DEPARTMENT OF ORTHODONTICS

AND DENTOFACIAL ORTHOPAEDICS

GENETICS
 CONTENT
• DNA

C
• Gene
• Regulation of Gene Expression – Transcription factor
• Mutation
• Mendelian Genetics
• Modes of Inheritance
• Twin Studies
• Methods of Transmission of Malocclusion
• Molecular Approach to Growth
• Growth Factors
• Trilaminar Germ Disk
• Neural Crest Cells
• The Role of Homeobox Genes
• Craniofacial Development and defects
• Future of Molecular Research in Craniofacial Growth
Presentation title 2
 Introduction
Genetics
The branch of biology which deals primarily with the
principles of heredity & variation, & secondarily with the role
of environmental factors as they interact with genes in the
development of an individual.

Heredity
It can be defined as that force of nature which permits the
transmission of characteristics of a species from generation
to generation

3
 Common terminologies used
in Genetics
Recessive – an allele
exhibited only when Alleles– Alternate form of a
homozygous gene
Dominant – an allele
that completely DNA- Deoxyribonucleic acid; the
masks of the presence molecule that carries genetic
of the other information
Locus – A specific Chromosome – A linear or circular
Common terms strand composed of DNA that
location on a
chromosome contains genes
Homozygote- An indivual Autosomal Chromosome – A nonsex
with two identical alleles chromosome
of a given gene or locus Gene – The fundamental unit of heredity;
Heterozygote- An individual with two A specific section of DNA within a
different alleles of a given gene or chromosome
locus Genotype- The genetic makeup of an
individual; the allele possessed at a given
locus
Presentation title 4
Presentation title 5
 Transcription
• Genetic transcription results in the formation of
messenger RNA (mRNA) from the DNA.

• The nucleotide sequence in the mRNA is complementary to

the DNA.

• RNA polymerase is the enzyme catalyzing genetic

transcription.

• RNA polymerase binds to the promoter area of the gene to

initiate transcription. It reads the DNA template in the 3' to
5' direction and synthesizes RNA in the 5' to 3' direction. 6
 • The mRNA produced by transcription is known as
the primary transcript.

• It undergoes splicing of the introns (non coding

regions) before being exported to the cytoplasm.
This mechanism is known as the post-
transcriptional modification.

• A gene can generate different transcripts which

code for different proteins by a method called
alternate splicing.
Presentation title 7
 Translation
• Translation is carried out by cell organelles, the
ribosomes, which are large complexes of RNA and
proteins, responsible for carrying out the chemical
reactions to add new amino acids to a growing
polypeptide chain by the formation of peptide
bonds.

• They use mature mRNA molecule as a template for

synthesizing new proteins.

Presentation title 8
 • Specialized RNA molecules called transfer RNA
(tRNA) is involved in translation.

• Each tRNA has three unpaired bases known as the

anticodon that are complementary to the codon it
reads on the mRNA.

• The tRNA is also attached to the amino acid

specified by the complementary codon

Presentation title 9
 • When the tRNA binds to its complementary codon in a
mRNA strand, the ribosome ligates its amino acid to the
new polypeptide chain, thus elongating it.

• The polypeptide chain is synthesized from the amino

terminus to the carboxyl terminus.

• During and after its synthesis, the new protein achieves a

three-dimensional structure to obtain functional
competency.

Presentation title 10
REGULATION OF GENE EXPRESSION
 • The process by which the inheritable information in
a gene, such as the DNA sequence, is made into a
functional gene product, such as protein or RNA is
known as gene expression.

• Gene regulation refers to the cellular control of

the amount and timing of changes in the
appearance of the functional product of gene.

Presentation title 12
 • The majority of the known mechanisms regulate
the expression of protein coding genes instead of
the RNA coding regions.

• Any step of the gene's expression may be

modulated, from DNA structure to transcription to
the post-translational modification of a protein.

Presentation title 13
 Stages where gene expression is
regulated are as follows:

• Chemical and structural modification of DNA or chromatin

• Transcription
• Translation
• Post transcriptional modification
• RNA transport
• mRNA degradation
• Post translational modifications.

Presentation title 14
 • Gene regulation is the basis for cellular
differentiation, morphogenesis and the versatility
and adaptability of any organism.

• It gives the cell control over its structure and

function.

• Proteins involved in regulating gene expression

are called regulatory proteins.

Presentation title 15
 • Regulatory proteins are of two types,
• activators
• repressors.

Activators bind to switch a gene on and repressors bind

to shut off a gene.

• Generally, as the organism grows more

sophisticated, their cellular protein regulation
becomes more complicated and indeed, some
human genes can be controlled by many
activators and repressors working together.
Presentation title 16
 Inducible systems
• An inducible system is off, unless there is the
presence of some molecule (called an inducer)
which allows for gene expression.

• The molecule is said to "induce expression".

Presentation title 17
 Repressible systems
• A repressible system is one which except in the
presence of some molecule (called a core pressor),
suppresses gene expression.

Presentation title 18
 Transcription Factor
• A transcription factor is a protein that binds to specific
parts of DNA using DNA binding domains and is part of the
system that controls the transfer of genetic information
from DNA to RNA.

• Transcription factors do not need to perform this function

alone, they may use other proteins in a complex, by
increasing (as an activator), or preventing (as a repressor)
the presence of RNA polymerase, the enzyme which
activates the transcription of genetic information from
DNA to RNA.
Presentation title 19
 UP-REGULATION
• The process which occurs within a cell, triggered by a
signal originating internal or external to the cell, which
results in increased expression of one or more genes and
as a result, the proteins encoded by those genes, is called
up-regulation.

