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Non-syndromic amelogenesis imperfecta (AI) is a collection of isolated inherited enamel malformations that follow X-linked, autosomal-dominant, or autosomal-recessive patterns of inheritance. The AI phenotype is also found in syndromes. We hypothesized that whole-exome sequencing of AI probands showing simplex or recessive patterns of inheritance would identify causative mutations among the known candidate genes for AI. DNA samples obtained from 12 unrelated probands with AI were analyzed. Disease-causing mutations were identified in three of the probands: a novel single-nucleotide deletion in both KLK4 alleles (g.6930delG; c.245delG; p.Gly82Alafs*87) that shifted the reading frame, a novel missense transition mutation in both MMP20 alleles (g.15390A>G; c.611A>G; p.His204Arg) that substituted arginine for an invariant histidine known to coordinate a structural zinc ion, and a previously described nonsense transition mutation in a single allele of FAM83H (c.1379G>A; g.5663G>A; p.W460*). Erupted molars and cross-sections from unerupted parts of the mandibular incisors of Mmp20 null mice were characterized by scanning electron microscopy. Their enamel malformations closely correlated with the enamel defects displayed by the proband with the MMP20 mutation. We conclude that whole-exome sequencing is an effective means of identifying disease-causing mutations in kindreds with AI, and this technique should prove clinically useful for this purpose.

Ameloblastin null mice fail to make an enamel layer, but the defects could be due to an absence of functional ameloblastin or to the secretion of a potentially toxic mutant ameloblastin. We hypothesized that the enamel phenotype could be rescued by the transgenic expression of normal ameloblastin in Ambn mutant mice. We established and analyzed 5 transgenic lines that expressed ameloblastin from the amelogenin ( AmelX) promoter and identified transgenic lines that express virtually no transgene, slightly less than normal (Tg+), somewhat higher than normal (Tg++), and much higher than normal (Tg+++) levels of ameloblastin. All lines expressing detectable levels of ameloblastin at least partially recovered the enamel phenotype. When ameloblastin expression was only somewhat higher than normal, the enamel covering the molars and incisors was of normal thickness, had clearly defined rod and interrod enamel, and held up well in function. We conclude that ameloblastin is essential for dental enamel formation.

Amelogenin is the major enamel matrix component in developing teeth. In eutherian mammals, amelogenin is expressed from the X chromosome only, or from both the X and Y chromosomes. Two classes of porcine amelogenin cDNA clones have been characterized, but the chromosomal localization of the gene(s) encoding them is unknown. To determine if there are sex-based differences in the expression of porcine amelogenin, we paired PCR primers for exons 1a, 1b, 7a, and 7b, and amplified enamel organ-derived cDNA separately from porcine males and females. The results show that exons 1a/2a and 7a are always together and can be amplified from both males (XY) and females (XX). Exons 1b/2b and 7b are also always paired, but can be amplified only from females. We conclude that porcine amelogenin is expressed from separate genes on the X and Y chromosomes, and not, as previously proposed, from a single gene with two promoters.

Dental enamel formation depends upon the transcellular transport of Ca2+ by ameloblasts, but little is known about the molecular mechanism, or even if the same process is operative during the secretory and maturation stages of amelogenesis. Identifying mutations in genes involved in Ca2+ homeostasis that cause inherited enamel defects can provide insights into the molecular participants and potential mechanisms of Ca2+ handling by ameloblasts. Stromal Interaction Molecule 1 (STIM1) is an ER transmembrane protein that activates membrane-specific Ca2+ influx in response to the depletion of ER Ca2+ stores. Solute carrier family 24, member 4 (SLC24A4), is a Na+/K+/Ca2+ transporter that exchanges intracellular Ca2+ and K+ for extracellular Na+. We identified a proband with syndromic hypomaturation enamel defects caused by a homozygous C to T transition (g.232598C>T c.1276C>T p.Arg426Cys) in STIM1, and a proband with isolated hypomaturation enamel defects caused by a homozygous C to T transition (g.124552C>T; c.437C>T; p.Ala146Val) in SLC24A4. Immunohistochemistry of developing mouse molars and incisors showed positive STIM1 and SLC24A4 signal specifically in maturation-stage ameloblasts. We conclude that enamel maturation is dependent upon STIM1 and SLC24A4 function, and that there are important differences in the Ca2+ transcellular transport systems used by secretory- and maturation-stage ameloblasts.

Enamelin is likely to be essential for proper dental enamel formation. It is secreted by ameloblasts throughout the secretory stage and can readily be isolated from the enamel matrix of developing teeth. The gene encoding human enamelin is located on the long arm of chromosome 4, in a region previously linked to an autosomal-dominant form of amelogenesis imperfecta (AI). To gain information on the structure of the enamelin gene and to facilitate the future assessment of the role of enamelin in normal and diseased enamel formation, we have cloned and characterized the mouse and human enamelin genes. Both genes are about 25 kilobases long. The enamelin gene has 10 exons interrupted by 9 introns. Translation initiates in exon 3 and terminates in exon 10. All of the intron/exon junctions within the mouse and human enamelin coding regions are between codons, so there are no partial codons in any exon, and deletion of one or more coding exons by alternative RNA splicing would not shift the downstream reading frame.

