Candidate Gene Strategy Reveals ENAM Mutations

Abstract

Amelogenesis imperfecta (AI) is a genetically and phenotypically heterogeneous genetic disorder affecting tooth enamel without other non-oral syndromic conditions. Based on a review of the literature, the authors constructed a candidate-gene-based mutational analysis strategy. To test the strategy, they identified two Turkish families with hypoplastic enamel without any other non-oral syndromic phenotype. The authors analyzed all exons and exon/intron boundaries of the enamelin (ENAM) gene for family 1 and the DLX3 and ENAM genes for family 2, to identify the underlying genetic etiology. The analysis revealed 2 ENAM mutations (autosomal-dominant g.14917delT and autosomal-recessive g.13185–13186insAG mutations). A single T deletion in exon 10 is a novel deletional mutation (g.14917delT, c.2991delT), which is predicted to result in a frameshift with a premature termination codon (p.L998fsX1062). This result supports the use of a candidate-gene-based strategy to study the genetic basis for AI.

INTRODUCTION

Defects in tooth enamel can be due to both genetic and environmental factors. Hereditary enamel defects can be either syndromic or non-syndromic (Hu and Simmer, 2007). Mature enamel is formed by a series of events: the synthesis of enamel matrix, calcification, and maturation. The defects arising from errors in any of these steps can be categorized as one of the three major types of amelogenesis imperfecta (AI): hypoplastic, hypocalcified, or hypomatured (Witkop, 1988).

Hypoplastic AI is caused by mutations in enamelin (ENAM; OMIM 606585) (Kim et al., 2005a; Ozdemir et al., 2005b) or amelogenin X (AMELX; OMIM 300391) (Kim et al., 2004; Kida et al., 2007). Hypocalcified AI (OMIM 130900) is caused by FAM83H mutations (Kim et al., 2008; Lee et al., 2008a), and hypomatured AI is caused by recessive mutations in enam-elysin (MMP20; OMIM 604629) (Kim et al., 2005b; Ozdemir et al., 2005a; Papagerakis et al., 2008) or kallikrein 4 (KLK4; OMIM 603767) (Hart et al., 2004). ENAM mutations cause autosomal-dominant or -recessive AI (TC Hart et al., 2003; Gutierrez et al., 2007), while AMELX mutations cause X-linked AI, and FAM83H mutations cause autosomal-dominant AI.

Mutations have been identified in all genes encoding the enamel matrix proteins with the exception of the ameloblastin (AMBN; OMIM 601259) gene, making these genes the primary candidates for hypoplastic and hypomatured AI (Kim et al., 2006).

The hereditary patterns and clinical phenotypes presented in previous studies allow the present studies to focus on the molecular genetic etiology in persons newly identified as having AI. A careful examination of the clinical phenotype and hereditary pattern via a detailed family history can help prioritize candidate gene(s) for mutational screening. The present study reports a candidate-gene-based mutational analysis strategy and identification of ENAM frameshift mutations in nuclear families with hypoplastic AI.

MATERIALS & METHODS

Identification of Kindred and Enrollment of Human Participants

This study was independently reviewed and approved by the Institutional Review Board at the University of Istanbul and the Seoul National University Dental Hospital. The experiments were undertaken with the understanding and written consent of each person according to the Declaration of Helsinki.

Polymerase Chain-reaction (PCR) and Sequencing

Genomic DNA was isolated from peripheral whole blood by means of the QuickGene DNA whole blood kit S with QuickGene-Mini80 equipment (Fujifilm, Tokyo, Japan). The purity and concentration of the DNA were quantitated by spectrophotometry, as measured by the OD₂₆₀/OD₂₈₀ ratio. A mutational analysis of the exons and flanking intron sequences of the ENAM gene for both families and the DLX3 gene for family 2 was performed. PCR amplifications were conducted according to previous reports (Kim et al., 2005a; Lee et al., 2008b), with the HiPi DNA polymerase premix (ElpisBio, Taejeon, Korea), and the products were purified with a PCR Purification Kit (ElpisBio). DNA sequencing was performed at the DNA sequencing center (Macrogen, Seoul, Korea). Sequence analyses were performed with the help of Mutation Surveyor^® (SoftGenetics, State College, PA, USA).

