a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 0 9 – 2 1 7

Phenotype and enamel ultrastructure characteristics inpatients with ENAM gene mutations g.13185–13186insAGand 8344delG

Alenka Pavlic a,*, Milan Petelin b, Tadej Battelino c

aDepartment of Paediatric and Preventive Dentistry, Faculty of Medicine, University of Ljubljana, Hrvatski trg 6, 1000 Ljubljana, SloveniabDepartment of Oral Medicine and Periodontology, Faculty of Medicine, University of Ljubljana, Hrvatski trg 6, 1000 Ljubljana, SloveniacUniversity Children’s Hospital Ljubljana and Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia

a r t i c l e i n f o

Article history:

Accepted 7 October 2006

Keywords:

Dental enamel

Enamel ultrastructure

Amelogenesis imperfecta

Enamelin mutations

a b s t r a c t

Objective: The main clinical manifestations of amelogenesis imperfecta (AI) include altera-

tion in the quality and quantity of enamel. AI is associated with different mutations in four

genes: enamelin (ENAM), amelogenin (AMGX), kallikrein (KLK4) and enamelysin (MMP-20).

Seven different mutations have been identified in the enamelin gene (ENAM).

Design: In this paper, we describe the phenotype and ultrastructure of enamel observed

using scanning electron microscopy (SEM) in patients with two autosomal dominant (AD)

mutations in the ENAM gene: g.13185–13186insAG and g.8344delG, each in one of two

unrelated families. Mutations were confirmed by sequence analysis of PCR amplified

products of all 10 exons and exon/intron boundaries of the ENAM gene.

Results: Phenotypic diversity was observed in patients with ENAM gene mutations g.13185–

13186insAG with consecutive protein alteration designated as p.P422fsX488 within family 1.

In the proband, the enamel of his entire dentition was chalky white with only mild local

hypoplastic alteration, while the phenotypic appearance of his father’s dentition was that of

local hypoplastic AI. In patients with the ENAM gene mutation g.8344delG from family 2 with

consecutive protein alteration designated as p.N197fsX277, generalised hypoplastic AI was

observed.

Conclusions: Ultrastructural enamel changes in the patient with the autosomal dominant

ENAM g.13185–13186insAG mutation, described for the first time in this study, were less

pronounced compared to ultrastructural changes in patients with the autosomal dominant

ENAM mutation 8344delG. Ultrastructural characteristics of the g.13185–13186insAG muta-

tion revealed deformed prisms, an oval shape on the cross-section and wider interprism

spaces, while enamel with the ENAM mutation 8344delG was laminated, but prismless.

# 2006 Elsevier Ltd. All rights reserved.

avai lab le at www.sc iencedi rec t .com

journal homepage: www. int l .e lsev ierhea l th .com/ journa ls /arob

1. Introduction

Amelogenesis imperfecta (AI) is an inherited tooth disorder

solely affecting tooth enamel formation, with widely varying

phenotypes. Although the AI enamel defects can be broadly

* Corresponding author. Tel.: +386 1 522 42 68; fax: +386 1 522 24 94.E-mail address: alenka.pavlic@mf.uni-lj.si (A. Pavlic).

0003–9969/$ – see front matter # 2006 Elsevier Ltd. All rights reservedoi:10.1016/j.archoralbio.2006.10.010

divided into hypoplastic and hypomineralised phenotypes,

there are many subtypes of these two main entities. The

classification most used worldwide distinguishes 14 subtypes

of AI based on various phenotypic criteria and mode of

inheritance (autosomal dominant (AD), autosomal recessive

(AR) or X-linked).1

d.

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 0 9 – 2 1 7210

Enamel is the most mineralised tissue in the human body.

