GENETICS AND MALOCCLUSION

PART-1: BASIC GENETICS AND ITS SIGNIFICANCE IN MALOCCLUSION

AND CRANIOFACIAL ANOMALIES

CONTENTS
INTRODUCTION
HUMAN CHROMOSOMES
STRUCTURE AND CLASSIFICATION
MORPHOLOGY
CHROMOSOME NOMENCLATURE
METHODS OF CHROMOSOME ANALYSIS
DNA: THE HERIDITARY MATERIAL
TYPES OF DNA SEQUENCES
TECHNIQUES OF DNA ANALYSIS
GENETIC CODE
STRUCTURE OF GENES
DEVELOPMENTAL GENE FAMILIES
HOMEOBOX GENES AND ITS IMPORTANCE
TWIST GENES
GROWTH FACTORS AND ITS SIGNIFICANCE
MUTATION
TYPES OF MUTATIONS
FUNCTIONAL EFFECTS OF MUTATIONS ON THE PROTEIN
MUTATIONAL TRACKING AND MOLECULAR APPROACHES
PATTERNS OF INHERITANCE
AUTOSOMAL INHERITANCE
SEX LINKED INHERITANCE
POLYGENIC AND MULTIFACTORIAL INHERITANCE
SEGREGATION AND LINKAGE ANALYSIS
TWINNING AND SIGNIFICANCE OF TWIN STUDIES
SIGNIFICANCE OF GENETIC STUDIES FOR MALOCCLUSION AND
CRANIOFACIAL ANOMALIES

GENETICS AND MALOCCLUSION

INTRODUCTION

The human genome contains approximately 3 billion nucleotides, making up

about 100,000 alleles, which in turn are contained on 46 chromosomes. Transcription
of these chromosomes releases the information necessary to synthesize some 6000
proteins. These proteins make up the trillion cells giving rise to the nearly 4000
anatomical structures that constitute a single human being. Mutation, the accidental
alteration of the genome, may result in heritable conditions or syndromes affecting
any aspect of growth and development.
Several questions need to be answered before a complete understanding can be gained
about how genetic factors influence a feature or disorder. These include:

 How important are genetic effects on human differences?

 What kinds of action and interaction occur between gene products in the
pathways between genotype and phenotype?

 Are the genetic effects on a trait consistent across sexes?

 Are there some genes that have particularly outstanding effects when
compared with others?

 Whereabouts on the human gene map are these genes located?

HUMAN CHROMOSOMES
STRUCTURE AND CLASSIFICATION
In humans the normal cell nucleus contains 46 chromosomes, made up of 22 pairs of
autosomes and a single pair of sex chromosomes XX in the female and XY in the
male. The Y chromosome is much smaller than the X.
Each chromosome is composed of DNA double helix and the packaging of DNA into
chromosomes involves several orders of DNA coiling and folding. In addition to the
primary coiling of the DNA double helix, there is secondary coiling around spherical
histone beads forming what are called nucleosomes. There is a tertiary coiling of the
nucleosomes to form the chromatin fibres which form long loops on a scaffold of non-
histone acidic proteins, which are further wound in a tight coil to make up the
chromosome as visualized under the light microscope, the whole structure making up
the so-called solenoid model of chromosome structure.

MORPHOLOGY
Each chromosome consists of two identical strands known as chromatids, or sister
chromatids. These sister chromatids are joined at a primary constriction known as the
centromere. Centromeres consist of several hundred kilobases of repetitive DNA and
are responsible for the movement of chromosomes at cell division. Each centromere
divides the chromosome into short and long arms designated p (=petite) and q
(=grande) respectively.

Morphologically chromosomes are divided into,

Metacentric centromere located centrally
Acrocentric centromere located at terminal end
Submetacentric centromere in intermediate position

CHROMOSOME NOMENCLATURE
Each chromosome arm is divided into regions and each region is subdivided into
bands numbering always from the centromere outwards. A given point on a
chromosome is designated by the chromosome number, the arm (p or q), the region
and the band, e.g. 15q12. Sometimes the word region is omitted so that 15q12 would
be referred to simply as band 12 on the long arm of chromosome15.
METHODS OF CHROMOSOME ANALYSIS
CHROMOSOME BANDING
Most commonly circulating lymphocytes from peripheral blood are used for studying
human chromosomes. Several different staining methods can be utilized in identifying
individual chromosomes characterized by light and dark bands under microscope.

KARYOTYPE ANALYSIS
Detailed analysis of the banding pattern of the individual chromosomes is carried out.
The banding pattern of each chromosome is specific and can be shown in the form of
a stylized ideal karyotype known as an idiogram. A formally presented karyotype or
karyogram will show each chromosome pair in descending order of size.

NUCLEOTIDES
Nucleic acid is composed of a long polymer of individual molecules called
nucleotides. Each nucleotide is composed of a nitrogenous base, a sugar molecule and
a phosphate molecule. The nitrogenous bases fall into two types, purines and
pyrimidines. The purines include adenine and guanine; the pyrimidines include
cytosine, thymine and uracil. There are two different types of nucleic acid, ribonucleic
acid (RNA) and deoxyribonucleic acid (DNA). DNA and RNA both contain the
purine bases adenine and guanine and the pyrimidine cytosine but thymine occurs
only in DNA while uracil is only found in RNA.

DNA : THE HERIDITARY MATERIAL

DNA molecules have a very distinct and characteristic three-dimensional structure
known as the double helix. In 1953 the structure of DNA was discovered by Watson
and Crick working in Cambridge using x-ray diffraction pictures taken by Franklin
and Wilkins.
X-ray diffraction pictures of the double helix show repeated patterns of bands
that reflect the regularity of the structure of the DNA. The double helix executes a
turn every 10 base pairs. The pitch of the helix is 34A so the spacing between bases is
3.4A. The diameter of the helix is 20A. The double helix is said to be 3 antiparallel.
One of the strands runs in the 5’3’ direction and the other 3’5’ direction. The
double helix is not absolutely regular and when viewed from the outside a major
groove and a minor groove can be seen. These are important for interaction with
proteins, for replication of the DNA and for expression of the genetic information.

COMPLEMENTARY BASE PAIRING

The bases of the two polynucleotide chains interact with each other. Thymine always
interacts with adenine and guanine with cytosine (i.e. A-T and G-C). The way in
which the bases form pairs between the two DNA strands is known as complementary
base pairing. Complementary base pairing is essential for the expression of genetic
information and is central to the way DNA sequences are transcribed into mRNA and
translated into protein.

TYPES OF DNA SEQUENCES

Analysis of human DNA have shown that approximately 60-70% of the human
genome consists of single or low copy number DNA sequences. The remainder of the
genome, some 30-40% consists of either moderately or highly repetitive DNA
sequences.
NUCLEAR DNA
(A) Nuclear genes
(i) Unique single copy genes
(ii) Multigene families – e.g. the HOX homeobox gene family.
Classical gene families
Gene super families
(B) Extragenic DNA
(i) Tandem repeat
Satellite
Minisatellite
Telomeric
Hypervariable
Microsatellite
(ii) Interspersed
Short interspersed nuclear elements
Long interspersed nuclear elements
MITOCHONDRIAL DNA
Two rRNA genes
22 tRNA genes
13 genes coding for proteins involved in oxidative phosphorylation.

TECHNIQUES OF DNA ANALYSIS

Many methods of DNA analysis involve the use of nucleic acid probes and the
process of nucleic acid hybridization.

NUCLEIC ACID PROBES

Nucleic Acid probes are usually single stranded DNA sequences which have
been radioactively or non-radioactively labeled and can be used to detect DNA or
RNA fragments with sequence homology. DNA probes can come from variety of
sources including random genomic sequences, specific genes, cDNA sequences or
oligonucleotide DNA sequences produced synthetically based on the knowledge of
the protein amino acid sequence. A DNA probe can be labeled by a variety of
processes, including isotopic labeling with 32 P and non-isotopic methods using
modified nucleotides containing fluorophores, e.g.fluorescein of rhodamine.
Hybridization of a radioactively labelled DNA probe with complementary DNA
sequences on a nitrocellulose filter can be detected by autoradiography while DNA
fragments which are fluorescently labelled can be detected by exposure to the
appropriate wavelength of light, e.g. fluorescent in situ hybridization.

NUCLEIC ACID HYBRIDIZATION

Nucleic acid hybridization involves mixing DNA from two sources which have been
denatured by heat or alkali to make them single stranded and then, under the
appropriate conditions, allowing complementary base pairing of homologous
sequences. If one of the DNA sources has been labelled in some way, i.e. is a DNA
probe, this allows identification of specific DNA sequences in the other source.
The two main methods of nucleic acid hybridization most commonly used are
Southern and Northern blotting.
Southern blotting
Southern blotting, named after Edwin Southern who developed the technique,
involves digesting DNA by a restriction enzyme which is then subjected to
electrophoresis on an agarose gel. This separates the DNA or restriction fragments by
size, the smaller fragments migrating faster than the larger ones. The DNA fragments
in the gel are then denatured with alkali, making them single stranded. A ‘permanent’
copy of these single stranded fragments is made by transferring them on to a
nitrocellulose filter which binds the single-stranded DNA, the so called Southern blot.
A particular target DNA fragment of interest from the collection on the filter can be
visualized by adding a single stranded 32 P radioactively labelled DNA probe which
will hybridize with homologous DNA fragments in the Southern blot which can then
be detected by autoradiography.

Northern blotting
Northern blotting differs from southern blotting by the use of mRNA as the target
nucleic acid in the same procedure; mRNA is very unstable because of intrinsic
cellular ribonucleases. Use of ribonuclease inhibitors allows isolation of mRNA
which, if run on an electrophoretic gel, can be transferred to a filter. Hybridizing the
blot with a radiolabelled DNA probe allows determination of the size and quantity of
the mRNA transcript, a so called Northern blot. In a gene disorder in which a
mutation has not been identified in the coding sequences, an alteration in the size of
the mRNA transcript suggests the possibility of a mutation in a non-coding region of
the gene such as the splice junction of the intron-exon border. Northern blotting can
also be used to demonstrate the differential pattern of expression of a gene in different
tissues or at different times of development.

GENETIC CODE
The DNA sequence of a gene is divided into a series of units of three bases. Each set
of three bases is called a codon and specifies a particular amino acid. The four bases
in DNA and RNA can combine as a total of 43 = 64 codons which specify the 20
amino acids found in proteins. Because the number of codons is greater, all of the
amino acids, with the exceptions of methionine and tryptophan, are encoded by more
than one codon. This feature is referred to as the degeneracy or the redundancy of the
genetic code
Codons which specify the same amino acid are called synonyms and tend to be
similar. Variations between synonyms tend to occur at the third position of the codon,
which is known as wobble position. The degeneracy of the genetic code minimizes
the effects of mutations so that alterations to the base sequence are less likely to
change the amino acid encoded and possible deleterious effects on protein function
are avoided. Of the 64 possible codons, 61 encode amino acids. The remaining three,
UAG, UGA, and UAA, do not encode amino acids but instead act as signals for
protein synthesis to stop and as such are known as termination codons or stop codons.
The codon for methionine, AUG, is the signal for protein synthesis to start and is
known as the initiation codon. Thus all polypeptides start with methionine although
this is sometimes removed later.

