ENAM Mutations Can Cause Hypomaturation Amelogenesis Imperfecta.

Amelogenesis imperfecta (AI) is a diverse group of inherited diseases featured by various presentations of enamel malformations that are caused by disturbances at different stages of enamel formation. While hypoplastic AI suggests a thickness defect of enamel resulting from aberrations during the secretory stage of amelogenesis, hypomaturation AI indicates a deficiency of enamel mineralization and hardness established at the maturation stage. Mutations in ENAM, which encodes the largest enamel matrix protein, enamelin, have been demonstrated to cause generalized or local hypoplastic AI. Here, we characterized 2 AI families with disparate hypoplastic and hypomaturation enamel defects and identified 2 distinct indel mutations at the same location of ENAM, c588+1del and c.588+1dup. Minigene splicing assays demonstrated that they caused frameshifts and truncation of ENAM proteins, p.Asn197Ilefs*81 and p.Asn197Glufs*25, respectively. In situ hybridization of Enam on mouse mandibular incisors confirmed its restricted expression in secretory stage ameloblasts and suggested an indirect pathogenic mechanism underlying hypomaturation AI. In silico analyses indicated that these 2 truncated ENAMs might form amyloid structures and cause protein aggregation with themselves and with wild-type protein through the added aberrant region at their C-termini. Consistently, protein secretion assays demonstrated that the truncated proteins cannot be properly secreted and impede secretion of wild-type ENAM. Moreover, compared to the wild-type, overexpression of the mutant proteins significantly increased endoplasmic reticulum stress and upregulated the expression of unfolded protein response (UPR)-related genes and TNFRSF10B, a UPR-controlled proapoptotic gene. Caspase, terminal deoxynucleotidyl transferase UTP nick-end labeling (TUNEL), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays further revealed that both truncated proteins, especially p.Asn197Ilefs*81, induced cell apoptosis and decreased cell survival, suggesting that the 2 ENAM mutations cause AI through ameloblast cell pathology and death rather than through a simple loss of function. This study demonstrates that an ENAM mutation can lead to generalized hypomaturation enamel defects and suggests proteinopathy as a potential pathogenesis for ENAM-associated AI.

Recessive Mutations in ACP4 Cause Amelogenesis Imperfecta.

Amelogenesis imperfecta (AI) is an innate disorder that affects the formation and mineralization of the tooth enamel. When diagnosed with AI, one's teeth can be hypoplastic (thin enamel), hypomature (normal enamel thickness but discolored and softer than normal enamel), hypocalcified (normal enamel thickness but extremely weak), or mixed conditions of the above. Numerous studies have revealed the genes that are involved in causing AI. Recently, ACP4 (acid phosphatase 4) was newly found as a gene causing hypoplastic AI, and it was suggested that mutant forms of ACP4 might affect access to the catalytic core or the ability to form a homodimer. In this study, a Korean and a Turkish family with hypoplastic AI were recruited, and their exome sequences were analyzed. Biallelic mutations were revealed in ACP4: paternal (NM_033068: c.419C>T, p.(Pro140Leu)) and maternal (c.262C>A, p.(Arg88Ser)) mutations in family 1 and a paternal (c.713C>T, p.(Ser238Leu)) mutation and de novo (c.350A>G, p.(Gln117Arg)) mutation in the maternal allele in family 2. Mutations were analyzed by cloning, mutagenesis, immunofluorescence, immunoprecipitation, and acid phosphatase activity test. Comparison between the wild-type and mutant ACP4s showed a decreased amount of protein expression from the mutant forms, a decreased ability to form a homodimer, and a decreased acid phosphatase activity level. We believe that these findings will not only expand the mutational spectrum of ACP4 but also increase our understanding of the mechanism of ACP4 function during normal and pathologic amelogenesis.

FAM83H and Autosomal Dominant Hypocalcified Amelogenesis Imperfecta.

Autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI; OMIM #130900) is a genetic disorder exhibiting severe hardness defects and reduced fracture toughness of dental enamel. While the condition is nonsyndromic, it can be associated with other craniofacial anomalies, such as malocclusions and delayed or failed tooth eruption. Truncation mutations in FAM83H (OMIM *611927) are hitherto the sole cause of ADHCAI. With human genetic studies, Fam83h knockout and mutation-knock-in mouse models indicated that FAM83H does not serve a critical physiologic function during enamel formation and suggested a neomorphic mutation mechanism causing ADHCAI. The function of FAM83H remains obscure. FAM83H has been shown to interact with various isoforms of casein kinase 1 (CK1) and keratins and to mediate organization of keratin cytoskeletons and desmosomes. By considering FAM83H a scaffold protein to anchor CK1s, further molecular characterization of the protein could gain insight into its functions. In this study, we characterized 9 kindreds with ADHCAI and identified 3 novel FAM83H truncation mutations: p.His437*, p.Gln459*, and p.Glu610*. Some affected individuals exhibited hypoplastic phenotypes, in addition to the characteristic hypocalcification enamel defects, which have never been well documented. Failed eruption of canines or second molars in affected persons was observed in 4 of the families. The p.Glu610* mutation was located in a gap area (amino acids 470 to 625) within the zone of previously reported pathogenic variants (amino acids 287 to 694). In vitro pull-down studies with overexpressed FAM83H proteins in HEK293 cells demonstrated an interaction between FAM83H and SEC16A, a protein component of the COP II complex at endoplasmic reticulum exit sites. The interaction was mediated by the middle part (amino acids 287 to 657) of mouse FAM83H protein. Results of this study significantly extended the phenotypic and genotypic spectrums of FAM83H-associated ADHCAI and suggested a role for FAM83H in endoplasmic reticulum-to-Golgi vesicle trafficking and protein secretion (dbGaP phs001491.v1.p1).

Alteration of Exon Definition Causes Amelogenesis Imperfecta.

Amelogenesis imperfecta (AI) is a collection of genetic disorders affecting the quality and/or quantity of tooth enamel. More than 20 genes are, so far, known to be responsible for this condition. In this study, we recruited 3 Turkish families with hypomaturation AI. Whole-exome sequence analyses identified disease-causing mutations in each proband, and these mutations cosegregated with the AI phenotype in all recruited members of each family. The AI-causing mutations in family 1 were a novel AMELX mutation [NM_182680.1:c.143T>C, p.(Leu48Ser)] in the proband and a novel homozygous MMP20 mutation [NM_004771.3:c.616G>A, p.(Asp206Asn)] in the mother of the proband. Previously reported compound heterozygous MMP20 mutations [NM_004771.3:c.103A>C, p.(Arg35=) and c.389C>T, p.(Thr130Ile)] caused the AI in family 2 and family 3. Minigene splicing analyses revealed that the AMELX missense mutation increased exonic definition of exon 4 and the MMP20 synonymous mutation decreased exonic definition of exon 1. These mutations would trigger an alteration of exon usage during RNA splicing, causing the enamel malformations. These results broaden our understanding of molecular genetic pathology of tooth enamel formation.

WDR72 Mutations Associated with Amelogenesis Imperfecta and Acidosis.

