Journal of Dental Research

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Genes and Related Proteins Involved in Amelogenesis Imperfecta

G. Stephanopoulos, M.-E. Garefalaki and K. Lyroudia
J DENT RES 2005 84: 1117
DOI: 10.1177/154405910508401206

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 CRITICAL REVIEWS IN ORAL BIOLOGY & MEDICINE
Genes and Related Proteins
Involved in Amelogenesis Imperfecta
G. Stephanopoulos1, M.-E. Garefalaki2,
and K. Lyroudia3*
1Diploma in Dental Science, Aristotle University of Thessaloniki,
Greece; 2Diploma in Biology, Aristotle University of Thessaloniki,
Greece; and 3Department of Endodontology, Dental School, Aristotle
University of Thessaloniki, 23, Papafi Str., 54638 Thessaloniki,
Greece; *corresponding author, lyroudia@zeus.csd.auth.gr
J Dent Res 84(12):1117-1126, 2005
ABSTRACT
Dental enamel formation is a remarkable example of a
biomineralization process. The exact mechanisms
involved in this process remain partly obscure. Some of
the genes encoding specific enamel proteins have been
indicated as candidate genes for amelogenesis imperfecta.
Mutational analyses within studied families have
supported this hypothesis. Mutations in the amelogenin
gene (AMELX) cause X-linked amelogenesis imperfecta,
while mutations in the enamelin gene (ENAM) cause
autosomal-inherited forms of amelogenesis imperfecta.
Recent reports involve kallikrein-4 (KLK4), MMP-20, and
DLX3 genes in the etiologies of some cases. This paper
focuses mainly on the candidate genes involved in
amelogenesis imperfecta and the proteins derived from
them, and reviews current knowledge on their structure,
localization within the tissue, and correlation with the
various types of this disorder.
KEY WORDS: amelogenesis imperfecta, kallikrein-4,
enamelin, amelogenin, DLX3.
Received January 25, 2005; Accepted May 19, 2005
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INTRODUCTION
Ethenamel, dentin, and cementum are the three major constructive
components of human teeth (Schroeder, 1992). Tooth enamel is
most highly mineralized structure in the human body, with 85%
of its volume occupied by unusually large, highly organized
hydroxyapatite crystals (Robinson et al., 1979; Simmer and
Fincham, 1995). In spite of repeated frictional forces during
mastication, enamel is able to resist catastrophic wear. The physical
properties and physiological function of enamel are directly related
to the composition, orientation, disposition, and morphology of the
mineral components within the tissue (Mahoney et al., 2003).
During organogenesis, the enamel transitions from a soft and
pliable tissue to its final form, almost entirely devoid of protein
(Paine et al., 2001). Enamel's final composition is a reflection of the
unique molecular and cellular activities that take place. These
activities are controlled by a regulated expression of multiple genes
(Paine et al., 2001). Deviation from this pattern leads to
amelogenesis imperfecta.
AMELOGENESIS IMPERFECTA
The term 'amelogenesis imperfecta' (AI) represents a group of
inherited disorders which are clinically heterogeneous and exhibit
tooth enamel defects in the absence of systemic manifestations
(Witkop, 1988). Both primary and permanent dentitions are affected
(Aldred and Crawford, 1995). The predominant clinical
manifestations of affected individuals are enamel hypoplasia
(enamel is seemingly correctly mineralized but thin),
hypomineralization (subdivided into hypomaturation and
hypocalcification), or a combined phenotype, which is seen in most
cases (Backman and Holmgren, 1988; Witkop, 1988; Aldred and
Crawford, 1995). The trait of AI can be transmitted by either an
autosomal-dominant, autosomal-recessive, or X-linked mode of
inheritance (Backman, 1997; Aldred et al., 2003). The distribution
of AI types is known to vary among different populations. In a
study in Sweden, 63% of the cases were inherited as autosomal-
dominant (Backman and Holmgren, 1988). In contrast, in a study in
the Middle East, the most common prevalent type of AI was found
to be autosomal-recessive (Chosack et al., 1979; Nusier et al.,
2004).
FROM GENES TO PROTEINS
AND AMELOGENESIS IMPERFECTA
Amelogenin Gene and Protein
The amelogenin gene is a tooth-specific gene expressed in pre-
ameloblasts, ameloblasts, and in the epithelial root sheath remnants
(Hu et al., 2001b; Fong and Hammarström, 2000), while a low
expression of amelogenin mRNAs has been recently shown in
odontoblasts (Nagano et al., 2003; Papagerakis et al., 2003).
Human and bovine amelogenin is expressed by genes located in the
X and Y chromosomes (Nakahori et al., 1991; Gibson et al., 1992;
Salido et al., 1992), while, in the mouse and rat, by a gene located
1117
1118 Stephanopoulos et al. J Dent Res 84(12) 2005

exons and 6 introns (Brookes et

al., 1995) (Fig. 1). Exon 1
contains the 5⬘ untranslated region
of the mature mRNA. The
remaining part of the 5⬘
untranslated region is included in
exon 2, where translation initiation
and the signal peptide sequences
are also encoded. A unique exon
4, not homologous to or evolved
from the exon 4 segment
expressed in humans and rodents,
has been recently identified in the
pig amelogenin gene (Hu CC et
al., 2002). It is noteworthy that
exon 6 shows considerable
variation among species, due to
additional internal sequence
lengths (Brookes et al., 1995).