GENOTYPE AND PHENOTYPE

Consideration of the heritability of a particular feature or trait requires a consideration
of the relationship between genotype and phenotype. Genotype is defined as the
genetic constitution of an individual, and may refer to specified gene loci or to all loci
in general. An individual’s phenotype is the final product of a combination of genetic
and environmental influences. Phenotype may refer to a specified character or to all
the observable characteristics of the individual.

STRUCTURE OF GENES
A gene is a unit of information and corresponds to a discrete segment of DNA
with a base sequence that encodes the amino acid sequence of a polypeptide. Genes
vary greatly in size from less than 100 base pairs to several million base pairs. In
humans there are an estimated 50-100000 genes arranged on 23 chromosomes.
The genes are very dispersed and are separated from each other by sequences that do
not contain genetic information.; this is called intergenic DNA. The intergenic DNA
is very long, such that in humans gene sequences account for less than about 30% of
the total DNA. Only one of the two strands of the DNA double helix carries the
biological information and this is called the template strand or sense or coding strand,
which is used to produce an RNA molecule of complementary sequence which directs
the synthesis of a polypeptide. The other strand is called the nontemplate strand or
antisense or noncoding strand.
Gene promoters
Expression of genes is regulated by a segment of DNA sequence present upstream of
the coding sequence known as the promoter. Conserved DNA sequences in the
promoter are recognized and bound by the RNA polymerase and other associated
proteins called transcription factors that bring about the synthesis of RNA transcript
of the gene.

Introns and Exons

In genes coding information is usually split into a series of segments of DNA
sequence called exons. These are separated by sequences that do not contain useful
information called introns. The length of exons and introns varies but the introns are
usually much longer and account for the majority of the sequence of the gene. Before
the biological information in a gene can be used to synthesize a protein, the introns
must be removed from RNA molecules by a process called splicing which leaves the
exons and the coding information continuous.

DEVELOPMENTAL GENE FAMILIES

1) Segmentation genes
2) Paired-box genes (PAX)
3) Zinc finger genes
4) Signal transduction (‘Signalling’) genes
5) Homeobox genes (HOX)

SEGMENTATION GENES
Insect bodies consist of series of repeated body segments which differentiate
into particular structures according to their position. Three main groups of
segmentation determining genes have been classified on the basis of their mutant
phenotypes.
 (A) Gap mutants – delete groups of adjacent segments
(B) Pair-rule mutants – delete alternate segments
(C) Segment polarity mutants – cause portions of each segment to be
deleted and duplicated on the wrong side.
(i) Hedgehog (Vertebrates)
 Sonic Hedgehog
 Desert Hedgehog
 Indian Hedgehog
(ii) Wingless

Hedgehog morphogens are involved in the control of left-right asymmetry, the

determination of polarity in the central nervous system, somites and limbs, and in
both organogenesis and the formation of the skeleton.
In humans, Sonic hedgehog (SHH) plays a major role in development of the ventral
neural tube with loss-of-function mutations resulting in a serious and often lethal
malformation known as holoprosencephaly where the facial features shows eyes close
together and there is a midline cleft lip due to failure of normal prolabia development.

PAIRED-BOX GENES (PAX)

The mammalian Pax gene family consists of nine members that can be
organized into groups based upon sequence similarity, structural features, and
genomic organization. The four groups include
A) Pax1 and Pax9
B) Pax2, Pax5, and Pax8
C) Pax3 and Pax7 and
D) Pax4 and Pax6

ZINC FINGER GENES

The term zinc finger refers to a finger-like loop projection which is formed by a series
of four amino acids which form a complex with a zinc ion. Genes, which contain a
zinc finger motif, act as transcription factors through binding of the zinc finger to
DNA.

SIGNAL TRANSDUCTION GENES

Signal transduction is the process whereby extracellular growth factors
regulate cell division and differentiation by a complex pathway of genetically
determined intermediate steps. Mutations in many of the genes involved in signal
transduction can cause developmental abnormalities. Fibroblast growth factor
receptors (FGFRs) belong to the category of signal transduction genes.

HOMEOBOX GENES (HOX) AND ITS IMPORTANCE

Since their discovery in 1983, the homeobox genes were originally described as a
conserved helix-turn-helix DNA motif of about 180 base pair sequence, which is
believed to be characteristic of genes involved in spatial pattern control and
development. The protein domain encoded by the homeobox, the homeodomain, is
thus about 60 amino acids long. Proteins from homeobox containing, or what are
known as HOX genes, are therefore important transcription factors which specify cell
fate and establish a regional anterior/posterior axis. The first genes found to encode
homeodomain proteins were Drosophila developmental control genes, in particular
homeotic genes, from which the name "homeo"box was derived. However, many
homeobox genes are not homeotic genes; the homeobox is a sequence motif, while
"homeotic" is a functional description for genes that cause homeotic transformations.

Four homeobox gene clusters (HOXA, HOXB, HOXC, and HOXD) that comprise a
total of 39 genes have been identified in humans. Each cluster contains a series of
closely linked genes. In each HOX cluster there is a direct linear correlation between
the position of the gene and its temporal and spatial expression. These observations
indicate that these genes play a crucial role in early morphogenesis. Lower number
HOX genes are expressed earlier in development and more anteriorly and proximally
than are the higher number genes.

Homeobox gene clusters in humans

Cluster Number of genes Chromosome location

HOXA
11 (1-7, 9-11, 13) 7p
(=HOX1)
HOXB
10 (1-9, 13) 17q
(=HOX2)
HOXC
9 (4-6, 8-13) 12q
(=HOX3)
HOXD
9 (1, 3, 4, 8-13) 2q
(=HOX4)

HOMEODOMAIN
The homeodomain is a DNA-binding domain, and many homeobox genes have now
been shown to bind to DNA and regulate the transcription of other genes. Thus
homeodomain proteins are basically transcription factors, most of which play a role in
development.
The homeodomain is a common DNA-binding structural motif found in many
eukaryotic regulatory proteins. Homeodomain proteins are involved in the
transcriptional control of many developmentally important genes, and 143 human loci
have been linked to various genetic and genomic disorders. X-ray crystallographic
and NMR spectroscopic studies on several members of this family have revealed that
the homeodomain motif is comprised of three α-helices that are folded into a compact
globular structure. Helices-I and II lie parallel to each other and across from the third
helix. This third helix is also referred to as the “recognition helix”, as it confers the
DNA binding specificity if individual homeodomain proteins. The homeodomain has
been evolutionarily conserved at the structural level. This is most evident upon
examination of divergent members of the homeodomain family.

TWIST GENES
Chromosomal rearrangements and linkage analysis have mapped the locus for TWIST,
the human homologue of the Drosophila twist gene, located in chromosome 7p21-
p22. The TWIST gene contains a basic helix-loop-helix (bHLH) motif that suggests
that the TWIST gene product acts as a transcription factor. The HLH region of this
motif is important for homo- or heterodimerization, whereas the basic domain is
essential for binding of the dimer complex to a target DNA binding sequence(s).
TWIST genes were identified in Drosophila and Drosophila TWIST gene affects the
expression of a fibroblast growth factor receptor (FGFR) homologue DFR1. In
humans mutations in TWIST amino acid sequence and FGFRs results in
craniosynostosis.

GROWTH FACTORS
Growth factors constitute an important class of signaling molecules. The effects of
growth factors are always mediated through binding of the factor to specific cell
surface receptors. During embryonic development many growth factors have been
shown to act as signals between tissue layers and they also act as signals during
organogenesis. One mechanism whereby growth factors regulate development is
through stimulation of homeobox genes.

Growth factor families

1) Transforming growth factor- beta (TGF-β)
a) TGF-β 1-5
b) Bone morphogenetic protein (BMP) 2-8
c) Growth and Differentiation factor (GDF) 1-7
2) Epidermal growth factor (EGF)
a) EGF
b) TGF-α
c) Amphiregulin
d) HB-EGF
3) Fibroblast growth factor (FGF)
FGF 1-8
4) Insulin like growth factor (IGF)
IGF 1-2
5) Platelet derived growth factor (PDGF)
PGDF A, B
6) Neurotrophins
a) Nerve growth factor (NGF)
b) Brain-derived neutrotrophic factor (BDNF)
c) Neurotrophin (NT) 3-4

FIBROBLAST GROWTH FACTORS (FGFs) AND ITS RECEPTORS (FGFRs)

FGFs comprise a family of 22 genes encoding structurally related proteins (Ornitz
and Itoh 2001). Six subfamilies of FGFs, grouped by sequence similarities, tend to
share biochemical and functional properties and are expressed in specific spatial and
developmental patterns. Four distinct FGF receptor tyrosine kinase molecules bind
and are activated by most members of the FGF family. Alternative mRNA splicing
produces FGF receptors with unique ligand binding properties. Alternative splicing is
mostly tissue-specific, producing epithelial variants (b splice forms) and
mesenchymal variants (c splice forms). FGF activity and specificity are further
regulated by heparan sulfate oligosaccharides, in the form of heparan sulfate
proteoglycans. Heparin/ Heparan sulfate, FGF, and an FGF receptor (FGFR) associate
to form a trimolecular complex. Heparan chains, themselves, have unique tissue-
specific modifications that are required for, and may actually regulate, functional
ligand–receptor interactions.
The expression of FGFs and FGFRs is temporally and spatially regulated during
craniofacial development. FGFs and FGFRs play an important role in
intramembranous as well as endochondral bone formation. During intramembranous
bone formation, Fgf2, Fgf4, and Fgf9 are expressed in sutural mesenchyme in early
craniofacial skeletogenesis, suggesting that they may be involved in regulating
calvarial osteogenesis. Fgf18 and Fgf20 are also expressed in developing calvarial
bones.
Mutations in FGFs AND FGFRs tend to cause craniosynostosis

MUTATION
A mutation is defined as a heritable alteration or change in the genetic material. A
mutation arising in a somatic cell cannot be transmitted to offspring, whereas if it
occurs in gonadal tissue or a gamete it can be transmitted to future generations.

TYPES OF MUTATIONS
Mutations occur in two forms:
1) Point mutations - involve a change in the base present at any position in a gene
2) Gross mutations - involve alterations of longer stretches of DNA sequence.
The location of the mutation within a gene is important. Only mutations that occur
within the coding region are likely to affect the protein. Mutations in noncoding or
intergenic regions do not usually have an effect.

Point mutations
A) Missense mutations
B) Nonsense mutations
C) Frameshift mutations
D) Silent mutations
Gross mutations
A) Deletions
B) Insertions
C) Rearrangements

POINT MUTATIONS
Missense mutations
These point mutations involve the alteration of a single base which changes a codon
such that the encoded amino acid is altered. Such mutations usually occur in one of
the first two bases of a codon. The redundancy of the genetic code means that
mutation of the third base is likely to cause a change in the amino acid. 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.

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.