Dental enamel malformations, or amelogenesis imperfecta (AI), can be isolated or syndromic. To improve the prospects of making a successful diagnosis by genetic testing, it is important that the full range of genes and mutations that cause AI be determined. Defects in WDR72 (WD repeat-containing protein 72; OMIM *613214) cause AI, type IIA3 (OMIM #613211), which follows an autosomal recessive pattern of inheritance. The defective enamel is normal in thickness, severely hypomineralized, orange-brown stained, and susceptible to attrition. We identified 6 families with biallelic WDR72 mutations by whole exome sequence analyses that perfectly segregated with the enamel phenotype. The novel mutations included 3 stop-gains [NM_182758.2: c.377G>A/p.(Trp126*), c.1801C>T/p.(Arg601*), c.2350A>T/p.(Arg784*)], a missense mutation [c.1265G>T/p.(Gly422Val)], and a 62,138-base pair deletion (NG_017034.2: g.35441_97578del62138) that removed WDR72 coding exons 3 through 13. A previously reported WDR72 frameshift was also observed [c.1467_1468delAT/p.(Val491Aspfs*8)]. Three of the affected patients showed decreased serum pH, consistent with a diagnosis of renal tubular acidosis. Percentiles of stature and body weight varied among 8 affected individuals but did not show a consistent trend. These studies support that WDR72 mutations cause a syndromic form of AI and improve our ability to diagnose AI caused by WDR72 defects.

Hypoplastic AI with Highly Variable Expressivity Caused by ENAM Mutations.

Tooth enamel, the hardest tissue in the human body, is formed after a complex series of interactions between dental epithelial tissue and the underlying ectomesenchyme. Nonsyndromic amelogenesis imperfecta (AI) is a rare genetic disorder affecting tooth enamel without other nonoral symptoms. In this study, we identified 2 novel ENAM mutations in 2 families with hypoplastic AI by whole exome sequencing. Family 1 had a heterozygous splicing donor site mutation in intron 4, NM_031889; c.123+2T>G. Affected individuals had hypoplastic enamel with or without the characteristic horizontal hypoplastic grooves in some teeth. Family 2 had a nonsense mutation in the last exon, c.1842C>G, p.(Tyr614*), that was predicted to truncate the protein by 500 amino acids. Participating individuals had at least 1 mutant allele, while the proband had a homozygous mutation. Most interestingly, the clinical phenotype of the individuals harboring the heterozygous mutation varied from a lack of penetrance to a mild hypoplastic enamel defect. We believe that these findings will broaden our understanding of the clinical phenotype of AI caused by ENAM mutations.

Biomimetic Enamel Regeneration Mediated by Leucine-Rich Amelogenin Peptide.

We report here a novel biomimetic approach to the regeneration of human enamel. The approach combines the use of inorganic pyrophosphate (PPi) to control the onset and rate of enamel regeneration and the use of leucine-rich amelogenin peptide (LRAP), a nonphosphorylated 56-amino acid alternative splice product of amelogenin, to regulate the shape and orientation of growing enamel crystals. This study builds on our previous findings that show LRAP can effectively guide the formation of ordered arrays of needle-like hydroxyapatite (HA) crystals in vitro and on the known role mineralization inhibitors, like PPi, play in the regulation of mineralized tissue formation. Acid-etched enamel surfaces of extracted human molars, cut perpendicular or parallel to the direction of the enamel rods, were exposed to a PPi-stabilized supersaturated calcium phosphate (CaP) solution containing 0 to 0.06 mg/mL LRAP for 20 h. In the absence of LRAP, PPi inhibition was reversed by the presence of etched enamel surfaces and led to the formation of large, randomly distributed plate-like HA crystals that were weakly attached, regardless of rod orientation. In the presence of 0.04 mg/mL LRAP, however, densely packed mineral layers, comprising bundles of small needle-like HA crystals, formed on etched surfaces that were cut perpendicular to the enamel rods. These crystals were strongly attached, and their arrangement reflected to a significant degree the underlying enamel prism pattern. In contrast, under the same conditions with LRAP, little to no crystal formation was found on enamel surfaces that were cut parallel to the direction of the enamel rods. These results suggest that LRAP preferentially interacts with ab surfaces of mature enamel crystals, inhibiting their directional growth, thus selectively promoting linear growth along the c-axis of enamel crystals. The present findings demonstrate a potential for the development of a new approach to regenerate enamel structure and properties.

MMP20 Proteolysis of Native Amelogenin Regulates Mineralization In Vitro.

