0.05).

The whole mouth PD score of the control subjects was

statistically lower than those of the others (p < 0.05). CAL

Table 1 – Demographic characteristics and clinical periodontal data of the study groups (distribution or mean W SE).

Generalized aggressive periodontitisN = 72

Chronic periodontitisN = 78

ControlN = 149

Age 28.9 � 0.7* 47.6 � 0.8* 40.5 � 0.6

Gender (N)

Male 40 42 88

Female 32 36 61

PD 3.8 � 0.09 3.8 � 0.09 1.9 � 0.03*

CAL 4.5 � 0.13 4.7 � 0.13 0.07 � 0.008*

BOP % 71.4 � 2.3 82.8 � 2.1* 37 � 2.9*

PBI 1.9 � 0.12 2.2 � 0.07 1 � 0.08*

PI 2.6 � 0.13 3 � 0.09* 2.3 � 0.06*

PD = probing depth, CAL = clinical attachment level, BOP = bleeding on probing, PBI = papilla bleeding index, PI = plaque index, SE = standard

error.* Significant difference from the other groups ( p < 0.05).

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 5 7 3 – 5 7 9576

scores were similar in generalized aggressive periodontitis

and chronic periodontitis patients (p > 0.05). Periodontitis

patients (both aggressive and chronic) had significantly higher

CAL scores than those of control subjects (p < 0.05). Control

subjects had significantly less percentages of sites with BOP

when compared with the others (p < 0.05). Control subjects

had lower PBI and PI scores compared to the other groups

(p < 0.05) (Table 1).

3.2. Molecular genetic findings

Seventy-eight chronic periodontitis subjects, 72 generalized

aggressive periodontitis subjects, 149 unrelated controls were

Fig. 1 – Exon 1’s nucleotide sequences and amino acid codon ele

panel: mutant type.

analysed by the direct DNA sequencing method for the

mutation of CAMP gene. All four coding exons and exon–

intron bounder region of CAMP gene from all subjects were

sequenced. Twenty-five subjects had missense mutation in

exon 1 of the CAMP gene. Single nucleotide substitution in

exon 1 (c.101G>A) leads to missense amino acid mutation

(p.S34N) (AGC>AAC) (Fig. 1). Homozygous mutation p.N34N

was not observed in the study subjects. All amino acid

substitutions were heterozygous mutation (Table 2). The

carrier rate for the N34 allele was 8.4% (25 subjects) in whole

study population. The prevalence of the p.S34N mutation for

generalized aggressive periodontitis, chronic periodontitis,

and control groups were 16.7%, 6.4%, and 5.4%, respectively

ctropherogram of CAMP gene. Upper panel: wild type, lower

Table 2 – Distribution of genotypes and allele frequenciesof the study groups (N, %).

Generalizedaggressive

periodontitisN = 72

Chronicperiodontitis

N = 78

ControlN = 149

Genotype

Wild type 60 (83.3) 73 (93.6) 141 (94.6)

S34N 12 (16.7)* 5 (6.4) 8 (5.4)

N34N 0 (0.0) 0 (0.0) 0 (0.0)

Allele

S34 132 (91.7) 151 (96.8) 290 (97.3)

N34 12 (8.3)* 5 (3.2) 8 (2.7)

* Significant difference from the other groups ( p < 0.05).

Table 3 – Logistic regression analysis for an associationbetween the prevalence of p.S34N mutation and period-ontitis (adjusted for BOP, age, and gender).

OR 95% CI p

BOP 0.98 0.97–1 0.09

Age 0.97 0.91–1.04 0.47

Gender (reference male) 2.87 1.08–7.6 0.03

Generalized aggressive

periodontitis (reference control)

5.32 1.3–22.1 0.02

Chronic periodontitis

(reference control)

3.32 0.64–17.21 0.15

OR = odds ratio, CI = confidence interval.