Exon 7 codes for the final amino
acid, Asp. It also contains the stop
codon and, in humans, has been
shown to contain a putative
polyadenylation site (Salido et al.,
1992). In addition to these known
exons, Li et al. (1998) identified 2
additional exons at the 3⬘ end of
the amelogenin gene in genomic
DNA of the rat. Exons 8 and 9 are
45 and 110 bp long, respectively,
followed by a stop codon and a
polyadenylation site. It has been
confirmed that these two exons
code for proteins present in the
matrix of developing enamel in the
rat (Baba et al., 2002). The
presence of these exons in the
mouse and the human genome was
indicated by Southern blot
analysis (Li et al., 1998). Another
interesting feature is that of
alternative splicing of the pre-
mRNAs, resulting in multiple
transcript variants that encode
Figure 1. Human amelogenin gene. Mutations in amelogenesis imperfecta and phenotypic effect on different isoforms (Simmer, 1995).
enamel. The intron-exon structure of the human amelogenin gene based on the data of Salido et al. Amelogenins represent a
(1992). The numbered cylinders represent the exons, the line the introns. Translation of the signal
sequence initiates in exon 2 (view shape ◆ in diagram), and the translation stop codon is at the beginning family of proteins involved in the
of exon 7 (view shape ▼▲ in diagram). Vertical arrows show the location of the mutations, while the formation of the enamel matrix,
branched boxes include the genomic DNA (upper row) and the mutated protein (lower row). The before the initiation of enamel
phenotypic effects of these proteins, as well as the references where each mutation is described, are biomineralization, and constitute
depicted below the gene representation.
up to 90% of the enamel matrix.
Protein sequencing has been
reported for amelogenins isolated
from different species, including
only in the X chromosome (Chapman, 1991). In human males, humans (Seyer, 1972; Fincham et al., 1981; Zeichner-David et
90% of the amelogenin transcripts are expressed from the X al., 1988; Catalano-Sherman et al., 1993; Cerny and
chromosomal copy of the gene (AMELX) (Xq22), while only Hammarström, 1998, 1999; Lyaruu et al., 1998). A recent
10% is expressed from the gene (AMELY) located at the Y comparison of amelogenin sequences representative of the
chromosome (Yp11). Moreover, the X and Y copies are main mammalian lineages showed highly conserved residues in
processed differently, despite being expressed in the same cells the hydrophilic N- and C-terminal regions, while the central
(Salido et al., 1992). The amelogenin gene is composed of 7 region was more variable (Delgado et al. 2005). Amelogenins

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J Dent Res 84(12) 2005 Genes & Proteins in Amelogenesis Imperfecta
exhibit an unusual amino acid (aa) composition, represented by
relatively high amounts of histidine, glutamine, and leucine,
that confers considerable hydrophobicity to the molecule,
whereas the most abundant amino acid is proline (25-30%).
While, overall, it is hydrophobic, the 12-carboxy-terminal
residues present a hydrophilic motif providing the protein with
a bipolar nature (Snead et al., 1985). Amelogenin is secreted
primarily as a protein of 175 aa (isoform 1), while the signal
peptide consists of 16 aa. Signal peptides typically include
three distinct domains: an N-terminal positively charged (n-
region), a central hydrophobic part (h-region), and a more polar
C-terminal domain (c-region) (von Heijne, 1990). For splice
variants containing exon 4, the mature protein is composed of
189 aa (isoform 3) (Hu et al., 1997a), and for splice variants
lacking exons 3 and 4, the mature protein is composed of 159
aa (isoform 2). The percentages of isoforms 1, 2, and 3 are
80%, 16%, and < 1%, respectively. The functional significance
of these multiple isoforms remains obscure. Experiments in
mice showed that amelogenin is expressed by ameloblasts
throughout the secretory, transition, and early maturation stages
(Hu et al., 2001b). Another event that contributes to the
heterogeneity of the amelogenin molecules presented in the
matrix is the processing of the parent amelogenin molecule
soon after secretion (Simmer and Hu, 2002). The best-studied
models are the porcine and bovine. In the porcine model, the
first cleaving of the 25-kDa parent molecule creates the 23-kDa
peptide (Yamakoshi et al., 1994). A 20-kDa peptide, which has
the same localization pattern as the 25-kDa parent and the 23-
kDa amelogenin, can occur either as a single step, by direct
cleavage of the parent molecule, or via the 23-kDa intermediate
(Brookes et al., 1995). Subsequent cleavage creates the 5-kDa
peptide, called tyrosine-rich amelogenin protein (TRAP)
(residues 1-45) (Fincham and Moradian-Oldak, 1993;
Yamakoshi et al., 1994), and a larger, 13-kDa peptide (residues
46-148) (Tanabe, 1984). While TRAP is relatively insoluble (in
common with most other amelogenin-derived products), the
13-kDa peptide is believed to be unusually soluble and can
diffuse out of the enamel (Brookes et al., 1995). Finally, the
TRAP is degraded to create smaller fragments. Different
degradation products are observed in the bovine model
(Brookes et al., 1995). Amelogenin is found in all compart-
ments throughout the enamel. Intact amelogenins and their C-
terminal cleavage products are detected only in the outer
enamel within 40 ␮m of the surface. It is not known whether
specific amelogenin cleavage products, such as the TRAP,
concentrate within selected compartments in the inner enamel
(Fincham et al., 1994; Uchida et al., 1991b; Simmer and Hu,
2002).
