CONTENT

• Introduction
• What is GENE?
• Does every one has same genes?
• Functional components of gene
• Genetic basis of disease
• Methods of genetics analysis

• Evidence for role of genetic variants in aggressive

periodontitis & chronic periodontitis

• Polymorphism studies in periodontitis -- DISCUSSION

• Conclusion and future developments

 INTRODUCTION
• Periodontitis – only a portion of variability can be explained
by environmental factor
• Classic study by Loe et al 1986
• Host susceptibility -- defined in terms of genetic variation
• Recent focus on – genetic variants causing PDL disease
• Sufficient data – genetic factors are imp. determents
• To understand potential clinical relevance
genetic variability on periodontitis we must
understand how different genes can
contribute to disease
 What is GENE?
• Protein building blocks of body
• Direct structure and function of body
Protein :-- series
of amino acids in
a specific order
 Structure of DNA
DNA is a twisted ladder
double helix
DNA is a protein-building code library. A protein is
like a word built of letters (amino acids) which are
spelled out along the length of the DNA "page."
• This code or instruction making specific
protein is written on a specific segment of
DNA which is called “gene” for that protein

• All genes for structure and function of body –

“Human genome”
• Human genome is located in
 CHROMOSOME

• 23 pairs
• 22 autosome
• 1 sex chromosome
Does Every One Have Same Genes?
• All human being have same set of genes

• Same genes may differ in their nucleotide

sequences

• These different sequences are termed ALLELES

 Functional Components Of Genes
• Promoter :- Controls when and in what
tissue a gene is expressed

• Coding region:- Structure of protein

• Exon:- Expressed portion of DNA that will

ultimately translated into protein
• Genetic locus:- a specified nucleotide base
location in specific gene

• Identified physically by an address that represent the

no of nucleotide bases from the point on gene where
cell starts reading the protein instruction ie
transcription site.
• A locus of +3954 for IL 1B gene refers to the
3954 gene base pair of nucleotides past the
trancription start site of gene IL 1B
 GENETIC DISEASE PARADIGMS
• Genetic variance
• 25000-50000 genes
• Different forms of genes:-- alleles
• When specific alleles occurs in 1% of
population – polymorphism otherwise
mutation
• Triplet of nucleotide – codon – a. acid
• Alteration in codon → no change in
a. acid → silent
• Alteration in codon → change in a. acid →
dysfunctional protein → ↑or ↓ persons risk
for disease & can be enhanced by
environmental factors
 GENETIC BASES OF DISEASE

Based on pattern of transmission of disease genetic

diseases are classified in 2 broad groups as

1) Simple mendelian disease

2) Complex genetic disease

 Simple mendelian disease

• Diseases that follow predictable and generally simple

patterns of transmission have been called
"Mendelian" conditions

• occur in simple patterns in families,

• single gene locus is the major determinant of the

clinical disease phenotype

• Rare <0.1%
• Eg -- amelogenesis imperfecta

• possible to develop a diagnostic test

• to make fairly specific determinations of the

probability of the mutant gene being passed to a child

• to predict the course of clinical disease.

 COMPLEX GENETIC DISEASES

• More Prevalent >1%

• Result of the interaction of multiple different gene loci
• Environmental factors are important in the disease
process.
• Much less disruptive, and usually work within the
normal range of function.
• Occur in unaffected as well as affected individuals.
 Methods of Genetic Analyses

(A) FAMILIAL AGGREGATION

(B) TWIN STUDIES

(C) SEGREGATION ANALYSIS

(D) LINKAGE ANALYSIS

(E) ASSOCIATION STUDIES

 FAMILIAL AGGREGATION

• suggest genetic etiology

• common environment, including diet and
nutrition, exposures to pollutants, and
behaviors such as smoking
 TWIN STUDIES
• Most powerful

• Monozygous :- single fertilized ovum

genetically identical

• Dizygous :- from two different eggs

~ 50% genes in common

• Disconcordance:-- dissimilarity --

in characteristic
 SEGREGATION ANALYSIS
• Genes are passed from parents to children in a
predictable manner, and genes segregate in
families as predicted byMendel's Laws
• Used to determine if trait transmission
appears to fit a Mendelian or other mode of
genetic transmission