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 5 7 3 – 5 7 9 577

(Table 2). There was no significant difference in prevalence of

p.S34N mutation between chronic periodontitis and control

group (p > 0.05) (Table 2). The prevalence of p.S34N mutation

and N34 allele frequency in generalized aggressive periodon-

titis group was significantly higher compared to those of the

other groups ( p < 0.05) (Table 2).

The logistic regression analysis adjusted for BOP, age and

gender showed that generalized aggressive periodontitis was

significantly associated with p.S34N mutation ( p < 0.05) (Table

3). In addition, gender was also found to be associated with

p.S34N mutation (p < 0.05). The logistic regression analysis

demonstrated that generalized aggressive periodontitis

patients were 5.32 (95% CI = 1.3–22.1) times more likely to

have p.S34N mutation than control subjects (p < 0.02). Whilst

age and chronic periodontitis did not appear to be associated

with p.S34N mutation, females were 2.87 (95% CI = 1.3–22.1)

times more likely to have p.S34N mutation than male subjects

(p < 0.03).

4. Discussion

We report a novel missense mutation in CAMP gene in the

present study. Our results showed significant difference in

prevalence of p.S34N mutation between generalized aggres-

sive periodontitis and the other groups. p.S34N mutation

prevalence was higher in generalized aggressive periodontitis

group compared to chronic periodontitis and control groups.

However, no significant difference in prevalence of p.S34N

mutation between chronic periodontitis patients and controls

was presented. To the best of our knowledge, this is the first

study reporting a novel missense p.S34N mutation in CAMP

gene.

cDNA encoding region for four exons and exon–intron

bounder region of CAMP gene from 299 subjects were

sequenced in the present study. Twenty-five missense

p.S34N mutations in CAMP gene were found in the whole

study population. p.S34N mutation in CAMP gene includes a

single base substitution in exon 1. This single base substitu-

tion in exon1 (c.101G>A) alters the DNA codon from the amino

acid serine substitution (AGC) to asparagine substitution

(AAC). This nucleotide change (AGC/AAC) caused p.S34N

missense mutation.

In the present study, eight of 149 control subjects had

p.S34N mutation although they had no sign of periodontitis.

Periodontal diseases are multifactorial inflammatory diseases

caused by microbial dental plaque, genetic and environmental

factors,10,13,34,35 and there is no factor responsible for

periodontal disease alone. Periodontal tissue destruction

depends on the balance between host protective and

destructive mechanism.2 Our results demonstrated that

generalized aggressive periodontitis patients have significant-

ly higher prevalence of p.S34N mutation compared to chronic

periodontitis patients and control subjects. Similarly, N34

allele frequency was also significantly higher in generalized

aggressive periodontitis patients than the others. However,

the prevalence of p.S34N mutation of chronic periodontitis

group was similar to that of the control group. Logistic

regression analysis adjusted for BOP, age and gender showed

that there was a significant association between p.S34N

mutation and generalized aggressive periodontitis. General-

ized aggressive periodontitis patients were 5.3 times more

likely to have p.S34N mutation than control subjects.

Nevertheless, chronic periodontitis was not associated with

p.S34N mutation after logistic regression analysis. Host

immune reactions are in part genetically determined, and

genetic predisposition is an important factor for especially

aggressive periodontitis.2,34,35 Furthermore, familial aggrega-

tion is possible in aggressive periodontitis.8 In our previous

study, we demonstrated that generalized aggressive peri-

odontitis patients showed less immunostaining neutrophils in

gingival tissue samples, besides downregulated mRNA levels

for hCAP-18/LL-37 compared to healthy controls.27 We

suggested that the lack of hCAP-18/LL-37 might be an

important factor in the pathogenesis of the generalized form

of the aggressive periodontitis.27 Based on the present data, we

suggest that p.S34N mutation might be one of the contributing

factors for the pathogenesis of generalized aggressive peri-

odontitis.

