By safna c.t.k
Tooth agenesis is the most prevalent craniofacial congenital
malformation in humans. [1] Up to 25% of the population may lose at
least one-third molar. [1] Agenesis of other permanent teeth ranges
from 1.6% to 9.6% depending upon the population studied. [2]
Traditionally, tooth agenesis is classified depending on the number of
missing teeth as either hypodontia [3] (up to six missing teeth) or
oligodontia [4],[5] (more than six missing teeth). While tooth agenesis
may be associated with several syndromes, non-syndromic
hypodontia refers to the congenital absence of a few teeth in the
absence of any other deformity.
The genetic basis of hypodontia has been a topic that has been keenly
studied by investigators ever since early studies showed that
hypodontia tended to run in families. [6] However, identifying the
exact genes responsible for the development of teeth and tracing the
mutations that cause hypodontia has only been made possible with
the recent advances in molecular genetics. The aim of this paper is to
review the genetic basis of hypodontia, the methods used to study it,
and identify the genes that have been definitively implicated in the

Early studies: The use of gene locus models to explain inheritance
The inheritance patterns of genes was first described by Gregor Mendel in
1865 [7] and extensively studied in the early part of the 20th
century. [8] While the traditional Mendelian model of inheritance is
extremely useful in studying traits caused due to a single gene, it does not
give an accurate description of traits caused by multiple genes. [9] This
was the reason that early investigators found that hypodontia could be
either an autosomal dominant, [10] autosomal recessive, [11] or X-linked
trait. [12] The limitations of the Mendelian model also meant that the
genetic studies often focused on groups with a limited gene pool, such as
groups with a history of consanguineous marriage [13] or isolated island
populations. [11]
By the 1970s, researchers had understood that all diseases had some
component of genetic risk, leading to the advent of Genome Wide
Association Studies (GWAS) and the use of complex mathematical
models to predict genetic risk and transmission patterns. [14] Using these
models it was shown that hypodontia fit the multiple locus model rather
than a single locus model proving that the initiation of tooth development
was controlled by more than one gene. [6

Experimental studies: The use of mouse models to identify the genes
In 1913, Bridges showed that genes were located on chromosomes, [15] but it was not until the
early 1970s that a variety of cytogenetic methods were discovered that produced distinct bands
on each chromosome [16],[17] making it possible to give each gene a specific "genetic
address." [18] Even though the first draft of the entire human genome was completed only in
2001, [19] by the 1990s researchers in oral biology were studying the dental implications of the
decoding of the human genome. At the forefront of research into the genetics of tooth
development were the homeobox genes. [20]
Homeobox (Hox) genes are a set of genes that determine an organizational pattern in
vertebrates. [21] First isolated in the fly Drosophila melanogaster, the temporal and spatial control
of Hox gene expression is essential for correct patterning of many animals. [22] In mammals, 38
Hox genes have been identified which reside in four main chromosomal clusters, termed Hoxa,
Hoxb, Hoxc, and Hoxd, and define 13 paralogous groups. Other vertebrate genes, such as the
Msx gene family, also contain homeoboxes, but since these are dispersed to different
chromosomal locations in the genome, they are referred to as "homeobox genes" rather than
"Hox genes." [23] Remarkably, the relative order of mammalian Hox genes in each cluster
parallels the order of the related genes in the Drosophila HOM-C, a paralogous relationship
which permits the mammalian Hox genes to be grouped into the 13 discrete groups. [20] The
relative similarity of the homeobox system in all mammals meant that research into human
odontogenesis could be carried out on animal models. Due to its suitability for both genetic
and embryologic studies, the mouse emerged as the predominant system for contemporary
experimental studies on tooth development. [20] Most of the early genetic analyses of tooth
development, which have formed the basis of our understanding of the genetics of hypodontia,
were done by the use of mouse mutations. [24] These could be accomplished by the use of the
following methods:

