Amelogenesis imperfecta phenotype–genotype correlations with two amelogenin gene mutations

Abstract

Amelogenin, the predominant matrix protein in developing dental enamel, is considered essential for normal enamel formation, but its exact functions are undefined. Mutations in the AMELX gene that encodes for amelogenin protein cause X-linked amelogenesis imperfecta (AI), with phenotypes characterized by hypoplastic and/or poorly mineralized enamel. Eight different AMELX deletion and substitution mutations have been reported to date. The purpose here was to evaluate the genotype and phenotype of two large kindreds segregating for X-linked AI. Phenotypically affected males in family 1 had yellowish-brown, poorly mineralized enamel; those in family 2 had thin, smooth, hypoplastic enamel. Heterozygous females in both kindreds had vertical hypoplastic grooves in their enamel. DNA was obtained from family members; exons 1–7 of AMELX were amplified and sequenced. Mutational analysis of family 1 revealed a single-base-pair change of A→T at nucleotide 256, resulting in a His→Leu change. Analysis of family 2 revealed deletion of a C-nucleotide in codon 119 causing a frameshift alteration of the next six codons, and a premature stop codon resulting in truncation of the protein 18 amino acids shorter than the wild-type. To date, all mutations that alter the C-terminus of amelogenin after the 157th amino acid have resulted in a hypoplastic phenotype. In contrast, other AMELX mutations appear to cause predominantly mineralization defects (e.g. the mutation seen in family 1). This difference suggests that the C-terminus of the normal amelogenin protein is important for controlling enamel thickness.

Introduction

Amelogenesis imperfecta (AI) is a clinically and genetically diverse group of conditions affecting the development of dental enamel (Witkop, 1989). Depending on the imperfecta type, the enamel defects may be abnormalities of amount, structure and/or composition. The molecular basis of these conditions has so far only been elucidated for X-linked forms of AI, in which a variety of mutations in the X-chromosome amelogenin gene (AMELX) that lead to diverse phenotypes have been found (see Hart et al. (2000) for recent review). No cases of mutations in the Y-chromosome amelogenin gene have been identified. The more common, autosomal-dominant types of AI are also clinically and genetically heterogeneous. Amelogenin gene mutations identified to date include large deletions, single-base deletions, and mis-sense and non-sense mutations.

Amelogenin, the predominant extracellular matrix protein in developing enamel, is thought to form a scaffold for enamel crystallites and to participate in controlling their growth (Robinson et al., 1990, Fincham et al., 1992, Simmer and Fincham, 1995), but its exact functions are not fully known. It is processed in a highly controlled fashion that is apparently important for normal enamel formation (Robinson et al., 1990, Brookes et al., 1995). The study of enamel formation under the influence of genetically altered amelogenin should therefore provide unique insights into the function of the protein. The amelogenin mutations reported to date result in diverse phenotypes, ranging from a deficiency in the amount of enamel (hypoplasia) to defects in enamel mineralization (hypomineralization) (Hart et al., 2000).

Although there is demonstrable diversity in the expression of AI even within individuals from the same kindred, there is growing evidence that unique phenotypes could be associated with specific AMELX mutations (Aldred et al., 1992, Ravassipour et al., 2000). For example, three families with the same single-base C→A substitution in codon 40 of exon 6 causing a Pro→Thr change show strong conservation of phenotype in affected individuals (Ravassipour et al., 2000). All the affected males with this mutation who have been analyzed had what was considered to be the hypomaturational form of AI, with retention of amelogenin and hypomineralization but no evidence of hypoplastic defects. In contrast, other AMELX mutations are often associated with severely hypoplastic phenotypes (Lench and Winter, 1995).

We hypothesize that those mutations which cause changes in domains of the amelogenin protein that have different and unique functions will result in distinct and diverse phenotypes. Identifying different AMELX mutations and correlating them with the resulting phenotype should help to define the functions of the amelogenin protein and its functional domains. Therefore, our purpose now was to characterize the phenotype and genotype in two large families segregating for X-linked AI.