Amelogenin and X-linked Amelogenesis Imperfecta
Molecular studies and mutational analyses in patients with X-
linked AI have established its correlation with the amelogenin
gene. To date, the studies have identified two gene loci that are
correlated with this disorder (Xp22.1-Xp22.3 and Xq24-
Xq27.1) (Lau et al., 1989; Lagerström et al., 1990; Aldred et
al., 1992a). Thus, genetic heterogeneity in X-linked AI may
exist. To date, there are 14 AMELX-associated AI mutations
(Lagerström et al., 1991; Aldred et al., 1992b; Lench et al.,
1994; Lagerström-Fermer et al., 1995; Lench and Winter,
1995; Collier et al., 1997; Kindelan et al., 2000; Sekigushi et
al., 2001a,b; Hart et al., 2002b; Greene et al., 2002; Kim et al.,
2004). With respect to the nomenclature system devised for
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1119
AMELX-associated AI mutations, exon 4 is included in the
numbering scheme, although exon 4 is present in less than 1%
of all transcripts (Hart et al., 2002a). The above studies showed
that mutations that cause changes in domains of the protein
with different functions result in diverse and distinct clinical
manifestations (Wright et al., 2003). The observed phenotypes
in X-linked AI vary considerably in severity, as well as in their
primary features. Great variation also exists between male and
female patients, because males express only one mutant allele,
whereas females show a mosaic pattern of expression, due to
X-chromosome inactivation (Lyonization) (Crawford and
Aldred, 1992; Collier et al., 1997). With respect to the reported
mutations, four are considered to include the signal peptide
(Lagerström-Fermer et al., 1995; Sekigushi et al., 2001a; Kim
et al., 2004) (Fig. 1). A single base substitution (g.11G>A) that
resulted in a premature stop codon, two missense mutations in
exon 2 that affected the translation initiation codon and/or the
secretion of amelogenin (P.W4S and P.M1T), and a deletion of
9 nucleotides that replaced amino acids 5 through 8 with
threonine (p.I5_A8delinsT) resulted in a clinical manifestation
of hypoplasia. A combined phenotype of hypomineralization
with hypomaturation, as a result of a 5-Kb deletion
(g.1148_54del), including all genomic DNA from exon 3
through part of exon 7, was reported in two Swedish families
(Lagerström et al., 1991). There are five described mutations
concerning the C-terminal region of amelogenin (Fig. 1). All
these mutations introduce a premature stop codon (Lench and
Winter, 1995; Kindelan et al., 2000; Greene et al., 2002; Hart
et al., 2002b). Four of these are single deletions in exon 6. The
difference lies in the changes between the point of deletion and
the stop codon. The loss of the C-terminal region results in a
hypoplastic type of AI. The fifth mutation introduces a
premature stop codon late in exon 6 (p.E191X) and a single
nucleotide change (c.571G>T). There are four mutations that
involve the amino-terminal region of amelogenin (Fig. 1).
Three of them are single-nucleotide substitutions causing single
amino-acid changes. The one that occurred in exon 5
(g.3455C>T) resulted in a phenotype described as
hypomineralization/hypomaturation, while the other two,
involving exon 6, resulted in a phenotype of hypomaturation
with discolored enamel (Lench and Winter, 1995; Ravassipour
et al., 2000; Hart et al., 2002b). The fourth was a single-
nucleotide substitution that introduced a premature stop codon
in exon 5 (Lench et al., 1994). Individuals appeared with a
predominant combined phenotype of hypomineralization with
hypomaturation, accompanied by various degrees of
hypoplasia.
Enamelin Gene and Protein
The enamelin (ENAM) gene is a tooth-specific gene expressed
predominantly by the enamel organ and, at a low level, in
odontoblasts (Hu et al., 1998; Hu et al., 2001a; Nagano et al.,
2003). ENAM cDNAs have been isolated and characterized
from the mouse, the pig, and, more recently, from humans (Hu
et al., 1997c, 1998; Hu CC et al., 2000). The human ENAM
gene is localized on chromosome 4 (4q13.3) (Hu CC et al.,
2000) and consists of only 9 exons and 8 introns (Fig. 2A) (the
mouse and pig enamelin genes consist of 10 exons). Intron and
exon sizes vary from 61 to 4171 bp and from 42 to 4805 bp,
respectively. In humans, the sequence corresponding to mouse
exon 2 is absent (Hu CC et al., 2000; Hu et al., 2001a).