The process of crystal initiation and growth is tightly

controlled by temporal and locational regulation of secretion,

procession and degradation of unique enamel proteins and

proteinases that compose the extracellular matrix (ECM). The

major enamel matrix proteins contributing to enamel forma-

tion are amelogenin, enamelin and ameloblastin.2 The

formation of unique enamel ECM proteins requires the

expression of multiple specific genes.3 Only then will a mature

enamel structure, with its highly ordered prismatic pattern

packed by hydroxyapatite crystallites, be formed.

Enamelin is the largest enamel ECM protein (180–190 kDa),

with about a third of itsapparentmolecular weightcomingfrom

glycosylation. It is present in comparatively small amounts,

constituting only 1–5% of the total matrix protein.3 Enamelin

plays an important role in several different stages of enamel

formation, for example, in the initiation of mineralisation and

regulation of crystal growth.4–6 Enamelin is the protein product

of the enamelin gene (ENAM), located on chromosome 4q217,

which has 10 exons, 8 of which are coding.3

The AI-associated gene mutations identified to date involve

mutations in the enamelin gene (ENAM) (Table 1), the

amelogenin gene (AMGX),8 the serine proteinase kallikrein

gene (KLK4)9 and the metalloproteinase enamelysin gene

(MMP-20).10,11 However, many of the mutations responsible for

the majority of types of AI are still unknown.12

A correlation between different phenotypes of AI and

genotypes of specific mutations in the AMGX gene13 and the

ENAM gene14 is proposed. However, phenotypic diversity

associated with some cases of AI may represent environ-

mental influences, pleiotropic effects of the underlying gene

mutation or modifying gene effects.5

In this study, the phenotypic characteristics and detailed

ultrastructure of the enamel in two autosomal dominant

mutations in the ENAM gene: g.13185–13186insAG and

g.8344delG, with consecutive protein alteration designated

as p.P422fsX488 and p.N197fsX277, respectively, each in one of

two unrelated families, are reported. In both families affected

members for the ENAM mutations were heterozygous. To the

best of our knowledge, this is the first report of enamel

ultrastructure in the ENAM gene g.13185–13186insAG muta-

tion.

2. Material and methods

2.1. Pedigree and diagnosis

Affected members of two unrelated families with different

phenotypes of AI were examined clinically and radiographi-

cally to determine affection status and to characterise the

clinical phenotype. Any unusual oral findings in addition to

quality and quantity of enamel changes, malformations or

missing teeth and dental malocclusion, were evaluated.

Patients were also examined for any metabolic or endocrine

defects, generalised diseases, syndromes or fluorosis. Pedi-

grees of the AI families were constructed. Informed consent

was obtained from all patients and/or their parents. The study

was approved by the Slovenian Committee for Medical Ethics

(Nos. 24/12/04 and 86/02/06).

2.2. ENAM mutation analysis

Genomic DNA was isolated from 10 ml of peripheral blood. All

10 exons and exon/intron boundaries of the ENAM gene were

amplified using pairs of primers according to Hart and co-

workers.13 PCR was performed in a volume of 50 ml containing:

20 pM each of forward and reverse primers, 200 mM each of

dNTPs, 3 mM MgCl2, 5 ml PCR buffer, 0.4 ml Ampli Taq GoldTM

polymerase (PE applied Biosystems, Norwalk, CT, USA) and

200 mg DNA. PCR was performed in the GenAmp PCR System

9700 (PE Applied Biosystems, Piscataway, NJ, USA) by an initial

denaturation at 94 8C for 9 min, followed by 35 cycles of

denaturation at 94 8C for 30 s, annealing at 58 8C (ENAM 1–3,

ENAM 4–5, ENAM 6, ENAM 7, ENAM 10a) or 60 8C (ENAM 1,

ENAM 8, ENAM 9, ENAM 10b, ENAM 10c, ENAM 10d, ENAM 10e)

for 30 s, extension at 72 8C for 40 s, followed by a final

extension at 72 8C for 7 min. The PCR products were electro-

phoresed on 2% agarose gels dyed with 2 mg/ml Et-bromide

(45 min, 90 V). The amplicons were extracted using the Qiagen

extraction kit (QIAGEN GmbH, Hilden, Germany). After a

second electrophoresis of PCR products, the concentration of

amplified sequences was estimated. Sequencing PCR was

performed in a volume of 20 ml under the following conditions:

