2001 Oxford University Press Human Molecular Genetics, 2001, Vol. 10, No.

16 16731677
Mutation of the gene encoding the enamel-specific
protein, enamelin, causes autosomal-dominant
amelogenesis imperfecta
M. Helen Rajpar, Kathryn Harley
1
, Chris Laing
2
, Robin M. Davies
3
and Michael J. Dixon*
School of Biological Sciences and Department of Dental Medicine and Surgery, 3.239 Stopford Building,
University of Manchester, Oxford Road, Manchester M13 9PT, UK,
1
Department of Paediatric Dentistry,
Edinburgh Dental Institute, Lauriston Building, Lauriston Place, Edinburgh EH13 9YW, UK,
2
Centre for Nephrology,
University College London, Middlesex Hospital, Mortimer Street, London W1N 8AA, UK and
3
Dental Health Unit,
Manchester Science Park, Lloyd Street North, Manchester M15 4SH, UK
Received April 5, 2001; Revised and Accepted June 5, 2001
Amelogenesis imperfecta (AI) is a group of inherited
defects of dental enamel formation that shows both
clinical and genetic heterogeneity. To date, mutations
in the gene encoding amelogenin have been shown
to underlie a subset of the X-linked recessive forms of
AI. Although none of the genes underlying autosomal-
dominant or autosomal-recessive AI have been
identified, a locus for a local hypoplastic form has
been mapped to human chromosome 4q11q21. In
the current investigation, we have analysed a family
with an autosomal-dominant, smooth hypoplastic
form of AI. Our results have shown that a splicing
mutation in the splice donor site of intron 7 of the
gene encoding the enamel-specific protein enamelin
underlies the phenotype observed in this family. This
is the first autosomal-dominant form of AI for which
the genetic mutation has been identified. As this type of
AI is clinically distinct from that localized previously to
chromosome 4q11q21, these findings highlight the
need for a molecular classification of this group of
disorders.
INTRODUCTION
Dental enamel is a highly mineralized tissue with 85% of its
volume occupied by unusually large, highly organized, hydroxy-
apatite crystals. Enamel is unique among the mineralized tissues
in that it is produced by ameloblasts, which have an epithelial
origin. Amelogenesis imperfecta (AI) is a common group of
inherited defects of dental enamel formation that exhibit
marked genetic and clinical heterogeneity with at least 14
different sub-types being recognized on the basis of their
clinical appearance (1). Taking all forms of AI into account,
the prevalence has been reported to be as high as 1:700 in northern
Sweden (2). Affected individuals show either hypoplastic (thin
but seemingly correctly mineralized) or hypomineralized enamel,
but an overlap of these signs is seen in many cases (3). There is
no apparent correlation between the phenotype and the mode
of inheritance with autosomal-dominant, autosomal-recessive
and X-linked recessive forms being recognized. To date,
mutations in the gene encoding the protein amelogenin have
been shown to cause some X-linked recessive forms of AI.
This locus has been designated AIH1 (411). The hetero-
geneity that characterizes this group of disorders has limited
further progress to the identification of additional loci by
linkage analysis; however, the underlying genes have not been
identified. For example, a second locus for X-linked recessive
AI, AIH3, has been mapped to chromosome Xq24q27.1 (12).
Similarly, an autosomal-dominant, local hypoplastic form of
AI (AIH2) has been mapped to a 4 Mb region of human
chromosome 4q11q21 that encompasses the gene encoding
the ameloblast-specific protein ameloblastin, AMBN (1315).
Recently, the gene encoding a second ameloblast-specific
protein, enamelin, has been mapped to the AIH2 critical region
within 15 kb of AMBN (16,17). Nevertheless, the genetic muta-
tion underlying this form of AI has not been identified and
additional genetic linkage studies have indicated that other
genetic loci for autosomal forms of AI exist (18). In the current
study, we have shown that a clinically distinct form of AI, thin
and smooth hypoplastic AI, which has been estimated to
account for 1.5% of cases of AI (3), maps to the AIH2 critical
region and that this type of AI results from mutation of the
gene encoding enamelin.