Knockout mice
Transgenic Mice
In knockout technology, a known gene is selectively targeted
for disruption in embryonic stem (ES) cells by the principle of
homologous recombination. [25] Reconstitution of ES cells in
chimeric mice and germline transmission results in mice which
carry a loss-of-function, typically recessive, mutation in a
known gene. These mice can then be bred to homozygosity and
the phenotypic consequences of the mutation assessed during
embryogenesis. [20] The disadvantage of knockout mutations
was that the gene that is knocked out may have growth
implications beyond the development of the tooth resulting in
the failure of the embryo to form, a problem that was overcome
by the development of conditional knockout systems. [26]
In transgenic technology, mutations of the genes responsible for tooth development are chosen and
extracted. Next they are injected into fertilized mouse eggs. Embryos are implanted in the uterus of
a surrogate mother. The selected genes will be expressed by some of the offspring allowing
investigators to study the effects of the gene. [27]
Using mouse models, by late 1990s researchers had managed to identify the genes responsible for
mammalian tooth formation. [28] In 1998, Thomas and Sharpe proposed that the patterning of
murine dentition was regulated by a complex interaction of the homeobox genes and termed it the
"Odontogenic Homeobox Code." [29] It was found that the genes responsible for tooth formation
comprised of transcription factors, growth factors, and receptors. [30]
A transcription factor (sometimes called a sequence-specific DNA binding factor) is a protein that
binds to specific DNA sequences and thereby controls the transfer (or transcription) of genetic
information from DNA to mRNA. [31] The Msx [32] and Dlx [33] families of homeobox genes are
examples of transcription factors that control tooth genesis. A growth factor is a naturally occurring
substance capable of stimulating cellular growth, proliferation, and cellular differentiation, matrix
metalo proteinase (MMP) [34] and fibroblast growth factor (FGF) being an examples of such factors.
[35] Receptors are protein molecules, embedded in either the plasma membrane or the cytoplasm of
a cell, to which one or more specific kinds of signaling molecules may attach. The epithelial growth
factor receptor (EGFR) being an example of such a receptor. [3]
Using these mouse models Vieira in 2003 proposed that, "Specific genes were responsible for specific
missing teeth" in mice [Table 1]. [36] In order to know which of these genes to look for in humans
and ascertain the location of these genes, oral clefts and syndromic forms of tooth agenesis were
proposed as suitable genetic models. [36

The association between syndromic and non-syndromic hypodontia
Teeth develop in the context of the whole craniofacial region and recruit conserved developmental
cascades common to the morphogenesis of other oro-facial structures and ectodermal derivatives.
Hence, several syndromes involving hypodontia as a primary feature display various dysplasias and
syndromic clefts. [1]
It has long been known that individuals with cleft lip and palate (CLAP) often have missing teeth.
While some of these missing teeth may be a result of the cleft itself or the associated surgery, it has
been shown that the more severe the cleft the greater the number of missing teeth. [37] The MSX 1 gene
was the first gene to be studied for mutations linked to both CLAP and tooth
agenesis. [38],.[39],[40] Further evidence for the possible association of MSX1 as a cause of tooth agenesis
came when it was shown that a nonsense mutation in MSX1 causes the tooth agenesis and nail
dysgenesis associated with the Witkop tooth and nail syndrome. [41] The locus of the MSX1 gene is
located on the short arm of chromosome 4 (4p) the absence of which is manifested as the WolfHirschhorn syndrome. [42] The knowledge of genetic contribution to tooth agenesis was further
enhanced with the discovery of the RIEG gene. The RIEG gene causes Rieger syndrome, an autosomal
dominant condition associated with missing teeth and anomalies of the eyes and the
umbilicus. [43] While these mutations explained the autosomal forms of tooth agenesis, they did not
explain X-linked tooth agenesis.
The association between ectodermal dysplasia and tooth agenesis is a well-established one.
Hypohydrotic/anhydrotic ectodermal dysplasia is an X-linked recessive disorder that is caused by
mutations on the EDA gene. [44] Virtually all persons with hypohydrotic ectodermal dysplasia express
hypodontia. [45] However, mutations of the EDA gene may cause hypodontia without other syndromic
features. [46] This suggests that genes involved in hypodontia associated with other syndromic features
may be promising candidates for non-syndromic hypodontia. [1]

