The genetic basis of periodontitis

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The genetic basis of periodontitis

DE N I S F. KI N A N E, H I D E K I S H I B A & TH O M A S C. HA R T

It is now evident that there is a genetic basis for most

diseases including periodontitis. Elucidation of the

genetic basis of periodontitis should permit a better

understanding of disease etiology, allowing improved

classification, diagnosis, and treatment of periodon-

tal diseases. Thus there has been enormous effort

expended so far to identify gene polymorphisms

associated with the risk for the periodontal diseases.

Reports of genetic polymorphisms associated with

periodontal disease are increasing, yet the limitationsof these reports are not widely recognized. To

appreciate these limitations it is necessary to under-

stand the genetic basis of complex diseases. Thus this

review has two sections, the first aimed at introdu-

cing modern genetic principles and the second

focusing on the utility of genetics in periodontics.

It is increasingly evident that genetic variance is a

major determinant of the differential risk for many

human diseases (24, 49, 92). While microbial and

other environmental factors initiate and modulate

periodontal disease, individuals are known to

respond differently to common environmental chal-lenges, and this differential response is influenced by

the individuals genetic profile. Genes clearly play a

role in the predisposition to and progression of per-

iodontal diseases (62, 63, 73, 78, 122, 156). Support

for the idea that genetic factors are important deter-

minants of periodontitis susceptibility and progres-

sion comes from studies of humans and animals

which indicate that genetic factors which impair

inflammatory and immune responses in general,

affect periodontitis experience specifically. It is

hoped that identification of the specific genes caus-ing periodontal disease susceptibility may have

diagnostic and therapeutic value.

To understand the potential clinical relevance of

genetic variability on periodontitis it is necessary to

understand how different genes can contribute to

disease. There are estimated to be 20,00025,000

different genes in the human genome. Genes can

exist in different forms or states. Geneticists refer to

the different forms of a gene as allelic variants, or

alleles. Genetic alterations could also change the

transcript level of the protein, that is, the poly-

morphism may occur not in the protein coding

region but in the gene promoter region, and thus

influence the amount of protein produced by the

gene. Allelic variants of a gene differ in their nuc-

leotide sequence. Different allelic forms of a gene

can produce an identical protein or different iso-

forms of a protein. In the former case, the genetic

polymorphism does not change the amino acid andthe genetic change is said to be silent. In the latter

case, a nucleotide change alters the amino acid

composition of a protein. The results can range

along a continuum of functional consequences,

from no observable change in protein function, to a

minor change in function, to a dramatic change or

obliteration of function. Individual genetic changes

that are causal of disease are typically the result of

a genetic alteration that dramatically alters a pro-

teins function. Such genetic changes are typically

termed mutations and are rare on a population

level, typically being present in less than 0.1% ofthe population. When a specific allele occurs in at

least 1% of the population, it is said to be a genetic

polymorphism. In contrast to mutations, these

more common genetic polymorphisms are usually

considered normal variants in the population.

Common genetic polymorphisms may change the

function of a protein, but usually the change is

relatively minor. Consequently, the specific protein

products of different alleles may function differ-

ently. These differences in physiological functioning

of different proteins can be enhanced by certainenvironmental exposures, e.g. diet, smoking, or

microbial factors. If the affected protein functions

in a biological process, e.g. inflammatory response

to a specific microbial agent, certain polymor-

phisms may increase or decrease a persons risk for

a disease phenotype. Whether a particular gene

variant contributes to a disease phenotype depends

upon the magnitude of the effect in contributing to

the development of disease.

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Periodontology 2000, Vol. 39, 2005, 91117

Printed in the UK. All rights reserved

Copyright Blackwell Munksgaard 2005

PERIODONTOLOGY 2000


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To appreciate the contribution of a genetic variant

to a disease it is critical to understand how genes

contribute to genetic diseases (Fig. 1). The contribu-

tion of an allelic variant to a disease can vary from

being deterministic to having only a minor effect on

the etiology. The manner and extent to which genetic

factors play a role in disease have important impli-

cations for identifying the genetic basis of etiology

and for utilizing this information for diagnosis andtreatment. Geneticists have traditionally divided

genetic diseases into two broad groups: simple

Mendelian diseases and complex diseases. The dis-

tinction of these broad groups is based on the pattern

of transmission of the disease, which reflects the

manner in which genes contribute to each disease.

Simple Mendelian diseases

Diseases that follow predictable and generally

simple patterns of transmission have been called

Mendelian conditions. The name reflects the fact

that these diseases occur in simple patterns in

families and in most cases genetic alterations at a

single gene locus are the major determinant of the

clinical disease phenotype. These diseases follow a

classic Mendelian mode of inheritance (autosomal

dominant, autosomal recessive or X-linked). The

disease phenotype usually manifests over a broad

range of environments, and although environmental

factors and other genes can modify the clinical

presentation, in most cases the mutation will

manifest in a remarkably similar phenotype. Thepopulation prevalence of individual Mendelian

diseases is rare (typically much less than 0.1%),

with the exception of some unique populations

that have been isolated from other human popu-

lations. Examples of Mendelian type diseases

include amelogenesis imperfecta, Crouzon syn-

drome, cleidocranio dysplasia, and PapillonLefevre

syndrome (Table 1). When the gene responsible for

a Mendelian disease has been identified, it is often

possible to develop a diagnostic test to identify

individuals who carry a disease-causing mutation in

the responsible gene. Depending upon the mode of

transmission, it is also possible to make fairly

specific determinations of the probability of the

Phenotype Environment Genotype* Biological Interactions*= + +

Fig. 1. The classic relationship of phenotype (disease

absence or presence), environment, and genotype. Geno-

type represents strictly genetic effects, environment sim-

ply environmental effects and Biological Interactions*

includes gene gene (epigenetic interactions) and gene

environment interactions. For the periodontal disease

phenotype, environmental risk factors include smoking

status and plaque control.

Table 1. Examples of syndromic forms of periodontitis in which inheritance is Mendelian and due to a geneticalteration at a single gene locus

Condition Biochemicaltissue defect Inheritance OMIM

PapillonLefevre syndrome Cathepsin C Autosomal recessive 245000

HaimMunk syndrome Cathepsin C Autosomal recessive 245100

EhlersDanlos syndrome type IV Collagen Autosomal dominant 130050

EhlersDanlos syndrome 8 Collagen Autosomal dominant 130080

Cyclic neutropenia Neutrophil elastase Autosomal dominant 162800

Chronic familial neutropenia Defect unknown Autosomal dominant 162700

ChediakHigashi syndrome Lysosomal trafficking

regulator gene

Autosomal recessive 214500

Congenital disorder of glycosylation

type IIc (Leukocyte adhesion

deficiency type 2)

Glucose diphosphate-

fucose transporter-1


Leukocyte adhesion deficiency

type I

Leukocyte chain

adhesion molecule

CD18


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mutant gene being passed to a child and often it is

possible to predict the course of clinical disease.

Complex genetic diseases

Genetically complex diseases differ from simple

Mendelian diseases in several important ways. Gen-

etically complex diseases do not follow a simple

pattern of familial distribution or transmission. Incontrast to simple Mendelian traits, which are

caused by a single gene, these complex traits are the

result of the interaction of alleles at multiple different

gene loci. Environmental factors are usually etiolog-

ically important, and often necessary, in the devel-

opment of complex diseases. On a population level,

complex genetic diseases are much more common

than simple Mendelian diseases, and many occur

with a population prevalence of greater than 1%.

Unlike rare mutations responsible for simple genetic

diseases, the allelic variants important for many

complex genetic diseases are common in the popu-

lation. In contrast to mutations that can eliminate a

gene product or change the protein product of a gene

so significantly that it acts to disrupt other biological

processes, the individual genetic variants that are

important in complex diseases are much less dis-

ruptive, and usually function within the normal

range. Many disease-associated genetic polymor-

phisms are common in the population and can be

present at allele frequencies of >20%, with some

disease-associated alleles reported in >50% of

populations studied (38). The hypothesis that most ofthe genetic risk for common, complex diseases is due

to common polymorphisms at disease loci is known

as the Common DiseaseCommon Variant hypothesis

(142). Recently, the idea concept has emerged that at

least a portion of susceptibility variants for common

diseases are less common (510%) but more pene-

trative (141). The fact that these genetic alterations

are not sufficient alone to cause disease has import-

ant implications. Disease-associated alleles may be

present in a significant proportion of the unaffected

general population. Knowledge of the presence of

one disease-associated allele in an individual doesnot provide enough information to make a clinical

diagnosis. In contrast to genetic mutations that are

often diagnostic for simple Mendelian conditions, the

presence of a polymorphism in a complex trait can be

difficult to interpret, and must be assessed together

with other information. It is important to have

information about the allele frequency in the

population tested, and also to be able to quantify

in a meaningful way the magnitude of effect a

disease-associated allele has on the disease proces-

ses. For this reason some measure of the specificity

and sensitivity of a disease-associated allele to pre-

dicting disease is desirable. Currently, considerable

attention is being focused on the clinical validity and

clinical utility of genetic polymorphisms that have

been reported to be associated with a disease.

