The role of genetic polymorphisms in periodontitis

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The role of geneticpolymorphisms in periodontitis

H I R O M A S A YO S H I E, T E T S U O KO B A Y A S H I, H I D E A K I TA I & J O H N A H C. GA L I C I A

A detailed knowledge of the dynamic between the

host immune responses and oral bacteria is essential

to understand clearly the pathogenesis of periodontal

disease. Periodontopathic bacteria initiate and re-

peatedly attack the host, stimulating an immune re-

sponse, but the presence of pathogenic subgingival

flora alone does not always equate to periodontal

destruction. Although bacterial plaque is essential for

the initiation of periodontitis, the amount of plaque

does not necessarily correlate with disease severity

(178). Each person has an individual doseresponse

curve that defines host susceptibility to periodontitis

(180). Certain patients are disease-resistant and will

not progress beyond gingivitis or early periodontitis.

This places the emphasis on host response, rather

than bacterial etiology, as the principal determinant

of disease expression.

The vast number of scientific papers availablesupport the indispensable role of genes in host re-

sponses and, consequently, in progression of the

disease. Specifically, different allelic variants can re-

sult in variations in tissue structure (innate immu-

nity), antibody responses (adaptive immunity) and

inflammatory mediators (non-specific inflammation)

(126). Genetic variations may also act as protective or

risk factors for certain conditions, including perio-

dontitis (173). For these reasons, periodontitis is

considered as a complex genetic disease whose

phenotype is determined by both the genetic makeup

and the environmental influences on the affectedindividual.

It has already been established that interindividual

variations in the host immune responses and most

human diseases have genetic components in them.

In particular, genetic differences in immune-cell

development and antigen presentation may contrib-

ute to the susceptibility to autoimmune and infec-

tious diseases (93, 194). Identifying genes can

therefore result in novel diagnostics for risk assess-

ment, early detection and individualized treatment

approaches.

Like any other complex disease, genetic variants at

multiple loci associated with periodontitis synergis-

tically contribute to the overall disease process. Re-

sults obtained from numerous genetic reports may be

utilized to identify the candidate genes for perio-

dontal disease profiling in both aggressive and

chronic periodontitis. Identifying genes that con-

tribute to the pathogenesis of periodontitis can have

significant public health, therapeutic and scientific

repercussions. Loss of teeth induced by periodontitis

has a considerable effect on the homeostasis of cra-

niofacial and oral tissues, and consequently on the

general wellbeing of the individual. This global health

concern is instrumental in the continuous quest of

the scientific field to provide evidence linking gen-

etics and periodontitis.

Evidence linking genetics andperiodontitis

Heritability of aggressive periodontitis

Aggressive periodontitis is a specific type of perio-

dontitis with clearly identifiable clinical findings, ra-

pid attachment loss and alveolar bone destruction,

which make it distinct from chronic periodontitis.

Aggressive periodontitis is subdivided into prepu-bertal, juvenile and rapidly progressive periodontitis,

and early onset periodontitis. Familial aggregation

reports have shown clustering within families, which

strongly suggests a genetic component of the disease

(16, 17, 21, 22, 28, 65, 66, 121, 134, 149, 154, 156, 165,

181, 206, 231, 251). Although these numerous reports

support a genetic basis for aggressive periodontitis,

sufficient consideration must be placed on the extent

of the environmental effects and behavioral patterns

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Periodontology 2000, Vol. 43, 2007, 102132

Printed in Singapore. All rights reserved

2007 The Authors.

Journal compilation 2007 Blackwell Munksgaard

PERIODONTOLOGY 2000


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that families share. These include nutrition, socio-

economic status, sanitation and diseases like diabe-

tes, among others (126). In a large-scale segregation

analysis of more than 100 families, performed by

Marazita et al. (154), a 70% autosomal-dominant

transmission of periodontitis was found in both

Blacks and non-Blacks. Segregation analysis is a

method used to study families to assess the likelihood

that a certain disease is inherited as a genetic trait. Alinkage study has identified a gene locus responsible

for aggressive periodontitis on chromosome 4 [log-

arithm of odds (LOD) score 3.0] but this finding

was later refuted with suggestions on the genetic

locus heterogeneity of aggressive periodontitis (22,

88). This means that the disease may be a result of

mutations in several loci. Very recently, aggressive

periodontitis was linked to chromosome 1q25 (LOD

score 3.48) (146). The result was established after

performing linkage analysis in four multigenerational

families exhibiting the localized aggressive perio-

dontitis phenotype. Linkage analysis is a way of

localizing a trait to a specific location along a chro-

mosome.

Heritability of chronic periodontitis

Chronic periodontitis (former name; adult perio-

dontitis) is the most frequently occurring form of

periodontitis, characterized by slowly progressing

alveolar bone destruction and attachment loss. Genes

have also been implicated to play a role in chronic

periodontitis, but in contrast to aggressive perio-dontitis, chronic periodontitis does not typically

follow a simple pattern of familial transmission or

distribution. The twin study is probably the most

popular method that supports the genetic aspects of

chronic periodontitis. This study substantiates the

contribution that genes make vs. the environment in

a phenotypic expression. Monozygous twins, in

contrast to dizygous twins, come from a single ovum

and therefore share exactly the same genes. Dis-

cordance in the disease experience of monozygous

twins must be caused by environmental determi-

nants as seen in twins reared apart. In dizygous twins,differences could be a result of both genetic and

environmental differences. Michalowicz et al. (166)

reported the periodontal condition of 110 pairs of

twins from 16 to 70 years old. The mean probing

depth and attachment level scores were found to vary

less in monozygous twins reared together than in

dizygous twins reared together. In a subsequent

related study of 64 monozygous and 53 dizygous

adult twin pairs, the genetic and environmental var-

iances and heritability in chronic periodontitis,

according to path models, were estimated using

maximum likelihood estimation techniques (167).

Monozygous twins were found to be more similar

than dizygous twins for all clinical measures. Statis-

tically significant genetic variance was found for both

the severity and extent of disease. Adult periodontitis

was estimated to havec. 50% heritability, which was

unaltered following adjustments for behavioral vari-ables, including smoking. It was concluded that there

is indeed a substantial genetic basis for the risk of

chronic periodontitis. A study by Corey et al. (38)

among 4908 twin pairs revealed that of the 116

identical and 233 non-identical twins, 9% reported a

history of periodontitis. The concordance rates were

0.230.36 for monozygous twins and 0.080.16 for

dizygous twins. However, in this study, environ-

mental factors like smoking were not controlled,

thereby creating bias toward establishing a correla-

tion between twins.

Gene polymorphisms inperiodontitis: aiming at the righttargets

Most genetic research in periodontitis has focused on

gene polymorphisms that play roles in immunoreg-

ulation or metabolism, such as cytokines, cell-surface

receptors, chemokines, enzymes and others that are

related to antigen recognition (Table 1). The follow-

ing sections discuss the different studies that wereundertaken to further understand the roles of gene

polymorphisms in periodontitis.

Cytokine gene polymorphisms

Interleukin-1 family

The biological activity, molecular biology and clinical

relevance of the interleukin-1 family [interleukin-1ab,

interleukin-1 receptor I/II/a] have been studied

extensively and reviewed time and again (6, 46, 50,212, 261). Interleukin-1 is a potent pro-inflammatory

agent that is released by macrophages, platelets and

endothelial cells. The gene encoding this cytokine is

assigned to chromosome 2q1321 (10, 29, 155). Based

on the number of published reports, the interleukin-1

genotypes appear to be the most studied genetic

association with periodontal disease (Table 1).

However, carriage rates of interleukin-1 alleles differ

across populations (Fig. 1). In 1997, Kornman et al.

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Table

1.

Reportsontheassociationbetweenperiodontitisandpolymorphismsingenesaffectinghostresponseandmetabolism

Gene

P

osition

Aggressiveperiodontitis*

Chronicperiodontitis*

References

IL-1cluster

a

;)889/+4845,)511

1/8,1/3

1/11,0/2

(4,8,49,74,78,80,96,

136,141,147,151)

b

;)31,+3954RNPAG

)4/10,2/4,0/6

1/1,5/14,1/1,5/10

(157,164,185,187,196,203,

207,221,237,246,259)

Otherinterleukins

2

;)330

1/1

(208)

4

;)590,VNTR

0/3,0/3

0/3,0/2

(51,79,122,168,195,209)

6

;)597,)572,)373,)190,)174

0/2,1/2,1/1,0/1,1/3

(102,133,248)

1

0;)1087,)819,)592,VNTR

)0/1

1/3,1/3,1/3,

(18,77,125,210,274)

1

8;)656,)607,)137,+113,

+127,codon35/3

Allloci0/1

(64)

TNF

a

;)1031,)863,)857,)376,

)308,)238,+489,VNTR

0/1,0/1,0/1

0/30/1,0/1

1/1,1/1,1/1,0/1

1/7,0/3,0/1,

(39,51,53,57,61,73,98,125,

135,192,217)

b

;+252

2/3

(221)

TGF-b

)

988,)800,)509,L10P,R25P

)5091/2,others0/1

(99,222)

FCGR

IIA,IIIA,IIIB

2/4,1/3,2/4

2/7,3/6,1/7

(34,36,69,127,129,130,

150,162,229,271)

MMP

1

;)1607

1/4

(101,113,223,233)

3

;)1171

0/2

(113,233)

9

;)1562

0/2

(224,233)

2

;)1575,)1306,)790,)735

0/2

(103,233)

O

thers

0/1

0/1

(233)

HLA

A

,B,Cw

1/2,0/1,0/1

1/2,1/2,1/2

(12,19,96,182,226,233,239)

D

RB1,DRB345,DRBblank

2/3,0/1

1/2,1/1,1/1

D

QA1,DQB1,DR4

0/1,4/5,0/1

1/2,1/2

VDR

A

paI,BsmI,TaqI

0/1,2/2

1/1,1/1,4/5

(46,88,108,235,236,276)

FPR1

c

.301,306,329,348,378

0/1,1/1,1/1,1/1,1/1

(85,278)

5

46,568,576

0/1,1/1,1/1

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(137) described a composite genotype formed by the

two polymorphic loci interleukin-1A ()889) and

interleukin-1B (+3953) single nucleotide polymor-

phisms that carry a CT transition. This was later

known as periodontitis-associated genotype (136).