Gene mapping and familial studies: finding the genes responsible for
non-syndromic tooth agenesis
In order to locate a gene, it has to first be localized to a chromosome a
technique that is popularly referred to as gene mapping. The method that
has been extensively used in oral biology research is the genetic linkage
analysis. Genetic linkage analysis relies on the investigator's ability to
detect recombinations that occur during meiosis due to independent
assortment and crossings-over of homologous chromosomes. If two loci
are located close to each other in the same chromosome, the chance of a
recombination between them is low, and they are said to be linked. [3] The
genetic distance of a disease locus and a marker can be evaluated by the
amount of recombinations between them in a pedigree. The use of this
technique was greatly facilitated by the by the identification of highly
polymorphic microsatellite markers that could be easily analyzed by a
polymerase chain-reaction (PCR). [3] Using gene mapping techniques on
families known to have hypodontia and/or oligodontia investigators
have been able to definitively link several gene mutations with tooth
agenesis [Table 2].


MSX1, initially called homeobox 7 (HOX7), is a non-clustered homeobox protein which is
located on the small arm of chromosome 4 with the genetic address 4p16.3-p16.1. [47] This
gene encodes a member of the muscle segment homeobox gene family. The encoded protein
functions as a transcriptional repressor during embryogenesis through interactions with
components of the core transcription complex and other homeoproteins. [48] The gene has
been shown to have a considerable role in tooth development. [49] It was the first gene to be
definitively associated with human tooth agenesis. [38] While MSX1 mutations have generally
been associated with autosomal dominant inheritance of hypodontia [38] as well as
oligodontia, [50] recently an autosomal recessive type of hypodontia was shown to be
associated with an MSX1 mutation [51] [Table 2].
PAX9 is a member of the paired box (PAX) family of transcription factors. The PAX 9 gene is
located on the long arm of chromosome 14 and has the genetic address 14q12-q13. [52] These
genes play critical roles during fetal development and cancer growth. [53] The PAX9 gene was
first shown to be associated with autosomal dominant, non-syndromic, familial oligodontia. [5]
Since then several novel mutations in the gene have been discovered in families around the
world [54],[55],[56],[57] [Table 2]. In addition, a recent study has also found that families with
affected benign hereditary chorea show a deletion of the PAX9 gene resulting in oligodontia.
[58] Peg-shaped lateral incisors and other forms of microdontia have long been known to be
mild forms of hypodontia. [59],[60] PAX9 mutations have been associated with both
hypodontia and a generalized reduction of the size of the teeth. [61]

AXIN2 or axis inhibitor protein 2 is a gene located on the long arm of
chromosome 17 with a genetic address of 17q23-q24. [62] The association of
the gene to tooth agenesis was first found in a Finnish family with a
predisposition for colorectal cancer. [63] It has been shown that the AXIN2
mutations may also be responsible for sporadic forms of incisor
agenesis. [64] The mode of transmission of hypodontia due to defects in
the AXIN2 gene has not been definitively proved.
LTBP3 (latent transforming growth factor beta binding protein 3) is a
gene that modulates the bioavailability of TGF-beta. [65] Located on the
long arm of chromosome 11, it has the genetic address 11q12. [66] A study
on a Pakistani family with a history of consanguineous marriage found
that a mutation in the LTBP3 gene causes an autosomal recessive form of
familial oligodontia. [67]
EDA (ectodysplasin 1) is a gene located at Xq12-q13.1 that has been linked
to X-linked recessive ectodermal dysplasia. [44] A study of Chinese
families with non syndromic X-linked hypodontia showed that a
Thr338Met mutation of the EDA gene was responsible for the congenital
absence of maxillary and mandibular central incisors, lateral incisors, and
canines, with the high possibility of persistence of maxillary and
mandibular first permanent molars. [46]