Enamelin is the largest protein in the enamel matrix of developing teeth. In the pig, enamelin is secreted as 186-kDa phosphorylated glycoprotein, which is rapidly processed by enamel proteinases into smaller cleavage products. During the secretory stage of enamel formation, enamelin is found among the crystallites in the rod and interrod enamel and comprises roughly 5% of total matrix protein. Although the function of enamelin is unknown, it is thought to participate in enamel crystal nucleation and extension, and the regulation of crystal habit. Here we report the results of enamelin in situ hybridization in a day 1 mouse developing incisor that shows that enamelin is expressed by ameloblasts, but not by odontoblasts or other cells in the dental pulp. The restricted pattern of enamelin expression makes the human enamelin gene a prime candidate in the etiology of amelogenesis imperfecta (AI), a genetic disease in which defects of enamel formation occur in the absence of non-dental symptoms. We have cloned and characterized a full-length human enamelin cDNA and determined by radiation hybrid mapping and fluorescent in situ hybridization (FISH) that the gene is located on chromosome 4q near the ameloblastin gene in a region previously linked to local hypoplastic AI in six families. These findings will facilitate the search for specific mutations in the enamelin gene in kindreds suffering from amelogenesis imperfecta.

Tuftelin is a protein that has been suggested to function during enamel crystal nucleation. Published sequences for bovine tuftelin cDNA and genomic clones proposed different reading frames that radically affected the derived amino acid sequence of the tuftelin carboxyl-terminus. We have isolated and characterized a full-length mouse cDNA clone and a partial porcine cDNA clone that include the region of the proposed frame-shift. The mouse tuftelin clone is 2572 nucleotides in length, exclusive of the poly(A+) tail. Translation from the 5'-most ATG yields a protein of 390 amino acids with an isotope-averaged molecular mass of 44.6 kDa and an isoelectric point of 5.9. Comparison of the bovine, mouse, and porcine cDNAs supports the revised bovine tuftelin amino acid sequence and suggests that the bovine tuftelin translation initiation codon be re-assigned to a more 5' ATG. Re-assigning the translation initiation codon lengthens the tuftelin protein by 52 amino acids, 51 of which are identical between bovine and mouse. At the carboxyl-terminus, the revised bovine and the mouse sequences match at 39 of the final 42 amino acid positions, compared with 2 identities with the originally published bovine reading frame. Northern blot analysis reveals that tuftelin is not ameloblast-specific but is expressed in multiple tissues, including kidney, lung, liver, and testis. Two tuftelin RNA messages, of 2.6 and 3.2 kb, were detected. DNA sequence characterization of an RT-PCR amplification product confirmed expression of tuftelin in kidney, and identified an alternatively spliced mouse tuftelin mRNA lacking exon 2.

Dental enamel forms by matrix-mediated biomineralization. The components of the developing enamel matrix are generally specific for that matrix. The primary structures of three enamel proteins-amelogenin, tuftelin, and sheathlin (ameloblastin/amelin)—have been derived from cDNA sequences. Here we report the cloning and characterization of mRNA encoding a fourth enamel protein: enamelin. The longest porcine enamelin cDNA clone has 3907 nucleotides, exclusive of the poly(A) tail. The primary structure of the secreted protein is 1104 amino acids in length. Without post-translational modifications, the secreted protein has an isotope-averaged molecular mass of 124.3 kDa and an isoelectric point of 6.5. Polymerase chain-reaction phenotyping of enamelin cDNA suggests that porcine enamelin transcripts are not alternatively spliced and use a single polyadenylation/cleavage site. Immunohistochemical and Western blot analyses with an affinity-purified antipeptide antibody specific for the enamelin carboxyl terminus demonstrate that enamelin is synthesized and secreted by secretory-phase ameloblasts. The parent protein is a 186-kDa glycoprotein that concentrates along the secretory face of the ameloblast Tomes' process. Intact enamelin and proteolytic cleavage products containing its carboxyl terminus are limited to the most superficial layer of the developing enamel matrix, while other enamelin cleavage products are observed in deeper enamel.