The interleukin-1A ()889), however, was superseded

by analysis of the interleukin-1A (+4845) GT

dimorphism, which is technically more straightfor-

ward, as the two single nucleotide polymorphismsare in complete linkage disequilibrium with one an-

other. Thus, analysis of the interleukin-1A (+4845)

single nucleotide polymorphism provides the same

genetic information (135, 242). The two loci compri-

sing the periodontitis-associated genotype were

found to be in linkage disequilibrium (80).

Interleukin-1 genotypes associate more with chronic

periodontitis in Caucasians. Galbraith et al. (74) and

Gore et al. (80) concurred with the 1997 report by

Kornman et al. (136) that either the smoking status or

the interleukin-1 periodontitis-associated genotype

contribute to periodontitis disease severity. Papapa-

nou et al. (185) did not find statistical difference

in the periodontitis-associated genotype between

periodontitis patients and controls, but the period-

ontitis-associated genotype correlated with the

severity of disease when assessed by clinical attach-

ment level. In addition, periodontitis-associated

genotype-positive patients had a lower systemic

immunoglobulin G response to periodontal microb-

iota than periodontitis-associated genotype-negative

patients. Laine et al. (141) investigated the distribu-tion of polymorphisms in the interleukin-1 gene

family among periodontitis patients and controls,

taking into account both smoking and microbiological

parameters, including the presence ofPorphyromon-

as gingivalisandActinobacillus actinomycetemcomi-

tans. The results showed a higher frequency of allele 2

carriage in interleukin-1A ()889) and interleukin-1B

(+3954) single nucleotide polymorphisms and inter-

leukin-1RN variable numbers of tandem repeat poly-

morphisms in non-smoking periodontitis patients in

whom P. gingivalis and A. actinomycetemcomitans

could not be detected [odds ratio (OR) 5.7; 95%confidence interval (CI): 1.619.8]. These results pro-

vide evidence that polymorphisms in genes of the

interleukin-1 family are associated with severe adult

periodontitis in the absence of other risk factors tested

in the patient population. The subjects enrolled in all

the studies mentioned were Caucasians. In a mixed-

population study by McDevitt et al. (157), non-

smokers and former light smokers (


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increased OR of 3.75 of having moderate to severe

periodontitis (95% CI: 1.0413.50). Performing the

analysis in European Caucasians only increased the

odds ratio to 5.27. This study also demonstrated the

relationship of patients age and former smoking

history with severity of adult periodontitis. On the

other hand, Meisel et al. (164) concluded that the in-

terleukin-1 periodontitis-associated genotype in Ger-

man Caucasians displayed a strong interaction with

smoking, resulting in an increased chronic perio-dontitis risk (OR 2.50; 95% CI: 1.215.13). Non-

smokers, even genotype-positive, were not at any in-

creased risk. Studies in Chinese, Greek, Japanese and

Thai populations did not find a relationship between

interleukin-1 genotypes and chronic periodontitis

disease susceptibility or severity (4, 8, 203, 225).

One study investigated the incidence of the inter-

leukin-1 genotypes in a population-based sample of

26-year-old New Zealanders (246). After controlling

for gender, smoking status and plaque levels, the

authors identified a high-risk group consisting of

subjects with the interleukin-1B (+3953) CC/inter-leukin-1A (+4845) TT genotype. This high-risk

genotype was consistent with the genotype pattern

reported by Diehl et al. (49) for early onset periodon-

titis, although the New Zealand study found no

association between early onset periodontitis and

interleukin-1 genotypes. In a study of Chilean Cau-

casians, with ages ranging from 20 to 48 years, the

heterozygous form of the interleukin-1B (+3954)

genotype was significantly higher in patients than in

controls and was associated with periodontitis

(OR 3.12; 95% CI 1.596.09; P 0.001) (151).

Homozygosity for allele 1 of the interleukin-1B

(+3954) genotype was a protective factor for perio-

dontitis. The prevalence of positive genotype (at least

one allele 2 present at each locus) was significantly

higher in periodontitis patients than in controls and

was significantly associated with periodontitis, irres-

pective of the smoking status and periodontitis dis-

ease severity. A 5-year longitudinal study in a group ofsubjects of essentially European heritage showed an

interaction of the interleukin-1 periodontitis-asso-

ciated genotype with age, smoking and P. gingivalis

(40). The authors concluded that the interleukin-1

periodontitis-associated genotype was a contributory,

but non-essential, risk factor for periodontal disease

progression.

Studies suggest that periodontitis-associated genotype

does not correlate with aggressive periodontitis. Unlike

chronic periodontitis, reports of aggressive periodon-

titis indicate that periodontitis-associated genotype isnot associated with this disease (Table 2). Hodge et al.

(95) investigated interleukin-1A and interleukin-1B

gene polymorphisms in unrelated Scottish Caucasian

patients with generalized early onset periodontitis and

found no association between patients and controls

for the periodontitis-associated genotype. Other rela-

ted studies demonstrated similar results. For example,

the studies by Diehl et al. (49) and Parkhill et al. (187)

actually found that allele 1 rather than 2 of the

Carriage rate of IL-1 polymorphic alleles

IL-1A+4845

IL-1B -511

IL-1B +3954

IL-1RN VNTR17.9%

8.2%

20.4%

48.6%

2.1%

9.3%

27.0%

61.1%

70.5%

68.0%

59.4%

11.6%

18.6%

26.9%

37.5%

0% 10% 20% 30% 40% 50% 60% 70% 80%

CaucasianCaucasian

African-American

JapaneseJapanese

ChineseChinese

African-American

Fig. 1. Heterogeneity across the population is evident in the distribution of single nucleotide polymorphisms identified inthe interleukin(IL)-1 gene.

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Table

2.Interleukin-1(IL-1)geneclusterpolymorphismsrelatedtochronicandaggressiveperiodontitisrisk(case-controlstudy)

Disease

association

Ethnicity

IL-1A)889/

+4845GT

IL-1B

PAG+4845/

+3954

IL-1RN

VNTR

References

)511TC

)31CT

+3953/3954CT

Chronic/adultperiodontitis

Susceptibility

Caucasian

Yes(1)*;no(1)

Yes(1)*;no(2

)

Yes(1);no(1)

Insmoker

OR

2.5

Yes(1)*

(141,157,164,185)

Others

No(5)

No(5)

Yes(1);no(4)

OR

3.75

(4,8,42,203,221)

Severity

Caucasian

No(3)

No(1)

Yes(2);no(2)

OR

4.67,3.36

Yes(2);no(1)

Innon-smoker

OR

6.8

(74,80,136,185,203)

Japanese

No(1)

No(1)

No(1)

(221)

Others

No(1)

No(1)

No(1)

(4)

Aggressive/earlyonsetperiodontitis

Earlyonset

Caucasian

No(3)

No(1)

Yes(2);no(2)

Allele1:OR

2.22

No(2)

Yes(1);no(1

)

(49,79,96,187,203)

African-American

No(2)

Yes(1);no(1)

Allele1:risk

No(1)

(259)

Japanese

No(1)

No(1)

No(1)

No(1)

Yes(1)

(221,237)

Others

Yes(1);no(2)

InmalesOR

5.58

Yes(1)

Inmale

sOR

3.16

Yes(1);no(2)

OR

2.86

No(2)

No(1)

(147,196)

Numbersinparenthesesindicatethen

umberofreports.

PAG,allele2oftheIL1A+4845singlenucleotidepolymorphism(SNP)andallele2oftheIL1B+3954SNP.

*Allele2+non-smoker+noP.gingiva

lisandA.actinomycetemcomitansdetectedoddsratio(OR)

5.7.

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interleukin-1B (+3953) significantly correlated with

aggressive periodontitis. A significant difference was

found only in the interleukin-1B genotype distribu-

tion after comparison of aggressive periodontitis

smokers with control smokers. In contrast, Quappe et

al. (196) reported that Chilean subjects who were

heterozygous for allele 2 of the interleukin-1B (+3953)

showed a significant association with aggressive

periodontitis (OR 2.86; 95% CI: 1.067.71; P 0.030). In a wholly African-American population,

Walker et al. (259) concluded that because of the high

frequency of allele 1 of the interleukin-1B (+3954)

single nucleotide polymorphism, studies of this

polymorphism could not be carried out in this

population.