However, the search for the intron separating the first and
1120 Stephanopoulos et al. J Dent Res 84(12) 2005

initiation codon is assigned at the

third ATG from the 5⬘ end of the
cDNA (exon 3). The first ATG is
in the appropriate position for
translation, but is followed in 4
codons by a stop signal (TGA).
The second is not appropriate,
since it contains a pyrimidine in
the -3 position. The 3⬘ untranslated
region for humans is particularly
long and contains 4 putative
polyadenylation signals (Hu CC et
al., 2000). The cDNA differs from
the human gene at two positions
that affect the deduced amino acid
of the protein. These differences
appeared to be polymorphisms. In
contrast to what is observed in
ameloblastin and amelogenin, no
alternatively spliced enamelin
mRNAs have been reported (Hu et
al., 1997c; Simmer and Hu, 2002;
Hart et al., 2003a).
Enamelin is the largest and
least abundant protein in the
enamel matrix of developing teeth
and represents roughly 1 to 5% of
total protein amount (Fukae et al.,
1996; Hu et al., 2001a). It is
composed of 1103 aa and a signal
peptide of 39 aa (Hu CC et al.,
2000). The protein was originally
characterized when it was isolated
from unerupted pig teeth (Fukae
and Tanabe, 1987b). A protein
comparison of human, pig, and
mouse enamelin reveals greater
similarity and identity between
human and pig enamelin than that
observed between human and
mouse enamelin. Three sites of
putative phosphorylation and three
N-linked glycosylation sites are
Figure 2. Human enamelin, enamelysin, kallikrein-4, and DLX3 genes in amelogenesis imperfecta. conserved in the human, the
Mutations and phenotypic effects. The exons are represented by cylinders, and introns are represented mouse, and the pig. Also, 5 of 6
by bars. The translation initiation codon is depicted in the diagram as ◆, and the stop codon is depicted cysteines in humans are also
as ▼▲. Vertical arrows show the location of the mutations, while the branched boxes include the genomic
DNA (upper row) and the mutated protein (lower row). The phenotypic effects of these proteins, as well
conserved in pigs and mice (Hu
as the references where each mutation is described, are depicted below the gene representation. (A) The CC et al., 2000). Experiments in
genomic organization of the enamelin gene is based on data from Hu CC et al. (2000) and Hu et al. mice showed that enamelin is
(2001a). The translation start codon is in exon 3, and the translation stop codon is in exon 10. Exon 2 is expressed during the three stages
shaded more darkly, as a reminder that this exon is skipped in human cDNA. (B) The genomic of enamel formation, and its
organization of the human enamelysin gene, based on data from Llano et al. (1997), Simmer et al.
(2000), and Caterina et al. (2000). The translation start codon is in exon 1, and the translation stop expression terminates prior to the
codon is in exon 10. (C) The genomic organization of the human KLK4 gene, based on data by Hu JC et expression of amelogenin (Hu et
al. (2000). The translation start codon is in exon 2, and the translation stop codon is in exon 6. (D) DLX3 al., 2001b). The proteolytic
genomic organization, based on data from Price et al. (1998a). processing of enamelin is very
important for proper enamel
development. The cleaving
second exons in the human gene showed a sequence progresses toward the C-terminal, producing a 142-kDa
homologous to mouse exon 2, flanked by appropriate splice enamelin via a 155-kDa intermediate. Subsequent cleavage
junctions, raising the possibility that alternative splicing may generates the 89-kDa and 34-kDa polypeptides (Fukae et al.,
occur (Hu et al., 2001a; Hart et al., 2003a). The translation 1996). Further processing of the 89-kDa enamelin results in

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J Dent Res 84(12) 2005 Genes & Proteins in Amelogenesis Imperfecta
polypeptides of 32 and 25 kDa (Fukae et al., 1996). The best-
studied enamelin cleavage product is the 32-kDa enamelin
(Tanabe et al., 1990; Uchida et al, 1991a), whose precursor is
the 89-kDa enamelin. It undergoes phosphorylation and
asparagine-linked glycosylation (Fukae et al., 1996;
Yamakoshi et al., 1998). Immunohistochemistry showed that
enamelin is present in the dentino-enamel junction (DEJ)
throughout the entire thickness during the secretory stage and
disappears early in the maturation stage (Uchida et al., 1991b;
Hu et al., 2001a). However, uncleaved protein is found in the
surface enamel near the Tomes' processes of ameloblasts (Hu et
al., 2001a). Many enamelin cleavage products appear to be
rapidly degraded and are found only in the outer enamel. Stable
enamelin cleavage products, principally of 32 kDa, are found
throughout the entire thickness of developing enamel but are
concentrated in the rod and interrod enamel (Fukae et al., 1993;
Simmer and Hu, 2002).
