Relationship of phenotype and genotype in X-linked amelogenesis imperfecta

X-linked amelogenesis imperfectas (AI) resulting from mutations in the amelogenin gene (AMELX) are phenotypically and genetically diverse. Amelogenin is the predominant matrix protein in developing enamel and is essential for normal enamel formation. To date, 12 allelic AMELX mutations have been described that purportedly result in markedly different expressed amelogenin protein products. We hypothesize that these AMELX gene mutations result in unique and functionally altered amelogenin proteins that are associated with distinct amelogenesis imperfecta phenotypes. The AMELX mutations and associated phenotypes fall generally into three categories. (1) Mutations (e.g., signal peptide mutations) causing a total of loss of amelogenin protein are associated with a primarily hypoplastic phenotype (though mineralization defects also can occur). (2) Missense mutations affecting the N-terminal region, especially those causing changes in the putative lectin-binding domain and TRAP (tyrosine rich amelogenin protein) region of the amelogenin molecule, result in a predominantly hypomineralization/hypomaturation AI phenotype with enamel that is discolored and has retained amelogenin. (3) Mutations causing loss of the amelogenin C terminus result in a phenotype characterized by hypoplasia. The consistent association of similar hypoplastic or hypomineralization/hypomaturation AI phenotypes with specific AMELX mutations may help identify distinct functional domains of the amelogenin molecule. The phenotype-genotype correlations in this study suggest there are important functional domains of the amelogenin molecule that are critical for the development of normal enamel structure, composition, and thickness.     

Amelogenin Protein Exhibits a Modular Design: Implications for Form and Function

The most abundant protein of forming enamel is amelogenin, a protein capable of self-assembly to form nanospheres. Naturally occurring mutations in the human amelogenin gene are responsible for at least some of the disease entities known collectively as amelogenesis imperfecta (AI), although it is clear that the AI phenotype may be caused by alteration to other genes responsible for the biogenesis of the enamel extracellular matrix. Mutations that create changes in the functional domains of the amelogenin protein do adversely affect enamel biomineralization. Protein engineering of amelogenin that phenocopies several of the known AI mutations exhibits defects in self-assembly. Amino acid alterations that occur within a domain of amelogenin appear to cause "mineral defects," that is to say hypocalcification of the enamel, whereas mutations that occur elsewhere in another domain of the amelogenin molecule result in "hypoplastic defects," a decrease in thickness of the enamel. However, not all patients with AI phenotypes segregate precisely into these arbitrary designations [1, 2]. Nonetheless, correlating the domain of the amelogenin protein that contains a specific mutation with the type of enamel structural alteration suggests a modular design for amelogenin that is corroborated by protein engineering using recombinant DNA techniques and transgenic animal studies [3].     

Amelogenin protein exhibits a modular design: implications for form and function

The most abundant protein of forming enamel is amelogenin, a protein capable of self-assembly to form nanospheres. Naturally occurring mutations in the human amelogenin gene are responsible for at least some of the disease entities known collectively as amelogenesis imperfecta (AI), although it is clear that the AI phenotype may be caused by alteration to other genes responsible for the biogenesis of the enamel extracellular matrix. Mutations that create changes in the functional domains of the amelogenin protein do adversely affect enamel biomineralization. Protein engineering of amelogenin that phenocopies several of the known AI mutations exhibits defects in self-assembly. Amino acid alterations that occur within a domain of amelogenin appear to cause "mineral defects," that is to say hypocalcification of the enamel, whereas mutations that occur elsewhere in another domain of the amelogenin molecule result in "hypoplastic defects," a decrease in thickness of the enamel. However, not all patients with AI phenotypes segregate precisely into these arbitrary designations. Nonetheless, correlating the domain of the amelogenin protein that contains a specific mutation with the type of enamel structural alteration suggests a modular design for amelogenin that is corroborated by protein engineering using recombinant DNA techniques and transgenic animal studies.     