GROSS MUTATIONS
Deletions
These involve the loss of a portion of the DNA sequence. The amount lost varies
greatly. Deletions can be as small as a single base or much larger in some cases
corresponding to the entire gene sequence.

Insertions
In this case the mutation occurs as a result of insertion of extra bases, usually from
another part of a chromosome.

Rearrangements
These mutations involve segments of DNA sequence within or outside a gene
exchanging position with each other. A simple example is inversion mutations in
which a portion of the DNA sequence is excised then re-inserted at the same position
but in the opposite orientation.

Gross mutations, because they involve major alterations to gene sequences, invariably
have serious effect on the encoded protein and are frequently associated with a mutant
phenotype.

FUNCTIONAL EFFECTS OF MUTATIONS ON THE PROTEIN

Mutations exert their phenotypic effect in one of two ways, either through loss- or
gain- of function.
Loss-of-function mutations
Loss-of-function mutation can result in either reduced activity or complete loss of the
gene product. The former can be the result of either reduced activity or of decreased
stability of the gene product and is known as a hypomorph, the latter being known as
a null allele or amorph. Loss-of function mutations in the heterozygous state would, at
worst, be associated with half normal levels of the protein product.
Haploinsufficiency
Loss-of function mutations in the heterozygous state in which half normal levels of
the gene product result in phenotypic effects are termed haploinsufficiency mutations.
There are number of autosomal dominant disorders where the mutational basis of the
functional abnormality is the result of haploinsufficiency, in which, homozygous
mutations result in more severe phenotypic effects.

Gain-of-function mutations
Gain-of-function mutations, as the name suggests, result in either increased levels of
gene expression or the development of a new function(s) of the gene product.
Mutations which alter the timing or tissue specificity of the expression of a gene can
also be considered to be gain-of-function mutations. Gain-of-function mutations are
dominantly inherited and the rare instances of gain-of-function mutations occurring in
the homozygous state are associated with a much more severe phenotype, which is
often a prenatally lethal disorder.

MUTATION TRACKING AND MOLECULAR APPROACHES

With marked advances in molecular genetic technology in recent years, gene mapping
techniques are now providing powerful approaches for locating genes associated with
various diseases and disorders.
Functional cloning uses the protein sequence and thereby the putative corresponding
DNA sequence to clone the relevant gene, or by extracting the messenger RNA
(mRNA) from the tissue to produce a complementary DNA (cDNA). This cDNA
corresponds to the DNA sequence of the coding regions (exons) of a gene.
Positional cloning, also known as reverse genetics, is used to identify the location of
the mutant gene on a particular chromosome by virtue of its co segregation with
polymorphic DNA markers. The first generation of these markers was termed
restriction fragment length polymorphisms (RFLPs).

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLPs)

Variation in the nucleotide sequence of the human genome is common, occurring
approximately once every 200bp. RFLPs arise as a result of minor alterations in the
DNA sequence on pairs of chromosomes. The DNA, usually obtained from peripheral
blood leucocytes, is digested with a restriction enzyme, which recognizes particular
DNA sequences and cuts at a certain point in the sequence. The resulting DNA
fragments are then separated in an agarose gel where the distance they migrate
depends upon their size, shorter fragments migrating further than larger fragments
over a given period of time. The DNA is then transferred from the gel to a nylon
membrane (Southern blotting) where it can be probed by markers. The markers are
DNA fragments which have been mapped to parts of chromosomes. Because of the
variation in cutting sites, in an ideal situation the probe will bind to two different sized
fragments of DNA. The probe is labelled using a radioisotope and appears as one or
more bands on an autoradiograph.
The different bands are referred to as alleles, and by following the segregation of
these alleles with the disease, the position of the gene is established. The limitation of
RFLPs is that individuals are frequently homozygous at a given marker, that is, they
have two alleles of the same size. To establish linkage (the position of the diseased
gene in relation to the RFLPs) affected individuals need to be heterozygous, that is,
have two alleles of different sizes. Linkage analysis depends upon having a sufficient
number of meioses, either in one or more large pedigrees or multiple smaller
pedigrees. It is difficult to establish linkage without a number of three-generation (or
more) pedigrees. Linkage also relies upon the fact that, at meiosis, recombination
events occur on the chromosomes. Thus, some individuals will inherit exact copies of
their parents’ chromosomes while others will inherit chromosomes which represent
rearrangements of the original chromosomes. These recombination events are the key
to mapping of a gene.

HYPERVARIABLE TANDEM REPEAT DNA LENGTH POLYMORPHISMS

The different classes of tandemly repeated DNA sequences in the human genome
have been useful clinically in mutation tracking.

VARIABLE NUMBER TANDEM REPEATS (VNTRs)

More recently, a new generation of polymorphic markers has been employed. These
variable number of tandem repeat (VNTR) markers rely upon variations in the
number of repeat sequences in non-coding regions of chromosomes. The VNTRs may
be either dinucleotide repeats (repeats of two DNA bases, usually cytosine and
adenine) or tri-, tetra-, or penta-nucleotide repeats. VNTRs have an advantage over
RFLPs in that the number of repeats is (in theory) infinitely variable and these
markers are more likely to be heterozygous. VNTRs obviate the need for Southern
blotting. They are identified using the polymerase chain reaction (PCR) which uses
primer sequences flanking the variable segment to amplify the DNA using a thermal
cycler. The resulting amplified DNA fragments are then separated by electrophoresis
in a polyacrylamide gel and revealed by autoradiography. Detection systems other
than radioactive systems are now available and some of these processes can be
automated.

Cosegregation of a disease with one or more DNA markers can be confirmed by

statistical analysis. The measures of cosegregation are the LOD score (logarithm of
the odds for linkage as opposed to no linkage) with a value of three being regarded as
significant, this indicating a one-thousand-fold likelihood of linkage. The other
measure is the recombination fraction, which is an indication of the distance from the
marker to the gene. With a high LOD score and a low recombination fraction the
researcher can be fairly certain that the gene responsible for the disease has been
localized.

The next stage is to clone the gene and numerous techniques are available to
accomplish this. If the disease has been localized to a small area of a chromosome, the
current strategy would be to use the markers either side of the disease (flanking
markers) to probe a yeast artificial chromosome (YAC) library and this, in turn, can
be used to screen other libraries containing smaller fragments of DNA such as cosmid
libraries.
Typical YACs are considerably larger than cosmids so this approach enables the
relevant section of DNA to be analysed on a smaller scale. Other techniques that can
be used include identification of the coding parts at the beginning of genes (CpG
islands) and exon trapping. Once the gene has been isolated it can then be sequenced
and the coding regions (exons) and non-coding regions (introns) identified. Following
this, mutations in affected individuals can be identified using techniques such as
single strand chain polymorphisms (SSCP) or direct sequencing.
PATTERNS OF INHERITANCE
An important reason for studying the pattern of inheritance of disorders within
families is to enable advice to be given to members of a family regarding the
likelihood of their developing it or passing it on to their children.
A trait or disorder which is determined by a gene on an autosome is said to show
autosomal inheritance, whereas a trait or disorder determined by a gene on one of the
sex chromosomes is said to show sex-linked inheritance.
1) Autosomal inheritance
 Autosomal dominant
 Autosomal recessive
2) Sex-linked inheritance
 X-linked dominant
 X-linked recessive
 Y-linked inheritance

AUTOSOMAL DOMINANT INHERITANCE

An autosomal dominant trait is one which manifests in the heterozygous state, i.e. in a
person possessing both an abnormal or mutant allele and the normal allele. It is often
possible to trace a dominantly inherited trait or disorder through many generations of
a family. This pattern of inheritance is sometimes referred to as ‘vertical’
transmission. Features include,
1) Males and females affected in equal proportions
2) Affected individuals in multiple generations
3) Transmission by individuals of both sexes, i.e. male to male, female to female,
male to female and female to male
Variable expressivity
The clinical features in autosomal dominant disorders can show striking variation
from person to person. This difference in involvement between individuals is referred
to as variable expressivity.
Reduced penetrance
In some individuals heterozygous for certain autosomal disorders, the presence of the
mutation can be undetected clinically, representing so-called reduced penetrance or
what is known as the disorder ‘skipping a generation’. Reduced penetrance is thought
to be the result of the modifying effects of other genes, as well as being due to
interaction of the gene with environmental factors. An individual who is heterozygous
for a dominant mutation but has no features of the disorder is said to represent non-
penetrance.

AUTOSOMAL RECESSIVE INHERITANCE

Recessive traits and disorders are only manifest when the mutant allele is present in a
double dose, i.e. homozygosity. Individuals heterozygous for a recessive mutant allele
show no features of the disorder and are perfectly healthy, i.e. they are carriers. It is
not possible to trace an autosomal recessive trait or disorder through the family, i.e.
all the affected individuals in a family are usually in a single sibship, that is, they are
brothers and sisters. This is sometimes referred to as ‘horizontal’ transmission.
Mutational heterogeneity
Individuals have been identified who have two different mutations at the same locus
and are known as compound heterozygotes, constituting what is known as allelic or
mutational heterogeneity. Most individuals affected with an autosomal recessive
disorder are probably, infact, compound heterozygotes rather than true homozygotes,
unless their parents are related when they are likely to be homozygous for the same
mutation by descent, having inherited the same mutation from a common ancestor.
Three features, which suggest the possibility of autosomal recessive inheritance, were,
1) The disorder affects males and females in equal proportions.
2) It usually affects individuals in one generation in a single sibship, i.e.
brothers and sisters, and does not occur in previous and subsequent
generations.
3) Parents can be related, i.e. consanguineous.

SEX-LINKED INHERITANCE
Sex-linked inheritance refers to the pattern of inheritance shown by genes which are
located on either side of sex chromosomes. Genes carried on the X chromosome are
referred to as being X-linked, while genes carried on the Y chromosome are referred
to as exhibiting Y-linked or holandric inheritance.
X-linked dominant inheritance
These are disorders, which are manifest in the heterozygous female as well as in the
male who has the mutant allele on his single X-chromosome. X-linked dominant
inheritance superficially resembles that of an autosomal dominant trait because both
daughters and sons of an affected female have 50% chance of being affected.
Features include,
1) Males and females are affected but often females are affected in excess.
2) Severity is less in females compared to males.
3) Affected males can transmit the trait to all his daughters but to none of his
sons.

X-linked recessive inheritance

An X-linked recessive trait is one determined by a gene carried on the x chromosome
and usually only manifests in males. A male with a mutant allele on his single X-
chromosome is said to be hemizygous for that allele.
Features of X-linked recessive inheritance were,
1) Males usually only affected.
2) Transmitted through unaffected heterozygous carrier females affect males,
as well as by affected males to their obligate carrier daughters with a
consequent risk to male grand children through these daughters.
3) Affected males cannot transmit the disorder to their sons.

Y-linked inheritance
Y-linked or holandric inheritance implies that only males are affected. An affected
male transmits Y-linked traits to all his sons but to none of his daughters.