25 repeated cycles of denaturation at 96 8C for 10 s, annealing

at 50 8C for 5 s and extension at 60 8C for 4 min. Extracted

amplicons were sequenced using the ABI PRISM1 310 Genetic

Analyser (PE Applied Biosystems). Results were compared to

normal sequences of the ENAM gene accessible on the Internet

(http://www3.ncbi.nlm.nih.gov: Acc. No.: AY167999).

2.3. Scanning electron microscopy

For scanning electron microscopy (SEM) analysis, three

deciduous molars were prepared immediately after exfolia-

tion: a lower right deciduous second molar (tooth 85)

belonging to the boy from family 1, an upper left deciduous

first molar (tooth 64) and an upper right deciduous second

molar (tooth 55) belonging to each of the brothers from family

2. For comparison, normal enamel of the second left deciduous

mandible molar (tooth 75) from a healthy subject was utilised.

The teeth were cut in half in the bucco-lingual direction. The

halves were embedded in epoxy resin (Araldite, Ciba-Geigy,

East Lansing, MI, USA) with the cut side exposed. The exposed

axial cross-sections were polished, dehydrated with 70%

alcohol, dried and sputter coated in a vacuum with a thin

carbon layer (Vacuum Evaporator, Type JEE-SS, JEOL, Tokyo,

Japan) and examined with SEM (JEOL JSM—5610, JEOL, Tokyo,

Japan). Images were obtained at magnifications between

1000� and 1400�.

3. Results

3.1. Family pedigrees and phenotype determination

No metabolic or endocrine defects, generalised diseases,

syndromes or fluorosis were identified in either family in

which the ENAM mutation was detected. Pedigrees segregat-

ing for autosomal dominant amelogenesis imperfecta are

presented in Fig. 1.

Table 1 – Mutations described in the enamelin gene (ENAM)

Reference g DNAa Exon c DNAb Proteinc AI typesd Enamele Openbitef

Inheritance

Mardh et al.23 g.2382A > T Exon 5 c.157A > T p.K53X Local hypoplastic Horizontal rows of pits,

grooves or large hypoplastic

areas

� AD

Kim et al.12 g.2382A > T Exon 5 c.157A > T p.K53X Local hypoplastic Enamel missing, linear

horizontal defects at

the cervical third

� AD

Kim et al.10 g.4806A > C Intron 6 IVS6-2A > C p.M71–Q157del Generalised hypoplastic Severe hypoplastic enamel,

horizontal grooves,

incisal edges chipped easily

+ AD

Rajpar et al.4 g.6395G > A Intron 8 IVS7+1G > A p.A158–Q178del Generalised hypoplastic Thin enamel, smooth surface,

small, yellow teeth

� AD

Kida et al.16 g.8344delG Intron 9 IVS8 + 1delG;

c.588 + 1delG

p.N197fsX277 Generalised hypoplastic/

local hypoplastice

Thin, smooth surface,

yellowish teeth/horizontal

lesion, pittede

+/� AD

Hart et al.14 g.8344delG Intron 9 IVS8 + 1delG;

c.588 + 1delG

p.N197fsX277 Generalised hypoplastic Thin, smooth surface/thin,

horizontal furrows with pits

� AD

Kim et al.10 g.8344delG Intron 9 IVS8 + 1delG;

c.588 + 1delG

p.N197fsX277 Generalised hypoplastic Severely hypoplastic, rough,

horizontal grooves, yellowish

+ AD

This study (family 2) g.8344delG Intron 9 IVS8 + 1delG;