Amelogenesis imperfecta: genotype-phenotype studies in 71 families

Amelogenesis imperfecta (AI) represents hereditary conditions affecting the quality and quantity of enamel. Six genes are known to cause AI (AMELX, ENAM, MMP20, KLK4, FAM83H, and WDR72). Our aim was to determine the distribution of different gene mutations in a large AI population and evaluate phenotype-genotype relationships. Affected and unaffected family members were evaluated clinically and radiographically by one examiner. Genotyping was completed using genomic DNA obtained from blood or saliva. A total of 494 individuals were enrolled, with 430 (224 affected, 202 unaffected, and 4 not definitive) belonging to 71 families with conditions consistent with the diagnosis of AI. Diverse clinical phenotypes were observed (i.e. hypoplastic, hypocalcified, and hypomaturation). Genotyping revealed mutations in all 6 candidate genes. A molecular diagnosis was made in 132 affected individuals (59%) and in 26 of the families (37%). Mutations involved 12 families with FAM83H (46%), 6 families with AMELX (23%), 3 families with ENAM (11%), 2 families with KLK4 and MMP20 (8% for each gene), and 1 family with a WDR72 mutation (4%). Phenotypic variants were associated with allelic FAM83H and AMELX mutations. Two seemingly unrelated families had the same KLK4 mutation. Families affected with AI where candidate gene mutations were not identified could have mutations not identifiable by traditional gene sequencing (e.g. exon deletion) or they could have promoter sequence mutations not evaluated in this study. However, the results suggest that there remain new AI causative genes to be identified.     

The molecular etiologies and associated phenotypes of amelogenesis imperfecta

The amelogenesis imperfectas (AIs) are a clinically and genetically diverse group of conditions that are caused by mutations in a variety of genes that are critical for normal enamel formation. To date, mutations have been identified in four genes (AMELX, ENAM, KLK4, MMP20) known to be involved in enamel formation. Additional yet to be identified genes also are implicated in the etiology of AI based on linkage studies. The diverse and often unique phenotypes resulting from the different allelic and non-allelic mutations in these genes provide an opportunity to better understand the role of these genes and their related proteins in enamel formation. Understanding the AI phenotypes also provides an aid to clinicians in directing molecular studies aimed at delineating the genetic basis underlying these diverse clinical conditions. Our current knowledge of the known mutations and associated phenotypes of the different AI subtypes are reviewed.     

An atomic force microscopic study of the ultrastructure of dental enamel afflicted with amelogenesis imperfecta

The ultrastructure of human tooth enamel from a patient diagnosed to have amelogenesis imperfecta (AI) was investigated using atomic force microscopy (AFM) and compared with normal human tooth enamel. AI is a hereditary defect of dental enamel in which the enamel is deficient in either quality or quantity. Tissue-specific proteins, especially amelogenins, have been postulated to play a central role in amelogenesis. The secondary structure of amelogenin has been assigned an important role in directing the architecture of hydroxyapatite (HA) enamel crystallites and an alteration of the secondary structure of amelogenin is expected to result in an altered architecture of the mineral phase in human enamel. Previous studies have shown that the human amelogenin gene encodes for a mutant protein in which a conserved Pro is mutated to a Thr residue (Pro → Thr); such a mutation should be expected to cause a disoriented pattern of the mineral phase in enamel. AFM results presented for the AI tooth enamel clearly demonstrate that the apatite crystal morphology in AI tooth enamel is perturbed in the diseased state; this might result from a defective synthesis of the extracellular matrix proteins, e.g. amelogenin, by the ameloblasts.     

Human and mouse enamel phenotypes resulting from mutation or altered expression of AMEL, ENAM, MMP20 and KLK4

Amelogenesis imperfecta (AI) is caused by AMEL, ENAM, MMP20 and KLK4 gene mutations. Mice lacking expression of the AmelX, Enam and Mmp20 genes have been generated. These mouse models provide tools for understanding enamel formation and AI pathogenesis. This study describes the AI phenotypes and relates them to their mouse model counterparts. Human AI phenotypes were determined in a clinical population of AI families and published cases. Human and murine teeth were evaluated using light and electron microscopy. A total of 463 individuals from 54 families were evaluated and mutations in the AMEL, ENAM and KLK4 genes were identified. The majority of human mutations for genes coding enamel nonproteinase proteins (AMEL and ENAM) resulted in variable hypoplasia ranging from local pitting to a marked, generalized enamel thinning. Specific AMEL mutations were associated with abnormal mineralization and maturation defects. Amel and Enam null murine models displayed marked enamel hypoplasia and a complete loss of prism structure. Human mutations in genes coding for the enamel proteinases (MMP20 and KLK4) cause variable degrees of hypomineralization. The murine Mmp20 null mouse exhibits both hypoplastic and hypomineralized defects. The currently available Amel and Enam mouse models for AI exhibit enamel phenotypes (hypoplastic) that are generally similar to those seen in humans. Mmp20 null mice have a greater degree of hypoplasia than humans with MMP20 mutations. Mice lacking expression of the currently known genes associated with the human AI conditions provide useful models for understanding the pathogenesis of these conditions.     

