Amelogenesis Imperfecta Due to a Mutation of the Enamelin Gene: Clinical Case With Genotype-phenotype Correlations

Abstract

The major protein components of the enamel matrix include the most abundant amelogenin proteins as well as less plentiful proteins such as enamelin and ameloblastin. The enamel defect in amelogenesis imperfecta (AI) generally results in enamel that is too thin (hypoplastic) or too soft (hypocalcification or hypomaturation). Previous reports indicate that mutations in the human enamelin gene (ENAM) cause hypoplastic AI through autosomaldominant inheritance patterns and patients may also exhibit an anterior open bite. Although crown resorption of unerupted teeth occurs more frequently in AI patients, this finding has not been previously associated with known ENAM mutations. The purpose of this article was to report the genotype-phenotype correlations for a 9-year, 11-month-old boy with a homozygous ENAM mutation (c.1258_1259insAG).

At the beginning of the formation of dental enamel, ameloblast cells within the dental organ secrete an organic matrix which immediately begins to mineralize. The major enamel protein components of this matrix include the most abundant amelogenin proteins as well as less plentiful proteins such as enamelin and ameloblastin¹. Mutations in the genes encoding these and other proteins lead to the enamel defect amelogenesis imperfecta (AI).^2–4 The defect in AI generally results in enamel that is too thin (hypoplastic) or too soft due to abnormal mineralization (hypocalcification or hypomaturation).^5,6

AI has been classified according to both the appearance of the enamel and the pattern of genetic inheritance.^2,5–8 As sufficient information is learned about the genetic causes of AI, a gene-based classification system will most likely be adopted. In the past, treatment has generally been provided without knowledge of the genetic defect. With most AI cases now diagnosable at the molecular level, however, this will not be the case in the future.

The AI trait caused by mutations in the enamelin (ENAM) gene is transmitted predominantly through an autosomal-dominant mode of inheritance.^9–16 ENAM mutations generally result in hypoplastic AI, in which the hypoplastic teeth can have proximal spacing due to reduced enamel thickness. Depending on the specific ENAM mutation, the hypoplastic enamel may be localized as pits or horizontal ridges or can be generalized with the entire enamel thickness being markedly reduced. In addition, several of the reported cases of ENAM gene mutations report an anterior open bite malocclusion.^10,15,17

Crown resorption of unerupted teeth and delayed eruption occurs more frequently in AI patients than non-AI patients.^18,19 This has not, however, been specifically linked to patients with ENAM mutations. These defects have aesthetic implications with frequent psychosocial impact requiring extensive treatment of primary and permanent dentitions as well as counseling therapies.²⁰

A case of autosomal hypoplastic AI presented to the pediatric dentistry clinic at Children's Hospital of Philadelphia (CHOP), Philadelphia, Pa. In addition to the enamel defects usually associated with AI, there appeared to be evidence of hyperplastic gingival tissue and several impacted permanent molars. Unlike several previous reports on autosomal hypoplastic AI, the purpose of this report was to describe the phenotypical/clinical findings for this patient as well as the histological findings and mutational analysis.

Case description

The patient and parents provided consent for treatment and genetic analysis. Saliva collection and mutational analysis was approved by the Institutional Review Board of the University of Pennsylvania, Philadelphia, Pa. A 9-year, 11-month-old AI male was referred to CHOP by a private pediatric dentist for dental evaluation and treatment. His chief complaint was that the permanent posterior molars were not erupting. His prenatal and medical history was noncontributory. His mother reported a normal pregnancy and delivery without complications. His father indicated a history significant for AI, as reported to him by his family dentist, who had performed extensive dental rehabilitation on him over the years (Figure 1). The father also stated that his mother had received dentures at a young age and that another cousin (not included on the pedigree chart) and his niece had been told by their dentists that they had AI. The mother denied any history of AI.

Figure 1.

Pedigree chart. (Males are indicated by squares, females are circles, sex unknown are diamonds, affected shaded in, and non-penetrant indicated by dark circles in the unshaded symbols. Except for the proband (indicated by arrow), affected kindred were reported by the proband's father.