Recent studies have shown that native phosphorylated full-length porcine amelogenin (P173) and its predominant cleavage product (P148) can inhibit spontaneous calcium phosphate formation in vitro by stabilizing an amorphous calcium phosphate (ACP) precursor phase. Since full-length amelogenin undergoes proteolysis by matrix metalloproteinase 20 (MMP20, enamelysin) soon after secretion, the present study was conducted to assess the effect of amelogenin proteolysis on calcium phosphate formation. Calcium and phosphate were sequentially added to protein solutions without and with added MMP20 (ratio = 200:1) under physiological-like conditions of ionic strength (163 mM) in 50 mM Tris-HCl (pH 7.4) at 37 °C. Protein degradation with time was assessed by gel-electrophoresis, and mineral products formed were characterized by transmission electron microscopy (TEM). MMP20 was found to cleave P173 to primarily generate P148, along with P162, P46-148, and P63/64-148. In sharp contrast, MMP20 did not cleave P148. In addition, the formation of well-aligned bundles of enamel-like hydroxyapatite (HA) crystals was promoted in the presence of P173 with added MMP20, while only ACP particles were seen in the absence of MMP20. Although P148 was found to have a somewhat lower capacity to stabilize ACP and prevent HA formation compared with P173 in the absence of MMP20, essentially no HA formation was observed in the presence of somewhat higher concentrations of P148 regardless of MMP20 addition, due to the lack of observed protein proteolysis. Present findings suggest that ACP transformation to ordered arrays of enamel crystals may be regulated in part by the proteolysis of full-length native amelogenin, while the predominant amelogenin degradation product in developing enamel (e.g., P148) primarily serves to prevent uncontrolled mineral formation during the secretory stage of amelogenesis.

Fluoride affects enamel protein content via TGF-ß1-mediated KLK4 inhibition.

Dental fluorosis is caused by chronic high-level fluoride (F(-)) exposure during enamel development, and fluorosed enamel has a higher than normal protein content. Matrix metalloproteinase 20 cleaves enamel matrix proteins during the secretory stage, and KLK4 further cleaves these proteins during the maturation stage so that the proteins can be reabsorbed from the hardening enamel. We show that transforming growth factor ß1 (TGF-ß1) can induce Klk4 expression, and we examine the effect of F(-) on TGF-ß1 and KLK4 expression. We found that in vivo F(-) inhibits Klk4 but not Mmp20 transcript levels. LacZ-C57BL/6-Klk4 (+/LacZ) mice have LacZ inserted in frame at the Klk4 translation initiation site so that the endogenous Klk4 promoter drives LacZ expression in the same temporal/spatial way as it does for Klk4. KLK4 protein levels in rat enamel and ß-galactosidase staining in LacZ-C57BL/6-Klk4 (+/LacZ) mouse enamel were both significantly reduced by F(-) treatment. Since TGF-ß1 induces KLK4 expression, we tested and found that F(-) significantly reduced Tgf-ß1 transcript levels in rat enamel organ. These data suggest that F(-)-mediated downregulation of TGF-ß1 expression contributes to reduced KLK4 protein levels in fluorosed enamel and provides an explanation for why fluorosed enamel has a higher than normal protein content.

STIM1 and SLC24A4 Are Critical for Enamel Maturation.

Dental enamel formation depends upon the transcellular transport of Ca(2+) 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 Ca(2+) homeostasis that cause inherited enamel defects can provide insights into the molecular participants and potential mechanisms of Ca(2+) handling by ameloblasts. Stromal Interaction Molecule 1 (STIM1) is an ER transmembrane protein that activates membrane-specific Ca(2+) influx in response to the depletion of ER Ca(2+) stores. Solute carrier family 24, member 4 (SLC24A4), is a Na(+)/K(+)/Ca(2+) transporter that exchanges intracellular Ca(2+) 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 Ca(2+) transcellular transport systems used by secretory- and maturation-stage ameloblasts.