Enamelin and Autosomal-inherited
Amelogenesis Imperfecta
One autosomal-inherited form of AI, namely, autosomal-
dominant amelogenesis imperfecta (ADAI), was linked to a
4Mb region on 4q21 (Kärrman et al., 1997). The ENAM gene
has been mapped within this locus by radiation hybrid analysis
(RHA) and fluorescent in situ hybridization (FISH), and was
therefore considered a candidate gene for this type of AI (Dong
et al., 2000; Hu CC et al., 2000). The reported mutational
analyses of families with AI support the linkage between the
ENAM gene and autosomal amelogenesis imperfecta (AAI)
(Hart et al., 2003). With respect to the nomenclature system,
the ENAM exhibited 10 exons and 9 introns (Hart et al.,
2003a). Kida et al. (2002) first reported an autosomal-dominant
hypoplastic form of AI caused by a single G-deletion within a
series of 7 G residues at the exon 9-intron 9 boundary of the
ENAM gene (g.8344delG). The affected individuals were
heterozygous for the mutation. It was suggested that this
mutation resulted in an alteration of the reading frame from
exon 9 and the introduction of a premature stop codon in the 5⬘
region of exon 10. Hart et al. (2003a) reported a G-deletion in
intron 9, suggesting a potential shortening of exon 9 and the
introduction of a premature stop codon. Mårdh et al. (2002)
described a nonsense mutation in exon 5 (g.2382A>T). It was a
single-base substitution in position 438 that resulted in a
change of lysine to a stop codon, and subsequently to a
truncated protein comprised only of 52 aa, compared with the
wild type. The affected individuals exhibited local hypoplastic
ADAI. Rajpar et al. (2001) reported a heterozygous mutation in
the splice donor site of intron 8 (g.6395G>A). The most
possible scenario was that the mutation caused the skipping of
exon 8, resulting in an in-frame deletion of the amino acid
sequence encoded by this exon. The phenotype of the affected
individuals was described as thin and smooth hypoplastic
ADAI. This type has been shown to map to the critical region
of the ADAI local hypoplastic form, and therefore these two AI
subtypes were considered allelic. Recently, Hart et al. (2003)
described a 2-bp insertion mutation that resulted in a premature
stop codon in exon 10 (g.13185_131186insAG). All affected
individuals were homozygous for the mutation and presented
with open bite and generalized hypoplastic AI. The
heterozygous carriers had localized enamel-pitting defects, and
none had AI or open bite. The importance of this report is that
it is the first to describe the correlation between ENAM and
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1121
autosomal-recessive AI (ARAI). In the same report, genetic
linkage studies were consistent for localization of an ARAI
locus to the AI candidate region of chromosome 4q13.3. Kim et
al. (2005a) identified two ENAM mutations in kindreds with
hypoplastic ADAI, one novel (g.4806A>C) and one previously
identified (g.8344delG). The novel mutation alters the intron 6
splice acceptor site. Two defective splicing outcomes are
probable for this mutation. The first is the inclusion of intron 6
(1615bp). This would insert multiple, in-frame stop codons
preceding the most 3⬘ exon. Translation of this transcript would
add 8 novel aa to 70 aa of the wild-type protein, with the first
39 aa constituting the signal peptide. A second possible
scenario would be the skipping of exon 7. This would maintain
the reading frame, but would delete 87 aa (71-157) from the N-
terminal side of the enamelin protein.
Ameloblastin Gene and Protein
The ameloblastin (AMBN) gene is expressed at high levels by
ameloblasts (Cerny et al., 1996; Fong et al., 1996a; Lee et al.,
1996) and at low levels by odontoblasts and pre-odontoblasts
(Fong et al., 1998; Nagano et al., 2003), while moderate
expression is also observed in Hertwig's epithelial root sheath
(Fong et al., 1996b, 1998), and in odontogenic tumors, such as
in ameloblastomas (Toyosawa et al., 2000). The first reports of
cloning and characterization of cDNAs encoding ameloblastin
rat homologues appeared in 1996 (Krebsbach et al., 1996).
Later, cDNAs from other species, such as the pig and the
mouse, were also published (Hu et al., 1997b; Simmons et al.,
1998). The human AMBN is localized on chromosome 4 at
locus 4q21 (MacDougall et al., 1997), near other genes
associated with mineralized tissues: osteopontin, bone
sialoprotein, and bone morphogenetic protein 3. It consists of
13 exons and 12 introns varying in size from 39 to 1101 bp,
and from 105 bp to approximately 2300 bp, respectively
(Mårdh et al., 2001). The translation initiation codon is located
at the 3⬘ end of exon 1 (putative start site at 84 bp), and the
translation stop codon is located in the middle of exon 13.
Splice site sequences for each intron follow the 5⬘-gt...ag-3⬘
rule. The human AMBN transcript is alternatively spliced, since
cDNA clones of different sizes have been identified
(MacDougall et al., 2000). The clones differ in a 45-bp
fragment that is included or excluded. This stretch of amino
acids is also alternatively spliced in the rat, mouse, and pig
(Cerny et al., 1996; Hu et al., 1997b; Simmons et al., 1998).