• For example, when a cell is deficient in some kind of

receptor. In this case, more receptor protein is synthesized
and transported to the membrane of the cell and thus, the
sensitivity of the cell is brought back to normal, re-
establishing homeostasis.
Presentation title 20
 DOWN-REGULATION
• Down-regulation is a process resulting in decreased gene
and corresponding protein expression.

• For example, when a cell is overly stimulated by a

neurotransmitter, hormone, or drug for a prolonged period
of time and the expression of the receptor protein is
decreased in order to protect the cell.

Presentation title 21
 MUTATION
• Alterations in the base sequence of a particular gene arise
from a number of sources of which the more important are
the errors in DNA replication and the aftermath of DNA
damage.

• These errors are very rare and are called mutations.

• Some mutations in protein-coding genes are silent, or

produce no change in the amino acid sequence of the
protein for which they code; for example, the codons UCU
and UUC code for serine, so the U↔C mutation has no effect
on the protein.
Presentation title 22
 • Mutations that do have phenotypic effects are most often
neutral or deleterious to the organism.

• Sometimes, mutations confer benefits to the organism's

fitness.

• Mutations propagated to the next generation lead to

variations within a species' population.

• Variants of a single gene are known as alleles, and

differences in alleles may give rise to differences in traits.

Presentation title 23
 the mutant allele will
If mutation appear in a gamete and,
occurs during consequently, in the cells
gametogenesis throughout the body of
any resulting individual.

If mutation only a proportion of

occurs after the cells will be
fertilization affected.

Presentation title 24
 • LENGTH MUTATIONS - Gain or loss of genetic material.

1. Deletions
2. Insertions
3. Rearrangements

• POINT MUTATIONS - Alteration of the genetic code,

but no gain or loss of genetic material.

1. Missense mutations
2. Nonsense mutations
3. Frameshift mutations
4. Silent mutations
Presentation title 25
 Different Types of Mutations

Presentation title 26
Missense mutations
• These point mutations involve the alteration of a single base which changes a codon such that the
encoded amino acid is altered.
• The effect of a missense mutation on the organism varies. Most proteins will tolerate some
change in their amino acid sequence.
• However, alterations of amino acids in parts of the protein that are important for structure or
function are more likely to have a deleterious effect and to produce a mutant phenotype.

Nonsense mutations
• These are point mutations that change a codon for an amino acid into a termination codon.
• The mutation causes translation of the messenger RNA to end prematurely resulting in a
shortened protein which lacks part of its carboxyl-terminal region.
• Nonsense mutations usually have a serious effect on the activity of the encoded protein and often
produce a mutant phenotype.
27
Frameshift mutations
• These result from the insertion of extra bases or the deletion of existing bases from the DNA sequence of a
gene.
• If the number of bases inserted or deleted is not a multiple of three the reading frame will be altered and the
ribosome will read a different set of codons downstream of the mutation substantially altering the amino acid
sequence of the encoded protein.
• Frameshift mutations usually have a serious effect on the encoded protein and are associated with mutant
phenotypes.

Silent mutations
• Mutations may occur at the third base of a codon and, due to the degeneracy of the genetic code, the amino
acid will not be altered.
• Silent mutations have no effect on the encoded protein and do not result in a mutant phenotype.
• They tend to accumulate in the DNA of organisms where they are known as polymorphisms.
• They contribute to variability in the DNA sequence of individuals of a species. 28
Presentation title 29
 Gene Targeting
• Gene targeting commonly refers to the techniques used
for altering or disrupting mouse genes and provides the
mouse models for studying the roles of individual genes in
embryonic development, human disorders, aging and
diseases.

• The mouse models, in whom one or more of their genes

were deactivated or made inoperable, are called
knockout mice.

Presentation title 30
“
MENDELIAN GENETICS ”
 • The existence of genes was first suggested by Gregor
Mendel.

• He studied inheritance in pea plants and arrived at a

conclusion that traits are carried from the parent to the
offspring by specific mechanisms.

• According to the theory of Mendelian inheritance,

variations in phenotype—the observable physical
and behavioral characteristics of an organism—are
due to variations in genotype, or the organism's
particular set of genes, each of which specifies a
particular trait.
Presentation title 32
 These are pairs of chromosomes in the nucleus of a body
cell.
The chromosomes in a pair carry the same genes in the
same place
What are Different versions of the same gene are called alleles.
For e.g. the gene for eye colour has an allele for blue eyes
alleles? and an allele for brown eye colour.
For any gene, a person may have the same two alleles or
two different ones.

ALLELE
S

2 types Dominant allele : always expressed even

of alleles if the individual has one copy of it

Recessive allele: will only be expressed

if the individual has two copies of it.
Presentation title 33
 MENDEL’S LAWS

Law of
Law of Law of Independen
Dominanc Segregatio t
e n Assortment

A dominant allele
completely masks of a In diploid organisms,
chromosome pairs are Alleles on different
recessive allele. It chromosomes are
produces the same separated into individual
gametes(eggs or sperm) distributed randomly to
phenotype in individual gametes.
heterozygotes and in to transmit genetic
homozygotes. information to offspring

Presentation title 34
 Twin Studies
• The relative contribution of genes and the environment to the
etiology of malocclusion has been a matter of controversy.
• Twin studies provide important insights into how genetic and
environmental factors contribute to variation in dental and
craniofacial morphology.
• Began in 1870s when SIR FRANCIS GALTON (1822-1911)
published a series of articles arguing that heredity (nature)
was a stronger factor than the environment (nurture) in
determining the respective characteristics of the twins
 Why to Study Twins?
• Twin studies are one of a family of designs in population
genetics which aid the study of individual differences by
highlighting the role of environmental and genetic causes
on different traits.