An atomic force microscopic study of the ultrastructure of dental enamel afflicted with amelogenesis imperfecta

The ultrastructure of human tooth enamel from a patient diagnosed to have amelogenesis imperfecta (AI) was investigated using atomic force microscopy (AFM) and compared with normal human tooth enamel. AI is a hereditary defect of dental enamel in which the enamel is deficient in either quality or quantity. Tissue-specific proteins, especially amelogenins, have been postulated to play a central role in amelogenesis. The secondary structure of amelogenin has been assigned an important role in directing the architecture of hydroxyapatite (HA) enamel crystallites and an alteration of the secondary structure of amelogenin is expected to result in an altered architecture of the mineral phase in human enamel. Previous studies have shown that the human amelogenin gene encodes for a mutant protein in which a conserved Pro is mutated to a Thr residue (Pro-->Thr); such a mutation should be expected to cause a disoriented pattern of the mineral phase in enamel. AFM results presented for the AI tooth enamel clearly demonstrate that the apatite crystal morphology in AI tooth enamel is perturbed in the diseased state; this might result from a defective synthesis of the extracellular matrix proteins, e.g. amelogenin, by the ameloblasts.     

Mutational spectrum of FAM83H: the C-terminal portion is required for tooth enamel calcification

Dental enamel forms through the concerted activities of specialized extracellular matrix proteins, including amelogenin, enamelin, MMP20, and KLK4. Defects in the genes encoding these proteins cause non-syndromic inherited enamel malformations collectively designated as amelogenesis imperfecta (AI). These genes, however, account for only about a quarter of all AI cases. Recently we identified mutations in FAM83H that caused autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI). Unlike other genes that cause AI, FAM83 H does not encode an extracellular matrix protein. Its location inside the cell is completely unknown, as is its function. We here report novel FAM83H mutations in four kindreds with ADHCAI. All are nonsense mutations in the last exon (c.1243G>T, p.E415X; c.891T>A, p.Y297X; c.1380G>A, p.W460X; and c.2029C>T, p.Q677X). These mutations delete between 503 and 883 amino acids from the C-terminus of a protein normally comprised of 1179 residues. The reason these mutations cause such extreme defects in the enamel layer without affecting other parts of the body is not known yet. However it seems evident that the large C-terminal part of the protein is essential for proper enamel calcification.     

Mutation of the gene encoding the enamel-specific protein, enamelin, causes autosomal-dominant amelogenesis imperfecta

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 4q11-q21. 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 4q11-q21, these findings highlight the need for a molecular classification of this group of disorders.     

Ultrastructural Study of Tooth Enamel with Amelogenesis Imperfecta in Al-Nephrocalcinosis Syndrome

This paper describes the ultrastructure of the affected enamel and the clinical features in two siblings with the syndrome of nephrocalcinosis and amelogenesis imperfecta. Nephrocalcinosis was diagnosed by intravenous pyelography, and confirmed by ultrasonography and CT scan. Amelogenesis imperfecta AI was diagnosed clinically and histologically.

Light microscopy showed that the affected enamel surfaces were rough and the enamel was hypoplastic and mainly positively birefringent. Scanning electron microscopy revealed a rough and extensively cracked enamel surface covered with oval shaped blister-like protrusions. TEM snowed porous enamel consisting of loosely packed and randomly oriented thin ribbon-like crystals with little or no prismatic structure.

Observations showed that hypoplasia together with hypocalcification and/or hypomaturation defects were present in the same tooth, indicating the possibility of an abnormality in interstitial matrix, leading to dystrophic calcification in the kidney and abnormal tooth enamel formation, or alternatively an involvement of two separate but closely linked genes.     

