Validation of amelogenesis imperfecta inferred from amelogenin evolution.
ABSTRACT We used the evolutionary analysis of amelogenin (AMEL) in 80 amniotes (52 mammalian and 28 reptilian sequences) to aid in the genetic diagnosis of X-linked amelogenesis imperfecta (AIH1). Out of 191 residues, 77 were found to be unchanged in mammals, and only 34 in amniotes. The latter are considered crucial residues for enamel formation, while the 43 residues conserved only in mammals could indicate that they play new, important roles for enamel formation in this lineage. The 5 substitutions leading to AIH1 were validated when the mammalian dataset was used, and 4 of them with the amniote dataset. These 2 sequence datasets will facilitate the validation of any human AMEL mutation suspected of involvement in AIH1. This evolutionary analysis also revealed numerous residues that appeared to be important for correct AMEL function, but their role remains to be elucidated.
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ABSTRACT: Dental enamel is a product of ameloblast cells, which secrete a mineralizing organic matrix, composed primarily of amelogenin proteins. The amelogenins are thought to be crucial for development of normal, highly mineralized enamel. The X-chromosomal amelogenin gene is a candidate gene for those cases of amelogenesis imperfecta, resulting in defective enamel, in which inheritance is X-linked. In this report, a kindred is described that has a C to A mutation resulting in a pro to thr change in exon 6 of the X-chromosomal amelogenin gene in three affected individuals, a change not found in unaffected members of the kindred. The proline that is changed by the mutation is conserved in amelogenin genes from all species examined to date.Archives of Oral Biology 04/1997; 42(3):235-42. · 1.55 Impact Factor
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ABSTRACT: A method for sex identification of the Japanese black bear was examined using a polymerase chain reaction (PCR) and sequencing of a part of the amelogenin gene. This gene is located on the X and Y chromosomes, and there are 54 nucleotide deletions on the Y chromosome-specific gene. Forty-seven (26 male and 21 female) DNA samples and 23 (13 male and 10 female) DNA samples, respectively extracted from white blood cells and hairs of Japanese black bears were analyzed. The primers SE47 and SE48 from this X-Y homologous region were used in sex identification by PCR amplification. These primers amplified X- and Y-specific bands, which could be used to discriminate between sexes by a length polymorphism in all samples. We suggest that PCR amplification using the primers SE47 and SE48 is useful for sex determination of the Japanese black bear and could be applied to DNA analysis of small samples such as hairs.Journal of Veterinary Medical Science 07/2002; 64(6):505-8. · 0.88 Impact Factor
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ABSTRACT: The phylogeny and timescale of life are becoming better understood as the analysis of genomic data from model organisms continues to grow. As a result, discoveries are being made about the early history of life and the origin and development of complex multicellular life. This emerging comparative framework and the emphasis on historical patterns is helping to bridge barriers among organism-based research communities.Nature Reviews Genetics 12/2002; 3(11):838-49. · 41.06 Impact Factor
chromosomes, but in males, 90% of the transcripts are expressed from
AMELX (Salido et al. 1992). AMEL plays a crucial role in enamel
formation, but its exact functions are not totally understood (Paine et al.,
2003). Its importance is well-illustrated, however, by the occurrence of a
genetic disease, X-linked amelogenesis imperfecta (AIH1), resulting from
AMEL mutations leading to various hypoplastic and hypomature enamel
phenotypes. To date, 14 mutations leading to AIH1 are known (Hart et al.,
2002a; Kim et al., 2004). The characterization of these mutations helps in
identifying particular regions, or specific residues, that play a crucial role in
AMEL function (Collier et al., 1997; Ravindranath et al., 1999). However,
the few AMEL mutations known so far are insufficient to target all important
Mutational analyses are time-consuming and expensive: analysis of the
individual's pedigree, mapping the mutation on a chromosome to identify a
candidate gene, sequencing, and sequence analysis to validate the mutation.
Moreover, AMEL polymorphism could lead to diagnostic errors in the
clinical context, and this possibility is largely underestimated. Indeed, if a
person has an enamel defect, and there is a pedigree consistent with an X-
linked mutation, then a polymorphism in AMELX is unlikely to be the cause
of the defect.