RESULTS
We are taking a positional candidate approach to identify the
genetic mutations underlying the various forms of AI and have
investigated a family with an autosomal-dominant form of this
condition (Fig. 1A). All affected members of the family exhibit
enamel hypoplasia in both the deciduous and permanent
dentitions. Clinically, the teeth appear small, thin and yellow
due to a lack of enamel thickness (Fig. 1B and C). Such denti-
tions have been classified according to the clinical appearance
as thin and smooth hypoplastic AI (1). As nephrocalcinosis
may be associated with this dental phenotype (19,20), all
affected individuals were screened with renal ultrasonography,
but none was evident.
*To whom correspondence should be addressed. Tel: +44 161 275 5620; Fax: +44 161 275 5620; Email: mike.dixon@man.ac.uk
1674 Human Molecular Genetics, 2001, Vol. 10, No. 16
Although this form of AI is clinically distinct from that
previously localized to chromosome 4q, we commenced our
analysis in this region of the genome. Initially, we focussed our
attention on the AMBN locus and identified a short tandem
repeat polymorphism in intron 11 of this gene. Segregation
analysis revealed no evidence for recombination between
AMBN and the disease phenotype in this family (Z
max
= 2.06)
suggesting linkage to chromosome 4q11q21. Mutation
screening of all 13 exons of AMBN (21) by single-strand
conformation polymorphism (SSCP) analysis failed to reveal
any AI-specific mobility shifts in this gene. As these results
effectively excluded mutations in AMBN from a causative role
in the pathogenesis of the AI in this family, we extended our
studies to the gene encoding enamelin, which has recently been
shown to map within 15 kb of AMBN (16). RTPCR analysis
showed that in the panel of human cDNAs tested, enamelin
transcript could only be detected in cDNA derived from tooth
germs (Fig. 2A) extending previous results from the rabbit, pig
and mouse to man (17,22,23).
In silico screening of the genomic sequence of human
chromosome 4 with human cDNA sequence using the BLAST
algorithm subsequently indicated that the gene encoding
enamelin contains nine exons, separated by eight introns. All
of the intronexon boundaries were found to be type 0, with
splicing occurring between exons and the splice donor and
acceptor sites conforming to the published consensus
sequences (24) (Table 1). SSCP analysis performed using
primers designed to screen each exon and the associated splice
Figure 1. Clinical details of the AI family. (A) Pedigree of the family; filled symbols denote affected individuals. (B and C) Photographs of individual II.8 showing
the small, smooth, yellow teeth that result from the enamel hypoplasia.
Table 1. Genomic organization of the human enamelin gene
Intronic sequences are indicated in lower case, exonic sequences in upper case. The positions within the cDNA sequence are indicated
using the first nucleotide of the translation initiation codon as position 1. UTR, untranslated region.
Exon cDNA position Splice acceptor Splice donor
1 281 to 60 5-UTR TGG CAT TGG gtgagtattaga
2 61 to 54 tatattttag TTT CTT CTC GAT AAC TTG gtggccaaac
3 55 to 123 tttattctag GTA CCA AAA GCT ATG CCA gtgagtattt
4 124 to 168 ttacttccag ATG CAC ATG AGT GAG GAG gtatgtacgt
5 169 to 210 ttggttgcag ATG ATG CGG GGC CCA CAT gtaagttttt
6 211 to 471 accctttcag ATG GCA CAC CCC CCT CAG gtgagactgg
7 472 to 534 ttctctctag GCA TTC CCA ATT CCA CAG gtgagaaatt
8 535 to 588 cacttctcag AGG TTA CCA GAA GGG GGG gtaagtacaa
9 589 to 3-UTR tttcctacag AAT CCT TAC 3-UTR
Human Molecular Genetics, 2001, Vol. 10, No. 16 1675
junctions for mutations, revealed an abnormal conformer that
was present in all affected family members, but not the
unaffected individuals (Fig. 2B). Direct sequencing revealed a
heterozygous mutation in the splice donor site of intron 7,
nt841+1 (GA) (Fig. 2C). This mutation deletes an HphI site,
which allowed us to confirm that the mutation co-segregates
with the disease phenotype (Fig. 2D). The mutation was not
detected in 200 control chromosomes of similar ethnic origin.