A study by Tai et al. (237) showed that generalized

aggressive periodontitis in a Japanese population

failed to find any association between aggressive

periodontitis and genotypes with respect to the in-

terleukin-1B (+3954), interleukin-1B ()511) or the

interleukin-1A (+4845) single nucleotide polymorph-

isms, although there was a significant association

with the interleukin-1RN intron 2 variable numbers

of tandem repeat genotype (OR 3.81). Recently,

Scapoli et al. (207) indicated a positive association

between generalized aggressive periodontitis and the

interleukin-1RN (variable numbers of tandem repeat)

genotype in Italian Caucasians. The genotype distri-

bution was significantly different between general-

ized aggressive periodontitis and control subjects,

and the interleukin-1B (+3953) single nucleotide

polymorphism was associated with generalizedaggressive periodontitis. Moreover, a recombination

hot spot between the interleukin-1B (+3953) and in-

terleukin-1B ()511) single nucleotide polymorphisms

was identified. However, there was no similar

association with the interleukin-1RN (variable num-

bers of tandem repeat) genotype in study of a UK

population (187). Li et al. (147) investigated the

associations of interleukin-1 gene cluster polymor-

phisms with aggressive periodontitis in a Chinese

population. There was no significant association

of interleukin-1 polymorphisms with generalized

aggressive periodontitis in unstratified subjects.However, when subjects were stratified by gender,

the positivity and frequency of allele 2 at the inter-

leukin-1A (+4845) single nucleotide polymorphism

were significantly increased in male patients com-

pared with male controls (OR 5.58). The frequency

of the interleukin-1B ()511) heterozygote was also

significantly increased in a male generalized aggres-

sive periodontitis group. Moreover, a possible com-

bined effect of the polymorphism of interleukin-1B

()511) and smoking on generalized aggressive peri-

odontitis susceptibility was suggested.

Taken altogether, the interleukin-1 composite

genotype, along with other candidate genes, may

contribute to the pathogenesis of chronic periodonti-

tis, but seemingly not to aggressive periodontitis. The

increase in risk of the periodontitis-associated geno-

type-positive subjects was demonstrated, irrespective

of confounding factors such as smoking,P. gingivalisand age, although periodontitis-associated genotype

was shown to interact with these confounding factors.

Owing to ethnic differences, the global applicability of

the interleukin-1 composite genotype as a periodon-

titis marker may not be established.

Interleukin-1 genotype-positive subjects exhibited in-

creased interleukin-1 protein. The number of reports

describing the functional importance of interleukin-1

gene polymorphisms is relatively few. Thus, a defin-

itive cause and effect could not be pointed out. Sev-

eral studies, however, have shown some possible

functional implications. For example, carriage of al-

lele 2 in the ()889) locus resulted in an almost four-

fold increase of interleukin-1a protein levels in

chronic periodontitis patients (219). Furthermore,

patients who were positive to the composite inter-

leukin-1A (+4845) and interleukin-1B (+3954) peri-

odontitis-associated genotype had a higher level of

interleukin-1b in gingival crevice fluid but not in

gingival tissue before and after treatment (54).

However, positivity to periodontitis-associated gen-

otype did not correlate with higher gingival crevicefluid volume and percentage bleeding on probing in

experimental gingivitis (114). Periodontitis patients

carrying one or two copies of the rare allele of in-

terleukin-1A ()889), tumor necrosis factor ()308) or

interleukin-6 ()174) polymorphisms displayed signi-

ficantly higher serum interleukin-6 concentrations.

Similarly, subjects carrying one or two copies of the

allele 2 at interleukin-6 ()174) and interleukin-1A

()889) had significantly higher C-reactive protein

serum levels than subjects homozygous for allele 1.1

(43). The interleukin-1RN tandem repeat poly-

morphism, in contrast to the non-synonymous, in-terleukin-1B (+3954) polymorphism, contributed to

interleukin-1b regulation in differentiating mono-

cytes (193, 217).

Tumor necrosis factor

Tumor necrosis factor is a pro-inflammatory cytokine

that possesses a wide range of immunoregulatory

functions. Tumor necrosis factor has the potential to

stimulate the production of secondary mediators,

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including chemokines or cyclooxygenase products,

which consequently amplifies the degree of inflam-

mation (115, 179).

Tumor necrosis factor gene polymorphisms and

periodontitis studies display conflicting results. The

tumor necrosis factor gene (TNF) is located in chro-

mosome 6 within the major histocompatibility com-

plex, in the 6p21.3 Class III human leukocyte antigenzone (148, 197). Eight single nucleotide polymorph-

isms in the promoter region of this gene have been

described at positions )1031T/C,)863C/A,)857C/T,

)575G/A, )376G/A, )308G/A, )244G/A, and )238G/A

(25, 26, 45, 92, 250, 265). Casecontrol studies ofTNF

promoter variations revealed conflicting findings.

The )380 locus was linked to chronic periodontitis

susceptibility (P < 0.01) in Czech Caucasians, but not

in Dutch, Polish, Swedish or US Caucasians (33, 51,

57, 61, 74, 217). The same locus was associated with

chronic periodontitis severity in U.S. Caucasians

(OR 10.2), but an earlier study in the same popu-

lation reported contrasting results (74, 136). Aggres-

sive periodontitis was not linked to this locus (53,

192, 217). The rest of the promoter polymorphisms

were not associated with periodontitis, except in one

study that found an association between the )1031,

)863, )857 single nucleotide polymorphisms and

chronic periodontitis severity in Japanese people (39,

53, 74, 125, 221). On the other hand, the A/A genotype

of the +252 single nucleotide polymorphism in the

tumor necrosis factor-b gene served as a protective

factor against chronic periodontitis (OR

0.08) inone study of Caucasian subjects, but this result was

not in accordance with another study of similar

subjects (57, 74). It is not clear why these studies gave

conflicting results. However, performing another

approach, such as meta-analysis, which pools all the

data to extract a common conclusion, would be

helpful. The major cellular source of tumor necrosis

factor is the monocyte/macrophage cell group,

although other cells, such as T lymphocytes and

epithelial cells, can also produce tumor necrosis

factor (120, 191). Ex vivo studies, on the correlation

between single nucleotide polymorphisms andtumor necrosis factor production by lipopolysac-

charide stimulation of monocytes, produced varying

results. Some studies concluded that TNFpolymor-

phisms, particularly)308 G/A did not affect tumor

necrosis factor production (99, 119, 241). On the

contrary, other studies reported that the)308 A allele

correlated with higher levels of tumor necrosis factor

(58, 152, 240). The contradictory results could be

attributed to the differences in size and choice of

subjects, time periods after stimulation, concentra-

tions of stimuli used and the absolute values of tumor

necrosis factor (in pg/ml) reported (15). Tumor

necrosis factor-a production in TNF 1031/)863 or

)857 single nucleotide polymorphism variant allele

carriers tended to be elevated in healthy Japanese

subjects (221). Gene polymorphisms and periodon-

titis association ofTNFand other cytokines, and their

receptors, are summarized in Table 3.

Other cytokines

Interleukin-2. Interleukin-2 is a pro-inflammatory

cytokine produced by T-helper 1 cells. It mediates the

cellular immune response by participating in B lym-

phocyte activation and macrophage stimulation, as

well as in natural killer and T lymphocyte prolifer-

ation (186, 243, 266). Periodontitis patients have been

reported to show higher interleukin-2 levels in the

serum compared with healthy subjects (164). In

addition, interleukin-2 was found to be produced by

lymphocytes cultured from chronically inflamed

periodontal tissues (215). The interleukin-2 gene is

located in chromosome 4q26 (214). Polymorphism at

positions ()330) and (+166), relative to the tran-

scription start site, were identified by John et al.

(116). The (+166) change occurs within the leader

peptide and does not affect the amino acid sequence.

The ()330) polymorphism has two common alleles (T

and G), making it an ideal marker for genetic

association. Homozygotes of the G-allele were found

to be at increased risk of hay fever (175). In a perio-

dontal study in Caucasian subjects, the T-allelecarriers seem to be approximately half as likely to

develop severe periodontal disease (OR 1.99; 95%

CI 1.073.7). Moreover, individuals with the TT

genotype seem to be 2.5-times less likely to develop

severe periodontitis than individuals who are het-

erozygous or GG homozygous (OR 2.57; 95%

CI 1.155.73) (208).

Interleukin-4. Multiple roles have been identified

with interleukin-4. This cytokine can rescue B

lymphocytes from apoptosis and enhance their sur-

vival, thus playing a role in promoting B-lymphocyte-mediated autoimmunity (107, 170, 220). Interleukin-4

is also a potent down-regulator of macrophage

function (56, 87). In established and advanced

periodontitis lesions, the presence of interleukin-4-

producing cells and the percentage of interleukin-4+

cells were significantly higher in periodontitis than in

gingivitis tissues (271). Interleukin-4 levels in the

serum of patients was higher in chronic periodontitis

than in controls, although these levels did not cor-

109



9/31

Table

3.