• Segregation analysis does not find or aim to

find a specific gene responsible for a trait.
 (D) LINKAGE ANALYSIS
• Linkage analysis is a technique used to localize the
gene for a trait to a specific chromosomal location

• Based on the fact that syntenic gene loci in proximity

tend to be passed together from generation to
generation (i.e.,segregate), as a unit. Such genes are
said to be "linked" and violate Mendel's law of
independent assortment
 Law of Independent Assortment

• States that the inheritance pattern of one trait

will not affect the inheritance pattern of another
• The goal -- to identify a piece of DNA of known location
that is inherited by all family members affected by the
disorder being studied, and is not inherited by any of the
unaffected family members.

• This piece of DNA is said to be to 'co-segregate' with the

disease phenotype.

• Once this piece of DNA is found, one knows that the

disease gene must lie somewhere close by.

• Determining the location of the disease gene is the first

step toward identifying the gene itself.
 ASSOCIATION STUDIES

• Linkage analyses may not be a useful strategy for the

detection of modifier genes or genes that exert small
effects—precisely those genes which might be
operating in chronic periodontitis and many other
complex disorders
• Association studies can sometimes detect weaker
effects than can linkage analysis (Hodge, 1994)
• Two types:-
1)Population-based 2) Family-based

• Population-based approach utilizes a standard

case-control design, in which marker allele
frequencies are compared between cases and
controls
• EVIDENCE FOR ROLE OF GENETIC
VARIANTS IN AGGRESSIVE
PERIODONTITIS & CHRONIC
PERIODONTITIS
AGGRESSIVE PERIODONTAL DISEASE
 ASSOCIATIONS WITH GENETIC AND INHERITED
CONDITIONS

• Aggressive periodontitis is consistent feature

in several inherited or genetic disorder
HYPOPHOSPHATASIA
• Mutation in tissue nonspecific alkaline
phosphatase gene 1p36.1-p34

• Deficiency in alkaline phosphatase activity

• Both forms :-- autosomal dominant

&recessive
 Deficiency in ALP

Abnormal bone Skeletal Cementum

mineralization anomalies hypoplasia
Premature loss
of deciduous
teeth

Cementum
Hypoplasia
• Infantile form is fatal

• Premature loss of primary teeth &

occasionally permanent teeth

• In adolescents --- This disease resembles

aggressive periodontitis
PAPILLON-LEFÈVRE SYNDROME

Palmoplanter
hyperkeratosis &
Aggressive periodontitis
• Mutation in Cathepsin C gene on chromosome 11 q14-
q21
• Cathepsin C present in --- 1) Epithelial cell
2) PMN cell
• Function :- 1] Degrading proteins
2] Activating proenzymes in
immune and inflammatory cell
• In PLS AP is associated with A.A.comitance

• Thus AP is not direct result of mutation but

specific bacterial infection in susceptible host
 Chediak-Higashi syndrome
• An autosomal recessive

• Abnormal transport of vesicles to and from

neutrophil lysosomes caused by mutation in
lysosomal trafficking regulator gene

• Generalized, severe gingivitis, extensive loss of

alveolar bone, and premature loss of teeth
 LEUKOCYTE ADHESION DEFICIENCY

LEUKOCYTE LEUKOCYTE
ADHESION ADHESION
DEFICIENCY TYPE I DEFICIENCY TYPE II
• CD18 – beta 2 • CD15 – neutrophil
integrin chain of ligand for E & P
LFA molecule selectin
 SEGREGATION ANALYSIS
• Melnick et al proposed X linked inheritance in
aggressive periodontitis because of preponderance
of female probands

• Saxen L 1984 proposed autosomal recessive model

for AP in finnish population

• In contrast in US marazita et al 1994 proposed

autosomal dominant mode
• Despite inconsistent conclusions regarding
their mode of inheritance, segregation
analyses consistently have supported the role
of a major gene in the etiology of the AP
disease
• Schenkein 1994 proposed a model of
inheritance which distinguishes between
etiologies of localized and generalized
aggressive periodontitis