DISCUSSION
Recent evidence from genetic studies has implicated a number
of molecules in the biomineralization events underlying tooth
development. For example, mutation of the dentine sialo-
phosphoprotein gene causes dentinogenesis imperfecta type II
(25,26). However, this condition is thought to be genetically
homogeneous. Conversely, the search for the genetic
mutations underlying AI has been frustrated by the clinical and
genetic heterogeneity observed in this condition. Progress in
this area has been limited to the finding that mutations in the
gene encoding amelogenin underlie a subset of cases of X-linked
recessive AI (411) and the delineation of at least three additional
genetic loci (12,13,18). Here we report the first genetic mutation
underlying an autosomal form of AI, thereby providing definitive
evidence that enamelin is essential for dental enamel formation.
Moreover, we demonstrate that the locus for smooth hypo-
plastic AI maps to the same genetic interval as that for the
clinically distinct local hypoplastic form. Therefore, our find-
ings suggest that these two AI subtypes are allelic and support
the need for a molecular classification of this heterogeneous
group of disorders (27).
The calcium phosphate hydroxyapatite crystals that
comprise the bulk of the mature enamel are unusually large,
uniform and regularly disposed within the tissue. This highly
organized and unusual structure is thought to be rigorously
controlled through the interaction of a number of organic matrix
molecules that include amelogenin, enamelin, ameloblastin,
tuftelin, dentine sialophosphoprotein and a variety of enzymes
(28). In porcine enamel, enamelin is synthesized and secreted
by ameloblasts as a 186 kDa precursor, the unprocessed form
concentrating along the secretory face of the ameloblast Tomes
process. Proteolytic processing towards the C-terminus
produces 142 kDa enamelin via a 155 kDa intermediate.
Subsequent cleavages generate an 89 kDa polypeptide encom-
passing the first 627 amino acids of the secreted protein and a
34 kDa polypeptide beginning at residue 632. Processing of
89 kDa enamelin results in polypeptides of 32 kDa (amino
acids 136238) and 25 kDa, commencing at residue 477 (29).
Intact enamelin and cleavage products containing the C-terminus
are limited to the most superficial layer of the developing
enamel matrix, while other cleavage products accumulate in
the deeper layers of this tissue in the maturing rod and inter-rod
enamel (30).
Due to the ameloblast-specific nature of enamelin and the
transient nature of ameloblasts during development, it was not
possible to prepare cDNA from affected individuals to confirm
the effect of the mutation on the enamelin transcript. Never-
theless, numerous studies have demonstrated that mutations of
the type observed in this study lead to aberrant splicing (31).
While skipping of exon 7 remains the most likely scenario, a
failure of splicing at the splice donor site of intron 7 with read
through into the intron remains a possibility. In the latter case,
this would result in the introduction of a termination codon
after nine amino acids with the likely loss of enamelin protein
function due to nonsense-mediated mRNA degradation (32).
Conversely, exon skipping would result in an in-frame deletion
of the 21 amino acids encoded by exon 7. Alignment of the
amino acid sequences of porcine and human enamelin indicates
that deletion of exon 7 would remove the 32 kDa proteolytic
cleavage site. Among the enamelin cleavage products, the
32 kDa polypeptide is the most characterized. For example, it
undergoes phosphorylation and asparagine-linked glycosylation
(29,33). Moreover, 32 kDa enamelin has been proposed to play
a role in amelogenin processing (34). Intriguingly, amelogenin
processing products have been suggested to inhibit hydroxy-
apatite crystal growth during amelogenesis, and removal of
Figure 2. Molecular analysis of the enamelin gene. (A) RTPCR analysis
indicates that, unlike the Treacher Collins syndrome gene, TCOF1, which is
widely expressed, the gene encoding enamelin is tooth-specific, a single PCR
amplicon of 797 bp being detected in cDNA generated from human tooth germ
RNA but not in other tissue sources. (B) SSCP analysis of genomic DNA from
the AI family reveals the presence of an abnormal conformer in individuals I.1,
II.1, II.3, II.6, II.8, III.2, III.5 and III.6, which is not detected in the unaffected
family members. (C) Sequencing of DNA from individual II.1 reveals the
presence of the mutation nt841+1 (GA) (arrowed), which is not present in a
control sample. (D) The mutation deletes an HphI site. Digestion of DNA from
affected individuals produces bands of 214, 116 and 98 bp, whereas the
214 bp band is not detected in unaffected individuals, confirming that the
mutation co-segregates with the disease phenotype.