Polymorphism vs. mutation

A major difference in the genetic basis for simple

Mendelian diseases vs. complex genetic diseases is

the number of genes involved and the contribution of

each gene to the overall disease phenotype. In Men-

delian disease, alteration of a single gene locus can

result in disruption of a protein product that has a

major physiological impact and therefore may be

considered to be deterministic of the disease. The

fact that the genetic alteration is predictably associ-

ated with a disease phenotype indicates that there is

no redundancy or compensation in the particular

biological system that can overcome the effect of the

underlying genetic defect. In contrast, the genetic

alterations that contribute to complex diseases are

individually responsible for much more subtle pert-

urbations of protein function. Consequently, it is

difficult to establish causal links with genetic poly-

morphisms that are associated with complex dis-

eases. There is not a one-to-one correlation of the

presence of a specific genetic allele and the occur-

rence of a complex disease, but rather specific alleles

are reported to be found more frequently in diseasedindividuals than in nonaffected controls. Often initial

support for the link is the statistical association of an

allele with a disease state. It is also important to

understand that disease alleles reported to be asso-

ciated with a complex disease are also found in

unaffected individuals. Because multiple (perhaps

58) different gene alleles can contribute to a com-

plex disease state, an individual with a complex

disease need not have all of the alleles reported to be

associated with that disease. Thus, the presence of a

disease-associated allele in an individual is not

diagnostic for a disease. Environmental factors arealso critical to the etiology of most common diseases.

Most chronic diseases are of adult onset, and there-

fore take many years to develop. Genes involved in

the innate, inflammatory, and immune responses are

often involved. These physiological and biological

pathways have many compensatory and redundant

aspects, and therefore it is extremely difficult to

quantify the effect of any one genetic variant to the

disease state. Consequently, if an individual is found

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to have a disease-associated genetic polymorphism,

it is often not clear what the clinical significance of

this is.

Single nucleotide polymorphisms

The most common type of genetic polymorphism is a

Single Nucleotide Polymorphism (SNP). There are an

estimated 10 million SNPs in the human genome.SNPs occur throughout the genome, in regions

encoding genes as well as in regions without identi-

fied open reading frames. SNPs in genes may occur in

protein coding regions (exons) and noncoding

regions (introns and regulatory regions). Many SNPs

that occur in genes have no effect on the encoded

protein, but a large number of SNPs do have an effect

on the gene product. Whether this change contri-

butes to a disease phenotype depends on the specific

consequence of the genetic variant on the function of

the gene and its protein product. An SNP is further

defined as occurring in at least 1% of the population,

and indeed several disease-related polymorphisms

occur at much higher frequencies (2050%).

Methods of genetic analyses

Clinical and scientific data from a variety of sources

suggest that genetic variants are major determinants

of syndromic and nonsyndromic periodontitis. To

evaluate the quality of supporting studies requires

an understanding of the formal genetic analyticalmethods that have been used. Geneticists use a vari-

ety of techniques to demonstrate the genetic basis of

disease. Some methods are general, whereas others

permit precise identification of genetic variants that

cause or contribute to disease. The methods sum-

marized below have been important in the evaluation

of genetic diseases including periodontitis.

Familial aggregation

Familial aggregation of a trait or disease can suggest

genetic etiology. However, families also share manyaspects of a common environment, including diet

and nutrition, exposures to pollutants, and behaviors

such as smoking (active and passive). Certain

infectious agents may cluster in families. Thus,

familial aggregation may result from shared genes,

shared environmental exposures and similar socio-

economic influences. To determine the evidence for

genetic factors in familial aggregation of a trait,

more formal genetic studies are required. There have

been many clinical reports suggesting a familial

aggregation of periodontitis, but until recently the

research tools to pursue these reports were lacking

(16, 66, 73).

Twin studies

Through the phenomenon of twins, in particular

monozygous twins, who arise from one fertilized egg,nature has provided a wonderful tool for the exam-

ination of genetic influences in disease and for par-

titioning the relative contribution of genes and

environment to a trait. Monozygous twins are gen-

etically identical. Dizygous twins are only as gen-

etically similar as brothers and sisters would be, on

average they share 50% of their genes in common

(dizygous twins are from two different eggs and two

different sperm). Discordance or differences in the

presence of disease between monozygous twins must

be due to environmental factors. Disease discordance

between dizygous twins could arise from both envi-

ronmental and genetic differences. The difference in

concordance between monozygous and dizygous

twins for a particular phenotype can be used to

estimate the relative contribution of genes (heredity)

and environmental factors to a disease and studying

disease presentation in twins is often a valuable first

step in this process.

Segregation analysis

Genes are passed from parents to children in a pre-dictable manner, and usually segregate in families as

predicted by Mendels laws (127). Geneticists can

study the pattern of disease transmission in families

using a method called segregation analysis. Segrega-

tion analysis evaluates the relative support for dif-

ferent transmission models to determine which

model can account for the observed segregation of a

trait through families. By sequentially comparing

models to each other, segregation analysis identifies

the model that best accounts for the observed

transmission of a trait in a given population. Genet-

icists generally apply segregation analyses to deter-mine whether a trait transmission appears to fit a

Mendelian or another mode of genetic transmission.

When comparing genetic models of transmission,

genetic characteristics including mode of transmis-

sion (e.g. autosomal, X-linked, dominant, recessive,

complex, multilocus, or random environmental),

penetrance, phenocopy rates and frequencies for

disease and nondisease alleles are some of the char-

acteristics included in the different models evaluated.

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But segregation analysis does not necessarily provide

the true model. Since it is a comparison of two

models, segregation analyses are only as good as the

models tested. If important assumptions of the model

tested are incorrect, this will limit the results. This

limitation of segregation analysis must be realized, as

it has resulted in inaccurate conclusions for the

transmission of at least one form of early onset per-

iodontitis (110). Segregation analysis tests alternativemodels to try to develop the best characterization of

transmission characteristics within a set of data. As

such, it is most appropriately applied to data sets of

many families to determine the best fitting model.

Segregation analysis does not find or aim to find a

specific gene responsible for a trait.

Linkage analysis

Linkage analysis is a technique used to localize the

gene for a trait to a specific chromosomal location.

Genetic linkage studies are based on the fact that

alleles at syntenic gene loci in close proximity on the

same chromosome tend to be passed together from

generation to generation (i.e. segregate), as a unit.

Such genes are said to be linked, and violate Men-

dels law of independent assortment. Geneticists can

apply quantitative analyses to detect this lack of

independent assortment of genetic loci, and can use

it to map (localize) genes to specific chromosome

locations. Over the past 15 years, genetic maps have

been developed that show the position of millions

of polymorphic genetic loci spanning the humangenome (58) (http://www.ncbi.nlm.nih.gov/genome/

guide/human/). Scientists can follow a specific trait

as it segregates through families of interest and

determine whether the trait appears to segregate with

a known genetic polymorphism that has been

localized to a specific chromosomal location. In this

manner, scientists can test whether a trait appears to

segregate in a manner consistent with linkage to a

known genetic marker. Because the precise chro-

mosomal location of the genetic marker is known,

when linkage is detected, the gene responsible for the

trait can be placed in the vicinity of the linked geneticpolymorphism. Linkage can therefore prove the

genetic basis of disease. Linkage is often used as a

first step to determine the approximate location of a

gene of interest, permitting subsequent studies to

identify the mutation responsible for a disease trait.

Linkage studies have been particularly effective in

identifying the genetic basis of simple Mendelian

traits (OMIM 2004), where mutation of a single gene

can cause a disease. Linkage studies of complex

genetic traits have not been as successful for a variety

of reasons (52, 171). A limiting factor in the tradi-

tional application of linkage to complex diseases is

that complex diseases are due the combined effect of

multiple genes of minor effect. When multiple genes

each contribute a small amount to the disease phe-

notype, traditional parametric linkage studies are

much less powerful. Fortunately, newer adaptations

of the linkage approach and the availability ofAssociation testing approaches offer a practical

alternative (106, 113, 186).