FAM20A mutations associated with enamel renal syndrome.

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 D(403) 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.

LAMB3 mutations causing autosomal-dominant amelogenesis imperfecta.

Amelogenesis imperfecta (AI) can be either isolated or part of a larger syndrome. Junctional epidermolysis bullosa (JEB) is a collection of autosomal-recessive disorders featuring AI associated with skin fragility and other symptoms. JEB is a recessive syndrome usually caused by mutations in both alleles of COL17A1, LAMA3, LAMB3, or LAMC2. In rare cases, heterozygous carriers in JEB kindreds display enamel malformations in the absence of skin fragility (isolated AI). We recruited two kindreds with autosomal-dominant amelogenesis imperfecta (ADAI) characterized by generalized severe enamel hypoplasia with deep linear grooves and pits. Whole-exome sequencing of both probands identified novel heterozygous mutations in the last exon of LAMB3 that likely truncated the protein. The mutations perfectly segregated with the enamel defects in both families. In Family 1, an 8-bp deletion (c.3446_3453del GACTGGAG) shifted the reading frame (p.Gly 1149Glufs*8). In Family 2, a single nucleotide substitution (c.C3431A) generated an in-frame translation termination codon (p.Ser1144*). We conclude that enamel formation is particularly sensitive to defects in hemidesmosome/basement-membrane complexes and that syndromic and non-syndromic forms of AI can be etiologically related.

Novel KLK4 and MMP20 mutations discovered by whole-exome sequencing.

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.

Structural changes in amelogenin upon self-assembly and mineral interactions.

Amelogenin, the major protein of forming dental enamel, plays a crucial role in the biomineralization of this tissue. Amelogenin is soluble at low pH and self-assembles to form higher order structures at physiological pH. To understand the mechanisms of its assembly and interactions with calcium phosphate mineral, we conducted FTIR spectroscopy (FTIRS) studies of pH-triggered assembly of recombinant porcine amelogenin rP172 and its interactions with mature hydroxyapatite and apatitic mineral formed in situ. Analysis of our data indicated that rP172 at pH 3.0 exists in an unfolded disordered state, while increases in pH led to structural ordering, manifested by increases in intra- and intermolecular ß-sheet structures and a decrease in random coil and ß-turns. Amelogenin assembled at pH 7.2 was also found to contain large portions of extended intramolecular ß-sheet and PPII. These FTIRS findings are consistent with those previously obtained with other techniques, thus verifying the validity of our experimental approach. Interestingly, interactions with mineral led to a reduction in protein structural organization. The findings obtained show that amelogenin has intrinsic structural flexibility to accommodate interactions with both forming and mature calcium phosphate mineral phases, providing new insights into the potential importance of amelogenin-mineral interactions in enamel biomineralization.

Leucine-rich amelogenin peptides regulate mineralization in vitro.

Amelogenin's capacity to regulate enamel formation is related to its conserved N- and C-terminal domains, its ability to self-assemble, and its ability to stabilize amorphous calcium phosphate (ACP) - a capacity enhanced by amelogenin phosphorylation. This in vitro study provides further insight into amelogenin function, using variations of the Leucine-Rich Amelogenin Peptide (LRAP), an alternative splice product comprised solely of amelogenin's N- and C-terminal domains. Peptide self-assembly was studied by dynamic light-scattering and transmission electron microscopy (TEM). TEM, selected area electron diffraction, and Fourier transform-infrared spectroscopy were also used to determine the effect of phosphorylated and non-phosphorylated LRAP on calcium phosphate formation. Results show that phosphorylated and non-phosphorylated LRAP can self-assemble into chain-like structures in a fashion dependent on the C-terminal domain. Notably, this capacity was enhanced by added calcium and to a much greater degree for phosphorylated LRAP. Furthermore, phosphorylated LRAP was found to stabilize ACP and prevent its transformation to hydroxyapatite (HA), while aligned HA crystals formed in the presence of non-phosphorylated LRAP. The N- and C-terminal amelogenin domains in non-phosphorylated LRAP are, therefore, sufficient to guide ACP transformation into ordered bundles of apatite crystals, making LRAP an excellent candidate for biomimetic approaches for enamel regeneration.