1676 Human Molecular Genetics, 2001, Vol. 10, No. 16
such inhibitory proteins is thought to be crucial to enamel
maturation (28). Although the 89 kDa precursor polypeptide is
also active, cleavage to the 32 kDa form in immature enamel to
from amelogeninase has been proposed to be essential for
full function in degrading amelogenins in maturing enamel
(34). The 32 kDa enamelin polypeptide may therefore play an
important role in enamel mineralization in vivo, and a failure of
enamelin cleavage with resulting reduced amelogeninase
activity may account for the mechanism underlying the amelo-
genesis imperfecta observed in this family. The delineation of
additional mutations in families with a history of AI and
complementary functional studies will be important in clarifying
this hypothesis.
MATERIALS AND METHODS
Family
The pedigree of the family is presented in Figure 1A. Each
member of the family was examined clinically and, where
appropriate, radiographically by K.Harley. Renal sonography
of affected individuals was performed by C.Laing.
RTPCR
Total RNA was either extracted according to the method of
Chomczynski and Saachi (35) or purchased commercially.
RTPCR analyses were performed and analysed as described
previously (36) using a forward primer in exon 6 (5-CATAA-
CAAGACTGATCAGACC-3) and a reverse primer in exon 9
(5-ACTGGATTTCCTGGACGAGC-3) of the enamelin
gene. These primers generate an amplicon of 797 bp from
cDNA.
Mutation analysis
SSCP analysis of genomic DNA extracted from peripheral
blood leucocytes was performed according to the method of
Orita et al. (37) using the primers 5-TTTTCAATACCACAT-
CACTCTG-3 and 5-ACTGATTGGTATATGGTATTCC-3.
To determine the sequence variant underlying the SSCP
mobility shift, PCR products were sequenced directly via the
dideoxy chain termination method using dye primer chemistry.
To confirm that the mutation co-segregated with the disease
phenotype, PCR amplified genomic DNA was digested with the
restriction enzyme HphI and the resulting products were resolved
on a 9% polyacrylamide gel.
ACKNOWLEDGEMENTS
We thank the test family for providing samples. This work is
supported by an MRC Industrial Collaborative Studentship
with Colgate (G216/4098) and by the Wellcome Trust
(051938).
REFERENCES
1. Witkop, C.J. (1988) Amelogenesis imperfecta, dentinogenesis imperfecta
and dentin dysplasia revisited: problems in classification. J. Oral Pathol.,
17, 547553.
2. Bckman, B. and Holm, A.-K. (1986) Amelogenesis imperfecta:
prevalence and incidence in a northern Swedish county. Community Dent.
Oral Epidemiol., 14, 4347.
3. Bckman, B. (1988) Amelogenesis imperfecta clinical manifestations in
51 families in a northern Swedish county. Scand. J. Dent. Res., 96,
505516.
4. Lagerstrm, M., Dahl, N., Nakahori, Y., Nakagome, Y., Backman, B.,
Landegren, U. and Pettersson, U. (1991) A deletion in the amelogenin
gene (AMG) causes X-linked amelogenesis imperfecta (AIH1). Genomics,
10, 971975.
5. Aldred, M.J., Crawford, P.J., Roberts, E. and Thomas, N.S. (1992)
Identification of a nonsense mutation in the amelogenin gene (AMELX)
in a family with X-linked amelogenesis imperfecta (AIH1). Hum. Genet.,
90, 413416.
6. Lench, N.J., Brook, A.H. and Winter, G.B. (1994) SSCP detection of a
nonsense mutation in exon 5 of the amelogenin gene (AMGX) causing
X-linked amelogenesis imperfecta (AIH1). Hum. Mol. Genet., 3, 827828.
7. Lagerstrm-Fermer, M., Nilsson, M., Backman, B., Salido, E., Shapiro,
L., Pettersson, U. and Landegren, U. (1995) Amelogenin signal peptide
mutation: correlation between mutations in the amelogenin gene (AMGX)
and manifestations of X-linked amelogenesis imperfecta. Genomics, 26,
159162.