Association studies

Genes contributing to common, complex diseases

such as periodontitis have proven more difficult to

isolate. When multiple, perhaps many, genes act

with environmental factors to contribute to disease

liability, it is difficult to formulate disease models. In

the absence of specific genetic models, the etiology

of complex diseases is often conceptualized as due

to multiple factors, i.e. several genetic loci interact-

ing with each other to produce an underlying sus-

ceptibility, which in turn interacts with additional

environmental factors to produce an actual disease

state. For complex traits, such as bipolar disorder

(11), diabetes (61), obesity (21), and oralfacial

clefting (18, 128), traditional parametric linkage

analysis has produced either negative results or a

plethora of weak, positive results not easily repli-

cated. Theoretical research suggests several reasons

for the ambiguity of the linkage results in thesecases. First, if a disease gene is neither necessary nor

sufficient to cause a disease, but rather is a modifier

gene that elevates a nonzero baseline risk, conven-

tional parametric linkage analysis may not detect

the gene (57). Second, if the relative contribution of

a gene to a disease phenotype is small, i.e. the dis-

ease susceptibility allele raises the risk by a factor of

< 2, linkage analysis using affected sibling pairs will

not be powerful enough to detect the gene, given

realistic sample sizes (143). Thus, linkage analyses

may not be a useful strategy to detect modifier

genes or genes that exert small effects preciselythose genes which might be operating in chronic

periodontitis and many other complex disorders.

Consequently, attention has shifted away from

model-dependent parametric linkage analysis to

model-free, nonparametric association analysis as

an alternative means of locating disease suscepti-

bility genes, especially since association studies can

sometimes detect weaker effects than can linkage

analysis (77).

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Two types of association analysis are commonly

employed in genetic studies: population-based and

family-based (76). The population-based approach

utilizes a standard case-control design, in which

marker allele frequencies are compared between

cases (affected individuals) and controls (either

unaffected individuals or individuals randomly cho-

sen from the population). When a positive associ-

ation is found, several interpretations are possible: the associated allele itself is the disease-predispo-

sing allele;

the associated allele is in linkage disequilibrium

with the actual disease-predisposing locus;

the association is due to population stratification;

the association is a sampling, or statistical, artifact.

The first two interpretations represent the alternat-

ive hypotheses of interest in a gene mapping context.

In the first case, the marker itself is the disease-sus-

ceptibility locus. This outcome is the rationale behind

candidate gene studies, in which alleles of the genes

being tested have some a priori expectation of being

directly involved in the disease process. Evidence of a

positive association can be followed up by investiga-

tions to establish a functional role. In the second case,

the associated allelic polymorphism itself does not

play a functional role in causing disease, but rather the

polymorphism is in close physical proximity to the

gene that does contribute to susceptibility. A classic

example is the human leukocyteantigen(HLA) system,

in which various HLA haplotypes are associated with a

number of diseases, including insulin dependent dia-

betes mellitus, rheumatoid arthritis, and ankylosingspondylitis (169). There is currently considerable

attention being directed towards the clinical use of

disease-associated genetic polymorphisms for genetic

testing. However, most initial reports of these poly-

morphisms have not been replicated (75), reinforcing

the need to develop acceptable criteria to determine

the clinical validity of such reports (52). Fortunately,

new approaches hold promise to identify significant

disease associations that may be important for

understanding susceptibility for complex diseases.

There is currently great interest in comprehensively

characterizing human SNPs to facilitate evaluation oftheir role in common diseases.

International HapMap project

The international HapMap project is being conduc-

ted to identify and catalog the common genetic

variants that occur in human beings (http://

www.hapmap.org). The project will describe each of

the common SNPs in the human genome. The goal is

to determine the genetic location of each SNP and

characterize how these genetic variants are distri-

buted in several different population groups. The

project is at present studying DNA samples repre-

sentative of African, Asian, and European ancestry.

By identifying most of the 10 million SNPs that occur

in humans, the HapMap project will identify the DNA

variants responsible for most of the genetic diversity

in humans. The project is not intended to identifyspecific disease-associated polymorphisms. Instead,

it is hoped that by providing such a catalog of com-

mon genetic variants, clinicians and scientists can

work together to identify SNPs with important dis-

ease associations to understand disease etiologies

and develop new diagnostic and treatment strategies.

Evidence for the role of geneticvariants in periodontitis

The risk for many diseases, including periodontal

diseases, is not borne equally by all individuals in a

population (88, 89). A variety of microbial, environ-

mental, behavioral, and systemic disease factors are

reported to influence the risk for periodontitis (132).

An individuals genetic makeup is a crucial factor

influencing their systemic or host response-related

risk.

There are reports in the literature on familial

aggregation of periodontal diseases, but it is difficult

to compare them. Although periodontal disease ter-

minology has changed many times over the years,most familial reports of early onset forms of perio-

dontitis are now referred to as aggressive periodon-

titis (8, 9, 14, 15, 17, 23, 46, 47, 90, 99, 110, 112, 121,

129, 147, 158, 161, 173). Reports of the familial nature

of chronic forms of periodontitis are less frequent but

the aggregation within families is consistent with a

genetic predisposition. We should remember, how-

ever, that familial aggregation of periodontal disease

may also reflect exposure to common environmental

factors. Shared environmental factors include edu-

cation, socioeconomic grouping, oral hygiene, shared

transmission of bacteria, diseases such as diabetesand environmental features such as passive smoking,

sanitation, etc. Some aspects of behavior are deter-

mined in part by genetics, which may influence

potential modifying factors such as education, life-

style and, of dental importance, oral hygiene. Com-

plex interactions between genes and environment are

difficult to quantify, but are likely to be important

when considering the familial risk for the periodontal

diseases.

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In chronic periodontitis, clinical disease charac-

teristics do not present until the third decade of life,

whereas in the aggressive forms of periodontal dis-

ease, the presentation can occur much earlier, in the

early teens or younger (38). This variability in pres-

entation makes diagnosis difficult, not only in

declaring disease but also in detecting patients who

are free of the disease and in differentiating between

chronic and aggressive forms of periodontitis. Theproblems associated with the clinical diagnosis of

periodontal disease are not uncommon in medical

genetics as similar problems arise when studying

other delayed onset genetics (16, 139).

The problems of genetic model testing in aggressive

periodontitis have been highlighted by Boughman

et al. (16) who noted that aggressive periodontitis has

a variable age of onset and is often not recognized

until after puberty. Diagnosis of aggressive perio-

dontitis in older individuals is problematic due to

the difficulties of distinguishing between aggressive

periodontitis and chronic adult onset forms of peri-

odontitis on the basis of the clinical signs. Similarly,

diagnosis in older edentulous individuals is also

problematic. The effects of the environment, for

example plaque accumulation and smoking, also have

major long-term influences on disease experience

(Fig. 1) and these confound the diagnosis of aggres-

sive periodontitis.

Diagnostic quandaries create significant problems

for genetic studies of periodontal disease, limiting

attempts to correlate cellular, functional, and

immune response variables with early onset perio-dontitis phenotypes in families. While there is

evidence for a gene of major effect in aggressive

periodontitis, early onset forms of periodontitis

appear to be etiologically complex and heterogene-

ous (6, 15, 139, 140, 158). Although bacterial trans-

mission between subjects has been suggested to

explain aggressive periodontitis clustering within

families, this observation alone is insufficient to

account for familial clustering (15). While the het-

erogeneity paradigm discussed by Potter (139) is

borne out in subsequent familial studies of what is

now classified as aggressive periodontitis, the strikingfamilial aggregation of the trait is consistent with a

significant genetic etiology. Characterization of the

specific genetic components in the etiology of this

disease requires more formal genetic analyses.

Twin studies

Twin studies have been invaluable in studying the

genetic basis of simple and complex traits. Large,

worldwide registers of data on twins and their rela-

tives have been established (13). Such twin studies

registers offer unique opportunities for selected

sampling of quantitative trait loci linkage and

association studies. Twin studies of periodontitis,

however, have generally been limited in scope and in

subject numbers. However, studies of concordance

for periodontitis and for clinical indices related to

periodontal health and disease generally support asignificant heritable component for periodontitis.

Most twin studies have studied the more prevalent

form of periodontitis, which is chronic periodontitis,

as well as chronic gingivitis.