POLYGENIC AND MULTIFACTORIAL INHERITANCE

POLYGENIC INHERITANCE
Polygenic or quantitative inheritance involves the inheritance and expression of a
phenotype being determined by many genes at different loci, with each gene exerting
a small additive effect. Additive implies that the effects of the genes are cumulative,
i.e. no one gene is dominant or recessive to another.
MULTIFACTORIAL INHERITANCE
As it is likely that many factors, both genetic and environmental, are involved in
causing these disorders they are generally referred to as showing multifactorial
inheritance. In multifactorial inheritance environmental factors interact with many
genes to generate a normally distributed susceptibility.

HERITABILITY
Heritability can be defined as the proportion of the total phenotypic variance of a
condition which is caused by additive genetic variance. Heritability is expressed either
as a proportion of 1 or as a percentage. Heritability is estimated from the degree of
resemblance between relatives expressed in the form of a correlation coefficient
which is calculated using statistics of the normal distribution. Alternatively,
heritability can be calculated using data on the concordance rates in monozygotic and
dizygotic twins.

SIB-PAIR ANALYSIS
If affected siblings inherit a particular allele more or less often than would be
expected by chance, this indicates that that allele or its locus is involved in some way
in causing the disorder.
The strength of this relationship can be quantified by determining the ratio of the
expected to observed proportions of affected sib-pairs which share zero alleles at the
relevant locus.

SEGREGATION AND LINKAGE ANALYSIS

Segregation analysis refers to the study of the way in which a disorder is transmitted
in families so as to establish the underlying mode of inheritance. It is a statistical
method for determining the mode of inheritance of a particular phenotype from family
data, particularly with the aim of elucidating single gene effects or so-called major
genes. With increasing computer power, models have been developed to detect the
contribution of individual genetic loci that have large effects against a background of
polygenic and environmental effects. Once evidence of major genes has been detected,
linkage analysis provides a means of determining where individual genes are located
within the genome. Until recently, however, application of these methods to clarify
the genetic basis of dental disorders has been limited by the difficulties of obtaining
data from large family pedigrees and also in identifying appropriate polymorphic
marker loci.

TWINNING
Twins can be identical or non-identical  monozygotic (MZ) or dizygotic
(DZ) – depending on whether they originate from a single conception or from two
separate conceptions.
MONOZYGOTIC TWINS
Monozygotic twins originate from a single egg which has been fertilized by a single
sperm. A very early division, occurring in the zygote before separation of the cells
which make the chorion, results in dichorionic twins. Division during the blastocyst
stage from days 3 to 7 results in monochorionic diamniotic twins. Division after the
first week leads to monoamniotic twins.
DIZYGOTIC TWINS
Dizygotic twins result from the fertilization of two ova by two sperm and are no more
closely related genetically than brothers and sisters. Hence they are sometimes
referred to as fraternal twins. Dizygotic twins are dichorionic and diamniotic although
they can have a single fused placenta if implantation occurs at closely adjacent sites.

SIGNIFICANCE OF TWIN STUDIES

The classical twin approach for separating the effects of nature and nurture involves
comparing identical (monozygous) twins and non-identical (dizygous) twins.
Differences between monozygous (MZ) twin pairs reflect environmental factors,
whereas differences between dizygous (DZ) twin pairs are due to both genetic and
environmental factors. Therefore, greater similarities between MZ twin pairs
compared with DZ twin pairs can be interpreted as reflecting genetic influences on the
feature(s) being studied.
Apart from comparisons of monozygous and dizygous twins, there are other twin
models that provide insights into the contributions of genetic and environmental
factors to observed variability. The monozygous co-twin model involves comparisons
of monozygous twins where each member of a pair has been exposed to different
environmental effects. For example, identical twins might be treated with different
appliances to correct similar malocclusions and the outcomes compared.
Monozygous twins are assumed to have identical genotypes, so their offspring are
genetically related as half-sibs but are socially first cousins. A nested analysis of
variance similar to that used in analysing data from half and full-sibling litters in
animal studies can be applied to provide estimates of genetic and environmental
effects.

SIGNIFICANCE OF GENETIC STUDIES FOR MALOCCLUSION AND

CRANIOFACIAL ANOMALIES
Dental occlusion reflects the interplay between a number of factors including
tooth size, arch size and shape, the number and arrangement of teeth, size and
relationships of the jaws, and also the influences of the soft tissues including lips,
cheeks and tongue.
The term ‘malocclusion’ is generally used to refer to variations from normal occlusal
development, and although in some instances it is possible to specify the cause of a
particular malocclusion, for example, genetic syndromes, embryological defects, or
trauma, most malocclusions represent variations from normal development for which
there is no apparent cause. There have been several excellent reviews of genetic
studies of craniofacial development and morphology.
Smith and Bailit (1977), in their comprehensive review of the problems and
methods in studies of the genetics of dental occlusion, listed five main research
objectives:
1) Elucidating modes of inheritance
2) Detecting the effects of admixture and inbreeding
3) Performing linkage analyses
4) Estimating heritabilities
5) Comparing population differences
Modes of inheritance
Occlusal variation appears to conform to a multifactorial mode of inheritance,
although strong familial similarities may be due to single major genes. For example,
the famous ‘Hapsburg jaw’ seen in consecutive generations of an Austrian royal
family may have been caused by a small number of segregating major genes. It is also
possible that epistatic factors, that is, the interaction between genes at different loci,
may play a more important role than most researchers have thought.

Admixture and breeding effects

Many research workers suggested that racial admixture increases the occurrence of
malocclusion. The notion that admixture might lead to an increased frequency of
malocclusion in humans appears to have originated from the work of Stockard and
Johnson (1941) in which gross deformities of the jaws of dogs were produced by
cross-breeding different inbred strains.

The X and Y-chromosomes

Pattern profiles of dental crown size show the dosage effect of the sex chromosomes,
with both the X and Y-chromosomes appearing to exert growth-promoting effects on
human tooth crown size.
The X chromosome appears to mainly regulate enamel thickness. On the other hand,
the Y chromosome seems to affect both enamel and dentine. The X and Y-
chromosomes also seem to influence craniofacial growth and development.
It is suggested that the X chromosome may alter morphology of the cranial base by
affecting growth at the synchondroses, that is, cartilaginous joints, and it also appears
to have a direct effect on mandibular shape.

Heritability
Early traditional twin studies (Lundstrom 1954) and intrafamilial comparisons (Stein
1956) indicated that occlusal traits were under reasonably strong genetic control.
However, more recent reports in twins (Corrucini 1980) and in first-degree relatives
(Harris 1991) have emphasized the importance of environmental factors.

Population differences
Variations in dental occlusion between different human populations have been
described and interpreted in genetic terms. Midfacial growth, alveolar bone
development and tooth migration associated with vigorous masticatory function tends
to provide space for unimpeded emergence and alignment of permanent teeth in
Aboriginals. Recent studies have shown that occlusal variation increased significantly
in the Yuendumu people within one generation after adoption of a more westernized
diet.

Manifestation of a malocclusion is the culmination of a hierarchy of subclinical

molecular, biochemical, physiologic, and metabolic markers of risk. Any one of these
can be modified by the environment, which makes the clinical expression remote
from gene action. This is the essence of why dentofacial structure is not suitable for
analyses with Mendelian models.
Heritability is a one-dimensional descriptive statistic that does not address the mode
of inheritance of malocclusions. The long-term goal should be to identify factors that
affect the frequency and/or severity of the phenotype. Calculation of heritability
estimates is a preliminary step that should be followed by tests for causative agents.
Within clinical orthodontics, the preliminary goal has been to define the relative
contributions of genetics and the environment.

GENETICS AND MALOCCLUSION

PART-2: DEVELOPMENTAL GENETICS AND ITS SIGNIFICANCE IN ORTHODONTICS

CONTENTS
 INTRODUCTION
 ROLE OF NEURAL CREST CELLS
 VERTEBRATE HOX GENES
 VERTEBRATE HOX CODE
 PATTERNING THE BRANCHIAL REGION OF THE HEAD
 PATTERNING OF FACE AND JAWS
 PATTERNING THE MIDLINE
 PATTERNING OF THE DENTITION
 HOX CODE
 ODONTOGENIC HOMEOBOX CODE
 MSX GENES
 DLXGENES
 BARX GENES
 PAX GENES
 HEDGEHOG GENES
 ODONTOGENIC EPITHELIAL – MESENCHYMAL
INTERACTIONS THROUGH GROWTH FACTORS
 FIBROBLAST GROWTH FACTORS
 BONE MORPHOGENETIC FACTORS
 GENETIC INFLUENCE ON TOOTH NUMBER, SIZE,
MORPHOLOGY, POSITION AND ERUPTION
 HERITABILITY OF MALOCCLUSION
 FAMILY AND TWIN STUDIES FOR HERITABILITY OF
DENTOFACIAL PHENOTYPES
 CLASS II MALOCCLUSION
 CLASS III MALOCCLUSION
 HERIABILITY OF LOCAL OCCLUSAL VARIABLES
 GENOMICS AND OROFACIAL CLEFTS
 CLEFT LIP AND CLEFT PALATE
 MEDIAN CLEFTS
 ALVEOLAR CLEFTS
 FACIAL CLEFTS
 CRANIOFACIAL SYNDROMES
 CROUZONS SYNDROME
 APERT’S SYNDROME
TREACHER COLLINS SYNDROME

PFIEFFER SYNDROME

 CRANIOFACIAL MICROSOMIA
 CONCLUSION
 GENETICS AND MALOCCLUSION

INTRODUCTION
The advent of molecular biology has allowed biologist to uncover, characterize, and
ultimately manipulate the genes. We can now study how genes and proteins operate
within their natural habitats.

This is significantly furthering our understanding of the fundamental principles of

development, how genes control cell behavior and thus, how they determine the
pattern and form of an embryo. Without this knowledge of gene activity and the
relevant cellular signaling pathway, elucidating the mechanism that control
development would be impossible. These advances are now influencing dentistry and
clinical genetics with almost daily progression in explaining the basis of multitude of
congenital malformation, and skeletal and dental abnormalities.

It is important that the clinicians attempt to keep abreast of these developments and
orthodontists are not immune.

Generation of craniofacial complex is a process that requires considerable

organization. The vertebrate head is a composite structure whose formation begins
early in development as the brain is beginning to form. Central to the development of
a head is a concept of segmentation; manifest in the hindbrain and brachial arch
systems. In conjunction with migrating neural crest cells these systems will give rise
to much of the head and neck and their associated, individual compartments. It is now
becoming clear that the molecular control of embryonic resides at the level of the
gene, in particular, within families of genes that encode transcription factors capable
of regulating downstream gene transcription.

ROLE OF THE NEURAL CREST

The neural crest is a highly pluripotent cell population that plays a critical role in
the development of the vertebrate head. Unlike most parts of the body, the facial
mesenchyme is derived principally from the neural crest and not the mesoderm of the
embryonic third germ layer. Neural crest cells migrates extensively throughout the
embryo in four overlapping domains (Cephalic, trunk, sacral and cardiac) and in the
developing head the cephalic neural crest migrates from the posterior midbrain and
hindbrain regions into the branchial arch system. The ectomesenchymal neural crest
cells that interact with epithelial and mesodermal population present within the
arches, leading to the formation of craniofacial bone, cartilage and connective tissue.

