FAM20A mutations can cause enamel-renal syndrome (ERS).

Enamel-renal syndrome (ERS) is an autosomal recessive disorder characterized by severe enamel hypoplasia, failed tooth eruption, intrapulpal calcifications, enlarged gingiva, and nephrocalcinosis. Recently, mutations in FAM20A were reported to cause amelogenesis imperfecta and gingival fibromatosis syndrome (AIGFS), which closely resembles ERS except for the renal calcifications. We characterized three families with AIGFS and identified, in each case, recessive FAM20A mutations: family 1 (c.992G>A; g.63853G>A; p.Gly331Asp), family 2 (c.720-2A>G; g.62232A>G; p.Gln241_Arg271del), and family 3 (c.406C>T; g.50213C>T; p.Arg136* and c.1432C>T; g.68284C>T; p.Arg478*). Significantly, a kidney ultrasound of the family 2 proband revealed nephrocalcinosis, revising the diagnosis from AIGFS to ERS. By characterizing teeth extracted from the family 3 proband, we demonstrated that FAM20A(-/-) molars lacked true enamel, showed extensive crown and root resorption, hypercementosis, and partial replacement of resorbed mineral with bone or coalesced mineral spheres. Supported by the observation of severe ectopic calcifications in the kidneys of Fam20a null mice, we conclude that FAM20A, which has a kinase homology domain and localizes to the Golgi, is a putative Golgi kinase that plays a significant role in the regulation of biomineralization processes, and that mutations in FAM20A cause both AIGFS and ERS.

Amelogenesis imperfecta in two families with defined AMELX deletions in ARHGAP6.

Amelogenesis imperfecta (AI) is a group of inherited conditions featuring isolated enamel malformations. About 5% of AI cases show an X-linked pattern of inheritance, which are caused by mutations in AMELX. In humans there are two, non-allelic amelogenin genes: AMELX (Xp22.3) and AMELY (Yp11.2). About 90% of amelogenin expression is from AMELX, which is nested within intron 1 of the gene encoding Rho GTPase activating protein 6 (ARHGAP6). We recruited two AI families and determined that their disease-causing mutations were partial deletions in ARHGAP6 that completely deleted AMELX. Affected males in both families had a distinctive enamel phenotype resembling "snow-capped" teeth. The 96,240 bp deletion in family 1 was confined to intron 1 of ARHGAP6 (g.302534_398773del96240), but removed alternative ARHGAP6 promoters 1c and 1d. Analyses of developing teeth in mice showed that ARHGAP6 is not expressed from these promoters in ameloblasts. The 52,654 bp deletion in family 2 (g.363924_416577del52654insA) removed ARHGAP6 promoter 1d and exon 2, precluding normal expression of ARHGAP6. The male proband of family 2 had slightly thinner enamel with greater surface roughness, but exhibited the same pattern of enamel malformations characteristic of males in family 1, which themselves showed minor variations in their enamel phenotypes. We conclude that the enamel defects in both families were caused by amelogenin insufficiency, that deletion of AMELX results in males with a characteristic snow-capped enamel phenotype, and failed ARHGAP6 expression did not appreciably alter the severity of enamel defects when AMELX was absent.

Enamel malformations associated with a defined dentin sialophosphoprotein mutation in two families.

Dentin sialophosphoprotein (DSPP) mutations cause dentin dysplasia type II (DD-II) and dentinogenesis imperfecta types II and III (DGI-II and DGI-III, respectively). We identified two kindreds with DGI-II who exhibited vertical bands of hypoplastic enamel. Both families had a previously reported DSPP mutation that segregated with the disease phenotype. Oral photographs and dental radiographs of four affected and one unaffected participant in one family and of the proband in the second family were used to document the dental phenotypes. We aligned the 33 unique allelic DSPP sequences showing variable patterns of insertions and deletions (indels), generated a merged dentin phosphoprotein (DPP) sequence that includes sequences from all DSPP length haplotypes, and mapped the known DSPP mutations in this context. Analyses of the DSPP sequence changes and their probable effects on protein expression, as well as published findings of the dental phenotype in Dspp null mice, support the hypothesis that all DSPP mutations cause pathology through dominant-negative effects. Noting that Dspp is transiently expressed by mouse pre-ameloblasts during formation of the dentino-enamel junction, we hypothesize that DSPP dominant-negative effects potentially cause cellular pathology in pre-ameloblasts that, in turn, causes enamel defects. We conclude that enamel defects can be part of the dental phenotype caused by DSPP mutations, although DSPP is not critical for dental enamel formation.

Enamel proteins and proteases in Mmp20 and Klk4 null and double-null mice.

Matrix metalloproteinase 20 (MMP20) and kallikrein-related peptidase 4 (KLK4) are thought to be necessary to clear proteins from the enamel matrix of developing teeth. We characterized Mmp20 and Klk4 null mice to better understand their roles in matrix degradation and removal. Histological examination showed retained organic matrix in Mmp20, Klk4, and Mmp20/Klk4 double-null mouse enamel matrix, but not in the wild-type. X-gal histostaining of Mmp20 null mice heterozygous for the Klk4 knockout/lacZ knockin showed that Klk4 is expressed normally in the Mmp20 null background. This finding was corroborated by zymogram and western blotting, which discovered a 40-kDa protease induced in the maturation stage of Mmp20 null mice. Proteins were extracted from secretory-stage or maturation-stage maxillary first molars from wild-type, Mmp20 null, Klk4 null, and Mmp20/Klk4 double-null mice and were analyzed by SDS-PAGE and western blotting. Only intact amelogenins and ameloblastin were observed in secretory-stage enamel of Mmp20 null mice, whereas the secretory-stage matrix from Klk4 null mice was identical to the matrix from wild-type mice. More residual matrix was observed in the double-null mice compared with either of the single-null mice. These results support the importance of MMP20 during the secretory stage and of KLK4 during the maturation stage and show there is only limited functional redundancy for these enzymes.

Target gene analyses of 39 amelogenesis imperfecta kindreds.

Previously, mutational analyses identified six disease-causing mutations in 24 amelogenesis imperfecta (AI) kindreds. We have since expanded the number of AI kindreds to 39, and performed mutation analyses covering the coding exons and adjoining intron sequences for the six proven AI candidate genes [amelogenin (AMELX), enamelin (ENAM), family with sequence similarity 83, member H (FAM83H), WD repeat containing domain 72 (WDR72), enamelysin (MMP20), and kallikrein-related peptidase 4 (KLK4)] and for ameloblastin (AMBN) (a suspected candidate gene). All four of the X-linked AI families (100%) had disease-causing mutations in AMELX, suggesting that AMELX is the only gene involved in the aetiology of X-linked AI. Eighteen families showed an autosomal-dominant pattern of inheritance. Disease-causing mutations were identified in 12 (67%): eight in FAM83H, and four in ENAM. No FAM83H coding-region or splice-junction mutations were identified in three probands with autosomal-dominant hypocalcification AI (ADHCAI), suggesting that a second gene may contribute to the aetiology of ADHCAI. Six families showed an autosomal-recessive pattern of inheritance, and disease-causing mutations were identified in three (50%): two in MMP20, and one in WDR72. No disease-causing mutations were found in 11 families with only one affected member. We conclude that mutation analyses of the current candidate genes for AI have about a 50% chance of identifying the disease-causing mutation in a given kindred.