• If we observe that children in a family are more similar

than might be expected by chance, this may reflect shared
environmental influences common to members of family
class, parenting styles, education, etc. but they will also
reflect shared genes, inherited from parents.
TWINS
For single gene
trait or For discontinuous
chromosomal multifactorial trait
disorder
Monozygotic Monozygotic
concordance concordance
rate = 100% rate =<100%

Dizygotic
concordance Dizygotic
rate = concordance
<100% rate = less
than that of
monozygotic
concordance
 Classical Twin Research Design

Involves comparison of

Monozygotic (MZ) or Dizygotic (DZ) or

identical twin pairs fraternal twin pairs

Quantify the relative contributions of genetic (nature) and environmental (nurture)

factors to variation in many human physical and behavioral features and disorders.
 Zygosity determination
• It aims toward investigating, if the twins are dizygotic (arising from two
zygotes) or monozygotic (arising from a single zygote),
Methods

Comparisons of
physical
features, for Blood groups
Questionnaire Highly
example body and enzyme
method (94% polymorphic
build, facial polymorphism
accuracy) DNA markers
appearance, s
eye color and
ear.
Different Types of Twin Designs
The MZ Co-twin Design

Twins Reared Apart

Opposite-sexed DZ
Twins
The Twin Half-sib Mode
 The MZ Co-twin Design
• Involves studying MZ twin pairs who show different
phenotypic expressions for a particular trait or disease
under investigation.
• Researchers can manipulate the environment so that
each member of a twin pair is exposed to different
environmental conditions.
• Monozygotic twin pairs are matched perfectly for age and
sex and, as they share the same genes except in very
rare circumstances, any differences between them will
normally reflect environmental effects.
Duffy DL. The co-twin control design. In: Spector TD, Sneider H, MacGregor AJ, eds. Advances in twin and sib-pair analysis.
London: Greenwich Medical Media Ltd, 2000:53-66.
• Martin et al. have pointed out, it is often
assumed that these environmental factors are
exogenous to the individual, e.g., related to
lifestyle, but a variety of endogenous events
leading to alterations in cellular development and
differentiation, e.g., from differences in the timing
or degree of methylation of autosomal genes, may
also lead to differences in phenotypic expression
between MZ twin pairs.
Examples:
Variation in expression of missing, peg-shaped, diminutive
and normally-formed upper lateral incisors within pairs of MZ
twins.
• These associated features conform to a multi-factorial threshold
model linking tooth size with presence and absence of teeth.
• Several examples have been reported in Australian MZ twin pairs
where one member of the pair displays agenesis of one or more
upper lateral incisors, whereas the other shows diminutive or peg-
shaped teeth.
Varying expression in the number of supernumerary teeth
Townsend G, Rogers J, Richards L, Brown T. Agenesis of permanent maxillary lateral incisors in South Australian twins.
 Twins Reared Apart
• Studies of Tom Bouchard and his colleagues in
Minnesota involved examination of MZ twin pairs who
were separated at birth and then reared apart in different
surroundings.
• This model overcomes one of the often mentioned
limitations of traditional twin studies – that MZ twin
pairs may be more similar than DZ pairs for
particular features, not because they share more genes,
but because they share more of the environmental factors
that influence the trait in question.

Bouchard TJ. Whenever the twain shall meet. The Sciences

• Over 80 pairs of MZ twins who were reared apart from
birth are enrolled in Bouchard’s study that addresses an
array of behavioral and biomedical features.
• Studies of MZ twin pairs reared apart have confirmed the
important role played by genes in both dental morphology
and caries experience.

Boraas JC, Messer LB, Till MJ. A genetic contribution to dental caries, occlusion, and morphology as demonstrated by twins
reared apart. J Dent Res 1988;67:1150-1155.
 Opposite-sexed DZ twins
• If zygosity and sex are considered, there are five main
groups of twin pairs that may be studied :-

MZ DZ
MZ male
female female
pair
pair pair
DZ male-
DZ male
female
pair
pair
• Comparison of correlations between same-sex DZ pairs
and opposite-sexed DZ pairs provide an opportunity to
investigate whether primary or secondary sexual
characteristics are affected by diffusion of hormones in
utero.

• There is indirect evidence that diffusion of hormones may

occur in humans via the maternal circulation.
 The Twin Half-sib Model
• This model involves making comparisons between the
offspring of MZ pairs and their partners.
• Potter emphasized the potential value of the twin half-sib
approach and indicated that it could be applied not only
for partitioning shared genetic and environmental risks
within families, but also for determining the involvement
of maternal effects or assortative mating.
• The advantage of this model is that the children of MZ
pairs who are born to different mothers are themselves
genetically half-siblings.
Potter RH. Twin half-sibs: a research design for genetic epidemiology of common dental disorders. J Dent Res
• Because MZ twin pairs have identical genotypes, their
children will share, on average, half their genes whether
they have the same twin as a parent or not.

• Monozygotic pairs, their spouses and their offspring share

different levels of genetic relationship, ranging from zero
for the spouses of twins in the absence of assortative
mating, to unity for the twins themselves.
 The Pioneer
• Galton in 1889 was the pioneer and first to discuss the merits of
twin investigations.
• Comparisons of the between twin pair differences yielded
relative assessments of the influence of heredity and
environment, if the twin pairs were raised in relatively identical
environments.