According to the human data, this fragment does not
correspond to an independent exon. The 5⬘ end of the 45-bp
sequence is adjacent to intron 5, while the 3⬘ end is within exon
6. This suggests that part of exon 6 can be excluded by the use
of an alternative splice site in exon 6. The above observations
differ from those of Toyosawa et al. (2000) with regard to: (a)
the location of the start site, suggesting that the putative start
codon is at 66 bp (exon 1); (b) the exon-intron sizes; and (c) the
number of putative polyadenylation signals.
Ameloblastin, or amelin (Cerny et al., 1996), or sheathlin
(Hu et al., 1997a) is present in the organic matrix and accounts
for about 5% of the total protein. During investigations of pig
enamel proteins, the N-terminal (Fukae and Tanabe, 1987b)
and C-terminal ends (Fukae and Tanabe, 1987a; Yamakoshi et
al., 2001) of ameloblastin were separately discovered due to
their different biochemical properties. A comparison of the
existing protein sequences for the human, pig, cattle, rat, and
mouse showed high similarity (MacDougall et al., 2000), and
1122
the sequences share several features, such as the presence of
potential phosphorylation sites, similar patterns of
hydrophilicity, and a high proportion of Pro (15.2%), Leu
(10.2%), and Gly (9%) residues. The human precursor protein
is composed of 447 aa, the signal peptide is composed of 26 aa,
and the mature protein of 421 aa. The fate of ameloblastin in
the matrix shares similarities with that of the other proteins.
Soon after secretion, the initial cleavages of the nascent
ameloblastin (65 kDa) generate relatively small polypeptides
containing the N-terminal and relatively large polypeptides
containing the C-terminal. The N-terminal polypeptides appear
to be rather stable and are gradually degraded, but not lost,
during the matrix formation stage, while the C-terminal large
polypeptides appear to be degraded rapidly and are soon lost
from the matrix (Uchida et al., 1997; Brookes et al., 2001).
Intact ameloblastin and its cleavage products containing the C-
terminal are found only in the outer enamel, concentrated
among the crystallites in the rod and interrod enamel
(Murakami et al., 1997). Ameloblastin cleavage products
containing the N-terminal side are found at all depths within
the enamel layer, but they do not present a random distribution
within the tissue; they concentrate in the sheath space (Uchida
et al., 1995). The functional significance of this spatial
distribution is not yet understood.
Enamelysin and Kallikrein-4 Genes and Proteins
The human enamelysin (MMP-20) gene, located in chromosome
11 (11q22.3-q23) (Llano et al., 1997), is comprised of 10 exons
and 9 introns (Caterina et al., 2000), with sizes varying from
104 to 310 bp and 806 to 14,296 bp, respectively. The start
codon (ATG) is located in exon 1, while the stop codon is
located in exon 10 (Fig. 2B). To date, MMP-20 is considered a
tooth-specific gene, since Northern blot analysis of RNAs that
were obtained from multiple human tissues failed to show any
positive hybridization signal with human enamelysin probes
(Llano et al., 1997). Additionally, no other intact,
physiologically normal, tissue has been demonstrated to express
MMP-20, apart from ameloblasts, pre-ameloblasts, and
odontoblasts (Bartlett, 2004), whereas the expression of MMP-
20 in human odontogenic tumors and carcinoma cell lines
originating from the tongue has recently been described (Bègue-
Kirn et al., 1998; Caterina et al., 2000; Takata et al., 2000;
Väänänen et al., 2001). The cloning and characterization of
cDNAs from different species, including humans, pigs, mice,
and cattle, has been reported (Llano et al., 1997; Bartlett et al.,
1998; Den Besten et al., 1998; Caterina et al., 2000).
MMP-20 gene codes for a calcium-dependent (Bartlett et
al., 1998) proteinase that is a member of the matrix
metallopeptidases family (MMPs) (Rawlings et al., 2004). This
protein family is characterized by a common domain structure,
which also applies in the case of MMP-20. MMP-20 is divided
into the following domains: signal peptide (1-22), propeptide
(23-107), catalytic domain (108-271), linker (272-295), and
hemopexin (296-483). The pre-proprotein has 483 aa, the
proprotein has 461 aa, while the active protein has 376 aa
(Simmer and Hu, 2002). However, MMP-20 possesses several
unique structural features that define it as a novel MMP. First,
the MMP-20 amino acid sequence contains no N-linked
glycosylation sites (Bartlett et al., 1996). Second, it lacks two
of the three residues important in defining the active site of a
collagenase, and lacks all three of the residues important in
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International and American Associations for Dental Research
Stephanopoulos et al. J Dent Res 84(12) 2005
defining the active site of a stromelysin (Bartlett et al., 1996;
Llano et al., 1997). Third, MMP-20, in contrast to stromelysins
and collagenases, has a unique hinge region of 24 residues that
connects the catalytic domain to the hemopexin-like domain
(Bartlett et al., 1996; Llano et al., 1997; Caterina et al., 2000).