Clinical examination revealed a mixed dentition. The patient denied any sensitivity of the teeth to thermal stimuli, eating difficulty, or pain. The teeth appeared smooth and lacking proximal contacts with a generalized yellow color and thin enamel consistent with the findings of hypoplastic AI. There was no clinical evidence of pitting, grooves, or other irregularities in the enamel (Figure 2). All of the first permanent molars were unerupted, despite having complete root formation. The primary maxillary posterior teeth appeared ankylosed, as they were well below the occlusal plane. This may have been due to the hyperplastic gingival tissue and uneven resorption of some of the primary roots. The primary mandibular central incisors were retained, and the permanent mandibular central incisors appeared to be congenitally missing. Soft tissues overlying the affected teeth were intact, and no periodontal pockets were detected on any adjacent teeth. There was enlargement of the gingival tissues in the molar region.

Figure 2.

Clinical presentation of patient (male: 9y/11m) with autosomal hypoplastic AI. Teeth are smooth and lacking proximal contacts with a generalized yellow color.

His occlusion appeared to be a bilateral Class I molar with an overbite of 100% (no anterior open bite) and an over-jet of 2mm. Radiographically, the unerupted permanent maxillary first molars appeared malformed with resorption of a portion of the clinical crowns (Figure 3). The permanent mandibular right first molar appeared to have a marked area of resorption on its mesial surface. There was no evidence of taurodontism. After consulting with an oral surgeon and an orthodontist, a treatment plan was formulated to surgically expose the 4 permanent molars and extract all remaining primary molars. Saliva samples were collected from the patient and his parents for DNA analysis.

Figure 3.

Panograph of patient (male: 9y/11m) with autosomal hypoplastic AI. Note the permanent molars are unerupted, with the maxillary first molars appearing externally resorbed.

The patient was taken to the operating room for oral rehabilitation under general anesthesia, and surgical exposure of the permanent first molars was performed. The 2 permanent maxillary molars appeared to have deformed dentin, a direct communication with the pulp chambers in both teeth, and little or no enamel (Figure 4). They were clinically non-restorable and subsequently extracted. The permanent mandibular molars were exposed, and an area of resorption was evident on the permanent mandibular right first molar's buccal surface. The mandibular tooth was restored, and the extracted teeth were preserved for evaluation by scanning electron microscopy (SEM).

Figure 4.

Extracted maxillary permanent first molars with crown resorption.

Histological evaluation

The teeth were photographed and then fractured, mounted on aluminum stubs with silver paint, and coated with Au-Pd for SEM.²¹ The fractured surfaces were evaluated to assess the enamel thickness and structure at various magnifications using a JEOL JSM 6300 SEM (JEOL USA, Peabody, Mass). The analysis revealed that the enamel lacked any prismatic architecture and was extremely thin (Figure 5).

Figure 5.

Scanning electron micrographs of fractured teeth. A) Normal enamel surface (E) and prismatic fractured enamel layer. B) In the proband's permanent maxillary molar, the enamel lacked any prismatic architecture and was extremely thin (Line ~10 μm) (E = Enamel, D = Dentin; original magnification (1400X).

Mutation analysis

Genomic DNA was isolated from saliva using the QIAamp kit (Qiagen, Santa Clara, Calif). The ENAM gene was sequenced as the most likely candidate gene in this AI family due to the generalized thin hypoplastic phenotype that has been previously associated with several different ENAM mutations. The exons and exon/intron boundaries of the ENAM gene were amplified by PCR using forward and reverse primers previously described.¹² Amplicons were electrophoresed through agarose gels, and the products were extracted using the Qiagen gel extraction kit. Extracted products were sequenced in both directions. The proband was homozygous for ENAM mutation c.1258_1259insAG, while the parents were both heterozygous (Figure 6). This insertion generates a premature stop codon at codon 448.¹⁷

Figure 6.

Chromatograms for Enam mutation (c.1258_1259insAG): A. DNA sequence of the proband, showing he is homozygous for the mutation. The AG insertion is indicated by a bracket, and generates a premature stop at codon 448. B. DNA sequence of the proband's father, who is heterozygous for the insertion, with the location indicated by an arrow.