Genepolymorphismsinothercytokines,cytokinerece

ptorsandperiodontitisassociation

Gene

Chromosome

Locus

Allele

Ethnicity

Association(no.ofreports)

References

Chronic

periodo

ntitis

Aggressive

periodontitis

TNFA

6p21.3

)1031

T/C

Japanese

Yes(1)*

No(1)

(53,221)

)863

C/A

Japanese

Yes(1)*

No(1)

(53,221)

)857

C/T

Japanese

Yes(1)*

No(1)

(53,221)

)376

G/A

Caucasian

No(1)

(39)

)308

G/A

Caucasian

Yes(1);

no(5)

OR10.2,GG:risk

No(1)

(57,61,73,74,217)

Japanese

No(1)

No(1)

(53,221)

Chilean

No(1)

(192)

)238

G/A

Caucasian

No(2)

(39,73)

Japanese

No(1)

No(1)

(53,221)

+489

G/A

Caucasian

No(1)

(39)

TNFB

6p21.3

+252

A/G

Caucasian

Yes(2);

no(1)

AA:protective,OR

0.08

(57,73,98)

TNF-a

6p21.3

Microsatellite

14alleles

Caucasian

No(1)

(125)

IL-2

4q2627

)330

T/G

Brazilian

Yes(1)

OR1.99

(208)

IL-4

5q31.1

)590

C/T

Caucasian

No(2)

(79,167)

Japanese

No(1)

(79)

Brazilian

No(1)

(209)

African-Brazilian

No(1)

(195)

Korean

No(1)

(122)

VNTR

VNTR(70bp)

Caucasian

No(2)

(79,168)

Japanese

No(1)

(79)

Brazilian

No(1)

(195)

African-Brazilian

No(1)

(209)

110

Yoshie et al.


10/31

Table

3.

Continued

Gene

Chromosome

Locus

Allele

Ethnicity


References

Chronic

periodontitis

Aggre

ssive

periodontitis

IL-6

7p21

)597

A/G

Caucasian

No(1)

(102)

Japanese

No(1)

(42)

)572

G/C

Caucasian

Yes

(1)

OR

0.27,GC:protective

(102)

Japanese

No(1)

(133)

)373(microsatellite)

AnTm

:3alleles

Japanese

Yes

(1)

OR

2.96,A9T11:protective

(133)

)190

C/T

Japanese

No(1)

(133)

)174

G/C

Caucasian

Yes

(1);no(1)

OR

3.0,Callele:protective

(102,248)

Japanese

No(1)

(133)

IL-10

1q3132

)1082/)1087

A/G

Caucasian

Yes

(1)

OR

2.58

(18)

Japanese

No(1)

No(1)

(273)

Brazilian

No(1)

(210)

)819/)824

C/T

Caucasian

No(1)

No(1)

(76)

Japanese

No(1)

No(1)

(273)

Brazilian

Yes

(1)

OR

3.78

(210)

)592/)597

C/A

Caucasian

No(1)

No(1)

(76)

Japanese

No(1)

No(1)

(273)

Brazilian

Yes

(1)

OR

3.35

(210)

111



11/31

relate with either the degree of bone loss or pocket

formation observed clinically (158). The gene for in-

terleukin-4 is found in chromosome 5q31.1 (232).

Promoter single nucleotide polymorphism at position

()590) and a 70-bp variable numbers of tandem re-

peat polymorphism at intron 2 have been identified

(173). Casecontrol reports relating to aggressive

periodontitis and chronic periodontitis susceptibility

and severity across several populations have failed toestablish a connection between these loci and peri-

odontal disease (79, 122, 168, 195, 209).

Interleukin-6. Essential biological activities depend

on interleukin-6. As a result of its wide range of ef-

fects, deregulated over-production of interleukin-6

causes various clinical symptoms and abnormal

laboratory findings in vivo (176). The interleukin-6

gene was demonstrated to be localized in chromo-

some 7p21 (23). Several studies have shown the

association of interleukin-6 gene polymorphisms

with some diseases or conditions such as rheumatoid

arthritis and bone mineral density (184, 189). In

periodontitis, the GC single nucleotide polymorph-

ism at the ()174) position correlated with chronic

periodontitis susceptibility in Brazilian Caucasians

(OR 3.0), but not in Czech Caucasians (57, 259).

With regard to the other interleukin-6 gene single

nucleotide polymorphism locations, the Czech study

suggested that the ()572) G/C polymorphism of the

IL-6 gene may be one of the protective factors asso-

ciated with lower susceptibility to chronic periodon-

titis (OR

0.27; 95%

CI: 0.120.61). Furthermore, thestudy did not find an association between the ()597)

G/A locus and chronic periodontitis susceptibility.

A recent report revealed that the ()174) G/C, ()190)

C/T and ()597) G/A loci were non-polymorphic in a

Japanese population (133). However, an over-repre-

sentation of the ()373) A9T11 allele was observed

in non-chronic periodontitis subjects (P 0.008;

OR 2.96; 95% CI 1.217.43). The authors also

reported that the interleukin-6 concentration in

subjects homozygous for the C[A10T10] haplotype of

the ()572) G/C and )373 AnTm loci was significantly

higher than in heterozygotes. In another study, peri-odontitis patients carrying one or two copies of the

rare allele in the interleukin-6 ()174) polymorphism

displayed significantly higher serum interleukin-6

and C-reactive protein concentrations (SE 0.8

1.1 ng/l, P < 0.001 for interleukin-6; SE 0.8

1.1 mg/l, P 0.023 for C-reactive protein) (43).

Cigarette smoking and carriage of the same allele was

associated with less reduction in probing depths

among chronic periodontitis patients after deliveryTable

3.

Continued

Gene

Chromosome

Locus

A

llele

Ethnicity


References

Chronic

periodontitis

Aggressive

period

ontitis

TGF-b1

19q13.113.2

)800

G

/A

Caucasian

No(1)

(99,222)

)509

C

/T

Caucasian

Yes(1);no(1)

+869

T

/C

Caucasian

No(1)

+915

G

/C

Caucasian

No(1)

TNFR2

1p36.236.3

+857

T

/G(Met196Arg)

Japanese

Yes(1)

OR

2.61

(218)

CCR5

3p21

Coding()?32)

3

2-bpdeletion

Caucasian

No(1)

(59)

IFN-cR1

6q2324

Intronicmicrosatellite

1

3alleles

Caucasian

Yes(1)

OR

5.56insmoker

(67)

IL,interleukin;IFN,interferon;OR,od

dsratio;TGF,transforminggrowthfactor;TNF,tu

mornecrosisfactor.

112

Yoshie et al.


12/31

of standard non-surgical periodontal therapy (44).

It appears that interleukin-6 gene polymorphisms

affect the serum levels of circulating interleukin-6,

and consequently modify the patients response to

periodontal treatment.

Interleukin-10. Interleukin-10 stimulates the pro-

duction of protective antibodies and down-regulates

pro-inflammatory cytokines produced by monocytes(200, 256). Periodontitis lesions demonstrated a

significantly higher messenger ribonucleic acid

expression for interleukin-10 than that in autologous

peripheral blood mononuclear cells (272). Moreover,

stimulation of peripheral blood mononuclear cells

with outer membrane antigen from P. gingivalis

induced variable but significantly higher interleukin-

10 messenger ribonucleic acid expression in perio-

dontitis patients than in healthy controls (5). These

data suggest that controlling mechanisms suppress

an excess production of inflammatory cytokines,

which could affect the inflammatory response in

periodontitis, resulting in different clinical manifes-

tations. The gene encoding interleukin-10 was map-

ped to chromosome 1q3132 (123). Three promoter

single nucleotide polymorphisms have been des-

cribed in this gene: ()1087) G/A; ()819) C/T; and

()592) C/A (139, 143). The three loci exhibit strong

LD (260). Microsatellite polymorphisms were also

identified in the 5-flanking region of the gene, but no

association with periodontitis has been established

(55, 125). The ()1087) G/A locus was not associated

with chronic periodontitis susceptibility in Japaneseand Brazilian subjects, but was linked to chronic

periodontitis severity in Swedish Caucasians

(OR 2.58) (18, 210, 273). The ()819) C/T and ()592)

C/A loci correlated with chronic periodontitis

susceptibility in Brazilians (OR 3.04; CI 1.31

6.91 and OR 3.38; 95% CI 1.298.82, respect-

ively) but not in Japanese and German Caucasian

subjects (77, 210, 273). The ()1087) single nucleotide

polymorphism falls within a putative transcription

factor binding site and was associated with high

in vitro interleukin-10 production (55, 139). The

()819) single nucleotide polymorphism may affect an

estrogen-responsive element, and the ()

592) singlenucleotide polymorphism lies within a region with a

negative regulatory function (139, 143).

Transforming growth factor-b1. One of the cytokines

released during tissue injury and by inflammatory

cells exposed to bacteria and their products is

transforming growth factor-b (258). This cytokine is

described as a double-edged sword, having both

therapeutic and pathologic potential (20). Perio-

dontopathic microorganisms have been reported to

induce the production, by mononuclear phagocytes,

of transforming growth factor-b in vitro, and the

transforming growth factor-b protein was detected

in the early and late stages of periodontal disease

(257).

The gene for transforming growth factor-b1

(TGFB1) is located in chromosome 19q13.1 (70). One

promoter polymorphism, the ()509) C/T, was asso-

ciated with chronic periodontitis severity in Brazilian

Caucasians (P 0.04) (224). However, this single

nucleotide polymorphism, together with the other

polymorphisms, was not related to chronic perio-

dontitis susceptibility in Czech Caucasians (100). The()509) C/T polymorphism was significantly associ-

ated with the plasma concentration of transforming

growth factor-b1 and osteoporosis in Japanese

subjects (82, 269).