c.588 + 1delG

p.N197fsX277 Generalised hypoplastic Severely hypoplastic, rough

surface, yellowish

+ AD

Ozdemir et al.15 g.12663C > A Exon 10 c. 737C > A p.S246X Local hypoplastic Hypoplastic enamel on occlusal

and palatal surface

� AD

Ozdemir et al.15 g.12946–12947ins

AGTCAGTACCA

GTACTGTGTC

Exon 10 c.1020–1021ins

AGTCAGTACCA

GTACTGTGTC

p.V340-M341

insSQYQYCV

Local hypoplastic Localised circumscribed enamel

pitting

� AD

Hart et al.5 g.13185–13186insAG Exon 10 c.1258–1259insAG p.P422fsX448 Generalised hypoplasticf,

generalised hypoplastic/

local hypoplastici

Generalised thin, yellowish

Severe hypoplastic and

under-mineralised enamel/

enamel pitting

�/+ AR/ADg

Ozdemir et al.15 g.13185–13186insAG Exon 10 c.1258–1259insAG p.P422fsX448 Local hypoplastic Local enamel-pitting � AD

This study (family 1) g.13185–13186insAG Exon 10 c.1258–1259insAG p.P422fsX448 Hypomaturation/local

hypoplastich

Chalky white enamel, minor

local hypoplastic alterations/

local hypoplastic, horizontal

groves

�/+ AD

AD, autosomal-dominant; AR, autosomal-recessive.a Reference sequencing for GenBank accession No. AY167999; the A of the initiation for ATG is taken as +1.b Reference sequencing for GenBank accession No. AF125373; the A of the initiation for ATG is taken as +1.c Reference sequencing for GenBank accession No. NP114095; the initiator methionine as +1 position.d Adopted from24.e In this kindred an obvious difference between phenotypes of heterozigotes affected members is reported.f AR is compound heterozygotes for the ENAM mutation g.12946–12947insAGTCAGTACCAGTACTGTGTC and ENAM mutation g.13185–13186insAG.g Homozygotes present generalised hypoplastic phenotype and heterozygotes local hypoplastic phenotype; the mutation is dose-dependent: generalised hypoplastic AI is transmited as AR and

localised enamel pitting as AD.h The proband and his father, both heterozygotes, presented different phenotypes.

ar

ch

iv

es

of

or

al

bio

lo

gy

52

(2

00

7)

20

9–

21

72

11

Fig. 1 – Pedigrees of (A) family 1 designated for the ENAM mutation g.13185–13186insAG and (B) family 2 designated for the

ENAM mutation g.8344delG; both pedigrees segregate with autosomal dominant AI. The filled symbols denote affected

individuals. An asterisk indicates individuals examined clinically and genetically.

Fig. 2 – Phenotypic differences in the two heterozygous patients for the 13185–13186insAG ENAM mutation in family 1. (A)

The 11-year-old proband (III-1) was presented with chalky whitish enamel and (B) only minor localised hypoplastic

alteration (arrows) with (C) normal enamel thickness, but a less distinguished difference between enamel and dentin on

dental panoramic tomograms was observed. (D) The proband’s father (II-3) had an open bite; the enamel was yellowish

with (E) horizontal grooves in the cervical half of the teeth crowns (arrows) and (F) poorly visible enamel on dental

panoramic tomograms.