hCAP-18/LL-37 is mainly released from neutrophils emi-

grating into inflammatory sites and plays an important role in

the balance between health and disease of the oral cavity.19,24

Dale et al.24 showed that antimicrobial peptides in the gingiva

are localized in specific sites, and have different roles in

various regions of the periodontium. Researchers stated that

hCAP-18/LL-37 was expressed from neutrophils in gingival

tissues and particularly in junctional epithelium which serves

as a route for neutrophil migration from the connective tissue

into the gingival crevice.24 Hosokawa et al.26 demonstrated

that neutrophils expressed hCAP-18/LL-37, and that expres-

sion was more prominent in the inflammatory lesions when

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 5 7 3 – 5 7 9578

compared to healthy gingiva. This higher expression of hCAP-

18/LL-37 in inflammatory lesions might be explained by the

fact that microbial dental plaque stimulates both inflamma-

tory and immune responses during the progression of

periodontal disease. Moreover, Puklo et al.36 stated that local

deficiency in hCAP-18/LL-37 could be considered as a

supporting factor in the pathogenesis of severe periodontitis.

After stimulation, neutrophils migrate into gingival crevice

through the junctional epithelium from connective tissue, and

this antimicrobial peptide plays a protective role in defense of

oral cavity against to microbial invasion. Putsep et al.28 have

suggested that a mutation in CAMP gene could prevent or shut

off production of the cathelicidin peptides and have been

suggested as the underlying mechanism in Morbus Kostman

which is characterized with severe neutrophil deficiency. A

mutation in CAMP gene encoding hCAP-18/LL-37 might

increase the severity of the disease by modifying host

responses. Our result shown that generalized aggressive

periodontitis was significantly associated with p.S34N muta-

tion whilst chronic periodontitis did not appear to be

associated with p.S34N mutation. Our results is supported

by the results of our previous study27 and the results of Puklo

et al.36

As a limitation of the present study, sixteen periodontally

healthy subjects who had CAL > 2 mm at <10% of measured

sites were included into the study. As it is well known,

clinically detectable attachment loss may occur as a result of

events other than periodontitis, such as traumatic tooth

brushing, tooth position or subgingival margins of restora-

tions, etc. In clinical view, it is very difficult to find

periodontally healthy subjects who have no clinical attach-

ment loss in her/his dentition, who are 35 years old and more.

The presence of CAL > 2 mm at <10% of sites in some control

subjects in the present study was not due to periodontitis but

to traumatic tooth brushing, overhanging fillings, orthodontic

therapy, etc., as judged by the examining clinician.

As a conclusion, we report a novel missense mutation in

CAMP gene, mutation p.S34N, which changes DNA codon for

the amino acid serine substitution (AGC) to asparagine

substitution (AAC). To best our knowledge, this is the first

study investigating the CAMP gene mutations in periodontal

diseases. Our results demonstrated that generalized aggres-

sive periodontitis was significantly associated with p.S34N

mutation, but not chronic periodontitis. Therefore, it might be

suggested that p.S34N mutation is a contributing factor for

developing generalized aggressive periodontitis, within the

limitations of the present study. The studies investigating the

improvement of the periodontal parameters after periodontal

treatment in periodontitis patients who have p.S34N mutation

would be interesting in view of evaluating of host response to

treatment in these patients. Further studies are necessary to

investigate if p.S34N mutations are associated with suscepti-

bility to periodontitis in combination with polymorphisms in

other genes.

Acknowledgements

We thank to the Scientific and Technological Research Council

of Turkey (TUBITAK).

Funding: This study was supported by grants from The

Scientific and Technological Research Council of Turkey

(TUBITAK, SBAG-105S463).

Competing interests: We do not have any conflict of interests.