Corey et al. (26) studied self-reported periodontal

health in 4908 twin pairs and found that 9% of sub-

jects, consisting of 116 identical and 233 nonidentical

twin pairs, reported a history of periodontitis. The

concordance rate, or level of similarity in disease

experience, ranged from 0.23 to 0.38 for monozygous

twins, and was much lower (0.080.16) for dizygous

twins. These findings suggest that heritable factors

are important in the reported periodontitis experi-

ence. Unfortunately, environmental factors such as

smoking status were not factored into this analysis

and could introduce a bias towards finding a corre-

lation between twins.

Michalowicz et al. (124) studied dizygous twins

reared apart (dizygous-A) and reared together (dizy-

gous-T) and monozygous twins reared apart

(monozygous-A) and reared together (monozygous-T).

The mean probing depth and clinical attachment

level scores were found to vary less for monozygous-T than for dizygous-T twin pairs, further supporting

the role of genetics in this disease. Michalowicz et al.

(123) investigated alveolar bone height and showed

significant variations related to genotype. The twin

groups had similar smoking histories and oral

hygiene practices. It was concluded that genetics

plays a role in susceptibility to periodontal disease.

In a subsequent study of 117 adult twin pairs,

Michalowicz and coworkers estimated genetic and

environmental variances and heritability for gingivitis

and chronic periodontitis (125). Genetic and envi-

ronmental variances and heritability were estimatedusing models with maximum likelihood estimation

techniques. Monozygous twins were found to be

more similar than dizygous twins for all clinical

measures. Statistically significant genetic variance

was found for both the severity and the extent of

disease. Chronic periodontitis was estimated to have

approximately 50% heritability, and was unaltered

after adjusting for behavioral variables, including

smoking. Monozygous twins were also more similar

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than dizygous twins for gingivitis scores but there

was no evidence of heritability for gingivitis after

behavioral covariates such as utilization of dental

care and smoking were incorporated. These results

confirm previous studies and indicate that approxi-

mately half of the variance for chronic periodontitis is

attributable to genetic variance. The basis for the

heritability of periodontitis appears to be biological

and not behavioral (125).

Segregation analysis

While familial aggregation suggests a heritable

component in the etiology of aggressive periodon-

titis, and twin studies support a genetic component

in chronic periodontitis, neither is appropriate to

identify the genetic model or specific gene loci

involved in these periodontal diseases. Segregation

analyses can evaluate the relative support for dif-

ferent models to identify the one that most closely

represents the clinical data. Segregation analysis is

very dependent upon the assumptions of the ana-

lyses and there have been few rigorous segregation

analyses of aggressive periodontitis. Many are

merely studies of one or more families and are

statistically underpowered. Genetic segregation

analysis needs accurate clinical identification of

affected individuals and familial relationships as

well as genetic assumptions of the analysis. If

inaccurate assumptions or data are used, the out-

comes will reflect this. Early studies of aggressive

forms of periodontitis were hampered by diagnosticclassification issues and by an overrepresentation

of affected females (67, 147), falsely supporting

X-linked transmission (68). Linkage reports of

aggressive periodontitis in an extended kindred

from Maryland supported an autosomal dominant

transmission (14). A segregation analysis of North

American families was performed by Marazita and

coworkers, who studied more than 100 families

segregating aggressive forms of periodontitis; their

results supported an autosomal dominant trans-

mission (112). They concluded that autosomal

dominant inheritance with approximately 70%penetrance occurred for both Blacks and non-

Blacks. The currently held theory on the genetics of

aggressive periodontitis is that prepubertal perio-

dontitis, localized aggressive periodontitis, and

generalized aggressive periodontitis are probably

due to a major gene locus transmitted in an auto-

somal manner with reduced penetrance; there is

evidence for both autosomal recessive (147) and

autosomal dominant forms (14, 112). It is likely that

these aggressive forms of periodontitis are genetic-

ally heterogeneous, meaning that while the mutated

gene responsible for the condition is likely to be

the same in any given family, there are probable sev-

eral different genetic loci that, if mutated, can cause

aggressive periodontitis. The expression reduced

penetrance means that some subjects with the geno-

type may not actually express the phenotype, i.e. the

clinical manifestations of aggressive periodontitis,whereas others may express it fully. Environmental

factors (such as smoking and plaquecontrol) as well as

epigenetic interactions of multiple susceptibility genes

may play a large role in whether the phenotype is

expressed clinically.

Clinical evidence as well as laboratory studies of

periodontitis patients suggest genetic heterogeneity

(6, 69). Consequently, the suggestion that aggressive

periodontitis may be transmitted in different ways

in different families is not a surprise. Some reviews

have reported this as problematic, but this is not

necessarily the case. There is a common precedent

in genetics for a heritable pathologic condition to

show different modes of inheritance in different

families. Often, once the genetic basis of the con-

dition is understood, it is found that mutations in

different genes can cause a clinically similar group

of conditions, as occurs in the EhlersDanlos syn-

dromes, a heterogeneous group of heritable con-

nective tissue disorders. These conditions involve

aberrations of collagen, and several are known to

have periodontal disease manifestation. More than

15 forms of EhlersDanlos syndromes are known,and these show different inheritance patterns

including autosomal dominant, recessive, and

X-linked forms. These findings reflect that different

genetic loci are capable of causing the disease in

dominant and recessive ways. Some of the genes

responsible are autosomal and others are X-linked,

accounting for the different observed modes of

transmission. For aggressive forms of periodontitis,

the preponderance of the evidence supports auto-

somal dominant transmission in North America and

autosomal recessive transmission in certain Euro-

pean populations (112, 146, 147). These differentmodes of transmission may reflect genetic hetero-

geneity such as that seen with EhlersDanlos

syndromes.

Linkage studies in aggressiveperiodontitis

Three linkage studies have been performed to date

on families with localized aggressive periodontitis.

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Boughman et al. (14) identified an autosomal

dominant form of localized aggressive periodontitis

in an extended family from Southern Maryland. In

this family, type III dentinogenesis imperfecta and

a localized form of aggressive periodontitis were

segregating as dominant traits. Since the gene for

dentinogenesis imperfecta-III had been previously

localized to chromosome 4, they performed a

linkage analysis on this chromosome and demon-strated a relatively close linkage with the suspected

locus for aggressive periodontitis (14). This was an

important study because it supported autosomal

dominant inheritance of a single major gene locus,

clearly indicating a major genetic component to the

aggressive periodontitis disease etiology. Hart et al.

(69) evaluated support for linkage to this region of

chromosome 4 in a different population of families

(14 African American and 4 Caucasian). Their

findings supported genetic locus heterogeneity of

aggressive periodontitis, as they excluded a chro-

mosome 4 major gene locus for aggressive perio-

dontitis in the families they studied. Thus, this

Brandywine population appears to have a different

form of periodontal disease with a different gene

being responsible compared with the African-

American and Caucasian families studied by Hart

and coworkers. These findings support genetic

heterogeneity, with at least one gene locus

responsible for aggressive periodontitis located

on chromosome 4. Recently, Li and coworkers (107)

reported evidence of a gene responsible for locali-

zed aggressive periodontitis located on chromo-some 1q25. To date, a gene of major effect for

aggressive periodontitis has not been identified.

Syndromic forms of periodontitis

Significant, irrefutable clinical and laboratory data

indicate that genetic variants do predispose to

disease states in humans. Severe periodontitis pre-

sents as part of the clinical manifestations of a

number of monogenic syndromes and the gene

mutation and biochemical defect is known for

many of these conditions. A commonality of theseconditions is that they are inherited as simple

Mendelian traits due to genetic alterations of a

single gene locus. Examples of these monogenic

conditions are given in Table 1. The significance of

these conditions is that they clearly demonstrate

that a genetic mutation at a single locus can impart

susceptibility to periodontitis. Additionally, these

conditions illustrate that this genetic susceptibility

may segregate by different transmission patterns.

Because altered proteins function in different

structural and immune pathways, genetic modula-

tion of a variety of different genes can affect a

variety of different physiological and cellular path-

ways (63). These conditions illustrate that the gen-

etic contribution to periodontitis susceptibility is

multifaceted, and may potentially involve many

different gene loci. However, in contrast to nons-

yndromic forms of periodontitis, these conditionshave periodontal disease manifestations as part of a

collection of syndromic manifestations. In most

cases of aggressive periodontitis, individuals present

with clinical manifestations of periodontitis, but do

not appear to have any other clinical disease

manifestations. This is not inconsistent with a

genetic disease etiology. Expression of genes can

vary in different tissues, and mutations of a ubiq-

uitously expressed gene can result in a tissue-spe-

cific condition. Recently, mutation of the SOS1

gene has been identified in individuals with her-

editary gingival fibromatosis (72). SOS1 is important

in determining whether cells grow, divide or dif-

ferentiate and is ubiquitously expressed. However,

the only clinical manifestation of this gene defect

appears to be enlargement of the periodontium, an

instance of the tissue-specific nature of many dis-

eases. A similar tissue-specific manifestation of a

gene defect may occur in nonsyndromic aggressive

periodontitis.