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 0 9 – 2 1 7212

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 0 9 – 2 1 7 213

3.1.1. Family 1The 11-year-old proband (Fig. 1A: III-1) was presented with

chalky whitish enamel (Fig. 2A) with yellow-brown coloured

fissures. Contacts between the teeth were normal. The enamel

surface was smooth at first glance, but roughness was felt on

exploration with a dental probe. The minor localised hypo-

plastic alterations were expressed predominantly in fissures

and thin anatomical parts (e.g. incisally) (Fig. 2B). On

Fig. 3 – In all four siblings in family 2 with heterozygous ENAM

alteration in the quality and especially in the quantity of enam

generalised hypoplastic AI and gingival inflammation due to in

enamel was visible on dental panoramic tomograms. (C) Similarl

old boy (III-2) and (D) similar enamel findings were observed on

profound tooth attrition in the brother, who was 2 years older (

the mixed dentition of the 7-year-old girl (III-3) and (F) her dental

and (G) phenotype of deciduous dentition of the 3-year-old girl (I

at the age of 4 and a half, revealed less altered deciduous teeth

The individual (III-3) was diagnosed on the basis of examination

was confirmed after the ENAM mutation was determined. Notic

tooth buds of permanent teeth presented on dental panoramic t

feeding in both individuals, (E) in the girl (III-3) photographed a

anterior open bite is present and the deciduous dentition of the

orthodontic assessment the estimated molar relations in

the saggital plane were normal (Angle class I). In the

intercanine region, however, an increased overbite was

observed. Dental panoramic tomograms revealed poorer

differentiation between dental enamel and dentin translu-

cency; the thickness of the enamel was estimated to be normal

(Fig. 2C). The intraoral examination of the proband’s father (II-

3) revealed poor dental status (Fig. 2D). Attrition was observed

mutation designated for the g.8344delG the determined

el was severe. (A) The 12-year-old boy (III-1) expressed

creased plaque retention on rough enamel surfaces. (B) No

y altered enamel was found in the dentition of the 10-year-

his dental panoramic tomograms. Notice the more

III-1) compared to the younger one (III-2). (E) Phenotype of

panoramic tomogram taken at the age of 8 and a half years

II-4) and (H) dental panoramic tomogram of her teeth taken

compared to permanent dentition of three older siblings.

of her permanent teeth and the individual (III-4) diagnose

e that on neither the deciduous tooth nor the developing

omogram, any enamel is visible. Due to a prolonged bottle

t the age of 8 years and (G) in her younger sister (III-4), the

girl (III-4) indicates severe anterior tooth decay.

Fig. 4 – Non-etched ultrastructure of enamel of the second right deciduous mandible molar (tooth 85) belonging to patient

(III-1) from family 1 viewed using scanning electron microscopy, revealing an empty and wider sheath space throughout

the thickness of the enamel. Supplementary (A) defective mineralised enamel with no prismless layer present on the

enamel surface (1200�), (B) areas with heavily deformed prisms (1200�) and (C) linking of oval cross-cut prisms together,

separated by an empty sheath space, which creates the impression of ribbons lining parallel to the DEJ (1000�) could be

observed. The first left deciduous maxillary molar (tooth 64) belonging to patient III-1 from family 2 showed: (D) heavily

reduced enamel thickness, unrecognisable prism ultrastructure and a rough enamel surface (1000�) with (E) defects on the

enamel surface (white arrow), defects in the bulk of the enamel (shorter white arrow) and a laminated appearance of the

inner third of the enamel (marked with an asterisk) (1400�). (F) The enamel of a second right deciduous maxillary molar

(tooth 55) belonging to patient III-2 from family 2 showed highly porous laminated enamel with a rough enamel surface

(1300�). For comparison, healthy enamel of the control teeth, also not etched, was evaluated; (G) the enamel of the second

left deciduous mandible molar (tooth 75) revealed well mineralised enamel prisms through the entire thickness of the

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 0 9 – 2 1 7214

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 0 9 – 2 1 7 215

on teeth with yellowish enamel. The enamel surface was

smoother compared to the boy’s (III-1), but in the cervical half

of the tooth crowns horizontal grooves were detected (Fig. 2E).

He had an anterior open bite and was prone to mouth

breathing. On dental panoramic tomograms, the difference in

translucency between dental enamel and dentin was poor

(Fig. 2F). Examination of the proband’s brother (III-2) and

mother (II-2) did not reveal any enamel alteration or mutations

in the ENAM gene.