Galton Francis; Natural Inheritance, London (1st

Heritability of
Dentofacial
Phenotypes
 Class II Malocclusion
Harris (1975)
cephalometric studies to determine the heritability of
certain craniofacial parameters in class II division 1
malocclusion.

His investigation have shown that in class II patients, the

mandible is significantly more retruded than in class I patients,
with the length of the body of the mandible and the overall
mandibular length reduced.

higher correlation between the patient and his immediate family

than data from random pairings of unrelated siblings, thus
supporting the concept of polygenic inheritance for class II
division 1 malocclusion.
 Markovic (1992])

Cephalometric study of 114 class II division 2

malocclusions, 48 twin pairs and six sets of triplets.

Concluded that Of the monozygotic twin pairs, 100

percent demonstrated concordance for the class II
division 2 malocclusion whilst almost 90 percent of the
dizygotic twin pairs were discordant.

This is strong evidence for the fact that genetics

is the main etiological factor in the development
of class II division 2 malocclusion.
 Class III Malocclusion
• The most significant example for hereditary
transmission of class III malocclusion is the
Hapsburgs jaw.

• Strohmayer concluded from his detailed pedigree

analysis of the Hapsburg Royal family (genetic
trait of large lower jaw passing through several
generations in the family), that the mandibular
prognathism was transmitted as an autosomal
dominant trait.
57
 Studied 1362 persons from 243 Japanese families and
noted that, while the index cases had mandibular
196 Suzuki prognathism; there was a significantly higher incidence
of this trait in other members of this family (34.4%) in
1 et al comparison to families of individuals with normal
occlusion (7.5%).

Studied mandibular prognathism in Schulze

monozygotic and dizygotic twins. They reported and
196
that the concordance in monozygotic twins was Weise 5
six times higher than among dizygotic twins.

Carried out an analysis of the literature to that date

and also analyzed a group of probands, siblings and
197 Litton parents with class III malocclusion. Both, autosomal
0 et al, dominant and autosomal recessive transmission
were ruled out and there was no association with
gender (male or female).
Presentation title 58
 Watnick, 1972, studied 35 pairs of monozygotic and 35
pairs of dizygotic like-sexed twins using lateral
cephalometry.

• He concluded that the analysis of unit areas with the

craniofacial complex represents local growth sites and revealed
different modes of control within the same bone.

• Certain areas, such as the lingual symphysis, lateral surface of

the ramus and frontal curvature of the mandible are
predominantly under genetic control.

• Other areas, such as the antegonial notch, are predominantly

affected by environmental factors
Presentation title 59
 Nakasima and Nakata, 1986, assessed the
craniofacial morphologic differences between the
parents of class II patients and the parents of class
III patients, as well as parent-offspring correlations,
and the genetic and environmental components of
variation within the craniofacial complex in these
malocclusions.

• The results showed that the parents of class II

patients had a convex profile with a distocclusion
type of denture pattern, while the parents of class
III patients had a concave profile with a
mesiocclusion type of denture pattern
Presentation title 60
 Heritability of Local Occlusal
Variables
• Lundstrom, 1948, studied 50 pairs of monozygotic and
50 pairs of dizygotic twins and concluded that heredity
played a significant role in determining, among other
factors, width and length of the dental arch, crowding and
spacing of the teeth and the degree of overbite

• Hu et al, 1992, also reported familial similarity in dental

arch form and tooth position.

Presentation title 61
 King et al, 1993, initial treatment records of 104
adolescent sibling pairs, all of whom subsequently
received orthodontic treatment, were examined.

• Heritability estimates for occlusal variations such as

rotations, crossbites and displacements were significantly
higher than in a comparable series of adolescents with
naturally good occlusion.

• The explanation offered was that genetically influenced

facial types and growth patterns of the siblings are likely
to respond to environment factors.
Presentation title 62
 Heritability of Tooth Number,
Size, Morphology, Position and
Eruption
• Hypodontia
• Supernumerary teeth
• Abnormal tooth shape
• Submerged primary molars
• Ectopic eruption and Transposition of canines

Presentation title 63
 Hypodontia
• The congenital absence of teeth may be referred to as
hypodontia, when one or several teeth are missing, or
anodontia when there is a complete absence of one or
both dentitions.

• Features include:
• They are more common in permanent than primary dentition.
• Absence of primary teeth associated with absence of permanent
successors.
• They may be associated with other developmental anomalies

Presentation title 64
 The hereditary nature of

Grahnen familial and

hypodontia - concluded that
in children with missing

, 1956 twin studies teeth, up to half of their

siblings or parents also had
missing teeth.

The tooth crown dimensions are

Osborne familial
strongly determined by heredity.
Clinical evidence suggests that

et al, and twin

studies
congenital absence of teeth and
reduction in tooth size, are associated,

1958,
e.g. hypodontia and hypoplasia of
maxillary lateral incisors are frequently
present simultaneously
 Butler's field theory
• As food habits are more defined with change to selective
pressure during chewing there is concomitant reduction in
tooth volume in the respective fields of incisors, premolars
and molars.

• Therefore, hypodontia involving third molars, second

premolars and lateral incisors are more common. This has
been referred to as "Butler's field theory".

Presentation title 66
 METHODS OF TRANSMISSION OF
MALOCCLUSION
• Repetitive: Recurrence of a single dentofacial deviation
within the immediate family and in the progenitors.

• Discontinuous: Recurrence of a tendency for a malocclusal

trait to reappear after few generations. Some generations will
be skipped and the tendency later reappears within family.