Potential role of the amelogenin N-terminus in the regulation of calcium phosphate formation in vitro.

N-terminal and C-terminal (CT) domains of amelogenin have been shown to be essential for proper enamel formation. Recent studies have also suggested that although the C-terminus plays an apparent role in protein-mineral interactions, other amelogenin structural domains are involved. The objective was to explore the role of the amelogenin N-terminus in the regulation of calcium phosphate formation in vitro. Spontaneous mineralization studies were carried out using the phosphorylated (+P) and nonphosphorylated (-P) N-terminus of the leucine-rich amelogenin peptide (LRAP) that lacks the hydrophilic CT domain. Mineralization progress was monitored via changes in solution pH. Mineral phases formed were characterized using TEM, selected area electron diffraction, and FT-IR. In controls, amorphous calcium phosphate was initially formed and subsequently transformed to randomly oriented hydroxyapatite (HA) plate-like crystals. In contrast to the control, LRAP(+P)-CT stabilized ACP formation for >1 day, while LRAP(-P)-CT accelerated the transformation of ACP to HA but had little effect on crystal shape or orientation. In conclusion, the N-terminal domain found in LRAP, as in amelogenins, appears to have the capacity to interact with forming calcium phosphate mineral phases. Results suggest that the N-terminal domain of amelogenin may play a direct role in early stages of enamel formation.

FAM83H mutations cause ADHCAI and alter intracellular protein localization.

Mutations in a family with sequence similarity 83 member H (FAM83H) cause autosomal-dominant hypocalcification amelogenesis imperfecta (ADH CAI). All FAM83H ADHCAI-causing mutations terminate translation or shift the reading frame within the specific exon 5 segment that encodes from Ser(287) to Glu(694). Mutations near Glu(694) cause a milder, more localized phenotype. We identified disease-causing FAM83H mutations in two families with ADHCAI: family 1 (g.3115C>T, c.1993 C>T, p.Q665X) and family 2 (g.3151C>T, c.2029 C>T, p.Q677X). We also tested the hypothesis that truncation mutations alter the intracellular localization of FAM83H. Wild-type FAM83H and p.E694X mutant FAM83H fused to green fluorescent protein (GFP) localized in the cytoplasm of HEK293T cells, but the mutant FAM83H proteins (p.R325X, p.W460X, and p.Q677X) fused to GFP localized mainly in the nucleus with slight expression in the cytoplasm. We conclude that nuclear targeting of the truncated FAM83H protein contributes to the severe, generalized enamel phenotype.

Transgenic rescue of enamel phenotype in Ambn null mice.

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.

Novel WDR72 mutation and cytoplasmic localization.

The proven candidate genes for amelogenesis imperfecta (AI) are AMELX, ENAM, MMP20, KLK4, FAM83H, and WDR72. We performed mutation analyses on seven families with hypomaturation AI. A novel WDR72 dinucleotide deletion mutation (g.57,426_57,427delAT; c.1467_ 1468delAT; p.V491fsX497) was identified in both alleles of probands from Mexico and Turkey. Haplotype analyses showed that the mutations arose independently in the two families. The disease perfectly segregated with the genotype. Only persons with both copies of the mutant allele were affected. Their hypomineralized enamel suffered attrition and orange-brown staining following eruption. Expression of WDR72 fused to green fluorescent protein showed a cytoplasmic localization exclusively and was absent from the nucleus. We conclude that WDR72 is a cytoplasmic protein that is critical for dental enamel formation.