Neutrophil functional disorders

Molecular biology has highlighted the importantrole of several receptors on the polymorphonuclear

leukocyte surface in adhesion, and emphasized that

defects in the number of these receptors may lead

to increased susceptibility to infectious disease (2).

Adhesion is crucial to the proper function of the

polymorphonuclear leukocyte because it affects

phagocytosis and chemotaxis which, if deficient,

might predispose to severe periodontal destruction.

Page et al. (131) proposed that the generalized form

of prepubertal periodontitis (this disease has gen-

eralized and localized forms) is a localized oral

manifestation of the leukocyte adhesion deficiencysyndromes. More recent studies have indicated that

generalized and localized prepubertal periodontitis

occur in otherwise healthy children (17, 151). Leu-

kocyte adhesion deficiency occurs in two forms,

leukocyte adhesion deficiency syndrome type 1 and

leukocyte adhesion deficiency syndrome type 2,

both of which are autosomal recessive traits. Cir-

culating leukocytes have reduced or defective sur-

face receptors and do not adhere to vascular

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endothelial cells; thus they do not accumulate in sites

of inflammation where they are needed. Reports of

leukocyte adhesion deficiency syndromes indicate

that although the blood vessels are full of neutrophils,

the disease sites lack sufficient leukocytes to combat

the microbial challenge and thus infections ensue

rapidly in these patients. Affected homozygotes suffer

from acute recurrent infections that are commonly

fatal in infancy. Those surviving will develop severeperiodontitis, which will begin as the primary denti-

tion erupts (174).

Other disorders of neutrophil function are associ-

ated with severe forms of periodontal destruction.

The ChediakHigashi syndrome is a rare disease

transmitted as an autosomal recessive trait. Those

affected are very susceptible to bacterial infections

due to alterations in the functional capacity of the

polymorphonuclear leukocyte. Humans (59) and

other animals (105) with ChediakHigashi syndrome

exhibit generalized, severe gingivitis and extensive

loss of alveolar bone and premature loss of teeth

(168). The polymorphonuclear leukocyte chemotactic

and bactericidal functions are thought to be abnor-

mal in these patients.

These diseases related to anomalies of leukocyte

and polymorphonuclear leukocyte function are

excellent examples of how monogenic defects can

cause periodontitis through a clearly attributable

mechanism. The host response is made up of a vast

number of processes, all of which are under genetic

control and all of which are feasible candidates for

irregularities that may result in variability in theclinical presentation of periodontal disease.

Deficiency in neutrophil numbers (neutropenias)

A further neutrophil deficiency is found in infantile

genetic agranulocytosis, a rare autosomal recessive

disease where polymorphonuclear leukocyte num-

bers are very low and which has been associated with

aggressive periodontitis (144).

Cohens syndrome is another autosomal recessive

syndrome and is characterized by mental retardation,

obesity, dysmorphia, and neutropenia. Individuals

with Cohens syndrome show more frequent andextensive alveolar bone loss than do age-, sex-, and

mental ability-matched controls (1).

Not all neutropenias result in periodontal disease.

Familial benign chronic neutropenia has variable

expressivity and although several individuals within a

family may be neutropenic, not all are affected by

recurrent infections or periodontal disease (34).

These findings might be explained by the variable

genetic expression of the disorder or by the variable

effects of the environment (such as plaque or smo-

king) on these patients.

Genetic defects of structural components

PapillonLefevre Syndrome is a condition in which

the cardinal clinical features are severe periodontitis

and great variation in the severity and extent of pal-

mar plantar hyperkeratosis (56, 60, 70). Geneticlinkage studies narrowed the PapillonLefevre gene

locus to chromosome 11 and subsequent mutational

analyses permitted identification of mutations in the

cathepsin C gene in patients with PapillonLefevre

syndrome (64, 170). Subsequent studies have identi-

fied more than 40 different cathepsin C mutations in

individuals from many different ethnic groups (65).

This is an excellent example of the success of genetic

studies in contributing to the identification of a gene

defect of periodontal importance. Genetic linkage

studies permitted localization of the gene defect to a

specific chromosome, permitting focused mutational

analyses on genes within an area of the chromosome.

These focused analyses uncovered gene mutations in

the cathepsin C gene. Mutations of this gene are

associated with the loss of protease activity of the

cathepsin C protein. Additional work has demon-

strated that PapillonLefevre syndrome and Haim

Munk syndrome (a slightly different clinical variant

within the PapillonLefevre syndrome group of dis-

orders) are allelic variants of cathepsin C gene

mutations, as predicted by Gorlin et al. (56, 65, 182,

183).EhlersDanlos syndrome refers to a collection of

connective tissue disorders characterized by defect-

ive collagen synthesis. EhlersDanlos types IV and

VIII are related to an increased susceptibility to per-

iodontitis (71, 108) and are inherited in an autosomal

dominant manner. Clinical characteristics of type

VIII EhlersDanlos syndrome include fragility of the

oral mucosa and blood vessels, and a severe form of

aggressive periodontitis (3).

Other genetic conditions related to defects in

structural components that maintain a healthy

periodontium include the very rare WearyKindlersyndrome and hypophosphatasia. Aggressive perio-

dontitis has beenreported in WearyKindler syndrome

where abnormalities of the epidermal keratinocytes

occur (176). Patients with hypophosphatasia have a

decreased serum alkaline phosphatase and the pres-

ence of phosphoethanolamine in the urine (48). In

these patients, there is severe loss of alveolar bone

and premature loss of the primary teeth (12, 19),

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particularly anteriorly (7). There is histologic evidence

of enlarged pulp chambers and a disturbance in

cementogenesis, the cementum being either absent

or hypoplastic. Gingival fibroblasts are also deficient

in alkaline phosphatase. The lack of connective

tissue attachment between the tooth and bone

accounts for the early spontaneous exfoliation of the

primary teeth. Baab et al. (7) described a family

where all three children manifested prematureexfoliation of the primary teeth similar to that seen

in prepubertal periodontitis (133). These children

were assigned a diagnosis of hypophosphatasia

on the basis of alkaline phosphatase and phospho-

ethanolamine levels. Baab et al. (7) noted that their

data suggest an autosomal dominant mode of

transmission and suggested that hypophosphatasia

might be considered in the etiology of some forms of

aggressive periodontitis.

Polymorphism studies inperiodontitis

In complex diseases, genetic variants at multiple

gene loci contribute to overall disease susceptibility.

As such, a simple cause and effect relationship

between a particular genetic allele and a disease is

not possible. Support for an etiologic role for genes

(Table 1), complex genetic diseases which are not

diachronic periodontitis, is based on the statistically

significant association of specific genetic alleles in

individuals with disease (cases) compared to indi-viduals without disease (controls). This mathemat-

ical association is not necessarily biological or

physiological. Studies reporting such associations

vary in design and rigor. Association studies ideally

should evaluate large numbers in population-based

studies and have the power to detect a significant

association. The issues of allele frequency in the

population studied, case control design, and pop-

ulation stratification are very important but are

unfortunately often omitted from dental studies.

This is not acceptable, and studies performed

without sufficient rigor need to be interpreted inthis light (52, 75, 109). The overstating of results

has become commonplace such that rigorous, sci-

entifically principled approaches are needed to

guard against unfounded and erroneous conclu-

sions. Tables 25 report a multitude of studies

evaluating associations of genetic polymorphisms

with periodontitis. The variation in design and

power of these studies raises questions regarding

the appropriateness of incorporating these studies

into rigorous analyses such as the meta-analysis

techniques used in Cochrane-style systematic

reviews.

Gene polymorphisms of host responseelements and periodontitis

The genetic basis of many aspects of the periodontalhost response has been discussed in reference to

genetic disorders predisposing to periodontal dis-

ease. The aim of this section is to generally

summarize the potential influence of innate,

inflammatory, and immunological genetic variations

and to consider where the most promising candi-

dates lie from the viewpoint of a genetic diagnostic

approach to periodontitis (Tables 25).