Fc R polymorphisms

FcRIIA

-H131 -R131

His131

Arg131

-158V -158F

FcRIIIA

Phe158

Val158

Cell membrane

-NA1 -NA2

FcRIIIB

Arg18

Ser18

Asn47

Asp64

Val

Ser47

Asn64

Ile88

High Low

(IgG2)

High Low

(IgG1, IgG3)

High Low

(IgG1, IgG3)

Receptor affinity

(Ligand)

GPI GPI

Fig. 2. Human FccR polymorph-

isms. FccRIIA alleles are distin-

guised by the presence of either Arg

or His at amino acid position 131,

whereas FccRIIIA alleles are

determined by either a Val or a Pheat position 158. The FccRIIIB-NA1/

NA2 polymorphism results in 4

amino acid substitutions, leading to

glycosylation differences (blue cir-

cles). The FccRIIA alleles interact

differntially with human immuno-

globulin G(IgG)2, whereas FccRIIIA

and FccRIIIB affinities are different

upon interaction with human IgG1

and IgG3. GPI glycosylphosphati-

dylinositol.

113



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Receptor and other gene polymorphisms

Fc receptor polymorphisms

Leukocyte receptors for the constant (or Fc-) part of

immunoglobulin (FcR) link cellular and humoral

branches of the immune system, which are consid-

ered essential for the host defense against bacteria.

Strong, specific immunoglobulin G responses against

periodontopathic bacteria are observed in the gingi-val tissue and gingival crevicular fluid (104, 160). FcR

for immunoglobulin G (FccR) may therefore play a

crucial role in the host defense against these bacteria.

FcR profile and gene polymorphisms. The human

leukocyte FccR family consists of three major classes,

and encompasses eight genes (CD64: FccRIA, IB, and

IC; CD32: FccRIIA, IIB and IIC; CD16: FccRIIIA and

IIIB), which have been mapped to the long arm of

chromosome 1 (1q21 and 1q2324) (144, 205, 238).

Most FccR subclasses consist of a separate ligand-

binding chain with an extracellular domain that

contains the immunoglobulin G-binding region, and

signaling chains essential for the initiation of signal

transduction. Neutrophil FccRIIIB is a notable

exception, as it is linked to the outer leaflet of the

lipid bilayer via a glycosyl phosphatidylinositol an-

chor.

Functional bi-allelic polymorphisms have been

identified for three FccR subclasses: FccRIIA, FccRI-

IIA, and FccRIIIB (Fig. 2) (183, 204, 267). FccRIIA

bears either an arginine (FccRIIA-R131) or a histidine

(FccRIIA-H131) at amino acid position 131 in thesecond extracellular immunoglobulin-like domain

(261, 262). This difference strongly affects the recep-

tor affinity for immunoglobulin G2 (188). FccRIIA-H/

H131 neutrophils internalize human immuno-

globulin G2-opsonized bacteria more efficiently than

FccRIIA-R/R131 neutrophils (24). The FccRIIIA-158V

allotype exhibits higher affinity for both monomeric

and immune-complexed immunoglobulin G1 and

immunoglobulin G3 than does FccRIIIA-158F (132).

The neutrophil-specific FccRIIIB bears the NA1-NA2polymorphism caused by four amino acid substitu-

tions within the first extracellular immunoglobulin-

like domain (183). Neutrophils from FccRIIIB-NA2

individuals bind immunoglobulin G1 or immuno-

globulin G3 less efficiently than those from FccRIIIB-

NA1 individuals (24) (Fig. 3A). In vitro findings have

suggested that inter-individual differences in the

efficacy of FccR-mediated effector functions depen-

ded on FccR polymorphisms.

Association of FcR polymorphisms with periodontitis

risk. Wilson and Kalmar (264) speculated that

FccRIIA-R/R131 subjects were more susceptible to

periodontitis as a result of the diminished capacity to

phagocytose immunoglobulin G2-opsonized perio-

dontopathic bacteria. It has been well documented

that FccR genes are associated with risk for various

types of periodontitis: aggressive periodontitis,

chronic periodontitis, and recurrent chronic perio-

dontitis (Table 4). Associations between FccR

polymorphisms and susceptibility to aggressive per-

iodontitis have been investigated in Caucasian, Asian

(Japanese and Taiwanese), and African-Americanpopulations. Loos et al. (150) studied FccRIIA, IIIA,

Table 4. FccR genes related to periodontitis risk

Periodontitis Population FccRIIA FccRIIIA FccRIIIB References

Aggressive

(early onset)

periodontitis

Caucasian Yes (H131: OR 3.7) Yes (158V: OR 2.6) No (150)

African-American No No Yes (NA2: OR 1.8) (69)

Japanese No No Yes (NA2: OR 2.0) (129)

Taiwanese Yes (R131: OR 2.4) Not tested No (34)

Chronic (adult)periodontitis

Caucasian Yes (H/H131) No No (150, 270)

Japanese No No No (127, 229)

Taiwanese No Not tested No (34)

Severe chronic (adult)

periodontitis

Caucasian Yes (H131: OR 2.4) Yes (158V: OR 2.9) No (150, 162, 270)

Japanese No Yes (158V: OR 2.0) No (130)

Recurrent chronic

(adult) periodontitis

Caucasian No No No (36)

Japanese No Yes (158F: OR 2.9) Yes (NA2: OR 3.3) (128, 230)

114

Yoshie et al.


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and IIIB genotypes in 12 patients with aggressive

periodontitis and 61 controls of Northern European

Caucasian background. The carriage rate of FccRIIA-

H131 and FccRIIIA-158V was higher in patients with

aggressive periodontitis than in controls, respectively

(P 0.013 and P 0.044), suggesting a putative

susceptibility factor for aggressive periodontitis.

Chung et al. (34) examined FccRIIA and FccRIIIB

genotypes in 30 patients with aggressive periodontitisand 74 healthy controls in Taiwan. FccRIIA-R131 was

found more frequently in the group with aggressive

periodontitis than in the group with chronic perio-

dontitis (P 0.01), but not in the control group. In

contrast, Japanese studies have demonstrated an

over-representation of the FccRIIIB-NA2 allele in

patients with aggressive periodontitis compared to

patients with chronic periodontitis and controls (129,

277), which was in accordance with the results of

African-American patients (69). From these results, it

is conceivable that the FccR-encoding genes related

to aggressive periodontitis risk are different among

ethnic populations.

Relationships between FccR polymorphisms and

chronic periodontitis risk have also been investigated

in detail. There is only one report showing a signifi-

cant association with susceptibility to chronic

periodontitis. Yamamoto et al. (270) indicated a

difference in the FccRIIA genotype distributions

between 213 Caucasian patients with chronic perio-

dontitis and 209 race-matched controls (P 0.036),

with enrichment of the FccRIIA-H/H131 genotype in

the patients compared with the controls. However,

other work failed to show a significant associationwith chronic periodontitis susceptibility (34, 127, 130,

162). Caucasian patients with chronic periodontitis

and FccRIIA-H/H131 were found to have more teeth

with severe periodontal breakdown than patients

carrying FccRIIA-R131 (150). Likewise, smokers with

FccRIIA-H/H131 exhibited a greater clinical attach-

ment loss than smokers with FccRIIA-R/H131 and

FccRIIA-R/R131 (270). On the other hand, Meisel

et al. (162) indicated a significant association of

FccRIIIA-158V with chronic periodontitis severity

(P 0.010), which was in accordance with a Japanese

study (130).

Three studies have investigated FccR genotype

distributions in patients with recurrent chronic per-

iodontitis. Colombo et al. (36) found no differences in

FcRIIIB polymorphism and neutrophil function

(%)50 75 100

Recurrent chronicperiodontitis (n=85)

22.3 45.9 31.8

Aggressive

periodontitis (n

=

38)

29.0 52.6 18.4

Healthy control(n=104)

11.5 52.9 35.6

Periodontitis -resistant (n=46)

43.5 56.5

Chronicperiodontitis (n=83)

48.2 36.115.7

250

5 15 25

0

20

40

60

Phagocytosis (%)

(min)

IgG1-opsonized

NA1/NA2NA2/NA2 NA1/NA1

A B

Fig. 3. (A) Diminished phagocytosis of immunoglobulin

G(IgG)1-opsonized Porphyromonas gingivalis by neu-

trophils expressing FccRIIIb-NA2/NA2 (closed circles)

compared with IIIb-NA1/NA1 (open circles) in six healthy

controls. Subjects were matched for their FccRIIA R/H131

genotypes. Values represent means standard error

(128). (B) Distribution of FccRIIIB genotypes in Japanese

patients with various types of periodontitis. FccRIIIB-NA2/

NA2 (closed bars) was found more frequently in aggressive

periodontitis patients than in healthy controls (127, 129,

230).

115



15/31

FccRIIA and IIIB genotype distributions among the

32 refractory, 54 successfully treated and 27 perio-

dontally healthy groups of Caucasian background.

On the contrary, a significant over-representation of

FccRIIIB-NA2 and FccRIIIA-158F was found in 85

Japanese patients with chronic periodontitis and

disease recurrence as compared with 15 race-mat-

ched chronic periodontitis patients without recur-

rence (P 0.0003 and P 0.009) (127, 229).