VERTEBRATE HOX GENES

In the early 1980s biologists began searching for genes containing the Drosophila
homeobox in vertebrates, reasoning that the highly conserved nature of the homeobox
between homeotic genes might have been preserved during evolution. In a landmark
evolutionary survey, using DNA from a variety of species, it was shown that the
homeobox is not confined to insects, but is also found in vertebrates.

The first vertebrate homeobox was rapidly cloned in the frog, Xenopus levis and this
was soon followed by the mouse. The degree of sequence similarity to the Drosophila
homeobox was remarkable, confirming that the genetic control of development was
more universal than previously imagined. These vertebrate genes are called Hox
genes, and as more were cloned it became clear that during the course of evolution
considerable duplication a divergence had occurred from the original ancestral cluster.

In the mouse and human genomes there are 39 Hox genes related to Drosophila
homeotic genes. These Hox genes are arranged in four clusters (instead of one in the
fruitfly) on four different chromosomes: Hox a-d in mice and HOZ A-D in man
(Scott, 1992)

VERTEBRATE HOX CODE

The expression of Hox genes in the vertebrate embryo can be seen along the dorsal
axis with the CNS from the anterior region of the hindbrain throughout the length of
the spinal cord. The patterns of these genes show a very precise spatial restriction.
Each Hox genes is expressed in and overlapping domain along the anterior-posterior
axis of the embryo, but each gene has a characteristic segmental limit of expression at
its anterior boundary.

In the developing head, this spatially restricted limits of Hox gene expression
corresponding to rhombomeres boundaries at two-segment intervals. As the neural
crest migrates from the rhombomeres into specific branchial arches it retains the
particular combination or code of Hox genes expression that is characteristic of the
rhombomeres from which it originated. Thus, the neural crest from each axial level
conveys a unique combinatorial Hox code.

It should be noted that the neural crest destined for the first brachial arch, from which
the maxillary and mandibular process develop, doesn’t express Hox genes related to
the homeotic homeobox (Hunt et al 1991).

It is subfamilies of homebox genes, more diverged from the ancestral Hox genes, that
are expressed in spatially restricted patterns within the first brachial arch (MacKenzie
et al 1992;Sharpe et al 1995).

PATTERNING THE BRANCHIAL REGION OF THE HEAD

Fundamental to the development of the craniofacial complex is the central nervous

system (CNS). The CNS arises from the neural plate, a homogenous sheet of
epithelial cells that forms the dorsal surface of the gastrula stage embryo.
As the neural plate rolls up along its AP axis to form the neural tube the enlarged
anterior end partitions into three vesicles. These vesicles are the primordial of the
developing forebrain (prosencephalon), midbrain (mesencephalon), and hindbrain
(rhombencephalon).

It is the rhombencephalic-derived neural crest that will give rise to the majority of the
brachial arch mesenchyme. Migration of these populations of the neural crest cells
from the regions of the rhombecephalon results in a ventral relocation to within the
brachial arches. Development of the mid-brain and lower regions of the craniofacial
complex is intimately associated with these branchial regions. It is clear, therefore,
that the neural crest derived from the hindbrain is essential for normal formation of
the face and neck.

The hindbrain itself is known to be a segment structure composed of eight subunits

called rhombomeres (Lumsden and Keynes, 1989). Rhombomeres are important
segmental unit of organization, which have distinct morphological properties that vary
with a two-segment periodically.
The neural crest cells that migrate and form the bulk of the facial mesenchyme arise
from the same axial level of neural tube as the rhombomeres whose neurons will
ultimately innervate that mesenchyme. Neural crest cells destined for the first
branchial arch migrate essentially from rhombomeres1 and 2, whilst those for the
second and third arched migrates from rhombomeres 4 and 6, respectively. The even
numbered rhombomeres 2, 4 and 6 contain the exit points for cranial nerves V, VII,
and IX nerves that will innervate branchial arches 1, 2 and 3. This leads to the concept
that an axial-level specific code exists which is established when the neural crest cells
still form part of the neural plate. Cells recognize each other and have a positional
identity. Following their migration into the arches, they produce the individual
structures that make up the composite head in an orderly and integrated manner.

These mechanisms of craniofacial development are under genetics control. How do

those genes that are involved produce the complex, recognized structures that form
the building blocks of the developing the head and neck? It is helpful to consider
those genes involved in embryogenesis as encoding a set of instructions or rules of
assembly.
Implementation of these one-dimensional rules, via genes expression and protein
interaction, produces the three-dimensional embryo (Thorogood and Ferretti, 1992).
In recent years, a number of genes and gene families have been identified that play a
critical role in establishing regional identity, including the various components of the
vertebrate head.

PATTERNING OF FACE AND JAWS

In humans a number of other homeobox-containing genes are expressed in the

maxillary and mandibular arches, and developing facial primoridia. These genes,
which all encode homeodomain–containing transcription factors, include Msx-1,
Msx-2, Dlx1-6 and Barx-1. Again many of these homeobox-containing genes are
related to the families of gene found in Drosophila. Knockout studies have confirmed
that these genes perform essential roles during the formation of the facial complex.

Members of the Msx gene family (Msx-1 and Msx-2) are normally expressed strongly
in the neural crest derived mesenchyme of the developing facial prominence, and
there is now strong evidence for a role of these genes in specification of the skull and
face (Ferguson 2000).

Targeted disruption of Msx-1 in the mouse produces a number of defects in facial

structures. There is cleft palate associated with a loss of the palatine bones, maxillary
and mandibular hypoplasia, and a highly penetrant arrest of tooth formation at the bud
stage of tooth development (Satokata and Maas).
In mice, defects in Msx-2 cause skull ossification with persistence of calvarial
foramen. This arises as a result of defective osteoprogenitor proliferation during
calvarial morphogenesis (Satokata et al, 2000).

Members of the multi-gene Dlx family are expressed in a complex pattern within the
embryonic ectoderm and mesenchyme of the maxillary and mandibular processes of
the first arch (Bulfone et al, 1993).

Targeted mutation in Dlx-1, Dlx-2 and Dlx 1/2 provide evidence that these genes are
required for the development of neural crest derived skeletal elements of the first and
second branchial arches (Qui et al, 1997).

Analysis of these mutations reveals that Dlx-1 and Dlx-2 regulate proximal first arch
structures and that, in the mandibular primordium, there is considerable functional
redundancy of Dlx-1 and Dlx-2 with other members of the Dlx family.

GOOSECOID GENE
Goosecoid is another homeobox-containing transcription factor, originally isolated in
Xenopus from a dorsal blastopore lip cDNA library. The dorsal blastopore lip has
long been known to be ultimately responsible for organization of the complete body
axis in the early embryo. However, when goosecoid was knocked out in transgenic
mice they formed a body axis normally, but exhibited a number of craniofacial defects
(Rivera- Perez et al; Yamada et al, 1995).

In wild type mice, goosecoid transcripts had been detected at later stages of
development in the osteogenic mesenchyme of the developing mandible, tongue and
middle ear. In mutants, the mandible was hypoplastic, and lacked coronoid and
angular process, whilst there were defects in several bones, including the maxillary,
palatine, and pterygoid. As a homeobox–containing transcription factor it would
appear that goosecoid is involved in essential inductive tissue interactions during the
formation of the head.

ENDOTHELIN
Another gene that has produced an even more perplexing phenotype is Endothelin-1
which encodes a vasoactive peptide expressed in vascular endothelial cells and is
thought to play a role in the regulation of blood pressure. Mice with targeted
disruption of Endothelin-1 have no abnormalities of their cardiovascular system but
do have a marked reduction in tongue size, micrognathia and cleft palate (Kurihara
et al, 1994).

One of the two G protein-coupled endothelin receptors, ET-A is expressed in the

neural crest derived ectomesechyme of the branchial arches, whilst its primary ligand,
ET-1 is expressed in arch epithelium, pharyngeal pouch endothelium, and arch core
paraxial mesoderm. The ET-A/ET-1 pathway appears to be important for proper
patterning of the caudal regions of the first arch (Tucker et al, 1999).

Target disruption of ET-A or ET-1 in mice produce craniofacial defects that resemble
a human condition called CATCH-22, which is characterized by abnormal facies and
cardiovascular defects (Wilson et al, 1993).

It has been recently been shown that the craniofacial defects in ET-A mice are, in
part, due to an absence of the goosecoid transcription factor (Cloutheir et al, 1998).

PATTERNING THE MIDLINE

Sonic hedgehog (Shh) is the vertebrate homologue of the Drosophila hedgehog

segment polarity gene. Hedgehog morphogens are involved in the control of left-right
asymmetry, the determination of polarity in the central nervous system, somites and
limbs, and in both organogenesis and the formation of the skeleton. In the vertebrate
embryo, Shh encodes a signaling peptide that is involved in a number of well-
characterized developmental signaling centers (Hammer Schmidt et al, 1997).

Recently, clues about the regulation of craniofacial morphogenesis have come from
studies of Shh gene.
Mutations of Shh in the mouse (Chiang et al 1996) and human (Belloni et al 1996)
leads to profound abnormalities in craniofacial morphogenesis.

Loss of Shh produces defective patterning of the neural plate resulting in

holoprosencephaly, a failure of cleavage in the midline forebrain and cylopia. Later in
development Shh is expressed in the ectoderm of the fronto-nasal and maxillary
processes and has been shown to be essential for their normal development (Wall and
Hogan, 1995; Helms et al 1997).
By manipulating developing chick embryos, it has been shown that a transient loss of
Shh signaling in these regions of the developing face can result in defects analogous
to hypotelorism and cleft lip/palate, which are characteristic features of the milder
form of holoprosencephaly. In contrast, excess Shh leads to medio-lateral widening of
the fronto-nasal process resulting in hypertelorism. In severe cases this can lead to
facial duplication (Hu and Helms, 1999).

PATTERNING OF THE DENTITION

HOX CODE

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

(Lumsden et al). 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.

However, the genes are not expressed in region rostral to rhombomeres 2, which

means that no Hox gene expression is seen in the neural crest that migrates to the

craniofacial region, including the first branchial arch (Hunt et al 1991). In terms of

patterning tooth development, we have to look at a subfamily of homeobox genes that

do show temporal and spatial patterns of expression within the first branchial arch.

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 (Sharpe et al 1995).

Various odontogenic homeobox genes identified were,

1) MSX genes  Msx-1, Msx-2
2) DLX genes  Dlx-1, Dlx-2
3) BARX genes  Barx-1, Barx-2

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 that regulate downstream gene transcription.
The proteins of this homeodomain act as transcription factors that result in activation
or inhibition of other genes. These homeobox genes also regulate the expression of
other target genes.

MSX genes
MSX is an important gene involved in tooth formation. MSX stands for muscle
segment homeobox gene. Campbell et al (1989) reported that MSX was homologous
to mouse Homeobox gene 7 (Hox 7) and Bell et al (1993) related it to the Drosophila
gene muscle-segment homeobox (msh).