Discussion

This report illustrates the diverse manifestations that can be associated with AI and is the first to document abnormal tooth eruption and coronal resorption with an ENAM mutation. Enamelin is a glycosylated protein, considered to be an enamel-specific protein secreted by ameloblasts in relatively low amounts (1–5% of the matrix).^22,23 While the specific role of enamelin in amelogenesis is unknown, it is thought to play a role in crystallite growth regulation and elongation. In enamelin mutations that result in a secreted but altered protein, a generalized thin hypoplastic phenotype is generally seen.² There is no known role of enamelin in tooth eruption, and abnormal tooth eruption is not generally reported with ENAM mutations.

The purpose of this paper was to illustrate how the clinical phenotype of generalized hypoplastic AI led us to suspect a mutation in the ENAM gene and the identification of the molecular defect. The ENAM mutation c.1258_1259insAG has been described previously and was associated with a generalized hypoplastic phenotype in individuals homozygous for the mutant allele.¹⁷ The child in this report also was homozygous for the ENAM mutant allele and had smooth but very thin enamel. It was, however, devoid of any pitting or grooving, as has been described with several previous cases.¹⁷ The unusual finding of unerupted permanent molars that undergo resorption has been reported in other cases of hypoplastic AI and is most commonly associated with autosomal recessive rough hypoplastic AI. The associated genetic mutation, however, has not been previously identified. The etiology of abnormal tooth eruption and coronal resorption remains unclear, but appears to be the result of abnormal function of the enamel epithelium and ameloblasts.

Failure of eruption in patients with this AI type may be a result of an abnormality in the molecular control of the eruption process that is known to be at least partially driven by the odontogenic epithelium. Some AI-associated, nonenamel manifestations could result from modifying genes or environmental effects.¹⁸ The marked phenotypic diversity observed in amelogenin gene mutations associated with AI illustrates how allelic mutations altering different functional domains of the amelogenin protein are responsible for markedly different enamel phenotypes.²⁴ The proband's mother reported not having AI's clinical manifestations. It is possible that, upon microscopic examination, however, she could have subclinical pitting or hypoplastic enamel defects that were not previously thought to be associated with AI, but which have been described in individuals who are heterozygous for the ENAM c.1258_ 1259insAG mutation.¹⁷ The father was also heterozygous for this mutation and appeared to have the same generalized thin enamel phenotype as seen in the proband. This suggests that: the generalized thin hypoplastic AI trait associated with the c.1258_1259insAG ENAM mutation is, indeed, transmitted in an autosomal dominant manner; and there is highly variable expression in people who are heterozygous for this allele. Whether this is correct remains to be validated by additional AI cases that are carefully phenotyped and evaluated at the molecular level.

Selecting appropriate treatment approaches for AI patients can be extremely complex, as demonstrated in this case. SEM analysis revealed a lack of prismatic enamel structure, leading one to question whether or not conventional bonding-based restorative therapies are appropriate. Seow and Amaratunge performed acid etching on the extracted teeth of patients with hypoplastic and hypomineralized AI. They concluded that the lack of typical etching patterns in these variants may be the result of abnormal prism structure, and the standard etching time and/or acid concentration may be inappropriate for the abnormal enamel.²⁵ Several studies have concluded that there is no relationship between bond strength to acid-etched enamel and etching conditions,^26–30 but these studies were all performed on sound enamel.

Acid etch bonding therapies are frequently applied in hypoplastic AI cases and generally appear to have a reasonable level of success, suggesting that even having a thin, nonpris-matic enamel layer could be sufficient to adequately retain bonded materials. Selecting appropriate treatment options and materials is ideally predicated on understanding the type of enamel defect that is present. For example, pretreatment with NaOCl may be used in hypomineralized AI types to improve bonding,³¹ but may not help in hypoplastic enamel that is well-mineralized and does not have increased protein content.

It may one day be practical and highly beneficial to determine the specific AI genotype and associated AI phenotype before rendering treatment to optimize preventive and restorative care. By studying the outcomes of various restorative procedures for each genotype/phenotype condition, practicing dentists will be able to use gene-based diagnoses to choose among various treatment options and thereby restore the dentition in a way that achieves the best results.⁴