There is a genetic basis to many aspects of the

periodontal host response. Mutations of genes

have been identified in Mendelian inherited syn-

dromes that have significant periodontitis findings

(Table 1), clearly indicating that single gene defects

can predispose to periodontitis. Data from human

and animal studies indicate that genetic variance

can influence the innate, inflammatory, and

immunological response to microbial infection. The

availability of the annotated human genome and

technological advances in genotyping have made it

possible to genotype allelic variants and to test for

association in casecontrol studies. This has facili-

tated studies to evaluate the support foragainst the

association of an array of genetic polymorphismswith periodontitis periodontitis (Tables 25). It is

important to realize that most of these studies are

underpowered to draw definitive conclusions.

Several features of the hosts innate immune sys-

tem which may contribute to genetic susceptibility to

aggressive periodontitis have been outlined already

and include epithelial, connective tissue, and fibro-

blast defects. Functional defects or a deficient num-

ber of polymorphonuclear leukocytes, as discussed

previously, have profound effects on the hosts sus-

ceptibility to periodontitis. Another aspect of the host

inflammatory response, proinflammatory cytokines,has attracted much attention as potentially crucial

variants influencing the host response in periodon-

titis. This review will not attempt to summarize the

plethora of SNP studies in periodontal disease but

will summarize tabularly (Tables 25) the areas of

current research and focus on the interleukin-1

polymorphism by way of example and illustration of

the SNP periodontal literature.

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Interleukin-1 gene polymorphisms inperiodontal disease

The interleukin-1 gene polymorphisms associatedwith periodontitis are a useful example for consid-

ering the strengths and limitations of using gene

polymorphisms in disease association studies in the

periodontal diseases. In 1997, Kornman et al. (100)

found an association between polymorphisms in the

genes encoding for interleukin-1a () 889) and inter-

leukin-1b (+ 3953) (termed the composite genotype)

and an increased severity of periodontitis. This initial

study spawned numerous publications and has been

highly influential in creating interest in gene poly-

morphisms and periodontal disease. The specific

genotype of the polymorphic interleukin-1 gene

cluster (periodontitis susceptibility trait, PST orcomposite genotype) was only associated with

severity of periodontitis in nonsmokers, and distin-

guished individuals with severe periodontitis from

those with mild disease (odds ratio 18.9 for ages

4060 years, but wide confidence intervals of

1.04343.05). Functionally, the specific periodontitis-

associated interleukin-1 genotype constitutes a vari-

ant in the interleukin-1b gene that is associated with

high levels of interleukin-1 production (136, 137).

Table 2. Cytokine polymorphism studies in periodontal disease

Gene Disease Association Authors (Ref.)

Interleukin (IL)

IL-2 ()330) ChP Assoc. Scarel-Caminaga et al. (149)

IL-4 (PP and IP) AgP No assoc. Gonzales et al. (53)

IL-4 ()590) ChP No assoc. Pontes et al. (138)

IL-4 ()

590) ChP No assoc. Kang et al. (91)

IL-4 ()590) ChP No assoc. Scarel-Caminaga et al. (150)

IL-4 (PP and IP) AgP Assoc. Michel et al. (126)

IL-6 ()572) ChP Assoc. (resist.) Holla et al. (85)

IL-6 ()174) ChP Assoc. Trevilatto et al. (172)

IL-10 (ATA haploytpe) ChP Assoc. Scarel-Carminga et al. (148)

IL-10 ()1087) ChP Assoc. Berglundh et al. (10)

IL-10 ()597, )824) AgP, ChP No assoc. Gonzales et al. (54)

IL-10 (gene promoter region) ChP No assoc. Yamazaki et al. (178)

IL-10 (R and G microsatellites) AgP No assoc. Kinane et al. (94)

Tumor necrosis factor-a (TNF-a)

TNF-a ()308) ChP No assoc. Folwaczny et al. (45)

TNF-a ()308) ChP No assoc. Fassmann et al. (41)

TNF-a ()308) and

Lymphotoxin (+252)

ChP Assoc. Fassmann et al. (41)

TNF-a () 1031, )863, )857) ChP Assoc. Soga et al. (157)

TNF-a ()376, )308, )238, +489) ChP No assoc. Craandijk et al. (27)

TNF-a ()1031, )863, )857, )308, )238) AgP No assoc. Endo et al. (39)

TNF-a ()

308) AgP No assoc. Shapira et al. (152)

TNF-a (microsatellite polymorphisms) AgP No assoc. Kinane et al. (94)

TNF-a ()238, )308, +252) ChP No assoc. Galbraith et al. (50)

TNF receptor 2 (+587) ChP Assoc. Shimada et al. (153)

ChP: chronic periodontitis. AgP: aggressive periodontitis. Assoc.: related to susceptibility to periodontitis. No assoc.: not related to susceptibility to periodontitis.Assoc. (resist.): associated with resistance to periodontitis.

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Kornman et al. (100) found that 86.0% of the severe

periodontitis in patients was accounted for by either

smoking or the interleukin-1 genotype. Similar

results were reported by McDevitt et al. (115) and

from smaller studies by McGuire et al. (117), Laine

et al. (101), and Gore et al. (55) (who suggested

that the two polymorphisms within the composite

genotype may be in linkage disequilibrium). Other

contradictory reports such as Meisel et al. (119) sta-

ted that the composite genotype showed a strong

interaction with smoking, an established risk factor

for periodontitis (93), whereas nonsmokers, even

when genotype positive, were not at any increased

risk. A similarly contradictory study (134) of 132

periodontitis patients who were age- and sex-matched

with controls, did not show any association between

Table 3. Receptor polymorphism studies in periodontal disease


TLR-2 (Arg753Gln, Arg677Trp) ChP No assoc. Folwaczny et al. (43)

TLR-4 (Asp299Gly, Thr399Ile) ChP No assoc. Folwaczny et al. (43)

FccRIIa (HH) ChP

smokers

Assoc. Yamamoto et al. (177)

FccRIIa (RR) ChP Assoc. Tang et al. (167)

FccRIIa(131R) AgP, ChP No assoc. Chung et al. (22)

Gm (23) allotypes ChP Assoc. Chung et al. (22)

FccRIIa (HH), FccRIIIa(158 V) AgP, ChP Assoc. Loos et al. (111)

FccRIIb (695) AgP Assoc. Yasuda et al. (180)

FccRIIb(646184, intron 4) Chp Assoc. Yasuda et al. (180)

FccRIIIb (NA2), FccRIIIa(158 V) ChP Assoc. Kobayashi et al. (97)

FccRIIIa (VV genotype) ChP

(Severity of

bone loss)

Assoc. Meisel et al. (118)

FccRIIIb (NA1) ChP Assoc. (resist.) Sugita et al. (159)

FccRIIIb (NA2), FccRIIIa (158F) AgP Assoc. Kobayashi et al. 2000 (95)

FccRIIa, FccRIIIb (Haplotypes),

Gm (23) allotype

ChP No assoc. Colombo et al. 1998 (25)

FccRIIIb (NA2) ChP Assoc. Kobayashi et al. (96)

Estrogen receptor-a (PvuII ER) AgP, Chp No assoc. Zhang et al. (184)

Estrogen receptor-a (XbaI ER) ChP Assoc. Zhang et al. (184)

FMLP receptor (329,378) AgP Assoc. Zhang et al. (185)

CCR5 (D32) ChP No assoc. Folwaczny et al. (42)

RAGE(1704) ChP Assoc. Holla et al. (84)

VDR (TaqI, BsmI) ChP Assoc. de Brito et al. (30)

VDR (TaqI) ChP Assoc. Tachi et al. (163)

VDR (TaqI) AgP Assoc. Sun et al. (160)

VDR (TaqI) AgP, ChP Assoc. Tachi et al. (164)

VDR (Bb) AgP No assoc. Yoshihara et al. (181)

VDR (Bb) and FccRIIIb (NA1NA2) AgP Assoc. Yoshihara et al. (181)

VDR (TaqI) AgP No assoc. Hennig et al. (74)

ChP: chronic periodontitis. AgP: aggressive periodontitis. Assoc.: related to susceptibility to periodontitis. No assoc.: not related to susceptibility to periodontitis.

Assoc. (resist.): associated with resistance to periodontitis.

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the composite genotype and periodontitis. The

prognostic utility of the interleukin-1 genotype on

chronic periodontitis progression following nonsur-

gical therapy was performed by Ehmke et al. (37). Ofthe 33 patients studied, 16 had the susceptible

composite genotype reported by Kornman et al.

(100). Following 2 years of periodontal maintenance

care, no differences in tooth or attachment loss were

detected between those with or without the genotype.