3.1.2. Family 2Mixed dentition of the 12-year-old boy (Fig. 1B: III-1) showed

generalised hypoplastic teeth. The enamel was thin, yellow

and hard, with a rough and tiny-grained enamel surface.

Attrition was observed (Fig. 3A). Orthodontic assessment

revealed Angle class I posterior occlusion with increased

overbite and overjet of 4 mm incisor relationship. On dental

panoramic tomograms no enamel could be seen (Fig. 3B). The

10-year-old brother’s (III-2) dental status revealed mixed

dentition. As in his brother, the enamel was thin and

yellowish, with a tiny-grained enamel surface (Fig. 3C).

Orthodontic assessment revealed dental malocclusion, with

Angle class II relation on the right side. Radiographically no

enamel was visible on the primary or permanent teeth

(Fig. 3D).

3.2. ENAM mutation analysis

Sequencing of the enamelin gene (ENAM) in family 1 revealed

the same heterozygous mutation in both the proband and his

father. According to a comprehensive nomenclature proposal

for ENAM mutations,14 the identified mutation was g.13185–

13186insAG. In family 2, a heterozygous ENAM mutation

g.8344delG was revealed in all four siblings and their father. No

other sequence alteration in the ENAM gene, including SNPs,

was found in any individual from either family.

3.3. Scanning electron microscopy

Enamel ultrastructure of the lower right deciduous second

molar (tooth 85) of patient (III-1) from family 1 revealed a

normal prismless structure only in a narrow ribbon near the

cervical part of the tooth crown. However, on the majority of

the enamel surface there was no prismless layer (Fig. 4A).

Longitudinal and cross-cut prisms were deformed, with wider

interprism spaces. The course of the enamel prisms was

abnormal, or could not be distinguished at all. Irregularly

shaped empty spaces were present. Borders of the enamel

prisms were undulated and structural shapes of prisms

unequal (Fig. 4B) as compared to normal cross-cut prisms of

healthy enamel (Fig. 4G and H). Cross-cut prisms of the (III-1)

patient’s teeth were oval in shape, as if compressed between

the enamel surface and the dentine-enamel junction (DEJ);

this phenomenon gave the impression of zones parallel to the

DEJ (Fig. 4C). The worse structured and the most porous

enamel was near the DEJ.

enamel from the enamel surface (1200�) towards (H) the DEJ (1

narrow sheath space, individual prisms are barely distinguisha

10 mm.

The enamel of the upper left deciduous first molar (tooth

64) of patient (III-1) from family 2 revealed an extremely thin

enamel layer with an approximate maximal thickness of

100 mm. No enamel was present occlusally. The enamel

surface was excessively rough (Fig. 4D), and defects were also

present in the bulk of the enamel. The ultrastructure of prisms

was unrecognisable. The laminated appearance, especially in

the inner third, gave the impression of layers parallel to the

DEJ (Fig. 4E). Empty spaces of different sizes were present

throughout the enamel thickness.

The enamel of the upper right deciduous second molar

(tooth 55) of patient (III-2) from family 2 also revealed a

markedly reduced thickness, it lacked a normal prismatic

structure and had a laminated appearance parallel to the DEJ

(Fig. 4F). In some layers, a higher degree of porosity was

expressed. The enamel showed severe porosity, especially

near the DEJ and was rough on the surface.

4. Discussion

Two distinct enamelin gene (ENAM) mutations were identified

in two unrelated families with different phenotypes of

autosomal dominant amelogenesis imperfecta.

Two clinically distinct forms of AI, smooth hypoplastic AI

and local hypoplastic AI, are described with their ENAM gene

mutations.13 However, the main clinical feature of the

proband (III-1) from family 1 in this study was chalky white

hypomaturated enamel with only mild local hypoplastic

alteration. On the other hand, clinical alteration of his father’s

enamel (II-3) clearly expressed features of local hypoplastic AI.