Ethical approval: The study protocol was approved by

Research Ethics Committee of Medical Faculty, Ege University

(# 05-9/6).

r e f e r e n c e s

1. Listgarten MA. Pathogenesis of periodontitis. J ClinPeriodontol 1986;13(5):418–30.

2. Kinane DF, Hart TC. Genes and gene polymorphismsassociated with periodontal disease. Crit Rev Oral Biol Med2003;14(6):430–49.

3. Boman HG. Innate immunity and the normal microfora.Immunol Rev 2000;173:5–16.

4. Ji S, Hyun J, Park E, Lee BL, Kim KK, Choi Y. Susceptibilityof various oral bacteria to antimicrobial peptides and tophagocytosis by neutrophils. J Periodont Res 2007;42(5):410–9.

5. Kornman KS. Mapping the pathogenesis of periodontitis: anew look. J Periodontol 2008;79(8):1560–8.

6. Lindhe J, Lamster RI, Charles A, Chung CP, Flemmig T,Kinane D, et al. Consensus report: chronic periodontitis. AnnPeriodontol 1999;1(4):38.

7. Flemmig TF. Periodontitis. Ann Periodontol 1999;4(1):32–8.8. Lang N, Bartold PM, Cullinan M, Jeffcoat M, Mombelli A,

Murakami S, et al. Consensus report: aggressiveperiodontitis. Ann Periodontol 1999;1(4):53.

9. Shapira L, Warbington M, Van Dyke TE. TNF alpha and IL-1 beta in serum of LJP patients with normal and defectiveneutrophil chemotaxis. J Periodontal Res 1994;29:371–3.

10. Page RC, Offenbacher S, Schroeder HE, Seymour GJ,Kornman KS. Advances in the pathogenesis of periodontitis:summary of developments, clinical implications and futuredirections. Periodontology 2000 1997;14:216–48.

11. Michalowicz BS, Wolff LF, Klump DG, Hinrichs JE, Aeppli D,Bouchard TJ, et al. Periodontal findings in adult twins. JPeriodontol 1991;62(5):293–9.

12. Gore EA, Sanders JJ, Pandey JP, Palesch Y, Galbraith GM.Interleukin-1beta13953 allele 2: association with diseasestatus in adult periodontitis. J Clin Periodontol1998;25(10):781–5.

13. Laine ML, Farre MA, Gonzalez G, van Dijk LJ, Ham AJ, WinkelEG, et al. Polymorphisms of the interleukin-1 gene family,oral microbial pathogens, and smoking in adultperiodontitis. J Dent Res 2001;80(8):1695–9.

14. Loos BG, Van der Velden U, Laine ML. Susceptibility. In:Lindhe J, Karring T, Lang NP, editors. Clinical periodontologyand implant dentistry. 5th ed. Oxford: Blackwell Munksgaard;2008. p. 328–46.

15. Marazita M, Burmeister J, Gunsolley J. Evidence forautosomal dominant inheritance and race-specificheterogeneity in early-onset periodontitis. J Periodontol1994;65(6):623–30.

16. Beaty TH, Boughman JA, Yang P, Astemborski JA, Suzuki JB.Genetic analysis of juvenile periodontitis in familiesascertained through an affected proband. Am J Hum Genet1987;40(5):443–52.

17. Hart TC, Marazita ML, Schenkein HA, Diehl SR. Re-interpretation of the evidence for X-linked dominantinheritance of juvenile periodontitis. J Periodontol1992;63(3):169–73.

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 5 7 3 – 5 7 9 579

18. Durr UH, Sudheendra US, Ramamoorthy A. LL-37, the onlyhuman member of the cathelicidin family of antimicrobialpeptides. Biochim Biophys Acta 2006;1758(6):1408–25.

19. Bals R, Wilson JM. Cathelicidins – a family ofmultifunctional antimicrobial peptides. Cell Mol Life Sci2003;60(4):711–20.

20. Frohm Nilsson M, Sandstedt B, Sorensen O, Weber G,Borregaard N, Stahle-Backdahl M. The human cationicantimicrobial protein (hCAP18), a peptide antibiotic, iswidely expressed in human squamous epithelia andcolocalizes with interleukin-6. Infect Immun 1999;67(5):2561–6.