• AP and IgG2 responsiveness to bacterial LPS

segregate independently as dominant and
codominant trait
• Subject with 1 AP disease allele and two
copies of the high IgG2 allele would develop
only localized disease

• In contrast subject with AP disease allele and

only 1 copy of IgG2 allele would develop more
widespread disease because their IgG2
response to LPS would be less robust
 LINKAGE STUDIES
• Boughman et al were the 1st to report linkage
between aggressive periodontitis and a specific
chromosomal region

• They found autosomal dominant AP to co-segregate

with dentinogenesis imperfecta

• AP gene found on chromosome 4 near DGI

• This study was contrasted by Saxen and Koskimies –

LAP found on chr. No 1
 ASSOCIATION STUDIES
• HLA – genetic risk marker for AP

• Genes for HLA class I and class II --- chr. No.6

• Nearby genes encode for complement fragments TNF-alfa

• Only few studies have consistent result.

• Sofaer JA 1990 – the risk of disease in subjects with HLA –

A9 or B- 15 is about 1.5 to 3.5 times greater than in those
lacking these antigens
• HLA class II DR4– associated with type 1 DM

• Katz et al – DR4 antigen to be more prevalent

in patients with AP disease than control
• Diehl SR 1999– IL 1 beta +3954 was reported in
linkage disequilibrium with GAP

• Hennig BJ 1999 – polymorphism of vit. D receptor

associated with AP

• Tai H 2002 -- polymorphism of IL-1 receptor

antagonist associated with AP

• Mutation in FMLP receptor gene associate with AP –

Gwinn MR 1999
 CHRONIC PERIODONTITIS
• TWIN STUDIES
• Michalowicz and Co-workers estimated
genetic and environmental variances and
heritability for gingivitis and CP.
• CP was estimated to have app.50%
heritability and was unaltered after adjusting
for behavioral variables Monozygous twins
were more similar than dizygous twins.
 ASSOCIATION STUDIES
• KORNMAN et al 1997 reported that composite IL-1
genotype consisting of at least one copy of the more
rare allele at both an IL 1 alfa and IL-1 beta was
associated sever periodontitis

• In contrast Gore et al found the more rare IL-1 beta

allele but not composite genotype to be more
prevalent in patient with advanced CP
• Diehl SR 1999 – risk for CP is conferred more
by the IL-1 beta than IL- 1 alfa allele

• Galbraith et al found no association between

CP and TNF alfa
• Kornman et al found the IL-1 composite
genotype was associated with severe disease
only in nonsmokers which supports the theory
that some harmful environments may
overwhelm any genetically determined
susceptibility or resistance to disease
• In contrast Meisel P 2003 found that IL-1
genotype was associated with disease in
smokers but not in non smokers
 POLYMORPHISM STUDIES IN
PERIODONTITIS -- DISCUSSION
 CYTOKINE GENE POLYMORPHISMS

• Interleukin-1 family
• Interleukin-1 is a potent pro-inflammatory
• agent that is released by macrophages,
platelets and endothelial cells
• IL-1 – stimulates bone resorption
inhibits collagen synthesis
up regulate MMP and Prostaglandins
• Gene encoding this cytokine is assigned to
chromosome 2q13–21
• Two forms IL 1-alfa and IL1- beta
• Kornman et al described a composite genotype formed by the
two polymorphic loci – interleukin-1A (889) and interleukin-
1B (+3953) – single nucleotide polymorphisms that carry a C–
T transition. This was later known as periodontitis-associated
genotype

• Interleukin-1 genotypes associate more with chronic

periodontitis in Caucasians.
• Meisel et al. concluded that the interleukin-1
periodontitis-associated genotype in German
Caucasians displayed a strong interaction with
smoking, resulting in an increased chronic
periodontitis risk
• Laine et al. (141) investigated the distribution of
polymorphisms in the interleukin-1 gene family among
periodontitis patients and controls, taking into account
both smoking and microbiological parameters, including
the presence of P. gingivalis and AAcomitans.

