GENETICS AND

MALOCCLUSION

PART-1 : BASIC GENETICS & ITS

SIGNIFICANCE IN MALOCCLUSION AND
CRANIOFACIAL ANOMALIES
 INTRODUCTION
HUMAN CHROMOSOMES
DNA: THE HERIDITARY MATERIAL
GENETIC CODE
STRUCTURE OF GENES
DEVELOPMENTAL GENE FAMILIES
HOMEOBOX GENES AND ITS IMPORTANCE
TWIST GENES
GROWTH FACTORS AND ITS SIGNIFICANCE
MUTATION AND ITS TYPES
MUTATIONAL TRACKING AND MOLECULAR APPROACHES
PATTERNS OF INHERITANCE
POLYGENIC AND MULTIFACTORIAL INHERITANCE
SEGREGATION AND LINKAGE ANALYSIS
TWINNING AND SIGNIFICANCE OF TWIN STUDIES
SIGNIFICANCE OF GENETIC STUDIES FOR MALOCCLUSION
AND CRANIOFACIAL ANOMALIES
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
X- Chromosome chromosome15.
short & long arm
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.

Gene map of the human genome
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
53 direction and the other 35 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 individuals
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.
1) Hedgehog (Vertebrates)
Sonic Hedgehog
Desert Hedgehog
Indian Hedgehog
2) 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
HOX Number of Chromosome
Cluster genes location
HOXA
(=HOX1)
11 (1-7, 9-11, 13) 7p
HOXB
(=HOX2)
10 (1-9, 13) 17q
HOXC
(=HOX3)
9 (4-6, 8-13) 12q
HOXD
(=HOX4)
9 (1, 3, 4, 8-13) 2q

The 39 human HOX genes are organized in four clusters on four

chromosomes. They are derived from a single ancestral cluster from
which the single HOM-C complex in Drosophila is also derived.
Antennapedia = labial, proboscipedia, Deformed, Sex combs
reduced, Antennapedia
Bithorax = Ultrabithorax, abdominalA, AbdominalB
Cluster duplication during evolution has led to the concept
of paralogous groups of HOX genes. Thus, groups of up to
four genes derived from a common ancestral gene in the
primitive cluster can be identified based upon sequence
homology. The paralogues can exhibit similar experssion
domains along the antero-posterior axis of the embryo
leading to the concept of functional redundency between
genes.
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 -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-)
a) TGF- 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-
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

Of all these factors, FGFs and BMPs are important

for craniofacial development.
FIBROBLAST GROWTH FACTORS (FGFs) AND ITS
RECEPTORS (FGFRs)

FGFs comprise a family of 22 genes encoding structurally

related proteins (Ornitz and Itoh 2001).
FGF1 (acidic-FGF, FGF) and FGF2 (basic-FGF, FGF) are
the main factors.
Studies of human disorders & gene knock-out studies in mice
show the prominent role for FGFs is in the development of the
skeletal system in mammals. 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 (FGFR1,
FGFR2, FGFR3 and FGFR4) 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).

GENE Chromosome location

FGFR1 8p11

FGFR2 10q25

FGFR3 4p16
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 ligandreceptor 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
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.
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
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
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
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.