Sheath proteins designate low-molecular-weight non-amelogenin enamel polypeptides and their parent protein, which concentrate in the sheath space separating rod and interrod enamel (Uchida et al., 1995). Two porcine sheath proteins, with apparent molecular weights of 13 and 15 kDa, are characterized by protein sequencing. The primary structures of these polypeptides match a portion of the derived amino acid sequences of clones isolated from a porcine enamel organ epithelia-specific cDNA library. Sheath protein RNA messages differ by the inclusion or deletion of a 45-nucleotide segment and by the use of three alternative polyadenylation/cleavage sites. The secreted proteins are 395 and 380 residues in length, with molecular masses of 42,358 and 40,279 Daltons and calculated isoelectric points of 6.3 and 6.7, respectively. Polyclonal antibodies were raised against a synthetic peptide having the sheathlin-specific sequence EHETQQYEYSGGC. Immunohistochemistry with this antibody demonstrates that the protein encoded by the sheathlin cDNA is preferentially localized in the sheath space. We propose that the porcine sheath proteins and their proteolytic cleavage products be designated "sheathlin".

The formation of dental enamel is dependent upon amelogenins, a family of proteins constituting most of the developing enamel matrix. Depending upon the species, these enamel proteins are expressed from either one or two copies of the amelogenin gene. Each gene directs the synthesis of a variety of amelogenin isoforms through alternative splicing of their pre-mRNA transcript(s). Before the role of amelogenins in dental enamel formation can be better understood, one must know the isoforms that are secreted and their biochemical properties. Previously, we cloned and characterized 7 mouse amelogenin RNA messages generated by alternative splicing. The largest amelogenin cDNA encoded a 194-residue amelogenin isoform which was the only clone to contain the 42-nucleotide exon 4 segment. Anti-peptide antibodies raised against the derived translation of this exon revealed an unexpectedly diverse assortment of murine amelogenins, suggesting that additional spicing variants could contain the exon 4 coding region. Using exon-4-specific oligonucleotide primers, we have amplified, cloned, and characterized three different amelogenin RNA messages. These messages encode amelogenin polypeptides (exclusive of signal peptides) 194, 170, and 73 amino acids in length. The isotope-averaged molecular weights for the deduced, single-phosphorylated, proteins are 21,897.1, 19,113.9, and 8176.5 Daltons, respectively. Splice-site selection for the generation of these mRNAs was identical to that of the previously characterized messages for the M180, M156, and M59 except for the inclusion of exon 4. The exon-4-containing amelogenin isoforms were heterologously expressed in E. coli by means of the pET11 expression system (Novagen, Madison, WI)

We identified two families with an autosomal-recessive disorder manifested by severe enamel hypoplasia, delayed and failed tooth eruption, misshapen teeth, intrapulpal calcifications, and localized gingival hyperplasia. Genetic analyses identified novel FAM20A mutations associated with the disease phenotype in both families. The proband of Family 1 had an altered splice junction in Intron 1 (g.502011G>C; c.405-1G>C) and a missense mutation in Exon 8 (g.65094G>A; c.1207G>A; p.D403N). The missense mutation is notable because D403 is strictly conserved among FAM20A homologues, and the corresponding defect in FAM20C caused osteosclerotic bone dysplasia and a loss of kinase activity. The proband at age 12 yrs tested negative for nephrocalcinosis. The proband and her affected father in Family 2 were homozygous for a single nucleotide deletion that altered a splice junction in Intron 10 (g.66622del; c.1361+4del). Minigene analyses demonstrated that this alteration precluded normal splicing. Immunohistochemistry (IHC) of mouse maxillary first molars localized FAM20A in secretory-stage ameloblasts, in odontoblasts, and in the eruption pathway. IHC of kidneys localized FAM20A in the renal tubules. We conclude that FAM20A is likely a secretory pathway kinase and that loss-of-function mutations cause pathology where its phosphorylations are necessary for normal development or homeostasis.

The enamel layer that covers the surfaces of teeth is thickest and most highly mineralized in mammals. The durability of mammalian enamel may have allowed for selection against the lifelong replacement of teeth that is observed in other vertebrates. Variation in enamel structure among animals is thought to be the result of evolutionary changes in the constituents of the developing enamel matrix. In placental mammals, the principal component of this matrix is amelogenin. We have determined the complete primary structures of two opossum amelogenins through a combination of protein sequencing, cloning, and DNA sequencing. RNA messages were cloned that encode 202- and 57-residue amelogenins, which are presumed to be expressed from the same gene but differ due to alternative splicing of identical pre-mRNAs. Edman degradation of the larger amelogenin ran for 42 cycles and yielded the sequence: IPLPPHPGHPGYINFS YEVLTPLKWYQSMMRQQYPSYGYEPM. The derived 202-residue amelogenin, assuming that serine 16 is phosphorylated, has an isotope-averaged molecular mass of 23,023.75 Daltons and a pI of 6.2. This is the largest amelogenin yet characterized. The increase in length is due to the presence of a 30-residue tandem repeat of QP(I/M) in exon 6 in the same position as a similar, but shorter, repeat expressed from the bovine X-chromosome. The 57-residue amelogenin, which is known from other organisms as the leucine-rich amelogenin protein (LRAP), has an isotope-averaged molecular mass of 6764.75 Daltons and a pI of 5.5. The opossum enamel protein is highly homologous to those previously characterized in eutherians and demonstrates that amelogenins were refined structurally prior to the metatherian/eutherian divergence between 100 and 150 million years ago.