• Variable: Occurrence of different, but related, types of

malocclusion within the several generations of the same
family
Presentation title 67
MOLECULAR APPROACH TO
GROWTH
 GROWTH FACTORS
• Growth factors are proteins that bind to the receptors on
the cell surface, with the primary result of activating
cellular proliferation and/or differentiation.

• The effect of a growth factor is mediated through surface

receptors on the target cells by activating intracellular
phosphorylating enzymes, which in turn, induce an
intracellular signaling pathway by aggregation of co-
factors and other proteins which migrate to the nucleus.

69
• Together with other transcription factors they activate a set
of genes, which then exert the specific changes in cellular
activity or phenotype.

• In vivo, the effect of growth factors is regulated through a

complex system of feedback loops, which involve other
growth factors, enzymes and binding proteins

70
 Transforming Growth Factor
(TGF)
• Transforming growth factor is used to describe two classes
of polypeptide growth factors, TGFα and TGFβ.

• The two classes of TGFs are not structurally or genetically

related to one another, and they act through different
receptor mechanism.

• TGFα is upregulated in some human cancers. It is

produced in macrophages, brain cells, and keratinocytes,
and induces epithelial development.
71
• TGFβ exists in three known subtypes in humans, TGFβ1,
TGFβ2, and TGFβ3.

• These are upregulated in some human cancers, and play

crucial roles in tissue regeneration, cell differentiation,
embryonic development, and regulation of the immune
system.

• Isoforms of transforming growth factor-beta (TGF-β1) are

also thought to be involved in the pathogenesis of pre-
eclampsia, TGFβ receptors are single pass serine/threonine
kinase receptors
72
 Fibroblast Growth Factors (FGF)
• Fibroblast growth factors (FGF) are a family of growth
factors involved in angiogenesis, wound healing, and
embryonic development.

• The FGFs are heparin-binding proteins and interactions with

cell-surface associated heparin sulfate proteoglycans have
been shown to be essential for FGF signal transduction.

• FGFs are keyplayers in the processes of proliferation and

differentiation of cells, particularly endothelial cells; they
(especially FGF-1) promote angiogenesis.
73
• In humans, 23 members of the FGF family have been
identified, all of which are structurally related signaling
molecules.

• The fibroblast growth factor receptor family consists of 4

members, FGFR1, FGFR2, FGFR3, and FGFR4.

• One of the most important functions of aFGF (FGF-1)

and bFGF (FGF-2) is the promotion of endothelial cell
proliferation and the physical organization of endothelial
cells into tube-like structures.

74
• It thus, promotes angiogenesis, the growth of new blood vessels
from the pre-existing vasculature.

• aFGF is a more potent angiogenic factor than VEGF (vascular

endothelial growth factor) or PDGF (platelet-derived growth
factor).

• As well as stimulating blood vessel growth, aFGF and bFGF are

important players in wound healing.

• They stimulate angiogenesis and the proliferation of fibroblasts

that give rise to granulation tissue, which fills up a wound
space/cavity early in the wound healing process.
75
• Fibroblast growth factors are associated with many
developmental processes including mesoderm induction,
anteroposterior patterning, neural induction, angiogenesis,
axon extension and limb formation.

• FGFs are crucial for the normal development of both

vertebrates and invertebrates and any irregularities in
their function leads to a range of developmental defects.

• Craniosynostosis syndromes have been shown to

result from mutations in FGFR1, FGFR2 and FGFR3.
Sometimes, the same mutation can cause two or
more different craniosynostosis syndromes.
76
 Bone Morphogenetic Proteins
(BMPs)
• Bone Morphogenetic Proteins (BMPs) are a group of growth
factors and cytokines known for their ability to induce the
formation of bone and cartilage.

• Originally, seven such proteins were discovered. Of these,

six of them (BMP2 through BMP7) belong to the
Transforming growth factor beta superfamily of
proteins.

• Since then, thirteen more BMPs have been discovered,

bringing the total to twenty.
77
• BMPs interact with specific receptors on the cell surface, referred to as
bone morphogenetic protein receptors (BMPRs).

• They have an important role during embryonic development on the

embryonic patterning and early skeletal formation.

• As such, disruption of BMP signaling can affect the body plan of the
developing embryo. For example, BMP4 and its inhibitors noggin and
chordin help in regulating the polarity of the embryo.

• Mutations in BMPs and their inhibitors (such as sclerostin) are

associated with a number of human disorders which affect the skeleton.

• Several BMPs are also named 'cartilage-derived morphogenetic

proteins' (CDMPs), while others are referred to as 'growth
differentiation factors' (GDFs).
78
 TRILAMINAR GERM DISK
• Development begins with fertilization, the process by
which male gamete, the sperm, and the female gamete,
the oocyte, unite to give rise to the zygote.

• Establishment of body axes take place during the third

week of development (trilaminar germ disc stage).

• The three germ layers in the trilaminar disk, namely

ectoderm, endoderm and mesoderm are formed by a
process called gastrulation.
79
• The cells at the anterior margin of the embryonic disk are
called the anterior visceral endoderm. The cells at this
region express genes OTX2, L1M1, and HESX1 and they
signal the anteroposterior axis even before
gastrulation.

• Gastrulation begins with the formation of primitive streak.

Primitive streak is initiated and maintained by nodal which is
a member of TGF-β family.

• BMP-4 is secreted throughout the embryo at this stage.

Ventralisation of the mesoderm to form kidneys, blood and
body wall take place under the influence of BMP-4 in the
presence of FGF.
80
• Chordin, noggin and follistatin are expressed in the
primitive node. They oppose the activity of BMP-4 and
dorsalise the cranial mesoderm to form notochord,
somites and somitomeres. Inhibition of BMP-4 is facilitated
by Goosecoid.