An amino acid sequence comparison between human MMP-20
(Llano et al., 1997) and that reported for porcine MMP-20
revealed a high percentage of identity (89%). The active
protease migrates as a doublet at 46 kDa and 41 kDa on
zymograms (Yamada et al., 2003). MMP-20 is the early
protease, and it is expressed throughout the secretory stage and
part of the maturation stage (Bartlett et al., 1996; Bègue-Kirn et
al., 1998; Bartlett and Simmer, 1999). Immunohistochemical
studies have showed the presence of enamelysin within the
secretory enamel, with the greatest staining occurring adjacent
to the secretory face of Tomes' process (Fukae et al., 1998).
Kallikrein-4 (KLK4), or prostase, or EMPS1, or KLK-L1, or
PRSS17 (serine proteinase 17) was first extracted in 1977 from
developing porcine enamel (Fukae et al., 1977), and the first
cDNA and protein sequence was deduced from a cDNA library
(Simmer et al., 1998). Different groups have isolated the
human cDNA from a variety of tissues, such as the central
nervous system and the prostate (Nelson et al., 1999;
Stephenson et al., 1999), and, more recently, from developing
human tooth tissues (Simmer et al., 2000). The KLK4 gene is
located near the telomere of chromosome 19 (19q13.3-19q13.4)
downstream of the KLK2 gene, and is considered a member of
the human tissue kallikrein gene family (Du Pont et al., 1999;
Diamantis et al., 2000; Hu JC et al., 2000). A further
characterization of the KLK4 gene, extending its 3⬘ downstream
sequence and identifying potentially important polymorphisms,
has been reported (Hu JC et al., 2000). These investigators also
identified an additional exon. The human DNA sequence is of
7115 bp and consists of 6 exons (5 of which are coding) and 5
introns. The exons vary in sizes from 72 to 251 bp, and intron
sizes vary from 83 to 1357 bp. The translation initiation codon
(ATG) is located in exon 2 (first coding exon) (660-662 bp),
and the termination codon (TGA) is located in exon 6 (fifth
coding exon) (5108-5110) (Fig. 2C). The sequences encoding
the amino acids of the catalytic triad presented in the enzyme
are located at coding exons 2, 3, and 5, respectively. KLK4 is
expressed by both ameloblasts and odontoblasts (Hu JC et al.,
2000; Nagano et al., 2003). A splice variant lacking exon 6 has
been described in skin and endometrial tumor cultures (Myers
and Clements, 2001; Komatsu et al., 2003), but its role has not
yet been defined.
KLK4 protein is a calcium-independent serine protease.
KLK4 is secreted as an inactive zymogen of 230 aa that
becomes the active protein (224 aa) with the removal of a 6 aa
propeptide by MMP-20 (Ryu et al., 2002). Human KLK4 has
six disulfide bridges and one potential N-linked glycosylation
site away from the active site of the enzyme. Of great
importance for KLK4's function is a triad of catalytic amino
acids (H71, S207, and D116) (Komatsu et al., 2003). Despite
its most recent official designation, KLK4 is no more closely
related to the original kallikreins than it is to the trypsins. The
kallikrein loop, a structural feature common to the kallikrein
proteases, is absent in KLK4. A single amino acid insertion that
is common to the pig, mouse, and human gene is absent from
the kallikreins (Hu JC et al., 2000; Ryu et al., 2002). KLK4 is
the late protease; its expression by ameloblasts begins in the
J Dent Res 84(12) 2005 Genes & Proteins in Amelogenesis Imperfecta
transition stage and continues throughout enamel maturation
(Hu JC et al., 2000, 2002). The KLK4 activity observed in the
highly mineralized enamel at the dentino-enamel junction
during the secretory stage is due to the gene's expression from
the odontoblasts (Fukae et al., 2002). KLK4 is responsible for
the degradation of the TRAP amelogenin cleavage product to
smaller fragments.
DLX3 Gene and Protein
The DLX3 gene is a member of the family of homeobox genes
that are homologous to the distal-less (Dll) gene of Drosophila,
known to be expressed during development of the
chondrocranium, dermatocranium, sensory organs, brain, limbs,
and appendages, and in the processes of osteogenesis and
hematopoiesis (Robinson and Mahon, 1994; Weiss et al., 1998).
The mammalian genes take the form of bigene clusters, namely,
Dlx2-1, Dlx5-6, and Dlx3-7. Scherer et al. (1995) mapped the
human homologue of the mouse Dlx3 gene to 17q21.3-q22,
which consists of 3 exons, with the homeodomain contained in
exons 2 and 3. Exons 1 and 2 are separated by a 1.1-kb intron;
exons 2 and 3 are separated by a 1.6-kb intron (Fig. 2D). The
start codon is located in exon 1, while the stop codon is in exon
3 (Price et al., 1998a). The encoded DLX3 human protein
(GenBank accession number NP_005211) is a 31738-Da protein
composed of 287 aa with a 60 aa homeodomain (129-188 aa).