Interestingly, FccRIIB polymorphisms may play

an important role in the pathogenesis of periodon-titis, because of a large numbers of FccRII-bearing

B lymphocytes in the periodontal lesion. Yasuda

et al. (275) found a significant over-representation

of the FccRIIB-232T allele in 32 patients with

aggressive periodontitis, as compared with 72

patients with chronic periodontitis and 72 controls

(P 0.039 and P 0.006), suggesting an association

with susceptibility to aggressive periodontitis in

Japanese subjects.

Hypothetical role of FcR risk allele in periodonti-

tis. The most convincing FccR genes related to

periodontitis risk might be supported by significant

evidence provided in more than two ethnic popu-

lation studies. FccRIIIB-NA2 seems to constitute a

gene-predisposing susceptibility to aggressive peri-

odontitis (Fig. 3B), which might be explained by the

hypothesis demonstrated in Fig. 4. FccRIIIB is a

neutrophil-specific receptor and plays a crucial

role in the control of specific immunoglobulin

G-opsonized pathogens in the gingival crevice.Immunoglobulin G1- and immunoglobulin G3-ops-

onized P. gingivalis may be more effectively

phagocytosed and killed by FccRIIIB-NA1 than

FccRIIIB-NA2 neutrophils (128). Therefore, ineffi-

cient clearance of periodontopathic bacteria by

neutrophils in FccRIIIB-NA2 subjects may result in

an increased level of bacteria in the gingival crevice,

finally leading to be relatively at high risk of

aggressive periodontitis.

Hypothetical role of FcR genotypes in susceptibility to periodontitis

cihtapotnodoireP

airetcab

ospO zin a noit

iw th IgG

sisotycogahpdetaidem-GgI

slihportuenybAggressive periodontitis

btneiciffE y

sllec-1AN lamroN

ksiRIn ybtneiciffesllec-2AN

noitagiL of

egahporcam/etyconom

etycohpmyldnaF-NT noitcudorp1-LI/

PC

dorp-lamroN noitcu

in R1 -13 1ro lec-F85 ls

dorp-revO noitcu

in -131H or sllec-V851

lamroN

ksiR

Fig. 4. Hypothetical role of FcR genotypes in suscept-

ibility to periodontitis. Inefficient clearance of perio-

dontopathic bacteria by neutrophils expressing the

low-affinity genotype FccRIIIB-NA2/NA2 may result in an

increased level of bacteria in the gingival crevice, leading

to high susceptibility to periodontitis in younger adults.

The high affinity genotype FccRIIA-H131/H131 and

FccRIIIA-158V/158V may contribute to a strong proin-

flammatory cytokine release by monocytes/macrophages

and lymphocytes upon interaction with immunoglobulin

G(IgG), possibly leading to an increased risk for period-

ontitis in adults.

116

Yoshie et al.


16/31

Other promising genes influencing susceptibility to

chronic periodontitis are FccRIIA-H131 and FccRI-

IIA-158V. The significance of these two genes might

be related to a hypothetical role of FccRIIA-bearing

monocytes/macrophages and FccRIIIA-expressing

lymphocytes in the periodontium (Fig. 4). FccRIIAand IIIA cross-linkings have been shown to produce

pro-inflammatory cytokines, such as tumor necrosis

factor-a and interleukin-1b (47, 156). It is therefore

proposed that the high affinity of FccRIIA-H131 and

FccRIIIA-158V to immunoglobulin G contributes to a

high level of pro-inflammatory cytokine release by

monocytes/macrophages and lymphocytes, possibly

leading to an increased risk for chronic periodontitis.

Cytokine and chemokine receptors

Receptors are important constituents of the whole

cytokine system. Through these membrane-bound orcirculating proteins, cell responses to various cytok-

ines, such as interleukin-6 and tumor necrosis factor-

a, are either elicited or blocked (2, 72). The soluble

form of tumor necrosis factor-receptor 2 (tumor

necrosis factor-R2), which is shed from the cell sur-

face, significantly reduced the loss of connective tis-

sue and alveolar bone in experimental periodontitis

(8, 9, 48). The tumor necrosis factor-R2 (+587) T/G

polymorphism was associated with chronic perio-

dontitis severity in Japanese patients (P 0.0097;

OR 2.61) (218). Given this scenario, this variation

in the tumor necrosis factor-R2 gene could have an

effect on the ability of the receptor to block efficiently

the activity of tumor necrosis factor-a. Using an

intronic (CA)n

polymorphic microsatellite markerwithin the interferon-c receptor 1 (IFNGR1) gene,

Fraser et al. (67) found a correlation between genetic

polymorphisms and periodontitis in Caucasians

(P 0.014; OR 5.56). It was reported that a strong

correlation exists among the IFNGR1 genotype, cel-

lular responsiveness to interferon-c and clinical dis-

ease features (52). On the other hand, the C-C

chemokine receptor 5 delta32 polymorphism was not

related to periodontitis susceptibility in German

Caucasians (59). Recent reports indicate the role of

interleukin-6 receptor gene polymorphisms in dia-

betes, obesity and serum soluble interleukin-6receptor level (75, 86, 268). The possible role of var-

iations in the interleukin-6 receptor gene in perio-

dontitis could therefore be investigated in the future.

Metabolism-related polymorphisms

Cathepsin C polymorphisms. Cathepsin C is a prote-

inase, and is expressed in the hyperkeratotic epithe-

lial lesions such as palms, knees and oral keratinized

Possible concept for common polymorphisms

AggressiveAggressive

periodontitisperiodontitis

ChronicChronic

periodontitisperiodontitisPeriodontitisPeriodontitis

SystemicSystemic

diseasesdiseases

Master gene Minor genes Common/shared genes

Fig. 5. Both aggressive and chronic periodontitis may have shared susceptibility genes, in the same manner as period-

ontitis may share susceptibility genes with other complex, inflammatory or systemic diseases.

117



17/31

gingiva (87). Hart et al. (89, 90) analyzed two con-

sanguineous Jordanian families, and identified a gene

on chromosome 11 (11q14) containing the cathepsin

C gene, responsible for prepubertal periodontitis as

well as Papillon-Lefevre Syndrome. All patients with

pre-pubertal periodontitis were found to be homo-

zygous for an AG mutation at gene position +1040,

resulting in a substitution of the amino acid tyrosine

by a cysteine. This gene polymorphism was shown tobe functional as there was a diminished activity of

cathepsin C in Papillon-Lefevre Syndrome (247).

Interestingly, other mutations found in the cathepsin

C gene have been linked to Papillon-Lefevre Syn-

drome (41, 89). Recently, Cury et al. (41) demon-

strated a novel TC mutation at gene position +587 in

exon 4, causing substitution of conserved leucine, at

position 196, by a proline. Another study, by Noack

et al. (177), reported two novel gene mutations at

positions 947 and 1268, which was associated with

these two diseases. Functional mutations in the

cathepsin C gene may therefore be considered as

causative for prepubertal periodontitis and Papillon-

Lefevre Syndrome.

Vitamin D receptor polymorphisms. Vitamin D plays

a role in the metabolism of calcium and phosphorus.

The human vitamin D receptor gene is localized in

chromosome 12q12q14 (169), and exhibits func-

tional polymorphisms associated with osteocalcin

levels and bone mineral density (137, 171). Vitamin D

receptor polymorphisms may therefore play a role in

the destruction of alveolar bone. Hennig et al. (91)studied a restriction fragment length polymorphism

(RFLP) for TaqI in exon 9 in 69 Caucasian patients

with aggressive periodontitis and 72 race-matched

controls. The authors indicated a higher prevalence

of TaqI RFLP (t) in the patients with loca-

lized aggressive periodontitis than in the controls

(P 0.017). In contrast, Yoshihara et al. (276) indi-

cated no association between the vitamin D receptor

genotypes and risk for aggressive periodontitis in the

Japanese. Tachi et al. (236) found an increased fre-

quency of the allele TaqI RFLP (T) in chronic perio-

dontitis patients compared with controls (P 0.04).Another polymorphism for BsmI (B/b) in exon 8 was

analyzed by de Brito et al. (27), who showed that the

vitamin D receptor haplotype was a risk factor for

chronic periodontitis. Inagaki et al. (108) examined

an association of the ApaI (A/a) and TaqI (T/t)

polymorphisms and periodontal disease progression

in 125 subjects for 23 years, and showed that theApaI

A/A genotype subjects exhibited the highest rate of

the disease progression.

Matrix metalloproteinase polymorphisms. Matrix

metalloproteinases play an important role in con-

nective tissue destruction in periodontitis. Increased

levels of the gene transcript and protein for matrix

metalloproteinase-1 (interstitial collagenase) and

matrix metalloproteinase-3 (stromelysin-1, activator

of collagenase) were found in the periodontitis le-

sions. Therefore, polymorphisms in the promoter

region of matrix metalloproteinase-1 and matrixmetalloproteinase-3 genes may contribute to sus-

ceptibility to periodontitis. de Souza et al. (223)

showed that the matrix metalloproteinase-1 ()1607)

2G/2G genotype was more frequently observed in 26

patients with severe chronic periodontitis than in 24

patients with moderate chronic periodontitis and 37

controls, all with a Brazilian background. The authors

also examined the matrix metalloproteinase-9 ()1562

C/T) promoter polymorphism, which was not asso-

ciated with susceptibility to chronic periodontitis

(224). Holla et al. (101) indicated that three matrix

metalloproteinase-1 polymorphisms ()

1607 1G/2G,

)519 A/G, and )422 A/T) showed only a small

effect on chronic periodontitis in the Czech popula-

tion. Recently, Itagaki et al. (113) studied matrix

metalloproteinase-1 ()1607 1G/2G) and matrix

metalloproteinase-3 ()1171 5A/6A) polymorphisms

in 37 patients with aggressive periodontitis, in 205

patients with chronic periodontitis, and in 142 heal-

thy controls, all with a Japanese background, but

failed to show a significant association with perio-

dontitis.