• The results showed a higher frequency of allele 2

carriage in interleukin-1A (889) and interleukin-1B
(+3954) single nucleotide polymorphisms and
interleukin-1RN variable numbers of tandem repeat
polymorphisms in non-smoking periodontitis patients in
whom P. gingivalis and A. A.comitans could not be
detected
• These results provide 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.
• Lopez MJ 2005 -- -Homozygosity for allele 1 of the
interleukin-1B (+3954) genotype was a protective
factor for periodontitis.

• 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,
irrespective of the smoking status and periodontitis
disease severity
• Unlike chronic periodontitis, reports of aggressive
periodontitis indicate that periodontitis-associated
genotype is not associated with this disease
 SUMMARY OF THE FINDINGS
ON THE IL-1 COMPOSITE GENOTYPE IN
PERIODONTITIS
 -
(i) It is unlikely to be relevant in aggressive periodontitis;

(ii) It is, at best, in linkage disequilibrium with the gene

contributing susceptibility to chronic periodontitis;

(iii) It confers risk independent of that attributable to smoking;

(iv) The polymorphism is at best one of several involved in

the genetic risk to chronic periodontitis, which is likely
to be a disease in which multiple genes may confer
risk;
(v) The polymorphism is a useful marker in only defined
Populations, is relatively absent in some (armitage et
Al., 2000), and is too prevalent (walker et al., 2000) in others
to be a genetic marker with utility;

(vi) Demonstration of the functional significance of this gene

polymorphism has yet to be confirmed; and

(vii) Clinical utilization of these composite polymorphisms

for risk assessment and prognostic determination is
currently premature.
• Homozygosity for allele 1 of the interleukin-1B
(+3954) genotype was a protective factor for
periodontitis
 Interleukin-1 Genotype-positive Subjects
Exhibited Increased Interleukin-1 Protein

• Shirodaria S,2000--carriage of allele 2 in the

(889) locus resulted in an almost fourfold
increase of interleukin-1a protein levels in
chronic periodontitis patients.
 Tumor necrosis factor
• pro-inflammatory cytokine

• wide range of immunoregulatory functions

• Tumor necrosis factor gene polymorphisms and

periodontitis studies display conflicting results
 OTHER CYTOKINES
• Interleukin-2
• mediates the cellular immune response by participating
in B lymphocyte activation and macrophage stimulation,
as well as in natural killer and T lymphocyt proliferation

• in Caucasian subjects, with the TT genotype seem to be

2.5-times less likely to develop severe periodontitis than
individuals who are heterozygous
or GG homozygous
• Interleukin 4 :- studies failed to show
assiciation
• Interleukin 6 :- Czech study suggested that the G/C
polymorphism of the IL-6 gene may be one of the
protective factors associated with lower
susceptibility to CP
• study did not find an association between the G/A
locus and chronic periodontitis susceptibility.
• IL 10:- stimulates the production of protective
antibodies and down-regulates pro-inflammatory
cytokines produced by monocytes

• Three promoter single nucleotide polymorphisms

have been described in gene: 1087 G/A;819 C/T; and
592 C/A

• G/A locus was not associated with chronic

periodontitis susceptibility in Japanese and Brazilian
subjects, but was linked to chronic periodontitis
severity in Swedish Caucasians
 Transforming growth factor-b1.

• Has both therapeutic and pathologic potential

• The gene for TGFB1 is located in chromosome

19q13.1

• One promoter polymorphism, the 509 C/T,

was associated with chronic periodontitis
severity in Brazilian Caucasians
 Receptor And Gene Polymorphism

• 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 considered essential for the host
defense against bacteria.
• Strong, specific IgG responses against
periodontopathic bacteria are observed in the
gingival tissue and gingival crevicular fluid. FcR for
immunoglobulin G may therefore play a crucial role
in the host defense against these bacteria
 FcR profile and gene polymorphisms
• The human leukocyte Fcν R family consists of three
major classes,

• It encompasses eight genes (CD64: FcrRIA, IB, and IC; CD32:

FcrRIIA, IIB and IIC; CD16: FcrRIIIA and IIIB), which have been
mapped to the long arm of chromosome 1 (1q21 and
1q23–24)
 Association of FcR polymorphisms with
periodontitis risk