Ivens et al (1990) localized this gene to chromosome 4p16 and mutation of this gene
has been associated with facial and dental abnormalities.
MSX-1 and MSX-2 genes:
MSX 1 gene is expressed in migrating neural crest cells and later in mesenchymal
cells of dental papilla and follicle. MSX-2 genes are involved in signaling
interactions, which are essential for the tooth development.

Prior to the initiation of odontogenesis both Msx-1 and Msx-2 exhibit very specific
horseshoe-shaped fields of corresponding mesenchymal expression in the anterior
regions of the first arch (McKenzie et al 1992). 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 Msx-1 expression
corresponds to the position of the future primary epithelial thickening. 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. FIGURE 3

DLX genes
Dlx genes are expressed in migrating neural crest cells and in the first brachial arch.
DLX stands for distal-less homeobox gene. McGuiness et al (1996) first reported the
distal-less Homeobox gene (Dlx2), which was localized at chromosome 2q32 loci.
The DLX genes have also been conserved during evolution and bear homology to the
distal-less gene of Drosophila (Porteus et al, 1991).

DLX-1 and DLX-2 genes:

The expression of Dlx-1 and Dlx-2 in the maxillary and mandibular arch mesenchyme
is restricted to the proximal regions where the future molar teeth will develop.

BARX genes
BARX stands for Bar class Homeobox gene that includes Barx-1 and Barx-2. Barx-1
is homeobox containing transcription factor that exhibits regionalized expression
within the ectomesenchyme of the first branchial arch (Tissier-Seta et al, 1995). Bar
class homeobox 2 genes (Barx-2) is also a group of homeodomain transcription
factors. This group of Homeobox genes was first located in Drosophila in the locus
11q25.
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 Barx-1 expression in the anterior
regions. As tooth development proceeds, Barx-1 expression becomes localized
exclusively to the mesenchymal regions around the developing molars (Thomas and
Sharpe, 1998).

Jones et al (1999) suggested that the mutation of these genes could be associated with
facial and dental anomalies.
PAX genes
Paired-box homeotic gene (PAX) is found in 2q35 locus. PAX gene products function
by binding enhancer DNA sequences and they modify 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.
Nebuser et al (1997) associated PAX9 transcription factor with tooth bud positioning
at the mesenchymal level and mutations in this gene results in conditions such as
hypodontia, transposition etc.

HEDGEHOG genes
Sonic Hedgehog gene (Shh) is located in 7q36 and is the vertebrate homologue of
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 initiation of tooth
development, epithelial signaling and cuspal morphogenesis. 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, Gli-2 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.

Inger kjaer EJO (2001) reported that less severe cases of holoprosencephaly is
characterized by a solitary median maxillary central incisor (SMMCI)

ODONTOGENIC EPITHELIAL - MESENCHYMAL INTERACTIONS THROUGH

GROWTH FACTORS

The molecular basis for odontogenesis is dependent upon many of the diffusible
protein signaling molecules and growth factors that are known to mediate reciprocal
signaling between cells groups in epithelium and mesenchyme during tooth
development.

A number of intercellular protein molecules have been identified in the developing

tooth germ at various stages of development. Among those factors, Fibroblast growth
factors (FGFs) and Bone morphogenetic protein (BMPs) are essential for
development of teeth.

FIBROBLAST GROWTH FACTORS (FGFs)

FGFs are proteins involved in the growth and differentiation of odontogenic cells
during tooth development. FGF is a family of heparin binding proteins, which is
expressed in tooth germs and regulates epithelial-mesechymal interactions. FGF-4,
FGF-8 and FGF-9 play an important role in odontogenesis. FGF-4 and FGF-9 are
essential for determing the coronal morphology and FGF-8 and 9 are vital for
initiation of tooth development. FGF-4 has been suggested to play a key role in
stimulating the proliferation of dental epithelium and mesenchyme.

BONE MORPHOGENETIC PROTEIN (BMPs)

Bone morphogenetic protein is a group of dimeric proteins, which come under the
classification of Transforming Growth factor β. Bone Morphogenetic Proteins, are
responsible for osteoinductive activity in bone matrix and cartilage. BMPs are
expressed in the condensed mesenchymal cells of bone primordial, and appear that
different BMPs are expressed in different bones.
Nearly 20 modifications of BMPs with slightly different modifications in small
secondary structure elements have been identified.
In odontogenesis, epithelial-mesenchymal interactions play a paramount role in the
formation of hard tissue. BMP’s are known to have a broad range of signaling
functions involving mediation of tissue interactions. BMP-2, BMP-4 and BMP-7 have
been associated with epithelial mesenchymal interaction during the morphogenesis
stage of tooth formation. BMP-4 is capable of inducing the expression of MSX-1 and
MSX-2. BMP-4 also determines the positions of future forming tooth germs.

GENETIC INFLUENCE ON TOOTH NUMBER, SIZE, MORPHOLOGY,

POSITION, AND ERUPTION.

Various developmental dental disorders, which are under the influence of genes,
include,
1) Hypodontia
2) Supernumerary teeth
3) Abnormal tooth shape
4) Submerged primary molars
5) Ectopic eruption and Transposition of canines

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,
 More common in permanent than primary dentition
 Absence of primary teeth associated with absence of permanent
successors
 May be associated with other developmental anomalies

Grahnen (1956) in his familial and twin studies revealed the hereditary nature of
hypodontia and concluded that in children with missing teeth, up to half of their
siblings or parents also had missing teeth.
Osborne et al (1958) in his twin studies have shown that tooth crown dimensions are
strongly determined by heredity. The molecular genetics of tooth morphogenesis with
the homeostatic Hox 7 and Hox 8 (now referred as Msx-1 and Msx-2) genes are being
responsible for stability in dental patterning.

Clinical evidence suggests that congenital absence of teeth and reduction in tooth size
are associated e.g., hypodontia and hypoplasia of maxillary lateral incisors frequently
present simultaneously. Numerous pedigrees have been published linking the two
characteristics and implying that they are different expressions of the same disorder.

Gruneberg (1965) suggested that a tooth germ must reach a critical size during a
particular stage of development or the structure will regress, and Suaraz and Spence
(1974) showed that hypodontia and reduction in tooth size are in fact controlled by the
same or related gene loci. It is apparent from all the evidence in this respect that tooth
size fits the polygenic multifactorial threshold model.

Markovic (1982) found a high rate of concordance for hypodontia in monozygous

twin pairs, while zygous twin pairs he observed discordant. These and other previous
studies concluded that a single autosomal dominant gene could explain the mode of
transmission with incomplete penetrance.
Vastardis (Nature Genetics 1996) studied the cause for selective tooth agenesis in
human, where missense mutation occurred in the MSX-1 homeodomain. This occurs
as a consequence of replacement of arginine with proline protein (Arg196Pro
mutation) in the homoedomain of MSX-1. Tooth agenesis was reported in a family
with a ser 105stop mutation of MXS-1 gene.

Dermaut and Smith (AJO1997) studied the prevalence of tooth agenesis correlated
with jaw relationship and dental crowding in 185 patients and found that,
 Hypodontia occurred more often in girls than in boys.
 The upper lateral incisors and lower premolars were the most frequently
missing teeth.
 Class I skeletal relationships were found more often in patients with agenesis
than in patients without missing teeth and are associated with deep-bite growth
patterns.

Research work by Cobourne (BJO 1999) on families affected with hypodontia has
revealed that it is transmitted as an autosomal dominant disorder with variable
expressivity and incomplete penetrance. Missing maxillary laterals and mandibular
second premolars have been associated with defects in MSX-1and MXS-2 genes.

Van den Boogard et al (Nature Genetics 2000) observed a genetic

aberration in a Dutch family with tooth agenesis. A stop codon in
MSX-1 mutation was identified implying the involvement of this gene
in tooth agenesis.

Nieminen (Eu J of Human Genetics 2001) found that, a non-sense mutation in the
PAX-9 gene was associated with molar tooth agenesis in a Finnish family. The
A340T transversion creates a stop codon at lysine 114, and truncates the coded PAX-
9 protein at the end of the DNA-binding paired box. The tooth agenesis phenotype
involved all permanent second and third molar and most of the first molars.

Lidral (JDR 2002) concluded that a mutation in MSX-1 gene in chromosome 4 has
been identified as the causative factor for oligodontia involving the absence of all
second premolar and third molar. Missing first molar and second molars have been
linked with a substitution mutation of MSX-1 gene.

With the help of molecular genetics techniques, Peck and Peck (AJO 2002) assessed
a family exhibiting an autosomal dominant trait of missing second premolar and third
molars. The affected chromosome was isolated to be in a chromosome 4p and many
genes were considered to be responsible for this tooth agenesis. A point mutation was
detected in the MSX 1 gene in all affected family. Also mutation of PAX-9
transcription factors has been observed in familial tooth agenesis and also in missing
mandibular second premolars and central incisors.

Recently Viera (JDR 2003) suggested that a fourth mutation has been
found in MXS-1 gene, which was Met611Lys and was associated with
missing second premolar and third molars.

SUPERNUMERARY TEETH

These are teeth additional to those of the normal series. A mesiodens is a

supernumerary tooth occurring between the maxillary central incisors and is the most
common of all supernumerary teeth. Supernumerary teeth most frequently seen in the
pre-maxillary region and with a male sex prediction also appear to be genetically
determined.

Niswander and Suguku (1963) analyzed the data from family studies and have
suggested that, like hypodontia, the genetics of the less prevalent condition of
supernumerary teeth in under the control of number of different loci.

Brook (1980) found that mesiodens is more commonly present in parents and siblings
of patients who present, although inheritance does not follow a simple Mendelian
pattern. Evidence from twins with supernumerary teeth also supports this theory
(Jasmin et al 1993).

ABNORMAL TOOTH SHAPE

Alvesalo and Portin (1969) provided substantial evidence supporting the view that

missing and malformed lateral incisors may be the result of a common gene defect.

Abnormalities in the lateral incisor region varies from peg shaped to microdont to

missing teeth, all of which have familial trends, female preponderance, and
association with other dental anomalies, such as other missing teeth, ectopic canine,

and transpostion, suggesting a polygenic etiology.

Aspects of tooth morphology such as the Carabelli trait also seem to be strongly

influenced by genes as evidenced by Australian twin study (Townsend and Martin,

1992).

SUBMERGED PRIMARY MOLARS

Primary molar submergence occurs most often in the mandibular arch with a
wide variation in the reported population (Kurol, 1980).
Helpin and Duncan (1986) found that, the siblings of children with submerged
primary molars are likely to also be affected and in monozygous there is a high
rate of concordance indicating a significant genetic component in the etiology. It
is also of interest that a variety of abnormalities are also associated with tooth
submergence with a suggestion that this may encompass different manifestations
of one syndrome, each manifestation having incomplete penetrance and variable
expressivity.

ECTOPIC ERUPTION AND TRANSPOSITION OF CANINES

Various studies in the past have indicated a genetic tendency for ectopic maxillary

canines.

Zilberman et al (1990) and Peck et al (1994) concluded that palatally ectopic

canines were an inherited trait, being one of the anomalies in a complex of genetically

related dental disturbances often occurring with missing teeth, tooth size reduction,

and other ectopically positioned teeth.