8. Lench, N.J. and Winter, G.B. (1995) Characterisation of molecular defects
in X-linked amelogenesis imperfecta (AIH1). Hum. Mutat., 5, 251259.
9. Collier, P.M., Sauk, J.J., Rosenbloom, S.J., Yuan, Z.A. and Gibson, C.W.
(1997) An amelogenin gene defect associated with human X-linked
amelogenesis imperfecta. Arch. Oral Biol., 42, 235242.
10. Ravassipour, D.B., Hart, P.S., Hart, T.C., Ritter, A.V., Yamauchi, M.,
Gibson, C. and Wright, J.T. (2000) Unique enamel phenotype associated
with amelogenin gene (AMELX) codon 41 point mutation. J. Dent. Res.,
79, 14761481.
11. Kindelan, S.A., Brook, A.H., Gangemi, L., Lench, N., Wong, F.S., Fearne, J.,
Jackson, Z., Foster, G. and Stringer, B.M. (2000) Detection of a novel mutation
in X-linked amelogenesis imperfecta. J. Dent. Res., 79, 19781982.
12. Aldred, M.J., Crawford, P.J.M., Roberts, E., Gillespie, C.M., Thomas, N.S.,
Fenton, I., Sandkuijl, L.A. and Harper, P.S. (1992) Genetic heterogeneity
in X-linked amelogenesis imperfecta. Genomics, 14, 567573.
13. Forsman, K., Lind, L., Backman, B., Westemark, E. and Holmgren, G.
(1994) Localization of a gene for autosomal dominant amelogenesis
imperfecta (ADAI) to chromosome 4q. Hum. Mol. Genet., 3, 16211625.
14. Krrman, C., Backman, B., Dixon, M., Holmgren, G. and Forsman, K.
(1997) Mapping of the locus for autosomal dominant amelogenesis
imperfecta (AIH2) to a 4-Mb YAC contig on chromosome 4q11q21.
Genomics, 39, 164170.
15. MacDougall, M., DuPont, B.R., Simmons, D., Reus, B., Krebsbach, P.,
Karrman, C., Holmgren, G., Leach, R.J. and Forsman, K. (1997) Ameloblastin
gene (AMBN) maps within the critical region for autosomal dominant
amelogenesis imperfecta at chromosome 4q21. Genomics, 41, 115118.
16. Dong, J., Gu, T.T., Simmons, D. and MacDougall, M. (2000) Enamelin
maps to human chromosome 4q21 within the autosomal dominant
amelogenesis imperfecta locus. Eur. J. Oral. Sci., 108, 353358.
17. Hu, C.-C., Hart, T.C., DuPont, B.R., Chen, J.J., Sun, X., Qian, Q.,
Zhang, C.H., Jiang, H., Mattern, V.L., Wright, J.T. et al. (2000) Cloning
human enamelin cDNA, chromosomal localization, and analysis of
expression during tooth development. J. Dent. Res., 79, 912919.
18. Krrman, C., Bckman, B., Holmgren, G. and Forsman, K. (1996) Genetic
heterogeneity of autosomal dominant amelogenesis imperfecta demonstrated
by its exclusion from the AIH2 region on human chromosome 4q.
Arch. Oral Biol., 41, 893900.
19. MacGibbon, D. (1972) Generalizedenamel hypoplasia and renal dysfunction.
Aust. Dent. J., 17, 6163.
20. Lubinsky, M., Angle, C., Marsh, P.W. and Witkop, C.J. (1985) Syndrome
of amelogenesis imperfecta, nephrocalcinosis, impaired renal concentration,
and possible abnormality of calcium metabolism. Am. J. Med. Genet., 20,
233243.
21. Toyosawa, S., Fujiwara, T., Ooshima, T., Shintani, S., Sato, A., Ogawa, Y.,
Sobue, S. and Ijuhin, N. (2000) Cloning and characterization of the human
ameloblastin gene. Gene, 256, 111.