• FGF-8 expresses nodal in the left side of the embryo and is

stimulator for the left and right sidedness. Nodal
upregulates PITX2 which is responsible for the left
sidedness.

81
 THE ROLE OF HOMEOBOX GENES
• A homeobox is a DNA sequence found within genes which
are involved in the regulation of development
(morphogenesis) of animals, fungi and plants.

• Genes that have a homeobox are called homeobox

genes and form the homeobox gene family.

• Homeobox genes are highly conserved throughout the

evolution of diverse organisms and are now known to play
a role in patterning the embryonic development.
82
 • These can also be regarded as the master genes of the head
and face controlling patterning, induction, programmed
cell death, and epithelial mesenchymal interaction during
the development of the craniofacial complex.

• Those of particular interest in the craniofacial development

include the HOX group, MSX1 and MSX2 (muscle segment), DLX
(distalless), OTX (orthodontical), GSC (goosecoid), and SHH
(sonic hedgehog).

• There are four HOX gene copies in humans, existing as HOXA,

HOXB, HOXC and HOXD. Each copy lies on a separate
chromosome and the genes in each copy can be numbered
from 1 to 13.
Presentation title 83
 • Homeobox genes have the following properties:
• Encode transcription factors.
• Each has a DNA-binding homeodomain.
• Act in sequential zones of the embryo in the same order that they
occur on the chromosome.

• Homeobox genes encode proteins named transcription

factors which control the transcription of RNA from the
DNA template within the cell nucleus.

• Transcription factors can turn genes on and off by

activating or repressing gene expression.

Presentation title 84
 • They, therefore, control other genes producing a co-
ordinated cascade of molecular events which, in turn,
control patterning and morphogenesis.

• At the cellular level this control is expressed through two

main groups of regulatory proteins, the growth factor
family steroid/ thyroid/retinoic acid super family.

Presentation title 85
 • These regulatory molecules in the mesenchyme such as
fibroblast growth factor (FGF), epidermal growth factor
(EGF), transforming growth factor alpha (TGFα),
transforming growth factor beta (TGFβ), and bone
morphogenetic proteins (BMPs) are the vehicles through
which the homeobox gene information is expressed in the
co-ordination of cell migration and subsequent cell
interactions that regulate growth.

• From this, it can be infered that the different parts of the

DNA are activated in different cells, regulating the
different proteins, enzymes, etc. produced by different
tissues and organs.

Presentation title 86
 • Patterning of the pharyngeal arches, except the first arch,
is regulated by the HOX genes carried in the neural crest
cells.

• HOX gene expression in the hind brain take place in

overlapping patterns. These expression patterns
determine organization of cranial ganglia and nerves.

• Crest cells express HOX gene from the segment of origin

but the maintenance of this expression is dependent upon
the interaction with the cells of mesoderm in the
respective pharyngeal arches.

Presentation title 87
 • Sonic hedgehog (SHH) has been shown to regulate HOX
gene expression in the arches, as is the case with retinoic
acid.

• Retinoic acid regulates HOX genes through retinoic acid

response elements (RARE) in a concentration dependent
manner.

Presentation title 88
 Patterning of the Dentition
• The hindbrain region of the developing neural tube from which the
neural crest migrates is segmented into eight rhombomeres.
Segment specific combinatorial HOX gene expression specifies
each rhombomeres identity.

• The migrating neural crest carries this HOX code defined patterning
which is transferred to the branchial arches. The HOX code, thus
sets up regional diversity within the branchial arch system.

• It is plausible therefore, that the HOX code of those cells migrating

to the tooth forming regions is responsible for specifying and
patterning the dentition.
Presentation title 89
 • However, the genes are not expressed in region rostral to
rhombomeres, which means that no HOX gene expression
is seen in the neural crest which migrates to the
craniofacial region, including the first branchial arch.

• In terms of patterning tooth development, we have to look

at a subfamily of homeobox genes that show temporal and
spatial patterns of expression within the first branchial
arch.

Presentation title 90
 Odontogenic Homeobox Code
• Based upon such highly specific domains of expression, it
has been suggested that these odontogenic homeobox
genes provide a homeobox code that specific regions of
the developing jaws to assume odontogenic potential.

• Various odontogenic homeobox genes identified were:

• MSX genes → MSX-1, MSX-2
• DLX genes → DLX-1, DLX-2
• BARX genes → BARX-1, BARX-2

Presentation title 91
 • Each specific region of the homeodomain expresses a
unique combination of homeobox genes, which monitor
the development of specific teeth.

• The molecular basis of this patterning is the differential

expression of the coded homeobox nuclear proteins which
regulate downstream gene transcription.

• The proteins of this homeodomain act as transcription

factor which that result in the activation or inhibition of
other genes. These homeobox genes also regulate the
expression of other target genes.

Presentation title 92
 MSX Genes
• MSX is an important gene involved in tooth formation. MSX
stands for muscle segment homeobox gene.

• Mutation of this gene has been associated with facial and

dental abnormalities.

• MSX-1 gene is expressed in migrating neural crest cells and

later, in the mesenchymal cells of dental papilla and follicle.

• MSX-2 genes are involved in signaling interactions, which are

essential for the tooth development.
Presentation title 93
 • Prior to the initiation of odontogenesis both MSX1 and
MSX-2 exhibit very specific horseshoe-shaped fields of the
corresponding mesenchymal expression in the anterior
regions of the first arch.

• These expression patterns are coincident, except along

their posterior border where the expression of MSX-1
extends further than MSX-2.