Previous studies by Mossey et al (1994) have also shown an association between

ectopic-maxillary canine and Class II div 2 malocclusion, a genetically inherited trait.

Peck et al (1997) classified a number of different types of tooth transposition in both

maxillary and mandibular arches, with maxillary caninefirst premolar class position
being the most common.
They also provided strong evidence of a significant genetic component in the cause of
this most common type of transposition in that there was
 A familial occurrence
  Bilateral occurrence in a high percentage of cases
 Female predominance and a difference in different ethnic groups
An increased frequency of associated dental anomalies; tooth agenesis and peg-
shaped maxillary lateral incisors were also reported. Neubuser et al (1995) found that
PAX-9 transcription factor is associated the genetic mechanism for tooth
displacement anomalies, such as palatally displaced canines and canine transposition.

HERITABILITY OF MALOCCLUSION

Malocclusion may be defined as a significant deviation from what is defined as

normal or ideal occlusion – Andrews 1972.

Many components are involved in normal occlusion. The most important are
1) The size of the maxilla
2) The size of the mandible
3) The factors, which determine the relationship between the two skeletal
bases such as cranial base and environment.
4) Arch form
5) Size and morphology of teeth present, and
6) Soft tissue morphology
There is dental anthropological evidence that population groups that are genetically homogenous tend to have normal
occlusion. In pure racial stocks, such as a Melanesians of the Philippine islands, malocclusion is almost non-existent.
However, in heterogeneous population the incidence of jaw discrepancies and occlusal disharmonies is significantly
greater.

Stockard 1941 carried out breeding experiments with dogs and produced gross
orofacial deformities and associated malocclusions. He concluded that individual
features of the craniofacial complex could be inherited independently of other
portions of the skull, and that jaw size and the tooth size could be inherited
independently, and as genetically dominant traits.

Fernex et al 1967 found boys to show more similarities to their parents than girls.
Facial skeletal structures were more frequently transmitted from mothers to sons than
from mothers to daughters.
Female twins showed greater concordance in facial features than male twins. While
the profile outline coincided most frequently, this was not true of the cranial base and
differences increased with age.

Littons et al 1970 concluded that siblings usually show similar types of malocclusion
and examination of older siblings can provide a clue to the need or interception and
early treatment of malocclusion.

Harris et al 1963 recommended that any study of genetic examination using line and
angles requires the use of multitriate analysis in order to identify relationship while
Kraus et al 1959 criticized the use of lines and angles to study heredity, and preferred
superimpositions of bony profiles to illustrate genetic control of craniofacial
morphology. Their study involved superimposition of lateral cephalograms of a
sample of identical twins and showed that many bony contours are in almost perfect
concordance. This applied equally to contours across sutures and to individual bony
contours such as the mandible.

FAMILY AND TWIN STUDIES FOR HERITABILITY OF DENTOFACIAL

PHENOTYPES

The twin method, when appropriately applied, provides geneticists with one of the
most informative technique available for analysis of complex genetic traits.
Alternative method for investigating the role of heredity in determining craniofacial
and dental morphology is by familial studies. Heritability in such studies is normally
expressed in terms of parent/offspring correlation coefficients or correlation
coefficients with sibling pairs, of which twins are a special kind.

The study of craniofacial relationship in twins has provided much useful

information concerning the role of heredity in malocclusion. The procedure is based
on the underlying principle that observed differences within a pair of monozygotic
twins (whose genotype is identical) are due to environment and those differences
within a pair of dizygotic twins (who share 50% of their total gene complement) are
due to both genotype and environment.
A comparison of the observed within-pair differences for twins in the two
categories should be provide a measure of the degree to which monozygotic twins are
more alike than dizygotic twins. The larger this differences between the two twin
categories, the greater the genetic difference effect on variability of the trait. This
model implies the zygosity is accurately determined and that environment effects are
equal in the two twin categories

The bulk of the evidence for the heritability of various types of malocclusion
arises from family and twin studies.

CLASS II MALOCCLUSION

Class II Division I Malocclusion:

Extensive cephalometric studies have been carried out to determine the heritability
of certain craniofacial parameters in class II division I malocclusion (Harris 1975).
These investigation have shown that in the class II patients, the mandible is
significantly more retruded than in class I patients, with the body of the mandible
length smaller and overall mandibular length reduced.
These studies also showed a higher correlation between the patient and his immediate
family that data from random pairings of unrelated siblings, thus supporting the
concept of polygenic inheritance for class II division I malocclusion.

Class II Division 2 malocclusion:

Class II division 2 is a distinct clinical entity and is a more consistent collection of

definable morphometric features occurring simultaneously i.e., syndrome than the
other malocclusion types put forward by Angle in the early 1900’s.
Class II division-2 malocclusion along with characteristic skeletal features is often
accompanied by particular morphometric dental feature also, such as a poorly
developed cingulum on the upper incisors and a characteristic crown angulation.
Markovic 1992 carried out a clinical and cephalometric study of 114 Class II
division-2 malocclusions, 48 twin pairs and six sets of triplets. Intra- and Inter- pair
comparisons were made to determine concordance-discordance rate for monozygotic
and dizygotic twins. Of the monozygotic twin pairs, 100% demonstrated concordance
for the Class II division-2 malocclusion, whilst almost 90% of the dizygotic twin pairs
were discordant. This is strong evidence for genetics as the main etiological factor in
the development of class II division2 malocclusion.

The studies point to incontestable genetics influences probably autosomal dominant

with incomplete penetrance and variable expressivity. It could also possibly be
explained by a polygenic model with a simultaneous expression of a number of
genetically determined morphological traits acting addictively, rather than being the
effect of a single controlling gene for the entire occlusal malformation.

Aspects of skeletal and muscle morphology are genetically determined and there is
some recent experiment evidence from a twin study (Lauweryns et al 1995)
indicating strong genetic factors in certain aspects of masticatory muscle behavior.

CLASS III MALOCCLUSION

Probably the most famous example of a genetic trait in humans passing through
several generations is the pedigree of the so-called Hapsburg jaw. This was the
famous mandibular prognathism demonstrated by several generations of the
Hungarians/Austrian dual monarchy.

Strohmayer (1937) concluded from his detailed pedigree analysis of the Haspburg
family line that the mandibular prognathism was transmitted as an autosomal
dominant trait. This could be regarded as an exception and in itself, does not provide
sufficient information to predict the mode of inheritance of mandibular prognathism.

Suzuki (1961) studied 1362 persons from 243 Japanese families and noted that, while
the index cases and mandibular prognathism; there was a significantly higher
incidence of this trait in other members of his family (34.4%) in comparison of
families of individuals with normal occlusion (7.5%).

Schulze and Weise (1965) also studied mandibular prognathism in monozygotic and
dizygotic twins. They reported that concordance in monozygotic twins was six times
higher than among dizygotic twins.

Both of the above studies reported a polygenic hypothesis as the primary cause for
mandibular prognathism (Litton et al 1970).

A class III malocclusion resulting from a skeletal imbalance between the maxillary
and mandibular bases may result from deficiency in maxillary growth, excessive
mandibular growth, or a combination of both. Various studies have also highlighted
the influence of a distinct cranial base morphology with a more acute cranial base
angle and shortened posterior cranial base resulting in a more anterior position the
gleniod fossa, thus contributing to the mandibular prognathism (Ellis and
Mcnamara, 1984; Singh et al 1997).
Various models have been suggested, such as autosomal dominant with incomplete
penetrance (Stiles and Luke1953), simple recessive (Downs 1928), variable both in
expressivity and penetrance with differences in different racial populations (Kraus et
al 1959).

Litton et al (1970) carried out an analysis of the literature to that date and also
analyzed a group of probands, siblings and parents with Class III malocclusion, and
analyzed the results in an effort to determine a possible mode of transmission.
Both autosomal dominant and autosomal recessive transmission were ruled out and
there was no association with gender (male or female).

The polygenic multifactorial threshold model put forward by Edward et al 1960,

however, did fit the data and accordingly proposed a polygenic model with a
threshold for expression to explain familial distribution, and the prevalence both
within general population and in siblings of affected persons.

Soft tissues do not generally play a part in the etiology of Class III malocclusion, and
in fact there is a tendency for lip and tongue pressure to compensate for a skeletal
Class III discrepancy by retroclining lower incisors and proclining upper incisors.

Polygenic inheritance implies that there is scope for environmental modification and
many familial and twin studies bear this out.

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.

Hughes and Moore 1942 suggested that the mandible and maxilla are under separate
influence of genetics control, and that certain portions of individual bones, such as the
ramus, body, and symphysis of the mandible are under different genetic and
environmental influences.

Nakasima and Nakata (AJO 1982) assessed the craniofacial morphologic

differences between parents of Class II patients and 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 distoclusion type
of denture pattern, while the parents of Class III patients had a concave profile
with a mesioclusion type of denture pattern. This suggests that both Class II
and Class III malocclusions have a genetic basis.
 The skeletal pattern was more directly related to genetic factors.
 Parent-offspring correlation data were in good agreement with the expected
level under the polygenic model of inheritance.
  Upper incisor proclination, gonial angle and the ramal height were considered
to be related to environmental factors.

HERITABILITY OF LOCAL OCCLUSAL VARIABLES

It has been thoroughly documented that measurements of the skeletal craniofacial

complex have moderate to high heritability, while measures of the dento-alveolar
portions of the jaws i.e., tooth position and dental relationships are given much less
attention in the literature.

Because of the adaptability of the dentoalveolar region when subjected to

environmental factors, local malocclusions are primarily acquired and would be
expected to have low heritablities.

In an analysis of the nature versus nurture in malocclusion Lundstrom (1984)

concluded that the genetic contribution to anomalies of tooth position and jaw
relationship overall is only 40%, with a greater genetic influence on the skeletal
pattern than on the dental features.

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
degree of overbite.

A study by Hu et al (1992) also reported familial similarity in dental arch form and
tooth position.

In a recent study by King et al (1993), initial treatment records of 104 adolescent

sibling pairs, all 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 occurring occlusions. The explanation offered was that a
genetically influenced facial types and growth patterns of the siblings are likely to
respond to environment factors e.g., chronic mouth breathing and reduced masticatory
stress similar fashions.
It is also important to remember the soft tissue morphology and behaviour have a
genetic component and they have a significant influence on the dentoalveolar
morphology.

Van der Linden (1966) described the concept that, the balance between the internal
and external functional matrices existed. For example, in a Class II division 1
malocclusion a short upper lip and low lip level with flaccid lip tone will reduce the
external influence and balance will favour proclination of the upper incisors. On the
other hand, a high lip level and more expressive lip behaviour will tend to produce a
Class II division-2 incisior relationship.
This external matrix is thought to be strongly genetically determined. The internal
matrix determined mainly by tongue posture and behavior that can be influenced by
environmental, as well as genetic factors.