Enamel protein regulation and dental and periodontal physiopathology in MSX2 mutant mice

Signaling pathways that underlie postnatal dental and periodontal physiopathology are less studied than those of early tooth development. Members of the muscle segment homeobox gene (Msx) family encode homeoproteins that show functional redundancy during development and are known to be involved in epithelial-mesenchymal interactions that lead to crown morphogenesis and ameloblast cell differentiation. This study analyzed the MSX2 protein during mouse postnatal growth as well as in the adult. The analysis focused on enamel and periodontal defects and enamel proteins in Msx2-null mutant mice. In the epithelial lifecycle, the levels of MSX2 expression and enamel protein secretion were inversely related. Msx2+/- mice showed increased amelogenin expression, enamel thickness, and rod size. Msx2-/- mice displayed compound phenotypic characteristics of enamel defects, related to both enamel-specific gene mutations (amelogenin and enamelin) in isolated amelogenesis imperfecta, and cell-cell junction elements (laminin 5 and cytokeratin 5) in other syndromes. These effects were also related to ameloblast disappearance, which differed between incisors and molars. In Msx2-/- roots, Malassez cells formed giant islands that overexpressed amelogenin and ameloblastin that grew over months. Aberrant expression of enamel proteins is proposed to underlie the regional osteopetrosis and hyperproduction of cellular cementum. These enamel and periodontal phenotypes of Msx2 mutants constitute the first case report of structural and signaling defects associated with enamel protein overexpression in a postnatal context.     

Subunit Structures in Hydroxyapatite Crystal Development in Enamel: Implications for Amelogenesis Imperfecta

Previous freeze-etching studies of developing enamel [21] revealed collinear arrays of spherical structures (~50 nM dia) of similar width to the crystals of mature tissue. Concomitant with matrix degradation/processing, spherical structures became less distinct until, coincident with massive matrix loss, only crystal outlines were seen. More recently, using Atomic force microscopy technology [22], early crystals exhibited topology reminiscent of these collinear spherical structures. After matrix loss these were replaced by similarly sized bands of positive charge density on the crystal surfaces [28]. The data suggest enamel crystals may form from mineral-matrix spherical subunits. Matrix processing may generate mineral nuclei and lead to their fusion and transformation into long apatite crystals. Support for this view derives from the appearance of short crystal segments in amelogenesis imperfecta (hypoplastic AI) or abnormally large crystals alongside 50 nM diameter spherical mineral subunits (hypomaturation AI). Mutation of matrix or processing enzymes leading to defective processing may have impaired mineral initiation, fusion, and subsequent growth.     

Subunit structures in hydroxyapatite crystal development in enamel: implications for amelogenesis imperfecta

Previous freeze-etching studies of developing enamel revealed collinear arrays of spherical structures (approximately 50 nM dia) of similar width to the crystals of mature tissue. Concomitant with matrix degradation/processing, spherical structures became less distinct until, coincident with massive matrix loss, only crystal outlines were seen. More recently, using Atomic force microscopy technology, early crystals exhibited topology reminiscent of these collinear spherical structures. After matrix loss these were replaced by similarly sized bands of positive charge density on the crystal surfaces. The data suggest enamel crystals may form from mineral-matrix spherical subunits. Matrix processing may generate mineral nuclei and lead to their fusion and transformation into long apatite crystals. Support for this view derives from the appearance of short crystal segments in amelogenesis imperfecta (hypoplastic AI) or abnormally large crystals alongside 50 nM diameter spherical mineral subunits (hypomaturation AI). Mutation of matrix or processing enzymes leading to defective processing may have impaired mineral initiation, fusion, and subsequent growth.     

Compositional,structural and mechanical comparisons of normal enamel and hypomaturation enamel

Hypomaturation amelogenesis imperfecta is a hereditary disorder of the enamel that severely influences the function, aesthetics and psychosocial well-being of patients. In this study, we performed a thorough comparison of normal and hypomaturation enamel through a series of systematical tests on human permanent molars to understand the biomineralization process during pathological amelogenesis. The results of microcomputed tomography, scanning electron microscopy, Fourier transform infrared, Raman spectroscopy, microzone X-ray diffraction, thermal gravimetric analysis, energy diffraction spectrum and Vickers microhardness testing together show dramatic contrasts between hypomaturation enamel and normal enamel in terms of their hierarchical structures, spectral features, crystallographic characteristics, thermodynamic behavior, mineral distribution and mechanical property. Our current study highlights the importance of the organic matrix during the amelogenesis process. It is found that the retention of the organic matrix will influence the quantity, quality and distribution of mineral crystals, which will further demolish the hierarchical architecture of the enamel and affect the related mechanical property. In addition, the high carbonate content in hypomaturation enamel influences the crystallinity, crystal size and solubility of hydroxyapatite crystals. These results deepen our understanding of hypomaturation enamel biomineralization during amelogenesis, explain the clinical manifestations of hypomaturation enamel, provide fundamental evidence to help dentists choose optimal therapeutic strategies and lead to improved biofabrication and gene therapies.     