• Wilson and Kalmar speculated that FcrRIIA-R/R131

subjects were more susceptible to periodontitis as a
result of the diminished capacity to phagocytose
immunoglobulin G2-opsonized periodontopathic
bacteria.
• It has been well documented that FcrR genes are
associated with risk for various types of
periodontitis: aggressive periodontitis, chronic
periodontitis, and recurrent chronic periodontitis
 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

• The human leukocyte antigen region is mainly divided into

two classesMHC class I molecules (human leukocyte antigen-
A, -B, and -C) are expressed on most nucleated cells
• MHC class II molecules (HLA-DP, -DQ, -DR) are
expressed on B- and T lymphocytes, and on
macrophages.

• Shapira et al. found that human leukocyte antigen-A9

and -B15 antigens were elevated in patients with
aggressive periodontitis as compared with healthy
controls.
• Takashiba et al. indicated a significant association
of the human leukocyte antigen-DQa gene with
susceptibility to aggressive periodontitis in the
Japanese.

• Hodge et al. reported no association between the

human leukocyte antigen-DQa gene and aggressive
periodontitis in Caucasians.
Conclusion And Future Developments

• With increasing knowledge of major and modifying

disease genes it is conceivable that no. of gene tests
will diagnose the degree of genetic predisposition at
an early age when periodontitis has not yet
developed
• Depending on degree of predisposition of a child the
appropriate preventiobn measure can be taken.

• However the use of genetic tests only makes sense if

enough majors and modifying disease genes have
been identified.

• In addition before genetic test can be used data of

the test for various ethnic groups/races should be
available.
 PROTOCOL
• Currently there are several reports on distribution of IL
composite genotypes in different ethnic and racial groups
such as Europeans, Africans, Americans, Chinese, Greek,
and Thai.
• Every population differs in its ethnic make up and
genetic association found in one population need not
hold true in populations with different ethnic
backgrounds. There are no reports on distribution of
IL gene polymorphism from South Indian population
which is the motive of study.
Aim:

• To study distribution of IL gene polymorphism

in study population.

• To determine association between gene

polymorphism and periodontal disease
severity.
• Objective: -
• To determine association of genotype status
of individuals with different variables such as
age, gender, plaque index, GBI, CAL, tooth
mobility with severity of chronic periodontitis
• Materials and methods:
• Study population: - Total no. 100
Sample from OPD of
GDC, Calicut.
• Subject belonging to South Indian ethnicity
only
• Study design: - Case Control Design
• Cases: - Chronic periodontitis: -
1) mild
2) moderate
3) severe
4) Aggressive periodontitis
• Control: - Healthy population
• Inclusion criteria

• 1) All ages

• 2) Both genders

• 3) Good generalised medical health

• 4) Chronic periodontitis or healthy

• Exclusion criteria

• Systemic disease

• Bleeding disorder

• Immunosuppressive disease

• Tobacco users

• Pregnant females
• Armamentarium:-
• Mouth mirror
• William’s periodontal probe
• Method
• Patients with chronic periodontitis identify and classify.
• Evaluation of periodontal parameters:-
• Plaque index
• Gingival index
• PPD
• CAL
• Mobility
• PCR technique for identification of gene polymorphism
 REVIEW OF LITERATURE

• Moreina PR et al 2007 studied 163 Brazilian individuals and

concluded that there is association of IL-1A -889
polymorphism with chronic periodontitis in Brazilian
individuals.

• Armitage GC et al 2000 studied Chinies population and

concluded that IL-1 and 1B is dramatically lower in Chinies
population with adult periodontitis.
• Scott R. Diehl et al 1999 concluded EOP as a
complex, oligogenic disorder, with IL-1 genetic
variation contributing an important but not
exclusive influence on disease risk.
• Gore EA et al 1998showed that the increased frequency
of the IL-1β 3953 1/2 and 2/2 genotypes in patients with
advanced periodontitis compared to those of the early
and moderate stages suggests that allele 2, previously
correlated with increased IL-lβ production, may
predispose an individual to increased severity of
periodontal disease.