GENOMICS AND OROFACIAL CLEFTS

Orofacial clefts, the most common craniofacial malformation ranks second among all
the craniofacial anomalies, among all the congenital malformation affecting human.
These include,
1) Cleft lip and Cleft palate
a) Cleft lip with or without cleft palate
b) Cleft palate only
2) Median clefts
3) Alveolar clefts
4) Facial clefts
Etiology of orofacial clefts appears to be complex with involvement of genetic,
environmental and tetragenic factors complicating the process.

CLEFT LIP AND CLEFT PALATE

Etiological factors:
1) Monogenic or single gene disorder
2) Polygenic or multifactorial inheritance
3) Chromosomal abnormalities
4) Familial
5) Sex predominance
6) Racial incidence

Monogenic or single gene disorders

Approximately half of the recongnized 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.

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 confirm to simple
Mendelian inheritance patterns.

Chromosomal abnormalities
Chromosomal abnormalities account for 18% of the clefting syndromes and would
invariably be associated with other malformations, delayed development and poor
prognosis. Chromosomal abnormalities notably trisomy D and also less frequently
trisomy E, may cause multiple malformations including cleft lip (palate).

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.

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 or not
significant.

Cleft lip and cleft palate can be broadly categorized as,

1) Non-syndromic CLP/CP
2) Syndromic CLP/CP
3) Syndromic isolated CP
4) Sex-linked CP (CPX)
5) Congenital healed cleft lip (CHCL)

Non-syndromic CLP/CP
Non-syndromic CLP/CP in humans seems to be etiologically distinctive and still
constitute majority of all classes with 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.
GROWTH FACTORS
TRANSCRIPTION FACTORS
Genes Loci
Genes Loci
Transforming Growth
Homeobox genes 2p11-13
Factor α (TGF α)
Muscle segment Transforming Growth
4p16.1 14q23-24
(MSX1) Factor β (TGF β)
Lim Homeobox Retinoic Acid Receptor
4q25-31 17q21
(Lhx8) Alpha (RARA)
Bar class GABA Receptor β 3
11q25 15q11.2-12
(Barx) (GABRB3)
Distal less B-cell leukemia/
2q32 19q13
(Dlx2) Lymphoma (3 BCL3)
Other Genes Jagged 2 14q32
Endothelin 1 6p23-24 (Jagg2)
Glutamate Decarboxylase Apolipoprotein C II
2q31 19q13.1
(GAD 67) (APOC2)
Syndromic CLP
Over 300 syndromes are known to have clefting of the lip or palate as an associated
feature. As with all clinically recognizable syndromes, cases of syndromic CLP or CP
can be broadly subdivided into,
1) Those that occur as part of characterized Mendelian disorder (Single Gene
defects)
2) Those arising from structural abnormalities of the chromosomes
3) Syndromes associated with known Teratogens
4) Those whose causation remains obscure and are therefore currently
uncharacterized.

One of the most common human autosomal dominant disorders associated with CLP
is van der Woude syndrome. Twin studies by Kondo et al (2002) 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,

1) Pierre Robin syndrome
2) CLP-ectodermal dysplasia syndrome (CLPED-1)
3) Ectrodactyly, ectodermal dysplasia, orofacial cleft (EEC syndrome)

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,
1) Mandibulofacial dysostosis (Treacher Collins syndrome)
2) Holoprosencephaly, type-3
3) Stickler syndrome

Sex-linked CP (CPX)
Philip Stainer and Gudrun Moore (1995) 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.

Laugier et al (2000) through silico analysis identified the gene loci at chromosome
Xq12-q21.

Bay brook et al (2001) identified six different mutations including missense, splice
site and non sense in the TBX22 gene families segregating X-linked cleft palate and
ankyloglossia.

Congenital healed cleft lip (CHCL)

CHCL is an unusual anomaly consisting of paramedian scar of upper lip with
appearance suggestive of typical cleft lip corrected in utero. It is usually associated
with an ipsilateral notch in the vermillion border and a collapsed nostril.
Castilla presented 25 cases of CHCL and suggested this condition to be most
common among males and preferentially affects left side. They further suggest a
familial predisposition to this phenomenon and may result from a defective fusion of
frontonasal and maxillary process or from spontaneously repaired open cleft with
visual scar later on development.

MEDIAN CLEFTS
True median cleft lip occurs with premaxillary agenesis and failure of completion of
the nose. Median clefts may occur with holoprosencephaly or may occur as an
isolated malformation (Cohen 1997). Other types of median clefts are associated with
syndromes such as,
1) Treacher Collins syndrome
2) Stickler dysplasia

ALVEOLAR CLEFTS
Alveolar clefts are associated with oral-facial-digital syndromes

CRANIOFACIAL SYNDROMES

A syndrome is recognised to represent multiple malformations occuring in

embryonically non-contigeous areas. Some of the syndromes with dental importance
are,
1) Crouzons syndrome
2) Aperts syndrome
3) Treacher Collins syndrome
4) Pfeiffer syndrome
5) Craniofacial microsomia

CROUZONS SYNDROME

 It is a frequent form of craniofacial dysostosis.

 It is characterized by multiple anomalies of the craniofacial skeleton with an
autosomal dominance inheritance pattern.
 Its manifestations are usually less severe than apert syndrome and there are no
malformations of the extremities.

Genetic etiology:
 Caused by multiple mutations in the fibroblast growth factor receptor2 gene
(FGFR2). Mutation in Tyrosine kinase receptor, at Ig II – Ig III domain
 Crouzons 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 associated
 coronal synostosis syndrome may present as bilateral coronal synostosis with
minimal midface involvement.
 Chromosome and region: 10q 253-q26

Clinical features:
 Premature synostosis of both coronal sutures with a resultant brachycephalic
shape to the skull.
 Cranial vault suture involvement other than coronal includes sagittal, metopic,
lambdoidal either in isolation or in combination.
 The cranial base and upper face sutures are variably involved resulting in a
degree of midface hypoplasia with an Angles class III malocclusion.
 Hypoplastic orbits with a proptosis
 Parrot beak nose.
 The anterior cranial base is short in the antero-posterior direction and wide
transversely.
 Cranial vault is high in the superoinferior dimension with anterior buldging of
the upper forehead resulting from compensatory growth through open metopic
and sagittal sutures.

APERT SYNDROME
Apert 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.

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 syndrome. Tyrosine kinase receptor is affected at
extracellular IgII-IgIII domain.
 Chromosome and region -10q 253 - q 26

Clinical Features:
 Bicoronal synostosis with a widely patent midline calvarial defect, allowing
the brain to expand anteriorly upto the metopic and anterior sagittal area with
increase in head breadth.
 In Apert Syndrome, the associated midface hypoplasia is thought to be
secondary to a cartilage maturation defect affecting the cranial base.
Patients have a high incidence of
 Cleft palate-30%
 Deafness-30%
 Bilateral complex syndactyly of the fingers and toes.

TREACHER COLLINS SYNDROME

Treacher collin syndrome,or mandibulofacial dysostosis,is an autosomal dominant
condition with variable expressivity. It is generaly characterised by bilateraly
symmetrical abnormalities of structures within the first and second branchial arches.
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
1million base pairs of DNA on chromosome 5.
A prenatal diagnosis requires blood sample from family members and a specimen for
culture and DNA isolation from an amniocentesis or chorionic villus sampling
procedure, performed at 16 to 17 weeks or 10 to11 weeks gestation, respectively.
Inheritance is autosomal dominant, and males and females are equally affected and
may be from one parent; multiple generation are affected, And an affected person met
with an unaffected person, each offspring has a 50% chance of inheriting the
condition.

Pathogenesis:
Theories include failure of differentiation of the branchial arch mesoderm, defective
facial bone ossification, and tissue ischemia resulting from stapedial artery
hypoplasia. Craig 1955 suggested that variability in the extent of the deformities with
this condition is due to the influence of “strong” or “weak” gene acting at an earlier or
later period of the embryo’s development. Behrents, McNamara, and Avery pointed
out that all major aspects of MFD are fully expressed by the 15 th week of embryonic
development.
Current research suggests that the abnormality may occur early as developmental
defects of the neural crest cells.

PFEIFFER SYNDROME

Features of Pfeiffer syndrome include craniostenosis, orbital dystopia, midface

hypoplasia, broad and medial deviated thumbs and great toes and partial soft tissue
syndactyly of the hands and feet.
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 (Pro 252 Arg)

of the FGFR1 gene and by several different mutations affecting FGFR2.

Clinical subtypes: Cohen has defined three distinct clinical subtypes:

 Pfeiffer syndrome type I
 Pfeiffer syndrome type II
 Pfeiffer syndrome type III

Pfeiffer syndrome type I:

 Generally compatible with life
 Normal or near normal intelligence in most patients.
 Bilateral coronal synostosis seen but with variable midface involvement.
 Near normal midface, normal mandibular growth, the airway is good.
 Anterior cranial base short an wide
 Cranial vault is tall, retruded with supero anterior bulging through open suture
or frontanelles.
  Posterior cranial vault is flat, lambdoid sutures are patent.
 Orbits are shallow; eyes are proptopic with a degree of hypertelorism.

Pfeiffer syndrome type II:

 Consists of cloverleaf skull.

 Severe midface deficiency.
 Ocular proptosis.
 Hydrocephalus.
 Limited life span.

Pfeiffer syndrome type III:

 Cohen has reported on several Pfeiffer patients without cloverleaf skull but
without a very poor prognosis.
 Hallmarks include severe ocular proptosis resulting from a markedly
decreased anterior cranial base length, shallow orbits and midface deficiency.
 Infants are neurologically compromised and have a limited life span

CRANIOFACIAL MICROSOMIA

Alternative Titles:
 First and Second Brachial arch Syndrome.
 Oral-mandibular-auricular Syndrome

The clinical expression of the syndrome was in the structures derived from the first
and the second brachial arch.

Jaw deformity: The most conspicuous deformity of unilateral microsomia is a

hypoplasia of the mandible on the affected side. The ramus is short or virtually absent
and the body of the mandible curves upward to join the short ramus. The chin is
deviated to the affected side; the body of the mandible on the normal or less affected
side is also characterized by abnormalities in the skeletal soft tissue anatomy. Body of
the mandible shows increased horizontal length and an increase in the gonial angle.
Ramus and the condyle malformations vary from minimal hypoplasia of the condyle
to its complete absence in association with hypoplasia or agenesis of the ramus.
Condylar anomalies are the pathognomic hallmark of the syndrome.

CONCLUSION

At the present time successful orthodontic interception and treatment of hereditary

malocclusion are limited by the extent of our knowledge because of,
1) lack of research dedicated to this particular problem e.g., prospective
randomized clinical trials
2) relative blunt measurement tools
 3) limited knowledge about the genetic mechanisms involved and the precise
nature and effects of environmental influences, we are unable to predict with a
satisfactory degree of certainty the final manifestation of the growth pattern or
the severity of the malocclusion conferred by a particular genotype.
On the genetics side the advent of diagnostic techniques in the field of molecular
genetics make it possible to identify relevant morphogenes or genetic markers such as
those for mandibular prognathism or to influence the development of malocclusion. It
is therefore incumbent on the orthodontic speciality to keep abreast of developments
in molecular genetics.