Phenotypic diversity associated with some cases of AI may

represent environmental influences, pleiotropic effects of the

underlying gene mutation or modifying gene effects.5 Differ-

ences in the clinical features, such as horizontal grooves

expressed on the father’s (II-3) but not on the proband’s

enamel (III-1), may have been either a genuine dental feature

of AI or environmentally induced enamel defects (e.g. tooth-

brush abrasion) in addition to localised enamel alteration

(Fig. 2E). However, as the grooves were distinct and ran around

the tooth, including interproximally, it seemed less likely that

the horizontal lines were due to toothbrush abrasion.

In both the proband and his father from family 1, the

heterozygous mutation of the ENAM gene g.13185–13186insAG

was identified. In the proband’s brother (III-2) and mother (II-

2), clinical examination did not reveal any enamel character-

istic of AI and no mutations in the ENAM gene were found. The

deduced mode of inheritance was therefore autosomal

dominant. The same kind of mutation has been reported

previously in three families.5 The authors concluded that the

phenotype associated with the g.13185–13186insAG ENAM

mutation is dose-dependent, such that the homozygote, with

a severe generalised hypoplastic phenotype and open bite

malocclusion, segregates as a recessive trait and the hetero-

zygote, with only localised enamel pitting, as a dominant trait.

In heterozygous patients no open bite has been observed.5 In

200�). Due to the high quality of mineralisation and very

ble. ES, enamel surface; DEJ, dentine-enamel junction; bar

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 0 9 – 2 1 7216

contrast to this previous report on the 13185–13186insAG

ENAM mutation, in the present study both heterozygous

members of family 1 were presented with orthodontic

malocclusion, with the father having an open bite. Therefore,

we also observed that a more profound clinical presentation

segregated as an autosomal dominant trait.

Although the reasons for these differences are not fully

understood and the meaning of the mutations in terms of the

protein are still unclear, variability in clinical appearance

among individuals with the same mutation can be explained

by translation of markedly different proteins or translation of

proteins of similar size with critical differences in functionally

important domains,14 influence of modifying genes or the

pleiotropic effect of gene(s),5 mosaicism16 and/or the influence

of environmental factors.5

In family 2, all four children (III-1, III-2, III-3 and III-4) and

their father (II-1) were presented with the generalised

hypoplastic AI phenotype in association with a heterozygous

ENAM mutation designated as g.8344delG. This mutation has

been previously reported in a Japanese family,16 in three

unrelated Lebanese families14 and in a family of Iranian

descent.10 It would appear that this particular site on the

ENAM gene is a ‘‘hot spot’’ for mutation14,17 with this occurring

in diverse populations around the world. Patients with the

g.8344delG ENAM gene mutation described in those reports

were presented with generalised or local hypoplastic AI,16

variable generalised hypoplastic AI with the teeth of some

individuals having a smooth, and others, a rough surface14 or

generalised hypoplastic AI with shallow horizontal grooves in

the middle 1/3 of the anterior teeth.17

Mutations in the ENAM gene were reflected not only in the

clinical phenotype but also at the ultrastructural level, where

both hypoplastic and hypomineralised features were present

in varying degrees in samples with g.13185–13186insAG or

g.8344delG ENAM mutations. This is in agreement with

previous findings that the hypoplastic enamel is not only

very thin but also generally porous, lacking the normal

prismatic structure in certain areas,18 with enamel surface

containing demineralised pores running perpendicular to the

surface.19 In addition to the deficiency in the quantity of

enamel, defective enamel quality also contributes to disco-

louration of the enamel and results in greater susceptibility to

tooth decay and loss of enamel due to occlusal stress. On

examination of teeth samples under SEM, no enamel was seen

occlusally in either of the ENAM mutations. We speculated

that enamel on these surfaces could not resist wear due to

repeated mastication forces.