Characterisation of molecular defects in X-linked amelogenesis imperfecta (AIH1)

Amelogenins are an heterogenous family of proteins produced by ameloblasts of the enamel organ during tooth development. Disturbances of enamel formation occur in amelogenesis imperfecta, a clinically heterogenous group of inherited disorders characterised by defective enamel biomineralisa-tion. An amelogenin gene, AMGX, has been mapped to the short of the X chromosome (Xp22.1—p22.3) and has been implicated in the molecular pathology of X-linked amelogenesis imperfecta (AIH1). We have identified three families exhibiting AIH1 and screened the AMGX gene for mutations using single-strand conformational polymorphism analysis and DNA sequencing. Three novel mutations were identified: a C-T substitution in exon 5, and a G-T substitution and single cytosine deletion in exon 6, confirming the existence of extensive allelic heterogeneity in this condition. The identification of family-specific mutations will enable early identification of affected individuals and correlation of clinical phenotype with genotype will facilitate an objective system of disease classification. © 1995 Wiley-Liss, Inc.     

Fluoride-induced alterations of enamel structure: an experimental study in the miniature pig

We studied the structural changes in the enamel of mandibular third molars of miniature pigs administered a daily oral dose of 2 mg NaF (approximately 0.9 mg of fluoride) per kg body weight (added to the feed) for 1 year. The treatment period covered most of the secretory stage and the entire post-secretory stage of amelogenesis of the M₃. The enamel of the molars from the fluoride-fed pigs appeared opaque and chalky, and the erupted portions were stained brown. The underlying histopathological change was a pronounced subsurface hypomineralization of the enamel beneath a thin surface rim of higher mineral content. This enamel hypomineralization was attributed to a fluoride-induced impairment of the process of enamel maturation. The most conspicuous finding in the fluorotic enamel was the presence of numerous pit-type hypoplastic defects, denoting a marked fluoride-induced disturbance also of the secretory stage of amelogenesis. Microradiography and scanning electron microscopy revealed an enhanced incremental pattern in the outer enamel of the fluorotic molars. Typically, the bottom of larger hypoplastic defects was underlain by a broad, grossly accentuated incremental line. Occurrence of larger hypoplasias was further associated with the presence of aprismatic enamel, the formation of which was attributed to a loss of the prism-forming (distal) portion of the Tomes processes of secretory ameloblasts. The findings in the miniature pigs closely parallel earlier observations on fluorotic enamel of free-ranging deer and wild boar from fluoride-polluted areas.     

Immunohistochemical and Western blot analyses of collar enamel in the jaw teeth of gars,Lepisosteus oculatus,an actinopterygian fish

Although most fish have no enamel layer in their teeth, those belonging to Lepisosteus (gars), an extant actinopterygian fish genus, do and so can be used to study amelogenesis. In order to examine the collar enamel matrix in gar teeth, we subjected gar teeth to light and electron microscopic immunohistochemical examinations using an antibody against bovine amelogenin (27?kDa) and antiserum against porcine amelogenin (25?kDa), as well as region-specific antibodies and antiserum against the C-terminus and middle region, and N-terminus of porcine amelogenin, respectively. The enamel matrix exhibited intense immunoreactivity to the anti-bovine amelogenin antibody and the anti-porcine amelogenin antiserum in addition to the C-terminal and middle region-specific antibodies, but not to the N-terminal-specific antiserum. These results suggest that the collar enamel matrix of gar teeth contains amelogenin-like proteins and that these proteins possess domains that closely resemble the C-terminal and middle regions of porcine amelogenin. Western blot analyses of the tooth germs of Lepisosteus were also performed. As a result, protein bands with molecular weights of 78?kDa and 65?kDa were clearly stained by the anti-bovine amelogenin antibody as well as the antiserum against porcine amelogenin and the middle-region-specific antibody. It is likely that the amelogenin-like proteins present in Lepisosteus do not correspond to the amelogenins found in mammals, although they do possess domains that are shared with mammalian amelogenins.