As with all DLX proteins, it shares similar DNA-binding sites
and is thought to act as a homeodomain transcription factor,
having N-terminal and C-terminal regions that act as
transcriptional activators (Bryan and Morasso, 2000). Therefore,
the presence of these proteins is considered critical for
craniofacial, tooth, brain, hair, and neural development.
Ameloblastin, Enamelysin, Kallikrein-4,
and DLX3 in Amelogenesis Imperfecta
AMBN gene loci are on chromosome 5 in the mouse (Krebsbach
et al., 1996) and chromosome 4q21 in humans (MacDougall et
al., 1997). AMBN maps within the critical region for autosomal-
dominant AI, and therefore is considered as a candidate gene.
However, it was excluded from a causative role by mutational
analyses within the families studied by Mårdh et al. (2001). As
far as KLK4 is concerned, Hart et al. (2004) identified the
mutation (g.2142G>A) (Fig. 2C) that resulted in a truncated
protein containing 152 aa and lacking the S207 site, which, as
we previously mentioned, is essential for the function of the
enzyme. Due to the abnormal enzymic activity, the crystallites
of the enamel grew to the normal length but incompletely in
thickness (Hart et al., 2004). This is the first report of mutation
in this gene found to cause ADAI. Kim et al. (2005b) identified
a homozygous mutation in the MMP-20 gene in two affected
members of a family with autosomal-recessive pigmented
hypomaturation AI. The mutation destroyed the splice acceptor
at the 3⬘ end of intron 6 (AG→ TG) and resulted in a
hypomatured enamel product. Two defective splicing outcomes
seem to be most probable, both of which would introduce an
upstream translation termination codon in the mRNA transcript.
In the first scenario, the retention of intron 6 in the mRNA
would generate a large mRNA with translation terminating in
the first complete codon of the retained intron (p.I1319X). In the
second splicing scenario, exon 7 would not be recognized as an
exon and would be skipped. Skipping exon 7 would shift the
reading frame after R318 and would introduce 19 extraneous aa
before terminating translation (p.I1319fs338X) (Kim et al.,
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International and American Associations for Dental Research
1123
2005b) (Fig. 2B). A comparison of the dental phenotypes of the
KLK4 and MMP-20 probands shows that they share many
similar features (Kim et al., 2005b).
Dong et al. (2005) demonstrated that a mutation within the
human DLX3 gene homeodomain is associated with
amelogenesis imperfecta (hypoplastic-hypomaturation type),
with taurodontism (AIHHT). This 2-bp deletion (CT) in exon 3
(Fig. 2D) causes a frameshift within the last two amino acids of
the homeodomain, with a premature stop codon truncating the
protein by 88 aa. This is the first report of a mutation within the
homeodomain of DLX3. Previous studies have shown a DLX3
mutation, outside the homeodomain, associated with
trichodento-osseous syndrome (TDO) (Price et al., 1998a,b).
Dong et al. (2005) suggested that TDO and some forms of
AIHHT are allelic. However, this requires further investigation.
However, AMELX, AMBN, ENAM, KLK4, and MMP-20
were excluded from a causative role in two families with
autosomal-dominant hypocalcified AI, suggesting that this type
of AI is caused by mutation of a gene that is either not known
or not considered to be a major contributor to enamel formation
(Hart et al., 2003b).
CONCLUSIONS
Amelogenesis imperfecta (AI) is a group of inherited defects of
dental enamel formation that shows both clinical and genetic
heterogeneity. Great progress has been made regarding the
definition of the genetic background in AI. To date, mutations
in 5 genes (AMELX, ENAM, KLK4, MMP-20, and DLX3) have
been found to cause AI. Alterations in the AMELX gene are
responsible for X-linked AI. The various enamel phenotypes
observed in families with X-linked AI correlate with the sites
of mutation within the coding region of the amelogenin gene.
Mutations in the ENAM, KLK4, and MMP-20 genes cause AI
with the autosomal pattern of inheritance. Recently, a mutation
within the DLX3 gene has been described and associated with
AIHHT. AMBN is another candidate gene for autosomal-
dominant amelogenesis imperfecta, but the involvement of this
gene in the disease has not yet been established. However,
genes that have so far been proposed as candidates for AI do
not cause some forms of autosomal-dominant hypocalcified AI.
It is expected that future research will help establish genotype-
phenotype correlations for all autosomal forms of AI. As the
knowledge regarding genetic mutations associated with the
various types of AI increases, our ability to make an accurate
diagnosis of AI will be remarkably improved. The similarity in
the clinical features of the different AI types makes the
differentiation among them difficult at the clinical level.
Correlation of the genetic mutations with the clinical
phenotypes will be extremely valuable for managing patients
with AI. Moreover, this knowledge allows us to make better
predictions of the AI types that are associated with problems
such as calculus formation and skeletal open bites, and to select
the most optimal treatment approaches for each case.
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