Antigen recognition-related polymorphisms.

Human leukocyte antigen polymorphisms. The major

histocompatibility complex system is a cluster of

genes encoding the human leukocyte antigens, which

are located on chromosome 6p21.3 (12). The human

leukocyte antigen region is mainly divided into two

classes: major histocompatibility complex class I

molecules (human leukocyte antigen-A, -B, and -C)

are expressed on most nucleated cells, whereas major

histocompatibility complex class II molecules (hu-

man leukocyte antigen-DP, -DQ, -DR) are expressed

on B- and T lymphocytes, and on macrophages.Shapira et al. (216) found that human leukocyte

antigen-A9 and -B15 antigens were elevated in

patients with aggressive periodontitis as compared

with healthy controls. Takashiba et al. (239) indicated

a significant association of the human leukocyte

antigen-DQa gene with susceptibility to aggressive

periodontitis in the Japanese. The DQB1 molecule

has been shown, by Ohyama et al. (182), to be critical

in the pathogenesis of aggressive periodontitis. In

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contrast, Hodge et al. (94) reported no association

between the human leukocyte antigen-DQagene and

aggressive periodontitis in Caucasians. Bonfil et al.

(19) demonstrated an important role of human leu-

kocyte antigen-DR4 in susceptibility to aggressive

periodontitis, in which subtypes 0401, 0404, 0405 and

0408 can be a risk factor for the disease. Recently,

Stein et al. (226) elucidated the variety of human

leukocyte antigen associations and the difficulty ofassigning single human leukocyte antigen markers to

periodontitis in Caucasians. A significant association

of certain human leukocyte antigen alleles (human

leukocyte antigen-A, -B, -Cw, -DRB1, -DRB3/4/5,

-DQB1) has been shown in aggressive periodontitis

and chronic periodontitis by Machulla et al. (153).

CD14 polymorphisms. The CD14 molecule is a

receptor for recognition of lipopolysaccharides, and

initiates the innate immune response to bacterial

invasion. The gene for the CD14 receptor is located

on chromosome 5q2123, consisting of c. 3900 bp

organized in two exons (81). A single nucleotide

polymorphism (CT) was identified in the promoter

region at position )159 upstream from the major

transcriptional site (110), affecting transcriptional

activity (145) and CD14 density (13). Four additional

polymorphisms have recently been identified at

positions )1619, )1359, )1145 and )809, which

influence soluble CD14 levels (254). Holla et al. (100)

found that in Czech patients, the )1359 C/T genotype

was associated with severity of chronic periodontitis,

while the)

159 G/T genotype was not. Yamazaki et al.(274) studied the )159 G/T genotype distribution in

163 Japanese patients and 104 race-matched con-

trols, and indicated that the )159 G/T genotype was

not associated with development of periodontitis, but

may be related to early disease activity. Folwaczny

et al. (62) reported that the )159 G/T genotype was

associated with periodontitis in women, but not in

men, in Germany. These results were consistent with

a recent study with Caucasian subjects by Donati

et al. (51).

n-Formyl-L-methionyl-L-leucyl-L-phenylalanine recep-tor polymorphisms. The n-formyl-L-methionyl-L-leu-

cyl-L-phenylalanine (fMLP) is a structural analogue of

bacterial products involved in neutrophil chemotaxis.

The fMLP receptors are also involved in the activation

and subsequent response to certain chemotactic

stimuli. The fMLP receptor genes are localized in

chromosome 19 (14). Gwinn et al. (85) examined the

fMLP receptor gene, and found that two single nuc-

leotide base alterations (329 T/C and 378 C/G) were

associated with aggressive periodontitis. These

alterations resulted in amino acid changes (110 Phe/

Ser and 126 Cys/Try) in the fMLP receptor, suggest-

ing a role in ligand binding and G-protein activation.

Differences in fMLP expression levels and chemotaxis

towards fMLP were examined between the fMLP

polymorphisms by Jones et al. (118). More defective

chemotaxis was found in the 126 Cys/Try mutant

than in the 110 Phe/Ser mutant, suggesting a moresevere form of aggressive periodontitis. In contrast,

Zhang et al. (278) found no association of these fMLP

receptor polymorphisms (329 T/C and 378 C/G) with

susceptibility to aggressive periodontitis.

Issues and concerns on thecandidate gene approach inperiodontitis

The candidate gene approach tries to identify one

allele of a gene that is more frequently seen in sub-

jects with the disease than in subjects without the

disease. Most of the studies mentioned above have

utilized this approach. These association studies,

which may include members of an affected family or

unrelated cases and controls, can be performed rel-

atively quickly and inexpensively and may allow

identification of genes with small effects (140). Au-

thors assert that candidate gene studies are better

suited for detecting genes underlying common and

more complex disease where the risk associated withany gene is relatively small (35).

Candidate genes are chosen on the basis of their

known or presumed functions that are thought to

have some plausible role in the disease. There are

three types of candidate genes: functional candidate

genes; positional candidate genes; and expressional

candidate genes (105). Functional candidate genes

are derived from an existing knowledge of the phe-

notype and the potential function of the gene in-

volved after clinical or physiological studies of

affected individuals. Positional candidate genes are

based on the involvement of the gene to a markedlocation after genetic linkage analyses. Expressional

candidate genes are determined through differences

in gene expression using microarrays.

For several genes, which have been individually

sequenced for association with periodontitis, we see

a scattered picture from different studies of varied

populations and ethnicities. To produce scientifically

sound and meaningful disease-association studies,

some issues and concerns that should be addressed

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when sequencing candidate genes are discussed

below.

Ethnic heterogeneity

In designing a casecontrol study, subjects should be

carefully matched by ethno-geographic origin in

addition to other potential confounding factors in

order to avoid systematic differences in geneticcomposition between the two groups (37). Failing to

do so could result in different frequencies of single

nucleotide polymorphism alleles and the unsuspect-

ing investigator might then draw unwarranted con-

clusions about localizations of susceptibility genes

(97). There is also a clear statement that in the pres-

ence of large biological and environmental variability,

genetic effects can differ across different populations,

or even among generations within the population

(111). Ideally, twin studies can be useful in sorting out

genetic heterogeneity and environmental factors, but

there are certain limitations in carrying out twin

studies in subjects with periodontitis (166).

Frequencies of the genetic marker of interest may

also show large heterogeneity between races (112).

Variation in genotype frequencies across diverse

populations may affect the number of individuals at

increased risk for a disease, and population substruc-

ture imbalances may create spurious differences in

genotype frequencies of the compared groupsin gene-

disease association studies (245). For example, specific

interleukin-1 gene variations of interleukin-1A+4845,

interleukin-1A-889 and interleukin-1B+3954 havebeen associated with increased severity of periodon-

titis in multiple Caucasian studies. However, these

interleukin-1 single single nucleotide polymorphisms

are found in low prevalence among Asians, including

Japanese, Koreans, and Chinese (Fig. 1). Moreover,

association studies of the same candidate genes in

supposedly related populations or ethnicities pro-

duced mixed or conflicting results (53, 95, 96,133, 136,

187, 234). Stephens et al. (227) reported that 3899 dis-

tinct genetic variations at 313 genes exist in unrelated

individuals, including Caucasians, African-Americans,

Asians and Latinos, and thus the diagnostic use ofgenes in periodontitis might be limited to a specific

population and may not apply globally or across an

ethnic group.

Considering the issues mentioned, it is prudent

to select a more homogenous population (age- and

race-matched), and to study, with caution, the

applicability of a certain gene marker before com-

mencing with any attempts to replicate the same

study in the population under investigation.

Clinical classification

Classifying periodontal diseases has been a long-

standing dilemma largely influenced by paradigms

that reflect the understanding of the nature of peri-

odontal diseases during a given historical period (7).

As a result of its familial tendency, aggressive perio-

dontitis generally appears in individuals before the

age of 35 years, but age alone is not sufficient toestablish diagnosis. On the other hand, chronic

periodontitis is quite complex and much more

dependent on environmental factors that confront

the patient during his lifetime. In addition, microbial

plaque deposition, smoking and systemic diseases

largely influence the phenotypic expression of the

disease. For these combined reasons, chronic perio-

dontitis is considered to appear later in life. The

periodontist is therefore challenged regarding into

which classification a patient would properly fall.

Another critical problem related to genetics re-

search in periodontitis is the similarity of the fol-

lowing clinical findings: deepening of the periodontal

pocket; and attachment and alveolar bone loss.