22. Zeichner-David, M., MacDougall, M. and Slavkin, H.C. (1983) Enamelin
gene expression during fetal and neonatal rabbit tooth organogenesis.
Differentiation, 25, 148155.
23. Dohi, N., Murakami, C., Tanabe, T., Yamakoshi, Y., Fukae, M.,
Yamamoto, Y., Wakida, K., Shimizu, M., Simmer, J.P., Kurihara, H. et al.
(1998) Immunocytochemical and immunochemical study of enamelins,
using antibodies against porcine 89-kDa enamelin and its N-terminal
synthetic peptide, in porcine tooth germs. Cell Tissue Res., 293, 313325.
Human Molecular Genetics, 2001, Vol. 10, No. 16 1677
24. Breathnach, R. and Chambon, P. (1981) Organization and expression of
eucaryotic split genes coding for proteins. Annu. Rev. Biochem., 50,
349383.
25. Zhang, X., Zhao, J., Li, C., Gao, S., Qiu, C., Liu, P., Wu, G., Qiang, B.,
Lo, W.H.Y. and Shen, Y. (2001) DSPP mutation in dentinogenesis
imperfecta Shields type II. Nat. Genet., 27, 151152.
26. Xiao, S., Yu, C., Chou, X., Yuan, W., Wang, Y., Bu, L., Fu, G., Qian, M.,
Yang, J., Shi, Y. et al. (2001) Dentinogenesis imperfecta 1 with or without
progressive hearing loss is associated with distinct mutations in DSPP.
Nat. Genet., 27, 201204.
27. Aldred, M.J. and Crawford, P.J. (1995) Amelogenesis imperfectatowards a
new classification. Oral Dis., 1, 25.
28. Robinson, C., Brookes, S.J., Shore, R.C. and Kirkham, J. (1998) The
developing enamel matrix: nature and function. Eur. J. Oral Sci., 106
(Suppl. 1), 282291.
29. Fukae, M., Tanabe, T., Murakami, C., Dohi, N., Uchida, T. and Shimizu, M.
(1996) Primary structure of the porcine 89-kDa enamelin. Adv. Dent. Res.,
10, 111118.
30. Hu, C.C., Fukae, M., Uchida, T., Qian, Q., Zhang, C.H., Ryu, O.H.,
Tanabe, T., Yamakoshi, Y., Murakami, C., Dohi, N. et al. (1997) Cloning
and characterization of porcine enamelin mRNAs. J. Dent. Res., 76,
17201729.
31. Krawczak, M., Reiss, J. and Cooper, D.N. (1992) The mutational
spectrum of single base-pair substitutions in mRNA splice junctions of
human genes: causes and consequences. Hum. Genet., 90, 4154.
32. Frischmeyer, P.A. and Dietz, H.C. (1999) Nonsense-mediated mRNA
decay in health and disease. Hum. Mol. Genet., 8, 18931900.
33. Yamakoshi, Y., Pinheiro, F.H., Tanabe, T., Fukae, M. and Shimizu, M.
(1998) Sites of asparagine-linked oligosaccharides in porcine 32 kDa
enamelin. Connect. Tissue Res., 39, 3946.
34. Moradian-Oldak, J., Leung, W., Simmer, J.P., Zeichner-David, M. and
Fincham, A.G. (1996) Identification of a novel proteinase (ameloprotease-1)
responsible for the complete degradation of amelogenin during enamel
maturation. Biochem. J., 318, 10151021.
35. Chomczynski, P. and Saachi, N. (1987) Single-step method of RNA
isolation by acid guanidinium thiocynatephenolchloroform extraction.
Anal. Biochem., 162, 156159.
36. Dixon, J., Hovanes, K., Shiang, R. and Dixon, M.J. (1997) Sequence
analysis, identification of evolutionary conserved motifs and expression
analysis of murine tcof1 provide further evidence for a potential function
for the gene and its human homologue, TCOF1. Hum. Mol. Genet., 6,
727737.
37. Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K. and Sekiya, T. (1989)
Detection of polymorphisms of human DNA by gel electrophoresis as
single-strand conformation polymorphisms. Proc. Natl Acad. Sci. USA,
86, 27662770.
1678 Human Molecular Genetics, 2001, Vol. 10, No. 16