Evolutionary analysis is an alternative for validating the AMEL
mutations responsible for AIH1, and for highlighting all the residues that
are important for the protein to function correctly (Delgado et al., 2005;
Sire et al., 2005, 2006). Such an analysis is based on the following
postulates: (i) Important residues must remain unchanged, because their
change or loss could lead to severe enamel defaults; (ii) conversely, less
important residues can be substituted without damage to enamel
structure and organization, and must therefore be considered
polymorphisms; and (iii) given the slow rate of mutations in most
lineages, the studied sample must cover a large evolutionary period and
must be representative of the various lineages in which the protein has
similar functions. This is the case in mammals and reptiles (amniotes), in
which enamel structure is roughly similar (Sander, 2000), although both
lineages separated approximately 310 million years (my) ago (Hedges,
2002). Nevertheless, in reptiles, teeth are continuously replaced during
life (polyphyodonty), and the constraints acting on enamel structure
could be less important than in mammals, which are diphyodont or
In the present study, we compiled 52 mammalian and 28 reptilian
AMEL sequences, with the aim of obtaining datasets that could be useful for
a rapid and accurate validation of the mutations responsible for AIH1.
melogenin (AMEL) is the major protein of forming enamel. In humans,
the amelogenin genes (AMEL) are located on the X and Y
We used the evolutionary analysis of amelogenin
(AMEL) in 80 amniotes (52 mammalian and 28
reptilian sequences) to aid in the genetic diagnosis
of X-linked amelogenesis imperfecta (AIH1). Out
of 191 residues, 77 were found to be unchanged in
mammals, and only 34 in amniotes. The latter are
considered crucial residues for enamel formation,
while the 43 residues conserved only in mammals
could indicate that they play new, important roles
for enamel formation in this lineage. The 5
substitutions leading to AIH1 were validated when
the mammalian dataset was used, and 4 of them
with the amniote dataset. These 2 sequence
datasets will facilitate the validation of any human
AMEL mutation suspected of involvement in
AIH1. This evolutionary analysis also revealed
numerous residues that appeared to be important
for correct AMEL function, but their role remains
to be elucidated.
KEY WORDS: amelogenin, amelogenesis
imperfecta, molecular evolution, enamel, teeth,
Received July 11, 2006; Last revision November 23, 2006;
Accepted November 29, 2006
A supplemental appendix to this article is published
electronically only at http://www.dentalresearch.org.
Validation of Amelogenesis
Imperfecta Inferred from
S. Delgado1, M. Ishiyama2, and J.-Y. Sire1*
1UMR 7138, Equipe "Evolution & Développement du
Squelette", Université Paris 6, Case 05, 7 quai St-Bernard,
75005 Paris, France; and 2Department of Histology, The
Nippon Dental University, School of Dentistry, Niigata,
Japan; *corresponding author, email@example.com
J Dent Res 86(4):326-330, 2007
J Dent Res 86(4) 2007Amelogenin Evolution and AIH1 327
MATERIALS & METHODS
In humans, AMELX is composed of 7
exons. Exon 1 is not translated; exon
4 is subjected to alternative splicing
(Hu et al., 1996; Yuan et al., 1996,
2001) and is absent in some mammals
and in all reptiles (Ishiyama et al.,
1998; Delgado et al., 2006); and exon
7 codes for a single amino acid. Nine
exons have been identified in rat and
mouse AMEL (Li et al., 1998), but
exons 8 and 9 are absent in all other
species studied so far. Therefore, only
AMEL exons 2, 3, 5, and 6 were
included in the present study. Because
the sequences of the small exons 2, 3,
and 5 (fewer than 60 bp each) are
well-conserved, we have concentrated
our efforts primarily on exon 6 (> 400
bp), which is more variable. AMELY
has evolved separately in various
mammalian lineages (Girondot and
Sire, 1998), in relation to the
particular pattern of Y chromosome
evolution (Iwase et al., 2001, 2003).
Therefore, AMELY was not included
in our study.
Several AMEL sequences were found
in GenBank, and one sequence was
obtained from the literature
(Yamamoto et al., 2002). We com-
pleted this dataset by blasting
sequencing AMEL in representative
species of most amniote lineages (Fig. 1). A dataset of 80 sequences
(52 mammals and 28 reptiles) was obtained. References to species
and sequences are found in APPENDIX 1. Taxa which have either
no teeth [e.g., baleen whales (Mysticeti), anteaters (Xenarthra),
pangolins (Pholidota)] or no enamel [e.g., armadillos (Xenarthra),
aardvarks (Tubulidentata)] were not included in this study.