Equivocal studies such as those of Cullinan et al. (28)

demonstrated an interaction of the interleukin-1

positive genotype with age, smoking, and P. gingi-

valis, which suggests that interleukin-1 genotype is a

contributory but nonessential risk factor for perio-

dontal disease progression in this population. Catta-

briga et al. (20) reported no significant differences in

tooth loss in patients with the interleukin-1 genotypeafter 10 years in a nonsmoking, well-maintained

periodontal population. De Sanctis et al. (31) dem-

onstrated that genotype expression did not affect

guided tissue regeneration treatment response at

1 year, but had a great impact on long-term stability

(year 4). In a 3-year period, patients with a positive

interleukin-1 genotype lost about 50% of the clinical

attachment level gained in the first year and were

about 10 times more likely to experience 2 mm

Table 4. Miscellaneous host response polymorphisms


CD14 ()1359) ChP Assoc. Holla et al. (82)

CD14 ()159) ChP No assoc. Yamazaki et al. (179)

NOD2CARD15(3020insC) ChP No assocn Folwaczny et al. (44)

NOD2CARD15(3020insC, C2104T) ChP No assoc. Laine et al. (102)

HLA-DQb1 (BamHI) AgP No assoc. Hodge et al. (79)

HLA-DRb1(1501) HLA-DQb1(0602) AgP Assoc. Takashiba et al. (166)

HLA-DQb1(0503, 0602) AgP Assoc. Ohyama et al. (130)

HLA-DQb (BamHI) AgP Assoc. Takashiba et al. (165)

MMP-1 ()1607, 1G) ChP

nonsmokers

Assoc. Holla et al. (85)

MMP-1()1607), MMP-3 ()1171) AgP, ChP No assoc. Itagaki et al. (86)

MMP-1 ()1607, 2G) ChP

nonsmokers

Assoc. de Souza et al. (32)

TGF-b1 ()509) ChP Assoc. de Souza et al. (33)

TGF-b1 ()800, )509, L10P, R25P) ChP No assoc. Holla et al. (83)

Fibrinogen (H1H2, H2H2) ChP Assoc. Sahingur et al. (145)

Plasminogen activator inhibitor-1(4G) ChP Assoc. Izakovicova et al. (87)

NAT2 (two mutated alleles) ChP

smokers

Assoc. Kocher et al. (98)

TGF: transforming growth factor. ChP: chronic periodontitis. AgP: aggressive periodontitis. Assoc.: related to susceptibility to periodontitis. No assoc.: notrelated to susceptibility to periodontitis. Assoc. (resist.): associated with resistance to periodontitis.

Table 5. Multiple gene polymorphism assessments

Gene Disease Association Authors

IL-1a ()889), TNF-a ()308), IL-6 ()174) ChP Assoc. with serum IL-6 DAuito et al. (29)

Comprehensive 125 Genes 310 SNPs ChP and AgP Assoc. noted Suzuki et al. (162)

TNF-b (NcoI bi), ACE (ID), Endothelin-1 (TaqI) ChP Assoc. Holla et al. (84)

IL, interleukin. TNF, tumor necrosis factor. ACE, angiotensin converting enzyme. ChP: chronic periodontitis. AgP: aggressive periodontitis. Assoc.: related tosusceptibility to periodontitis.

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clinical attachment loss when compared to oral

hygiene-matched genotype-negative patients. Indeed,

numerous studies, as depicted in Tables 25, show

associations and lack of associations across different

and similar populations for various forms of chronic

and aggressive periodontitis.

The polymorphisms in the interleukin-1 gene

cluster linked with periodontitis (100) are found in

approximately 30% of the European population.However, the prevalences are dramatically lower in

Chinese (2.3%) and thus the usefulness of the com-

posite genotype of allele 2 of both interleukin-1a

(+ 4845) and interleukin-1b (+ 3954) for determining

susceptibility in Chinese patients is dubious (5).

Interleukin-1 polymorphism inaggressive periodontitis

Hodge et al. (80) examined interleukin-1a and inter-

leukin-1b genetic polymorphisms in unrelated Euro-

pean white Caucasian patients with generalized early

onset periodontitis and found no significant differ-

ences between patients and controls for any of the

composite genotypes described by Kornman et al.

(100). No significant differences were found between

patients and controls whether smoking was included

as a covariate or not. It was concluded that there was

a lack of association between the interleukin-1 poly-

morphisms and aggressive periodontitis, which

questions the utility of these candidate genes as

markers of susceptibility. This was a relatively

homogeneous Scottish population and the results,although negative, merely reflect the lack of utility of

this composite genotype test in this population.

Other studies on the composite genotype reported

by Kornman et al. (100) and aggressive periodontitis

have had similarly mixed results. For example, the

studies by Diehl et al. (35, 36) actually found that

allele 1 rather than allele 2 of the interleukin-1b

+ 3953 exhibited polymorphism. Furthermore, Park-

hill et al. (135) investigated the frequency of poly-

morphisms in the genes encoding interleukin-1b in

Caucasians with aggressive periodontitis compared

to controls. The frequency of interleukin-1b geno-types homozygous for allele 1 of the interleukin-

1b + 3953 SNP was found to be significantly increased

in aggressive periodontitis patients (P 0.025). Upon

stratification for smoking status, a significant differ-

ence was found in the interleukin-1b genotype

distribution between aggressive periodontitis smo-

kers and control smokers (F-exact test, P 0.02), but

not between aggressive periodontitis nonsmokers and

control nonsmokers. The interleukin-1b 11 genotype

occurred at a higher frequency in aggressive perio-

dontitis smokers (odds ratio 4.9) than in control

smokers. These findings of Parkhill et al. (135), in

contrast to those of Hodge et al. (81), found that an

interleukin-1b genotype in combination with smo-

king is associated with aggressive periodontitis.

Similar negative findings for this composite

genotype and both chronic and aggressive perio-

dontitis populations from different racial and ethnicbackgrounds have been demonstrated and thus the

diagnostic utility of the composite genotype may be

restricted to specific populations, i.e. the results do

not appear to be applicable globally and across eth-

nic populations, and certainly not for aggressive

periodontitis.

Biological plausibility for the compositeinterleukin-1 polymorphisms

Many investigators have suggested a role for inter-

leukin-1 in the initiation and progression of perio-

dontitis and have quoted in vitro and in vivo studies

showing that interleukin-1 activates the degradation

of the extracellular matrix and bone of the perio-

dontal tissues. Elevated tissue or gingival fluid levels

of interleukin-1b have been associated with perio-

dontitis. Kornman et al. (100) quotes abstracts of

in vitro studies from Pociot et al. (137) and others

(104) which claim that the interleukin-1 poly-

morphism associated with severe periodontitis in

the Kornman study is also known to correlate with a

two- to fourfold increase in interleukin-1b produc-tion. A problem with this line of reasoning is that

interleukin-1 is a proinflammatory cytokine inti-

mately involved in all inflammatory reactions as well

as immune and reparative or healing responses and

any perturbation of its level so as not to be home-

ostatically controlled could have widespread conse-

quences not limited to periodontal disease. In

addition, interleukin-1 is one of many proinflam-

matory cytokines (interleukin-6, tumor necrosis

factor-a, etc.) with overlapping activities and thus

some redundancy exists in the cytokine system.

Furthermore, interleukin-1 has many controllingmechanisms which include inhibition of transcrip-

tion, release controls, and receptor antagonists, and

thus is highly regulated such that any polymorphism

coding for increased production of this molecule

could readily be controlled by the elaborate positive

and negative feedback loops associated with its

regulation.

The claimed association of severe periodontitis

with smoking and the interleukin-1 genotype (in

105



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that in smokers the composite interleukin-1 geno-

type did not influence susceptibility) poses further

problems. Do smoking and the overproduction of

interleukin-1 work along the same pathogenic

pathway and thus is the action of both factors not

additive but renders the other redundant, or is the

overall effect of smoking is so overriding that the

composite genotype has little or no effect? These

possibilities, while feasible, require much moremechanistic knowledge for both risk factors, but

especially for the genotype, given that the associ-

ation with periodontitis is not as established as that

found in the literature on smoking (93).

Socransky et al. (155) investigated the association

between the composite genotype and carriage of

periodontal species. They found the mean counts of

specific species were higher in general in interleukin-1

genotype positive than in negative subjects. The spe-

cies detected at higher levels were those frequently

associated with measures of periodontal inflamma-

tion. A further study aimed at studying the composite

genotype and inflammation was performed by Lang

et al. (103). Genotype-negative subjects had a signifi-

cantly lower percentage of bleeding on probing

(P 0.0097) and it was concluded that the increased

bleeding on probing prevalence and incidence ob-

served in interleukin-1 genotype-positive subjects

indicates that some individuals have a genetically

determined hyperinflammatory response that is

expressed in the clinical response of the periodontal

tissues.

