The Disease relevance of X chromosomal Amelogenin

• Amelogenesis imperfecta (AI) is a group of disorders of dental enamel formation, wherein various degrees and combinations of hypoplasia and hypomineralization lead to brown to yellow soft enamel. The deformation presents variably in primary and adult teeth. Depending upon the level of mineralization, the teeth are at related greater risk for caries infection (cavities).The treatment is full coverage dental restorations (e.g. crowns) for all teeth.
• Multiple cases of isolated Amelogenesis imperfecta have been correlated to various types of mutations in the Amelx gene, as well as others: ENAM (hypoplastic local), FAM83H (hypomineralized), MMP20 (pigmented hypomaturation; irregular greyish brown and glossy), KLK4 (pigmented hypomaturation; homogenous dark yellow); which strongly suggest rolls for these genes in enamel formation. Mutations in other genes have been related to AI in the presence of other diseases: DSPP (Dentinogenesis imperfecta), DLX (hypoplastic and Taurodontism or TrichoDentoOsseous Dysplasia), and Laminin alpha-3a (hypoplastic and LaryngoOnychocCutaneous syndrome).
• Missense coding mutations,
• Various types of mutations (for example nonsense mutations and frameshift deletions) induce early stop codons truncating the protein, removal of the secretion signal, and complete prevention of translation are noted in the literature. Also various missense coding mutations strongly correlated to AI have been noted in the literature. The effects of mutations in the Amelx gene are more diverse than those for the other genes. For example single missense mutations coding for adjacent amino acids have very different characteristics. Lyonization effects are observed commonly in women with an AI causing mutation in Amelx; the variable expression of the gene on each X-chromosome is patterened early in differentiation / proliferation events, which cause banding of enamel: normal enamel and deformed enamel alternate in elongated strips trailing from the gingival margin to the incisal edge / occlusal line angle, roughly 1/2 mm thick; swirls are not uncommon.
• Co-morbidity with open bite is prevelant at about 50%, which may or may not indicate a roll for occlusal forces in development of the mandible, maxilla, and/or alveolus.

Analytical, diagnostic and therapeutic context of Amelx

• The Y chromosomal Amelogenin gene is one of the most commonly used genes for paternity tests and investigation of archeological anthropology.

High impact information on Amelx

• Major portions of the Amelx protein have been conserved, for upwards of 400,000 years. Across 85 tetrapod known sequences for various portions of the protein, the translation of exons 2, 3, 5, and 7 are conserved greatly. Across all mammals, the exon 6, which codes for over half of the amino acids in the protein (full length mature AMELX is encoded by exons 2,3,4,5,6,7), is almost entirely conserved (with a couple varying pseduo-repeat insertions, for example cow).
• The Amelx protein coalesces into seemingly disorganized nanospheres, which are roughly 45nm in diameter and attach into elongated chains extending from the secretory end of Ameloblasts to the dento-enamel junction. These nanosphere chains guide the formation and elongation of hydroxyapatite into elongated ribbons, which grow into rods while the amelogenin proteins are broken down by MMP20 and KLK4. These rods are present only in mammals, give greater tensile strength to the enamel, are keyhole shaped in cross section, and seem to be produced each by one ameloblast.
• Amelogenin is a specific product of ameloblasts [1].
• The enamel matrix is comprised 95% by weight of multiple copies of AMELX protein.

Various other automatically curated information on Amelx

• Preliminary analyses of recombination between the distal X chromosome gene amelogenin ( Amelx) and the PAR loci for either TelXY or sex chromosome association (Sxa) suggest that the locus DXYRp1 maps to the distal portion of the PAR [2].
• Both fragments were closely linked to each other and located on the X chromosome distal to Amelx --in agreement with X or PA linkage [3].
• Recombinant plasmids containing amelogenin cDNA sequences were identified by differential hybridization, hybrid-selected translation, and blot hybridization analyses [4].
• This amelogenin cDNA probe now enables molecular investigations of a number of classical problems in developmental biology [4].
• We demonstrate that the underlying mutations are nested deletions which lie in the Phex-Amelx chromosomal segment conserved between man and mouse [5].
• LRAP down regulated OCN and up regulated OPN in a dose- and time-response fashion, and inhibited the capacity of mineral nodule formation [6].

• Mouse amelogenin cDNA was used in hybridization assays with genomic DNA, cut with the restriction enzyme Eco RI, from the edentulous chicken (Gallus domesticus), the monophyodont mouse (as control), diphyodont man, and the polyphyodont fishes Atlantic salmon (Salmo salar) and seawolf (Anarrhichas lupus) [7].

• The ribozymes, designed to cleave amelogenin mRNA, were injected close to developing mandibular molar teeth in newborn mice, resulting in a prolonged and specific arrest of amelogenin synthesis not caused by general toxicity [8].
• Specifically, the null mouse does not process amelogenin properly, possesses an altered enamel matrix and rod pattern, has hypoplastic enamel that delaminates from the dentin, and has a deteriorating enamel organ morphology as development progresses [9].
• The exon-4-containing amelogenin isoforms were heterologously expressed in E. coli by means of the pET11 expression system (Novagen, Madison, WI) [10].
• These data were interpreted to suggest that the polyoma virus truncated the differentiation pathway for these odontogenic tissues at an early stage of their development and retained the expression of basement membrane components and the enamelin-like polypeptides, yet excluded expression of amelogenin gene products [11].
• When Amelx null females were mated with P70T transgenic males, offspring developed structures similar to calcifying epithelial odontogenic tumors in humans [12] .