DNA and RNA Extraction
Genomic DNA was extracted (DNeasy tissue kit: Qiagen-GmBH,
Ilden, Germany) from soft tissues conserved in ethanol. mRNAs
were obtained from 4 lizards (RNeasy kit: Qiagen) and converted
into cDNAs (ReverAid kit: MBI Fermentas, Hanover, PA, USA).
Primers were defined from the alignment of known AMEL
sequences (see APPENDIX 2).
Genomic DNA or cDNA (1 ?L) was amplified in a mixture
composed of 5 ?L Taq buffer (10x) (pH 8.8), 3 ?L MgCl22 mM,
and 1 ?L dNTP 10 mM, in the presence of sense and antisense
primers, and 0.3 ?L Red Hot polymerase (Advanced
Biotechnologies Ltd., Foster City, CA, USA). Amplification was
performed in a thermocycler (Genius Techne) for 38 cycles, each
cycle consisting of 1 min of denaturation at 94°C, 1 min of
annealing at 59°C, and 1 min of extension at 72°C. The final
extension was for 20 min at 72°C.
One microgram of PCR product was isolated, ligated to pCR 2.1-
TOPO plasmid vector (Invitrogen SA, Carlsbad, CA, USA) by the
TA-cloning method, then used to transform competent E. coli
TOP10F bacteria. The transformed bacteria were grown overnight
at 37°C in Luria-ampicillin broth, and subjected to lysis in 200 ?L
of NaOH 0.2 M-SDS 1%, at 0°C for 5 min. Subsequently, a 150-
?L quantity of AcK 3 M was added at 0°C for 5 min to precipitate
the proteins. The plasmids were purified in a phenol/chloroform
mixture. Sequencing was done by Genome Express S.A (Meylan,
AMEL sequences were aligned via Clustal X 1.81 (Thompson et
al., 1997), and checked by hand with Se-Al v2.0 (available at
The Mammalian and Reptilian AMEL Datasets
Of the 250 amino acids (aa) in the alignment of the 52
Figure 1. Relationships of the amniote lineages (in bold) for which amelogenin was used in this study
(adapted from Madsen et al., 2001; Murphy et al., 2001; Janke et al., 2005; Vidal and Hedges,
2005). The number of species in each clade is indicated between the brackets. See APPENDIX 1 for
information on the species and sequences.
328Delgado et al.J Dent Res 86(4) 2007
mammalian AMEL sequences (including residue insertions), 77
were unchanged, and 30 were substituted by a residue from the
same group (APPENDIX 3A). Most of the conserved amino
acids were located in the N- and C-terminal regions [coded by
exons 2, 3, 5, and the begining of exon 6, up to the TRAP
(tyrosine-rich amelogenin peptide) proteolytic sites (aa 1-64)
and the end of exon 6 (aa 218-250), respectively]. In contrast,
the central region of exon 6 (aa 65-217) showed numerous
variations, with a particular region characterized by large
sequence deletions or insertions (aa 130-208). Twelve AMEL
sequences possessed triplet (PXQ or PXX) insertions (up to 10
in the water opossum), while 4 other sequences showed
deletions (up to 17 in the dolphin). All positions currently
considered important were unchanged, including the TRAP
proteolytic loci (aa 59 and 61) and the LRAP (leucine-rich
amelogenin peptide) intra-exonic splicing site (aa 223).
In crocodiles, the 6 AMEL sequences were highly similar
(APPENDIX 3B). Of 199 aa in the alignment, only 11 were
substituted, and most of these were by residues from the same
group. In squamates, the 22 AMEL sequences showed a high
degree of variation (APPENDIX
3C). Of 217 amino acids in the
alignment, 53 were unchanged, and
18 were substituted by a residue
from the same group. Most
unchanged residues were located in
the N (aa 1-64) and C (aa 192-217)
terminal regions; nearly all
positions in the variable region of
exon 6 (aa 65-191) were
When we considered the
complete alignment of amniote
AMEL, we could not align most
parts of exon 6 (from aa 68 onward
in our alignment), due to the high
number of variations (substitutions,
(APPENDIX 3D). Only the N- and
C- terminal regions could be
aligned. We found 34 unchanged
residues in these regions and 15
residues that were substituted by a
residue from the same group. The
proteolytic loci leading to TRAP
were conserved, while the intra-
exonic splicing site for LRAP
could not be identified in most
Validation of AIH1 Using Two
The results obtained from the
analysis of mammalian (52 AMEL)
and amniote (80 AMEL) sequences
were transposed onto the human
AMEL sequence, with indication of
residues that were unchanged,
substituted by an amino acid from
the same group, or variable (Figs.