Shirodaria et al. (154) have taken the research fur-ther by attempting an assessment of the functional

effect of the composite genotype in terms of the

quantity of interleukin-1a protein in gingival crevice

fluid of severe chronic periodontitis patients. These

researchers found that allele 2 at position ) 889 of the

interleukin-1a gene (one of the alleles linked with

susceptibility to periodontitis by Kornman et al.

(100)) was associated with a fourfold increase in

interleukin-1a as determined by enzyme linked im-

munoassay. This technique does not demonstrate

activity but merely protein presence or absence and

would not differentiate protein bound to receptors orinhibitors. Furthermore, it is feasible that inhibitors

of proinflammatory cytokines may concomitantly be

produced to dampen this effect. The authors noted

reduced levels of interleukin-1a protein in heavy

smokers regardless of genotype but this may be

related to the reduced gingival crevice fluid noted

in smokers (93). This is a useful study given

that it addresses the in vivo effects of the poly-

morphism on interleukin-1 protein quantities,

However, differences in local gingival crevice fluid

production among patients, sites, smokers, and

across gender add considerable variance to such a

study, and these factors have to be considered in the

interpretation of the data.

Engebretson et al. (40) also found elevated levels of

interleukin-1b in the gingival crevice fluid in shallow

sites of patients who were positive for the composite

genotype

reported by Kornman et al. (100). Smokingwas not considered in the study and no statistically

significant differences were noted for deeper pockets.

Interestingly, of the 22 chronic periodontitis patients

examined, only seven were positive for the suscept-

ible genotype.

Mark et al. (114) studied peripheral blood mono-

cytes from composite genotype positive and negat-

ive patients to examine whether the interleukin-1b

polymorphism was correlated with increased inter-

leukin-1b expression by monocytes in response to

periodontal bacterial stimulus. Contrary to previous

reports, these workers found no significant differ-

ences in interleukin-1b production in response to any

stimulant tested. They went on to report marked

interindividual variation in the production of inter-

leukin-1b in both the genotype positive and negative

patient groups. Clearly, either the genotype is not

important in monocyte production of interleukin-1

or other genetic loci may determine the monocyte

interleukin-1 responses.

Summary of the findings on the

interleukin-1 composite genotype inperiodontitis

It appears that the interleukin-1 composite geno-

type has an equivocal ability to detect susceptibil-

ity to periodontitis and may at best be limited in its

utility to only specific populations. It would appear

from the mixed reports on this composite geno-

type that:

it appears irrelevant in aggressive periodontitis;

it may be in linkage disequilibrium with the gene

contributing susceptibility to chronic periodontitis;

the composite polymorphisms may be part ofseveral involved in the genetic risk for chronic

periodontitis;

the polymorphism is only a useful marker in

defined populations (5, 175);

confirmation of the functional significance of this

gene polymorphism remains to be confirmed;

clinical utilization of the composite polymor-

phisms for risk assessment and prognostic deter-

mination is premature.

106

Kinane et al.


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General considerations

Clinical diagnoses

Many confounding factors have been recognized in

population association studies of periodontal dis-

ease. Only recently have more carefully defined

classifications of periodontal disease appeared (4).

Prior to the American Academy of PeriodontologyWorkshop in 1999, there was much variation in the

literature with respect to disease definition and

numerous differences in what we define as a case

and indeed what constitutes a control in perio-

dontitis studies. These variable definitions make

comparisons across studies virtually impossible.

Other complications arise related to the variable

presentation over time of periodontal diseases,

notably the aggressive forms of periodontitis, which

generally appear prior to the age of 35 years of age

but which are not confined to this age. In many

cases the aggressive forms will appear very early inthe teenage years or quite late in the early 30s.

Chronic periodontitis is similar in that its presen-

tation and its categorization are quite complex and

very much dependent on environmental exposures

of the patient over their lifetime. Exposures such as

smoking, microbial plaque deposition (oral hygiene)

and other factors such as systemic disease modifi-

ers (e.g. diabetes) or general factors (e.g. socioeco-

nomic status) have a large influence on the

expression of the phenotype.

An additional criticism arises in the suggested

utility of these population association studies in that

there are often methodologic problems within the

reported studies. These include underreporting,

where multiple polymorphisms may be tested but

few are reported and where, for those few reported,

there is a lack of understanding of the importance of

multiple range testing, i.e. the reduced statistical

inference that can be made in such exploratory

studies. On many occasions the results are reduced

such that only the positive ones are reported, with

obvious statistical implications. The bias against

publishingnegative

results is one that has a tre-

mendous impact on our literature and recognition of

the importance of such results is needed.

Many do not report the sensitivity and specificity

of tests or the many associated environmental

aspects which need to be considered. Most studies

have insufficient numbers of cases and controls to

permit robust pronouncements on the association

between polymorphisms and disease. A precise

account of the racial and ethnic background of

both cases and controls is necessary as such biases

within populations would render the inference

questionable.

Environmental variables

Numerous assumptions have been made regarding

the populations researched in periodontal disease

association studies. Are the controls truly resistantto the disease? Or are they performing high stand-

ards of oral hygiene such that environmental

factors required to interact with the genotype to

express the phenotype are missing? In addition,

these subjects may have excellent access to health

care and health education and may not smoke,

thus avoiding further known risk factors which may

be needed to reveal the genotype. In this situation,

misplacing the subject(s) in the control group

dilutes the chances of seeing the real disease

association. Similarly, it is accepted that periodon-

tal destruction is incremental and thus the diseaseexperience and the clinical features increase with

age. Thus, the age of the cases and controls have to

be considered and matched. Gender and, more

importantly in genetic studies, race have to be

considered and documented carefully. Thus, the

importance of environmental factors in revealing

the genotype can not be overstated in the categ-

orization of cases and controls, and knowledge of

the racial or genetic heterogeneity is needed. A

further difficulty is knowing which environmental

factors will exert an influence. Twin studies can be

particularly useful here in sorting out genetic and

environmental factors (discussed in detail later).

Smoking and susceptibility genes

An important environmental risk factor is smoking.

The interplay between smoking and genes in the risk

for periodontitis has been rightly discussed in most

studies on genetic risk. Interestingly, the attributable

risk for periodontitis from smoking may be influ-

enced by the polymorphism of the N-acetyltransf-

erase (NAT2) gene, which may change the subjectsmetabolism to produce more damaging smoking-

derived xenobiotics. Meisel et al. (120) hypothesized

that an NAT2 genotype could be a risk factor for

periodontal disease. Severe chronic periodontitis

patients were predominantly slow acetylators

(OR 2.385.02). Thus, the slow acetylator pheno-

type may be associated with a higher risk for

periodontitis. This is an example of a strong gene

environment interaction.

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Biologic plausibility

Researchers should always consider the biological

plausibility of the effect of the gene polymorphism in

the periodontal disease process. There are, however,

a great many unknown aspects which provide con-

siderable leeway for investigators in this area. The

pathogenesis of periodontal disease is to some extent

known. It is a chronic inflammatory condition whichprogresses to the destruction of bone and supporting

structures and ultimately the loss of teeth, but the

reason that some patients are more susceptible than

others is not understood. Any of the innate, inflam-

matory, or immune processes may be involved in the

etiology of periodontal disease and, in addition, it is

feasible that structural aspects and aspects which

may influence the healing process may be similarly

involved. This leaves an extremely wide range of

factors, which could be considered fortuitous from a

research justification viewpoint but frustrating in the

search for the true etiology of the periodontal dis-

eases.

Criticisms of the lack of logic inassociation studies

A hypothetical example of the logic in play when

studying gene polymorphism associations with dis-

eases might be the following. In periodontitis (con-

dition A), bone is lost (abnormality B), and since bone

is removed by osteoclasts, which are influenced by a

specific gene (gene C), we then study the associationof a known polymorphism (polymorphism D) of gene

C with periodontal disease. Obvious problems in this

logic are apparent even superficially. Whether the

osteoclast has a pivotal role in the disease process is

unknown and to assume it does would be purely

speculation. Nevertheless, the link between A and B

typically results in the search for a gene C and a

polymorphism D, which happens to be readily

investigatable. Linking gene C with periodontal dis-

ease is a task for molecular geneticists, who would

employ linkage analysis techniques and positional

cloning together with robust statistic

Documents

The genetic basis of periodontitis