In mammals, the organic matrix of developing enamel is dominated by amelogenins. To investigate the expression of proteins secreted into the developing enamel matrix, we have constructed a porcine enamel organ epithelia-specific cDNA library. The amelogenin fraction of the cDNA library was characterized by the cloning of amelogenin-specific polymerase chain-reaction (PCR) amplification products, 5' and 3' rapid amplification of cDNA ends (RACE), and by helper phage rescue of unamplified clones. Clones were characterized that encode porcine amelogenin isoforms 173, 157, 56, 41, and 40 amino acids in length. The structure of the porcine amelogenin gene differs from that of any of those yet described. There are two homologous but distinct exons 1, 2, and 7. One of the two exon 7s can vary in length depending upon the selection of either of two polyadenylation signal/cleavage sites. As a rule, a given exon 1 always pairs with the same exon 2 but can be associated with either exon 7. Despite significant sequence divergence within these exons, no differences are observed in exons 3, 5, and 6. We interpret these findings as evidence of a single amelogenin gene expressed from two promoters; however, the results do not exclude the existence of a second amelogenin gene. The variability generated through the use of alternate promoters and exon 7s primarily affects the non-coding regions of the message. A given amelogenin isoform expressed from the two promoters displays four amino acid differences within the signal peptide, while the secreted proteins are identical. Similarly, the alternative use of exon 7 does not alter the structure of the protein products. The pattern of RNA splicing of amelogenin pre-mRNAs is different for the transcripts expressed from the two promoters. The 173- and the 56-residue amelogenins can be expressed from either promoter, while the 157-residue amelogenin is generated by onlv one of the two promoters.

Enamelysin (MMP-20) is a tooth-specific matrix metalloproteinase that is initially expressed by ameloblasts and odontoblasts immediately prior to the onset of dentin mineralization, and continues to be expressed throughout the secretory stage of amelogenesis. During the secretory stage, enamel proteins are secreted and rapidly cleaved into a large number of relatively stable cleavage products. Multiple proteinases are present in the developing enamel matrix, and the precise role of enamelysin in the processing of enamel proteins is unknown. We have expressed, activated, and purified the catalytic domain of recombinant pig enamelysin, and expressed a recombinant form of the major secreted pig amelogenin rP172. These proteins were incubated together, and the digestion products were analyzed by SDS-PAGE and mass spectrometric analyses. We assigned amelogenin cleavage products by selecting among the possible polypeptides having a mass within 2 Daltons of the measured values. The polypeptides identified included the intact protein (amino acids 2-173), as well as 2-148, 2-136, 2-107, 2-105, 2-63, 2-45, 46-148, 46-147, 46-107, 46-105, 64-148, 64-147, and 64-136. These fragments of rP172 include virtually all of the major amelogenin cleavage products observed in vivo. We propose that enamelysin is the predominant proteinase that processes enamel proteins during the secretory phase of amelogenesis.

The maturation of dental enamel succeeds the degradation of organic matrix. Inhibition studies have shown that this degradation is accomplished by a serine-type proteinase. To isolate and characterize cDNA clones encoding this proteinase, we used two degenerate primer approaches to amplify part of the coding region using polymerase chain-reaction (PCR). First, we purified the proteinase from porcine transition-stage enamel matrix and characterized it by partial protein sequencing. The enzyme was isolated from the neutral soluble enamel extract by successive ammonium sulfate precipitations, hydroxyapatite HPLC, reverse-phase HPLC, DEAE ion exchange, and affinity chromatography with a Benzamidine Sepharose 6B column. The intact protein and lysylendopeptidase-generated cleavage products were characterized by amino acid sequence analyses. Degenerate oligonucleotide primers encoding two of the polypeptide sequences were synthesized. In a complementary strategy, degenerate oligonucleotide primers were designed against highly conserved active-site regions of chymotrypsin-like proteinases. Both approaches yielded PCR amplification products that served as probes for screening a porcine enamel organ epithelia-specific cDNA library. The longest full-length clone is 1133 nucleotides and encodes a preproprotein of 254 amino acids. We designate this protein enamel matrix serine proteinase 1 or EMSP1. The active protein has 224 amino acids, an isotope-averaged molecular mass of 24.1 kDa, and an isoelectric point of 6.0. Multiple-tissue Northern analysis indicates that EMSP1 is a tooth-specific protein. Gelatin enzymography shows a dramatic increase in EMSP1 activity in the transition-stage enamel matrix. EMSP1 is most homologous to kallikriens and trypsins.