2A, 2B). Of the 5 residues known
to lead to AIH1 when substituted
(M1, W4, T37, P56, and H63 in our sequence; p.M1T, p.W4S,
p.T51I, p.P70T, p.H77L, respectively, in the AIH1
nomenclature), 4 were validated (i.e., unchanged) in
mammalian and amniote sequence datasets, and all when only
AMEL sequences were used. Indeed, the p.H77L mutation was
not validated by the amniote dataset: Histidine (H: basic group)
was substituted by glutamine (Q: polar) in crocodiles and in a
snake. In humans, this AIH1 resulted from substitution by a
leucine (L: non-polar). Most residues known to be important
for a correct function of AMEL were conserved in amniotes. In
addition, the datasets revealed a high number of unchanged
A genetic diagnosis of AIH1 relies, eventually, upon the
sequencing of AMEL and comparison of the obtained sequence
with the reference sequence for humans. When an obvious
mutation is found (large deletions, reading frameshift leading to
a stop codon, etc.), it is considered to be responsible for the
Figure 2. Amino-acid sequence of human amelogenin with indication of important residues inferred
from the alignment of 52 mammalian sequences (A) and of 80 amniote sequences (52 mammals, 6
crocodiles, and 22 squamates) (B). (The alignments are presented in APPENDIX 2.) Exon 4 (14 residues)
was not included, because it was absent in most species studied. Signal peptide is on the grey
background. The protein sequence (191 amino acids) is numbered from methionine (1). Large
characters = residues unchanged; italics = residues that can be substituted for by an amino acid from
the same group only. Small characters = residues for which substitution can be made. Boldface
characters = the 5 residues known to lead to amelogenesis imperfecta after substitution.
J Dent Res 86(4) 2007Amelogenin Evolution and AIH1329
observed phenotype. When the mutation leads to a single
amino acid substitution, the genotype-phenotype relationship is
less obvious, and one could envisage this mutation as a
polymorphism, i.e., the disorder not being related to this
mutation. Of the 14 AMEL mutations identified for X-linked
AI (Hart et al., 2002a; Kim et al., 2004), 5 are single-residue
substitutions. If the mutation is in a position conserved in other
species, this feature supports the genetic diagnosis. Indeed, the
sites of crucial importance for AMEL must be kept unchanged
during evolution; otherwise, their substitution could lead to a
genetic disease. However, given the high sequence similarity of
AMEL in closely related mammalian species, it is difficult to
decide whether conserved sites are preserved because they are
highly constrained or because the evolutionary distance
between these lineages is too short to reveal all low-constrained
sites. Species that are too closely related are not relevant in a
decision of evolutionary conservation. To ensure that residue
conservation is related to a functional constraint, one needs to
know AMEL sequences in species that are more distantly
related. This is the reason we built these sequence datasets
based on mammalian and reptilian diversity, to help in AIH1
We have chosen to present 2 datasets, one based on AMEL
sequences of 52 mammals, and the other on a compilation of 80
amniote sequences. Indeed, although enamel structure is
roughly similar in mammals and reptiles, some enamel
specificities could have been selected for during the long
evolutionary period (310 my) that separates these lineages. In
contrast to reptiles, in which some ancestral characters, such as
polyphyodonty, have been conserved, mammals no longer
replace their teeth continuously throughout life. Furthermore,
from a structural viewpoint, Tomes' processes, a feature of
mammalian ameloblasts related to the prismatic structure of
enamel, do not exist in reptiles, in which enamel is non-
prismatic (Sander, 2000). These two mammalian novelties
could have led to new constraints in the AMEL sequence. We
hypothesized that the 34 AMEL residues which are unchanged
at the amniote level are essential for the correct formation and
mineralization of enamel, i.e., they are important for AMEL
interactions with the cell membrane and/or the mineral crystals.