RESULTS

Mutation Analyses

Family 1

In the present mutational analysis of the ENAM gene, a single nucleotide deletion in exon 10 (Figs. 1A, 1B) was discovered. This mutation was perfectly correlated with the disease phenotype among available family members. According to the nomenclature guidelines for ENAM mutations (PS Hart et al., 2003), this mutation was designated as g.14917delT (c.2991delT). This frameshift mutation (p.L998fsX1062) is predicted to result in the introduction of 64 novel amino acids, followed by a premature termination codon. Because the parents (individuals 9 and 10) of affected individual 16 were not available for mutational analyses, the spontaneity of the mutation in individual 16 could not be determined.

Family 2

Mutational analysis of the DLX3 gene showed no disease-causing sequence variation. Mutational analysis of the ENAM gene revealed a homozygous insertional mutation (g.13185–13186insAG, c.1258_1259insAG, p.P422fsX448) in the proband (Figs. 2A, 2B). Mother, father, brother, and sister of the proband were heterozygous carriers.

Clinical Findings

Family 1

The general clinical phenotype of this family was local hypoplastic. Typical hypoplastic horizontal grooves from the middle to the cervical third of the crown were found in the affected mother (Fig. 1D ), while the daughter presented with wide irregular hypoplastic regions with small hypoplastic spots (Figs. 1C, 1E ).

Family 2

The proband showed generalized hypoplastic enamel with anterior open bite (Figs. 2C, 2D ). Typical hypoplastic horizontal grooves were not noticed. Several second molars (#17, 27, and 37) showed taurodontism (Fig. 2E ). Other family members did not show the enamel phenotype related to AI in their remaining teeth.

DISCUSSION

Using an analysis of the current literature, the authors of the present study constructed a candidate-gene-based strategy for the molecular genetic analysis of persons with AI (Fig. 3 ). FAM83H, DLX3, and genes encoding enamel matrix proteins (AMELX, ENAM, MMP20, and KLK4) were included in the diagram. Careful examination of the dental phenotype as well as the hereditary pattern should be performed prior to the mutational analysis of candidate genes.

Analysis of known mutations has allowed for the possibility of establishing a genotype-phenotype correlation in AI cases caused by AMELX mutations (Wright, 2006). Reduced secretion due to mutations in the signal peptide region and truncated proteins lacking the C-terminus are associated with hypoplastic X-linked AI, while mutations in the N-terminus of AMELX are related to hypomatured X-linked AI in the affected males. Thus, the AMELX gene should be considered as a candidate gene in cases of X-linked hypoplasic or hypomatured AI.

Mutations in the MMP20 and KLK4 genes cause the autosomal-recessive form of hypomatured AI. The characteristic dental phenotype is a yellowish- to brownish-pigmented hypomatured enamel, which is less mineralized and does not contrast well with dentin in radiography. Thus, both genes should be considered as candidate genes for the autosomal-recessive form of hypomaturation type AI.

Tooth enamel in the Tricho-dento-osseous syndrome (TDO; OMIM 190320) is hypoplastic, and the molar teeth have a characteristic taurodontism. Non-oral phenotypes are kinky/curly hair at birth and increased cortical bone density (Wright et al., 1997). A 4-bp deletion (c.571_574delGGGG) in the DLX3 (OMIM 600525) gene has been identified in multiple TDO families (Price et al., 1998). In addition, a 2-bp deletion (c.561_562delCT) in the DLX3 gene has been reported to cause hypomaturation-hypoplastic type AI with taurodontism (AIHHT; OMIM 104510) (Dong et al., 2005). Recently, a new family with the same 2-bp deletion in the DLX3 gene was identified, and the affected members had curly hair at birth and brittle nails, but bone density was not increased (Lee et al., 2008b). The anterior teeth had enlarged pulp chambers instead of the taurodontism observed in the molar teeth. Clinicians should consider the DLX3 gene when enamel is hypoplastic and hypomatured with an enlarged pulp chamber, in addition to any of the TDO phenotypes.

The hypoplastic enamel with typical hypoplastic horizontal grooves (individual 16) in family 1 gave a priority order of ENAM gene screening. Furthermore, there was no Lyonization pattern, as shown in the previous studies of AMELX mutations (Kim et al., 2004; Wright, 2006). Family 2 was recruited during the revision of the manuscript. For this family, the DLX3 and ENAM genes were screened based on the taurodontic features of the second molars as well as hypoplastic enamel.