• This region of isolated mesenchymal MSX1 expression

corresponds to the position of the future primary epithelial
thickening
Presentation title 94
 • As tooth development progresses, the expression of MSX-1
becomes localized in the mesenchymal cells of the dental
follicle and papilla.

• The domains of expression of MSX-2 also become more

restricted to the dental follicle and papilla, but unlike MSX-
1, MSX-2, is also expressed strongly in the enamel organ.

Presentation title 95
 DLX Genes
• DLX genes are expressed in the migrating neural crest
cells and in the first brachial arch.

• DLX stands for distalless homeobox gene. The DLX

genes have also been conserved during evolution and bear
similarity to the distal-less gene of Drosophila
melanogaster.

• The expression of DLX-1 and DLX-2 in the maxillary and

the mandibular arch mesenchyme is restricted to the
proximal regions where the future molar teeth will develop
Presentation title 96
 BARX Genes
• BARX stands for Bar class Homeobox gene which includes
BARX-1 and BARX-2.

• BARX-1 is homeobox containing transcription factor which

exhibits regionalized expression within the
ectomesenchyme of the first branchial arch.

• Bar class homeobox 2 genes (BARX-2) are also a group of

homeodomain transcription factors. This group of
Homeobox genes was first located in Drosophila
melanogaster in the locus 11q25
Presentation title 97
 • Prior to the appearance of the primary epithelial thickening
BARX-1 (along with DLX-2) is expressed in the posterior
regions of the first branchial arch mesenchyme, the region of
future molar development.

• There is no BARX1 expression in the anterior regions.

• As tooth development proceeds, BARX-1 expression becomes

localized exclusively to the mesenchymal regions around the
developing molars.

• Mutation of these genes could be associated with facial and

dental anomalies.
Presentation title 98
 PAX Genes
• Paired-box homeotic gene (PAX) function by binding
enhancer DNA sequences and they modify the transcriptional
activity of downstream genes.

• There are nine PAX genes organized into four groups (Pax1 to
Pax9).

• Of these genes, Pax9 is associated with the development of

teeth. Mutations in this gene results in conditions such as
hypodontia, transposition, etc.

• Neubuser et al, found that PAX-9 transcription factor is associated

with the genetic mechanism for tooth displacement anomalies,
such as palatally displaced canines and canine transposition.
Presentation title 99
 Hedgehog Genes
• Sonic hedgehog gene (SHH) is the vertebrate homologue of the
Drosophilia hedgehog gene.

• SHH is expressed in the epithelial thickenings of the tooth

forming regions.

• SHH along with bone morphogenetic protein (BMP-4)

determines the position of future forming tooth germs.

• SHH is necessary for the initiation of tooth development,

epithelial signaling and cuspal morphogenesis.
Presentation title 100
 • The interaction of SHH gene with other target genes like GLI is
also imperative for tooth formation. GLI Zinc transcription
factors are known to act downstream of SHH gene.

• There are three subtypes namely GLI-1, GLI2 and GLI-3, which
play a vital role in tooth development.

• Mutant GLI-2 gene results in the formation of abnormal incisors.

• When GLI-2 and GLI-3 were affected, maxillary incisor

development was absent and sizes of mandibular incisors were
reduced. When GLI-3 alone was affected, there was no damage
in the development of incisors.
Presentation title 101
CRANIOFACIAL DEFECTS
 Cleft Lip and Cleft Palate

Cleft Lip
and Cleft
Palate

Non- Syndromic
Syndromic Sex-linked
syndromic isolated
CLP/CP CP (CPX)
CLP/CP CP

Presentation title 103

 Non-syndromic CLP/CP
• Non-syndromic CLP/CP in humans seems to be etiologically
distinctive and still constitute the majority of all classes of
clefting disorder.

• Various transcription factors and growth factors are

involved in non-syndromic cleft lip/cleft palate where
mutations in these factors results in the disorder

Presentation title 104

 Syndromic CLP
Broadly subdivided into—
• Those that occur as part of characterized Mendelian
disorder (single gene defects)
• Those arising from structural abnormalities of the
chromosomes;
• Syndromes associated with known teratogens
• Those whose causation remains obscure and are therefore,
currently uncharacterized.

Presentation title 105

 • One of the most common human autosomal dominant
disorders associated with CLP is van der Woude syndrome.

• Twin studies revealed that a non-sense mutation in the

interferon regulatory factor-6 (IRF6) gene resulted in van
der Woude syndrome.

• Some of the syndromes associated with CLP are, Pierre

Robin syndrome, CLP-ectodermal dysplasia syndrome
(CLPED-1), ectrodactyly, ectodermal dysplasia, orofacial
cleft (EEC syndrome)

Presentation title 106

 Syndromic CP
• In addition to syndromic CLP, progress has also been made
in elucidating the genetic mechanisms behind several
syndromic causes of isolated CP.

• Some of the syndromes associated with CP are—

• mandibulofacial dysostosis (Treacher Collins syndrome),
• holoprosencephaly,
• type-3 Stickler syndrome.

Presentation title 107

 Sex-linked CP (CPX)
• Philip Stainer and Gudrun Moore found the Sex (X)
chromosome linked form of cleft palate (CPX) and an
associated disorder ankyloglossia can occur due to
mutations in a particular gene T-Box 22.

• T-Box genes are members of a family of transcription

regulators that share a common DNA-binding domain, the
T-Box.

Presentation title 108

 Inheritance Patterns of Cleft Lip and Palate

Monogenic or single gene disorders: Approximately

half of the recognized syndromes associated with cleft lip
and palate are due to single gene disorders with equal
distribution between autosomal dominant and autosomal
recessive.