• The Grpr locus mapped between the Pdha-1 and Amelx loci on the mouse X chromosome [13].
• Alternative splicing of mouse amelogenin pre-mRNA leads to the production of more than 14 protein isoforms, the functions of which are not totally understood [14].
• Increased enamel formation in the former case resulted from an upregulation of amelogenin synthesis whereas delayed enamel formation in the later case was most probably an indirect consequence of the high porosity of the underlying dentin [15].
• Leucine-rich amelogenin peptide: a candidate signaling molecule during cementogenesis [6].
• RESULTS: LRAP had no effect on cell proliferation up to 6 days, with a decrease in cell growth observed at the highest dose by 9 days versus untreated cells [6].

• Southern blot analysis of genomic DNA from the mouse using this cDNA as a probe indicates that there is only one mouse amelogenin (Amel) gene [16].
• Using immunohistochemistry, we were not able to detect any differences in the spatial distribution of the four amelogenin epitopes investigated [17].
• Leucine-rich amelogenin peptide appears to have a direct effect on cementoblast activity that may prove significant during development as well as in regeneration of periodontal tissues [6].
• CONCLUSION: These data suggest that amelogenin may be a critical signaling molecule required for appropriate development of the periodontium [18].
• In situ hybridization and immunohistochemical analysis of sections from amelogenin null mice revealed a dramatic reduction in expression of BSP mRNA and protein in cementoblasts and surrounding osteoblasts in comparison to age-matched controls [18].
• Two related low molecular mass polypeptide isoforms of amelogenin have distinct activities in mouse tooth germ differentiation in vitro [19].

• The enamel protein amelogenin binds to GlcNAc (Ravindranath, R. M. H., Moradian-Oldak, R., and Fincham, A.G. (1999) J. Biol. Chem. 274, 2464-2471) and to the GlcNAc-mimicking peptide (GMp) (Ravindranath, R. M. H., Tam, W., Nguyen, P., and Fincham, A. G. (2000) J. Biol. Chem. 275, 39654-39661) [20].
• During tooth development, enamel organ epithelial cells express a tissue-specific gene product (amelogenin) which presumably functions to control calcium hydroxyapatite crystal growth patterns during enamel biomineralization [21].
• Furthermore, we showed the entry of exogenous fluorescein isothiocyanate-conjugated rm-amelogenin into the cytoplasm of HAT-7 cells [22].
• In addition, a proteolytic fragment of amelogenin, known as the tyrosine-rich amelogenin peptide or TRAP, is present in low but isolatable quantities [23].
• Intron length, therefore, is not the sole determinant controlling amelogenin exon 4 inclusion, and cis-acting inhibitory elements may also be involved in exon skipping [24].

Physical interactions of Amelx

• Functional antagonism between Msx2 and CCAAT/enhancer-binding protein alpha in regulating the mouse amelogenin gene expression is mediated by protein-protein interaction [25].

• In support of this interpretation, recombinant enamelysin was previously demonstrated to cleave recombinant amelogenin at virtually all of the precise sites known to occur in vivo [9].

• Signaling pathway assays suggested involvement of the MAPK pathway, since the addition of the MAPK inhibitor suppressed OPN expression in LRAP-treated cells [6].
• RESULTS: The lowest dose of amelogenin slightly enhanced BSP expression, whereas at the highest dose, a dramatic decrease (three-fold) in BSP expression was observed [18].
• Taken together, these data indicate that protein-protein interaction rather than competition for overlapping binding sites results in the functional antagonism between Msx2 and C/EBPalpha in regulating the mouse amelogenin gene expression [25].

• The two telomeric loci, Glra2 and Am, had higher ratios of polymorphism to divergence than the two loci experiencing lower recombination rates [26].
• Transcripts for OPG were increased in LRAP-treated cells compared to control, but RANKL mRNA levels were not affected [6].
• To further investigate the potential epithelial-mesenchymal interaction, an amelogenin knockout mouse model was used to examine expression of BSP and other markers, including Type I collagen, in tissue samples [18].
• In op/opCS mice, DMP1 in odontoblasts increased to near normal and dentin morphology was restored; amelogenin also increased [27].
• The position of the amelogenin locus (Amel) relative to the loci for alpha-galactosidase (Ags), proteolipoprotein (Plp), and the random genomic probe DXWas31 has been determined [16].
• Independent technical methods, consisting of the yeast two-hybrid (Y2H) assay and surface plasmon resonance (SPR), have been used to demonstrate the importance of amelogenin self-assembly domains [28].
• A yeast two-hybrid assay has been used to identify protein partners for amelogenin, ameloblastin, and enamelin [29].
• Antisense oligodeoxynucleotide strategy was used in a simple organ culture system to inhibit amelogenin translation [21].

References