21. Malm J, Sorensen O, Persson T, Frohm-Nilsson M, JohanssonB, Bjartell A, et al. The human cationic antimicrobial protein(hCAP-18) is expressed in the epithelium of humanepididymis, is present in seminal plasma at highconcentrations, and is attached to spermatozoa. InfectImmun 2000;68(7):4297–302.

22. Frohm M, Agerberth B, Ahangari G, Stahle-Backdahl M,Liden S, Wigzell H, et al. The expression of the gene codingfor the antibacterial peptide LL-37 is induced in humankeratinocytes during inflammatory disorders. J Biol Chem1997;272(24):15258–63.

23. Ciornei CD, Sigurdardottir T, Schmidtchen A, Bodelsson M.Antimicrobial and chemoattractant activity,lipopolysaccharide neutralization, cytotoxicity, andinhibition by serum of analogs of human cathelicidin LL-37.Antimicrob Agents Chemother 2005;49(7):2845–50.

24. Dale BA, Kimball JR, Krisanaprakornkit S, Roberts F,Robinovitch M, O’Neal R, et al. Localized antimicrobialpeptide expression in human gingival. J Periodontal Res2001;36(5):285–94.

25. Murakami M, Ohtake T, Dorschner RA, Gallo RL.Cathelicidin antimicrobial peptides are expressed insalivary glands and saliva. J Dent Res 2002;81(12):845–50.

26. Hosokawa I, Hosokawa Y, Komatsuzawa H, Goncalves RB,Karimbux N, Napimoga MH, et al. Innate immune peptide

LL-37 displays distinct expression pattern from beta-defensins in inflamed gingival tissue. Clin Exp Immunol2006;146(2):218–25.

27. Turkoglu O, Kandiloglu G, Berdeli A, Emingil G, Atilla G.Antimicrobial peptide hCAP-18/LL-37 protein and mRNAexpressions in different periodontal diseases. Oral Dis2011;17(1):60–7. 10.1111/j.1601-0825.2010.01704.x.

28. Putsep K, Carlsson G, Boman HG, Andersson M. Deficiencyof antibacterial peptides in patients with morbusKostmann: an observation study. Lancet 2002;360(9340):144–1149.

29. Sorensen OE, Follin P, Johnsen AH, Calafat J, Tjabringa GS,Hiemstra PS, et al. Human cathelicidin, hCAP-18, isprocessed to the antimicrobial peptide LL-37 byextracellular cleavage with proteinase 3. Blood2001;97(12):3951–9.

30. Ainamo J, Bay I. Problems and proposals for recordinggingivitis and plaque. Int Dent J 1975;25(4):229–35.

31. Quigley GA, Hein JW. Comparative cleansing efficiency ofmanual and power brushing. J Am Dent Assoc 1962;65:26–9.

32. Saxer UP, Muhlemann HR. Motivation and instruction. SSOSchweiz Monatsschr Zahnheilkd 1975;85(9):905–19.

33. Armitage GC. Development of a classification system forperiodontal diseases and conditions. Ann Periodontol1999;4(1):1–7.

34. Loos BG, John RP, Laine ML. Identification of genetic riskfactors for periodontitis and possible mechanisms of action.J Clin Periodontol 2005;32(6):159–79.

35. Shapira L, Wilensky A, Kinane DF. Effect of geneticvariability on the inflammatory response to periodontalinfection. J Clin Periodontol 2005;32(6):72–86.

36. Puklo M, Guentsch A, Hiemstra PS, Eick S, Potempa J.Analysis of neutrophil-derived antimicrobial peptides ingingival crevicular fluid suggests importance of cathelicidinLL-37 in the innate immune response againstperiodontogenic bacteria. Oral Microbiol Immunol2008;23(4):328–35.