• Single gene defect may give rise to Mendelian pattern of

inheritance, either of isolated cleft lip (palate) or in
multiple malformations associated with cleft lip with or
without cleft palate

Presentation title 109

 Polygenic or multifactorial inheritance:

• Several genes, each with a relatively small effect, act in

concert with poorly defined environmental triggering
mechanisms leading to the expression of the abnormality.

• Thus, such cases show a slight familial tendency but do

not conform to simple Mendelian inheritance patterns.

Presentation title 110

 Polygenic or multifactorial inheritance:

• Several genes, each with a relatively small effect, act in

concert with poorly defined environmental triggering
mechanisms leading to the expression of the abnormality.

• Thus, such cases show a slight familial tendency but do

not conform to simple Mendelian inheritance patterns.

Presentation title 111

 Familial:
• Fogh-Anderson's family studies showed that siblings of
patient with cleft lip had increased frequency of cleft lip
and cleft palate, but no increased frequency of cleft palate
alone.
• Siblings of patients with cleft palate had increased
frequency of cleft palate, but not CL and CP.

Sex predominance:
• More males are born with cleft lip and cleft palate than
females and more females than males have cleft palate
alone.
Presentation title 112
 Racial incidence:

• The incidence of cleft lip and cleft palate is greatest in the

Mongoloid population, being greater than that in the
Caucasian population, which is in turn greater than in the
Negroid population.

• In contrast, the racial differences for cleft palate alone are

not significant.

Presentation title 113

Craniofacial Syndromes
 Craniofacial Syndromes

• Crouzon’s syndrome
• Apert’s syndrome
• Treacher Collins syndrome
• Pfeiffer syndrome
• DiGeorge syndrome, velocardiofacial syndrome,
conotruncal anomalies face syndrome (CATCH 22
spectrum).

Presentation title 115

 Crouzon’s Syndrome
• It is a frequent form of craniofacial
dysostosis, characterized by multiple
anomalies of the craniofacial skeleton
with an autosomal dominance
inheritance pattern.

• Characteristic premature
synostosis of both coronal sutures
results, with a resultant
brachycephalic shape to the skull,
midface hypoplasia with an Angle's
class III malocclusion, hypoplastic
orbits with a proptosis, Parrot
beak nose and short anterior
cranial base. 116
Genetic etiology: It is caused by multiple mutations in the
fibroblast growth factor receptor2 gene (FGFR2).

• Mutation in tyrosine kinase receptor, at IgII–IgIII domain.

Crouzon’s with acanthosis nigricans has been described with a
specific Ala391Glu mutation in FGFR3.

• More recently, Muenke and co-workers reported that it is due

to an amino acid substitution (Pro250Arg) that results from a
single point mutation in FGFR3 on chromosome 4P.

• This new syndrome called FGFR3 syndrome and the associated

coronal synostosis syndrome may present as bilateral coronal
synostosis with minimal midface involvement.
117
 Apert’s Syndrome
• Apert’s syndrome (also known
as Apert-Crouzon disease) is
characterized by skull
malformation (acrocephaly of
brachysphenocephalic type)
and syndactyly of the hands
and feet of a special type
(complete distal fusion with a
tendency to fusion also of the
bony structures).

• It is associated with an
autosomal dominant
inheritance pattern. 118
Genetic etiology:

• At the molecular level, one of the two fibroblast growth factors

2 gene (FGFR2) mutations involving amino acids (ser 252 trp
and pro 253 Arg) are found to cause Apert’s syndrome.

• Tyrosine kinase receptor is affected at the extracellular IgII–

IgIII domain.

• In Apert’s syndrome, the associated midface hypoplasia is

thought to be secondary to a cartilage maturation defect
affecting the cranial base.
119
 Treacher-Collins Syndrome
• Treacher Collins syndrome, or
mandibulofacial dysostosis, is an
autosomal dominant condition with
variable expressivity.

• It is generally characterized by
bilaterally symmetrical abnormalities of
the structures within the first and
second branchial arches.

• It is characterized by malar hypoplasia,

mandibular hypoplasia, downward
palpebral fissures and coloboma of
lower eye lid and malformed external
ears. 120
 Genetic etiology:

• The gene for Treacher Collins syndrome has been mapped

to chromosome 5q31.3-q33.3.

• The Treacher Collins Syndrome gene resides between the

colony-stimulating factor receptor (CSFR) gene and the
osteorectin (SPARC) gene, a region of less than 1 million
base pairs of DNA on chromosome 5.

Presentation title 121

 Pfeiffer Syndrome
Features of Pfeiffer
syndrome include
• craniostenosis,
• orbital dystopia,
• midface hypoplasia,
• broad and medial
deviated thumbs and
great toes
• partial soft tissue
syndactyly of the hands
and feet.

Presentation title 122

 • Pfeiffer syndrome is said to have an autosomal dominance
inheritance pattern

• Genetic etiology: It is heterogeneous because it is

caused by a single recurring mutation of the FGFR1 gene
and by several different mutations affecting the FGFR2
gene.

Presentation title 123

 DiGeorge Syndrome
• DiGeorge syndrome, velocardiofacial
syndrome and conotruncal anomalies face
syndromes come under disorders in CATCH
22 spectrum.

• CATCH 22 is due to the deletion of long arm

of chromosome 22 (more specifically, foci
22q11).

• Cardiac defects, abnormal facies, thymic

hypoplasia, cleft palate and hypocalcemia
are the clinical features.

• The origin of defects is caused by abnormal

development of neural crest cells which
contribute to the formation of all the
affected structures. 124
Thank
you