Therefore, investigators should strictly adhere to the

classification set during the American Academy of

Periodontology workshop in 1999 (31). Moreover,

subjects falling into the gray zone between aggressive

and chronic periodontitis should be excluded in the

study. Aggressive and chronic periodontitis probably

share a common pathogenic pathway, so several

common polymorphisms may exist and/or overlap

between the two.

Functional polymorphisms and directevidence

In the 2001 report of the International SNP Map

Working Group, they approximated around

1.42 million single nucleotide polymorphisms in the

human genome (201). Sixty thousand of these sin-

gle nucleotide polymorphisms fall within exons.

Structural gene defects can affect the qualitative

response, and regulatory polymorphisms can alter

the response quantitatively. However, many studiesfail to provide functional evidence for gene poly-

morphisms and periodontal diseases. The majority

only statistically demonstrated an association be-

tween polymorphisms and periodontitis. Moreover,

some of the studied alleles may not be associated

with disease themselves, but instead may be in LD

with the disease-associated allele. If so, because LD

breaks down differently in different populations,

variations in the estimated OR may reflect variable

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LD rather than variation in the true genetic effect

(112).

Kinane et al. (126) outlined the requirements in

providing a disease-polymorphism association: the

polymorphism must influence the gene product;

biases in the study population should be recognized

and controlled for; confounders such as smoking and

socio-economic class must be sorted out; and the

affected gene product should be part of the diseaseetiopathology. Genetic markers for proposed gene-

disease associations vary in frequency across popu-

lations, but their biological impact on the risk for

common diseases may usually be consistent across

traditional racialboundaries (112).

Common guidelines for associationstudies

When performing candidate gene casecontrolstudies, factors such as study design, methods of

recruitment of case and controls, selection of can-

didate genes, functional significance of polymor-

phisms chosen for study, and statistical analysis

require close attention to ensure that only genuine

associations are detected (37, 83, 84, 109, 110).

These factors are frequently debated upon in cases

of conflicting results from a similar study.

Size of study groups

One major concern related to genotype studies is thesample size of the study subjects. Owing to limited

number of samples, most sample sizes in genetics are

small. This scenario describes very well the small

number of cases in aggressive periodontitis associ-

ation studies. The number of subjects in studies of

chronic periodontitis tends to be larger, but varia-

tions do occur. The results of small studies might

differ significantly from the results of larger studies,

but large studies with thousands of participants

might not be carried out (109). Experience from other

clinical domains suggests that small studies maymistakenly yield more favorable outcomes than lar-

ger studies (112). Wang et al. (260) demonstrated that

if susceptibility alleles have minor allele frequencies

(MAFs) of


21/31

accuracy. A commentary by Aderem and Hood (1)

summarized the impact of the completion of the

human genome project on biology in general. They

discussed the shifting of approaches in biology, the

effect to small university-based laboratories, aca-

demic vs. industrial strengths and integrating large-

and small-scale sciences. In the succeeding parts,

we discuss several genetic methods and research

advances that could be useful in clarifying the role ofgenetic polymorphisms in periodontitis.

Multigene model for periodontitis

From the overview of publications mentioned above,

it is reasonable to conclude that both aggressive

periodontitis and chronic periodontitis are not sin-

gle-gene but polygenic diseases. A polygenic model

for periodontitis is presented in Fig. 5. A good

example to describe this model is the risk for Alz-

heimers disease that has been considered to be

substantially influenced by a total of 10 genetic

polymorphisms of inflammation-related molecules,

including pro-inflammatory cytokines and protease

inhibitors (126). It is thought that several of these

relatively common high-risk polymorphisms may be

inherited by an individual, giving them a suscepti-

bility profile (159).

Similarly to other complex diseases, it is estimated

that between 10 and 50 genes with several major

master genes may be involved in periodontitis. So, it

would be more efficient to simultaneously analyze a

large sample size for multiple gene polymorphisms.Recently, a large-scale investigation of genomic

markers for severe periodontitis was performed (234).

In this study, subjects were genotyped at 637 single

nucleotide polymorphisms in 244 genes using Taq-

man polymerase chain reaction. Genes encoding

gonadotropin-releasing hormone 1, phosphatidy-

linositol 3-kinase regulatory 1, dipeptidylpeptidase 4,

fibrinogen-like 2, and calcitonin receptor were found

to be associated with severe periodontitis. A previous

study performed by the same group sequenced a

total of 310 single nucleotide polymorphisms in 125

candidate genes for their association with aggressiveperiodontitis and severe chronic periodontitis in a

Japanese population using the Taqman allelic dis-

crimination assay (233). Single nucleotide poly-

morphisms in genes encoding collagen type 4 a1,

collagen type 1 a1 and gp130, among others, were

associated with chronic periodontitis. On the other

hand, single nucleotide polymorphisms in cathepsin

D, collagen type 17 and others were linked with se-

vere chronic periodontitis. Polymorphisms in the

inflammatory mediators and structural factors of

periodontal tissues were associated with periodonti-

tis in Japanese.

Genome screening and linkage analysis

With the concerns for the candidate gene approach

and association studies (e.g. inter- and intra-popu-

lation heterogeneity of periodontitis genes), thesearch for genes contributing to periodontitis would

be more effective using genome-wide screening and

linkage analysis of affected families or ethnic groups.

In these approaches, all genes are systematically

scanned using panels of microsatellite or single nu-

cleotide polymorphism DNA markers uniformly dis-

tributed along the entire genome.

Numerous papers have employed genomic

screening and linkage analysis as common tools in

the search for genes of other complex diseases, such

as diabetes, inflammatory bowel disease and obesity

(11, 32, 33, 174, 263, 280). The results obtained from

the screenings and analyses of the disorders men-

tioned provide promising results that are worth

considering in the search for periodontitis genes in

the future. However, practical concerns may still

surface in using these methods: models of the allelic

architecture of common diseases, sample size, map

density and sample-collection biases (260).

Other questions that arise in linkage studies of

complex diseases include the number of marker loci

that should be included in the whole genome screen,

and whether single nucleotide polymorphisms or thehighly polymorphic microsatellite markers, which are

less common throughout the genome but are more

informative, should be preferred (198). Kruglyak (138)

concluded that single nucleotide polymorphisms

could be as informative as microsatellites when there

is a sufficiently dense map, and marker densities of

one per centimorgan or less were sufficient for an

initial screening for linkage. However, difficulties still

existed in linkage studies and the need for larger

samples was encouraged (3).

In 2003, localized aggressive periodontitis was

linked to human chromosome 1q25 (146). In thisstudy, a genetic linkage analysis was performed first

with four multigenerational families exhibiting the

localized aggressive periodontitis phenotype. Four-

teen chromosome locations were analyzed using a

directed genomic screening approach. The localized

aggressive periodontitis phenotype was linked to a

DNA marker, D1S492, with the LAP locus spanning

about 26 million DNA base pairs between D1S196

and D1S533. In addition, a gene of major effect for

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prepubertal periodontitis was localized by Hart et al.

(90) in chromosome 11q14.

Haplotypes and tag single nucleotidepolymorphisms

The human genome sequence is thought to con-

tain more than 10 million single nucleotide poly-

morphisms with a frequency of >1% (244). Thecombination of closely linked alleles observed on a

chromosome and inherited as a unit is called a

haplotype. Single nucleotide polymorphisms are

present in the millions but a limited number were

sufficient to define all of the common haplotypes,

and a collection of single nucleotide polymorphisms

can represent at least 95% of the haplotypes studied

(117). The selected limited number of single nucleo-

tide polymorphisms is termed haplotype-tagging

single nucleotide polymorphisms. Most of the genetic

variation represented by the 10 million common

single nucleotide polymorphisms in the population

could be provided by genotyping between 200,000

and 1,000,000 tag single nucleotide polymorphisms

across the genome (30, 71, 76, 190, 244). Some of the

studied alleles, however, may not be associated with

disease themselves, but instead may be in LD with

the disease-associated allele (111). Therefore, know-

ledge of LD may result in a substantial reduction in

the amount of genotyping, with little loss of infor-

mation. Wang et al. (260) placed the general con-

sensus of r2 (degree of LD between alleles) at 0.8,

which is sufficient for tag single nucleotide poly-morphism mapping to obtain a good coverage of

untyped single nucleotide polymorphisms, allowing

genotyping of a lower number of marked single nu-

cleotide polymorphisms with relatively small losses

in power. They added that if the LD between single

nucleotide polymorphisms is strong, this could result

in the need to carry out up to 7080% less genotyp-

ing. On the other hand, if LD in a region is low,

almost every single nucleotide polymorphism might

have to be genotyped to ensure comprehensive cov-

erage of the region.

Conclusion

With the completion of the human genome project

and the availability of cutting-edge technology,

researchers have turned from studying genetic se-

quences to analyzing a larger number of proteins

encoded by them. Recent studies from multiple

groups have pointed to genetics as an important

determinant of the severity and progression of peri-

odontitis. A number of aspects of the inflammatory

and immune response that are suspected to play a

role in the development of periodontitis have a

clearly defined genetic basis. Currently, the presence

of interleukin genetic variations appears to identify

individuals who are at increased risk for more severe

chronic periodontitis and for a less predictable re-

sponse to therapy. A correlation of genetic polymor-phisms with periodontitis, such as that demonstrated

in the Fc receptor gene, appear to provide the

promising use of genetic determinants in periodon-

titis.

Applying genetic information and tech

Documents

The role of genetic polymorphisms in periodontitis