The 1-bp deletion in the ENAM gene identified in family 1 results in a frameshift after a synonymous change at codon Ile⁹⁹⁷ (ATT to ATC). Normal enamelin protein is 1142 amino acids; however, this frameshift mutation truncates the protein, producing a 1061-amino-acid protein lacking the 145 C-terminal amino acids and replacing them with 64 novel amino acids after codon 997 (p.L998fsX1062).

Enamelin is a secretory protein with a 39-aminoacid signal peptide. The mutant enamelin with its intact signal peptide is believed to be secreted as a normal protein. Secreted enamelin is subjected to a series of proteolytic processes (Hu et al., 2005), whereby the C-terminal end of the protein is rapidly processed and degraded, thus restricting the expression of the full-length protein to the enamel matrix secretory front of the Tomes’ process.

One of the possible outcomes from this mutation is haplo- insufficiency due to the lack of a C-terminus, which may be involved in enamel crystallite elongation or initial crystallite calcification. The other possibility is a dominant-negative effect due to the novel 64 amino acids, which may resist degradation and interfere with normal enamel matrix formation and crystal growth.

Phenotypic variation associated with a single ENAM mutation has been reported in different families (Kida et al., 2002; PS Hart et al., 2003) and even in the same family (Kim et al., 2005a). In this study, the affected mother of family 1 had horizontal hypoplastic grooves, which is typical for enamelin mutation in several cases. However, the proband of family 1 did not have typical horizontal grooves, but rather had irregular hypoplastic regions with several discolored pits. This phenotypic variation in one family may be due to modifying gene effects and/or environmental factors. The g.13185_13186insAG ENAM gene mutation identified in family 2 has been reported previously (TC Hart et al., 2003; Pavlic et al., 2007). In this family, heterozygous carriers did not show enamel pitting in their remaining teeth. Reportedly, in a family with the g.13185_13186insAG ENAM gene mutation, the proband had an extremely mild phenotype, while the affected father had typical horizontal hypoplastic grooves (Pavlic et al., 2007). Furthermore, incomplete penetrance has been suggested in a family with a g.4806A>C ENAM gene mutation (Kim et al., 2005a).

In this study, mutational analyses of persons with AI were performed based on the candidate-gene-based strategy and the two ENAM gene mutations identified. This strategy, along with careful examination of the clinical phenotype and hereditary pattern of persons with AI, will help clinicians and molecular geneticists determine the genetic etiology of the individual with AI and gain a better understanding of the mechanism of tooth enamel formation through genotype-phenotype correlations.

Figure 1.

Pedigree, clinical phenotype, and mutational analysis of family 1. (A) Pedigree of family 1 with the g.14917delT (c.2991delT) ENAM mutation. (B) DNA sequencing chromatogram of affected individual 20. Upper panel is an actual chromatogram. The red arrow indicates the position of a T deletion. Middle and lower panels are chromatograms of a normal and mutated allele, respectively. (C) Frontal view of affected individual 20. (D) Frontal view of affected individual 16. (E) Panoramic radiograph of affected individual 20.

Figure 2.

Pedigree, clinical phenotype, and mutational analysis of family 2. (A) Pedigree of family 2 with a homozygous g.13185_13186insAG ENAM mutation. (B) DNA sequencing chromatograms. Upper panel is a chromatogram of a normal control individual. Middle panel is a chromatogram of the heterozygous g.13185_13186insAG ENAM mutation (individuals 15, 20, 24, and 25). Lower panel is a chromatogram of the homozygous g.13185_13186insAG ENAM mutation (proband). The inserted AG is indicated by a black bar. (C) Frontal view of the proband. (D) Mandibular occlusal view of the proband. (E) Panoramic radiograph of the proband.

Figure 3.

Strategy diagram of candidate-gene-based mutational analysis of persons with AI.

Footnotes

Acknowledgements

The authors thank all the family members involved in this study for their cooperation. This work was supported by a grant from the Korea Science and Engineering Foundation (KOSEF) through the Biotechnology R&D program (No. M10646010003-08N4601-00310) and by the Korea Science and Engineering Foundation (KOSEF) Science Research Center grant funded by the Korean Ministry of Education, Science and Technology (MEST) through the Bone Metabolism Research Center (No. R11-2008-023-02003-0).

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