This hypothesis was well-supported: All these conserved
positions were found at the N- and C-terminal regions, which
are known to exert such functions (Paine et al., 2003; Snead,
2003). We hypothesized also that the 43 residues that are
conserved only in mammals are related to the peculiar features
of enamel that were selected for during mammalian evolution
(180 my). Half of the unchanged positions were found in the N-
and C-terminal regions, reflecting a possible stronger constraint
on the AMEL sequence in these regions in mammals than in
reptiles. The other conserved positions were found in the region
known to be variable (Delgado et al., 2005; Sire et al., 2005,
2006), either close to the N- and C-terminal regions or in the
central region of exon 6. This could also reflect new constraints
in this region, but we can also envisage that these positions are
not really important for AMEL function. Perhaps 180 my are
insufficient for random substitution of amino acids that are not
The 5 amino acid substitutions known to lead to AIH1 were
validated by our method with the mammalian dataset, and 4 of
them with the amniote dataset. In reptiles, the substitution of
H63 in our alignment (p.H77L: Hart et al., 2002b) by a
glutamine (Q) could indicate that this locus has probably been
constrained during mammalian evolution only. The presence of
this basic residue probably plays a role in TRAP proteolysis by
enamelysin (MMP20). Does this mean that there is no TRAP in
crocodiles, or that a polar residue (Q) could replace a basic one
(H)? Amino acids that were replaced by residues from the same
group were also indicated in the human sequence. Indeed, if
one considers that only the biochemical characteristics of a
position are important, there would be no problem if the residue
were substituted by an amino acid from the same group.
Our evolutionary analysis of AMEL at the amniote level
confirmed our previous findings, inferred from the comparative
study of mammalian AMEL, i.e., highly conserved residues in
the N- and C-terminal regions, and a variable region in exon 6
(Delgado et al., 2005, 2006; Sire et al., 2006). In exon 6, the
intra-exonic splicing site, which releases LRAP (a short peptide
involved in cell signaling: Veis et al., 2000), was well-
conserved in mammals, but not in reptiles. The 'hot spot' of
mutation (i.e., large insertions and/or deletions located in the
central region of exon 6) in mammals (Delgado et al., 2005)
was found in the present study in a few newly sequenced
AMEL of mammalian species, but was absent in reptiles. These
features were acquired recently in mammalian evolution.
In addition to proposed sequence dataset, which will help in
the diagnosis of AIH1, this analysis has revealed 30 unchanged
residues with unknown, but certainly important, function.
These amino acids could be good candidates for AIH1 if they
were substituted, and their role in AMEL function should be
Our study showed how evolutionary analysis, when
conducted within a phylogenetic framework, could help both in
validating mutations in humans and in revealing amino acids
that could play important roles in enamel structure and
organization. In dental research, this method could be applied
to the study of other genes—for instance, enamelin, which is
known to be responsible for autosomal-dominant AI, and
dentin sialophosphoprotein, responsible for dentinogenesis
imperfecta. The large number of genomes currently being
sequenced in mammals could be taken as an opportunity to
build datasets that could be used to validate mutations
responsible for a genetic disease.
We are grateful to Prof. Ann Huysseune (Ghent University,
Belgium) for helpful criticism of the manuscript. We are
grateful to the following colleagues for sending either DNA or
tissue samples: F. Catzeflis (UMR 5554, Université de
Montpellier 2, France); L. Fougeirol and S. Martin (La Ferme
des Crocodiles, Pierrelatte, France); A. Lécu and F. Ollivet
(Zoo de Vincennes, MNHN, France); G. Véron, V. de Buffrénil
and N. Vidal (Muséum national d'Histoire naturelle, France);
W. Dabin (Muséum de la Rochelle, France); T. Robinson
(Stellenboch University, Afrique du Sud); and D.J. Harris
(Centro de Estudos de Ciência Animal, Vila do Conde,
Portugal). This work was financially supported by IFRO
(Institut Français de Recherche Odontologiques).
Since our article was in press, "A Novel Missense Mutation
(p.P52R) in Amelogenin Gene Causing X-linked Amelogenesis
Imperfectca" was published in JDR, 86:69-72, 2007, by M.
Kida et al. This substitution is validated by our evolutionary
analysis (exon5, position 38 in our alignment).
330Delgado et al.J Dent Res 86(4) 2007
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