Missing permanent teeth in a child are a matter of concern to most
parents. The ability to rebuild a tooth that is congenitally missing or lost
due to disease is therefore a great boon to the pediatric dentist. Although
the dream of genetically engineering teeth still remains a distant one, the
study of tooth agenesis and genes producing the molecules involved in
tooth agenesis have led to the development of experimental tooth
regeneration techniques such as tissue scaffolding [68] and tooth
engineering. [69] In addition, the association of genes such as AXIN2 and
PAX9 to both tooth agenesis and colorectal cancer has shown the
potential of using tooth agenesis as a genetic marker for the diagnosis of
cancer. [63],[64],[70],[71]
While this paper has reviewed the genes and their mutations, most likely
to cause tooth agenesis the list is far from complete. Several forms of
tooth agenesis remain unexplained. Research into tooth agenesis today is
increasingly relying on familial studies to locate mutations in the
genes. [38] As the person who first detects the problem the pediatric
dentist is in the best position to chart the pedigree of the family and
determine the type of inheritance pattern. An understanding of the basic
concepts of genetics will enable pediatric dentists to contribute greatly to
future research into the emerging fields of tooth regeneration and
molecular dentistry
.


To identify genes downstream of activin signaling in tooth development, the expression of well-characterized
mesenchymal and epithelial marker genes was examined in mutant embryo teeth. The expression patterns in
mutant mandibular molars were compared with those in the normal maxillary molar tooth germs. Expression
in the mutant incisors was compared with expression in incisors of wild-type or heterozygous littermates
(Fig. 8). Radioactive in situ hybridization was performed on wild-type, heterozygous, and homozygous mutant
embryos up to the bud stage. The expression patterns of genes examined included Barx1, Msx1, Dlx2, Pax9,
Gli3, Lef1, syndecan-1, Tgfβ1, Tgfβ3, Bmp4, Bmp7, Sonic hedgehog (Shh), CD44, and Otlx2.
In wild-type teeth at the bud stage, Barx1 gene expression is restricted to the molar region of the mandible and
maxilla but is present in a broad field of neural crest derived mesenchymal cells rather than being restricted to
dental mesenchyme (Fig. 8A;Tissier-Seta et al. 1995). Msx1, Lef1, and Bmp4 are expressed in the dental
mesenchyme (i.e., the condensing mesenchymal cells associated with invaginating incisor and molar epithelial
tooth buds) in response to epithelial signaling (Fig. 8B,F,J; Mackenzie et al. 1991; Vainio et al. 1993;Kratochwil
et al. 1996). It is thought that these genes are then involved in reciprocal signaling from mesenchyme to
epithelium, which is required for epithelial cell differentiation including the formation of the enamel
knot.Dlx2expression is restricted to mesenchymal cells immediately surrounding the epithelial bud, but is also
present in the dental epithelium on the buccal side of the buds (Fig. 8C; Thomas et al. 1995;Qui et al.
1997). Pax9 is expressed in early tooth mesenchyme prior to bud formation and subsequently in condensing
mesenchyme at the bud stage (Fig. 8D; Neubüser et al. 1997). Gli3 is expressed in the mesenchyme from E10.5.
At the bud and cap stageGli3 expression is slightly more localized than Pax9expression, and is restricted to the
dental papilla and dental follicle (Fig. 8E; Hardcastle and Sharpe 1998). Syndecan-1, a cell surface heparin–
sulfate proteoglycan is transiently expressed in the dental mesenchyme and is thought to regulate dental
mesenchymal cell condensation beneath the invaginating dental epithelium (Fig. 8G;Thesleff et al.
1996). Tgfβ1 is found in the dental mesenchyme and weakly in the epithelium of the incisors and only appears
in the molars in the dental epithelium at the cap stage (Fig.8H; Vaahtokari et al. 1991). Tgfβ3 expression is
widespread in the mesenchyme of the face but, interestingly, its expression appears to be excluded from the
condensin mesenchymal cells immeadiately adjacent to the epithelial buds of incisors and molars (Fig. 8I; Chai
et al. 1994).