Ultrastructure of the AI enamel has previously been

described primarily according to clinically determined phe-

notypes, with the specific ENAMmutation also being described

in only one report.14 The findings of this study were in

agreement with this previous report on ENAM mutation

g.8344delG ultrastructure, in which affected enamel is

markedly reduced in thickness, lacks a normal prismatic

structure and has a laminated appearance.14 The ultrastruc-

ture of the g.13185–13186insAG ENAM mutation has, to the

best of our knowledge, not been previously described.

Each ENAM gene mutation that results in a functionally

altered uncleaved protein influences crystal elongation. Intact

enamelin (186 kDa) and the large enamelin cleavage products

(155, 142 and 89 kDa) are present only near the enamel surface

and do not accumulate in the matrix.19 All of these proteins

contain the original enamelin N-terminus which does not bind

minerals and concentrates in the sheath space21 along with N-

terminal polypeptides from ameloblastin. 19 If 89 kDa enam-

elin function is altered, as is the case in ENAM mutations

g.13185–13186insAG and g.8344delG, the prism formation can

be altered and the sheath space appears empty. Indeed, in the

enamel of the boy from family 1, in which the 89 kDa cleavage

products were mutated but 32 kDa was normal, the non-

etched enamel ultrastructure revealed deformed prisms and

an empty and extremely wavy sheath space (Fig. 4A–C).

The 32 kDa enamelin cleavage product, which is the most

abundant in developing enamel,22 concentrates in the rod and

interrod enamel and shows a ‘‘reverse honeycomb’’ distribu-

tion pattern.20 The 32 kDa enamelin, which is resistant to

further proteolytic digestion and bind HA crystals in vitro, has

two phosphorylated serines (Ser191 and Ser216) and 3 glycosy-

lated asparagines (Asn245, Asn252 and Asn264) that are thought

to impart a high calcium affinity and possibly crystallite

binding properties to the protein.5,20 In the ENAM mutation

g.8344delG, alterations in codon 197 result in the abandon-

ment of Ser216 and abolition of all three N-glycosylation sites

on the 32 kDa enamelin.14 The enamel of both boys from

family 2 demonstrated a laminated appearance, but no prism

formation (Fig. 4D–F). The repeated process of aborting the

normal long axis growth of enamel crystallites could be the

cause of this phenotype, with new crystallites being initiated

until the entire process of amelogenesis terminates prema-

turely.14

Identification of the enamel ultrastructure in each defined

ENAM mutation will improve our understanding of the role of

enamelin in normal and AI enamel formation, add important

information for clarifying different AI types, and provide

additional information helpful for establishing the exact

diagnosis and treatment of these conditions.

In conclusion, phenotypic diversity of enamel was found in

patients with ENAM gene mutations g.13185–13186insAG

within the same family, and the clinical presentation in the

heterozygous state can be more pronounced than previously

described, indicating an autosomal dominant mode of

inheritance. Ultrastructural characteristics in the proband

with the ENAM g.13185–13186insAG mutation revealed

deformed prisms, an oval shape on the cross-section and

wider interprismal spaces. However, in the proband’s father

with the ENAM gene mutations g.13185–13186insAG, only

clinical enamel phenotypes but no ultrastructural changes

could be characterised. Ultrastructural enamel changes in the

proband with the ENAM mutation g.13185–13186insAG,

described for the first time in the present study, were less

pronounced compared to ultrastructural changes in patients

with the ENAM mutation 8344delG.

Acknowledgements

The study was supported in part by the Slovenian Research

Agency grant J3-6072. The authors would like to thank Prof.

Kosec and Dr. Skraba, Department of Materials Science and

Metallurgy, Faculty of Natural Sciences and Engineering,

a r c h i v e s o f o r a l b i o l o g y 5 2 ( 2 0 0 7 ) 2 0 9 – 2 1 7 217

University of Ljubljana, for their help in setting up the study

and also to Ms. Nika Breskvar and Ms. Jurka Ferran for their

expert technical assistance.

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