Tooth enamel is the most highly mineralized tissue in vertebrates. Enamel crystal formation and elongation should be well controlled to achieve an exceptional hardness and a compact microstructure. Enamel matrix calcification occurs with several matrix proteins, such as amelogenin, enamelin, and ameloblastin. Among them, amelogenin is the most abundant enamel matrix protein, and multiple isoforms resulting from extensive but wellconserved alternative splicing and postsecretional processing have been identified. In this report, we recruited a family with a unique enamel defect and identified a silent mutation in exon 4 of the AMELX gene. We show that the mutation caused the inclusion of exon 4, which is almost always skipped, in the mRNA transcript. We further show, by generating and characterizing a transgenic animal model, that the alteration of the ratio and quantity of the developmentally conserved alternative splicing repertoire of AMELX caused defects in enamel matrix mineralization.

ABSTRACT. The runt-related transcription factor 2 gene (RUNX2), which is also known as CBFA1, is a master regulatory gene in bone formation. Mutations in RUNX2 have been identified in cleidocranial dysplasia (CCD) patients. CCD is a rare autosomal dominant skeletal dysplasia that is characterized by delayed closure of cranial sutures, aplastic or hypoplastic clavicle formation, short stature, and dental anomalies, including malocclusion, supernumerary teeth, and delayed eruption of permanent teeth. In this study, we recruited three de novo CCD families and performed mutational analysis of the RUNX2 gene as a candidate gene approach. The mutational study revealed three diseasecausing mutations: a missense mutation (c.674G>A, p.Arg225Gln), a frameshift mutation (c.1119delC, p.Arg374Glyfs*), and a nonsense mutation (c.1171C>T, p.Arg391*). Clinical examination revealed a unique dental phenotype (no typical supernumerary teeth, but duplication of anterior teeth) in one patient. We believe that this finding will broaden the understanding of the mechanism of supernumerary teeth formation and CCD-related phenotypes.

In order to achieve highly mineralized tooth enamel, enamel proteinases serve the important function of removing the remaining organic matrix in the mineralization and maturation of the enamel matrix. Mutations in the kallikrein 4 (KLK4), enamelysin (MMP20), and WDR72 genes have been identified as causing hypomaturation enamel defects in an autosomal-recessive hereditary pattern. In this report, 2 consanguineous families with a hypomaturation-type enamel defect were recruited, and mutational analysis was performed to determine the molecular genetic etiology of the disease. Whole exome sequencing and autozygosity mapping identified novel homozygous mutations in the KLK4 (c.620_621delCT, p.Ser207Trpfs*38) and MMP20 (c.1054G>A, p.Glu352Lys) genes. Further analysis on the effect of the mutations on the translation, secretion, and function of KLK4 and MMP20 revealed that mutant KLK4 was degraded intracellularly and became inactive while mutant MMP20 was expressed at a normal level but secreted only minimally with proteolytic function.

Amelogenesis imperfecta is a heterogeneous group of genetic conditions affecting enamel formation. Recently, mutations in solute carrier family 24 member 4 (SLC24A4) have been identified to cause autosomal recessive hypomaturation amelogenesis imperfecta. We recruited a consanguineous family with hypomaturation amelogenesis imperfecta with generalized brown discoloration. Sequencing of the candidate genes identified a 10-kb deletion, including exons 15, 16, and most of the last exon of the SLC24A4 gene. Interestingly, this deletion was caused by homologous recombination between two 354-bp-long homologous sequences located in intron 14 and the 3' UTR. This is the first report of exonal deletion in SLC24A4 providing confirmatory evidence that the function of SLC24A4 in calcium transport has a crucial role in the maturation stage of amelogenesis.

Amelogenesis imperfecta (AI) is a genetic disease affecting tooth enamel formation. AI can be an isolated entity or a phenotype of syndromes. To date, more than 10 genes have been associated with various forms of AI. We have identified 2 unrelated Turkish families with hypoplastic AI and performed mutational analysis. Whole-exome sequencing identified 2 novel heterozygous nonsense mutations in the ENAM gene (c.454G>T p.Glu152* in family 1, c.358C>T p.Gln120* in family 2) in the probands. Affected individuals were heterozygous for the mutation in each family. Segregation analysis within each family revealed individuals with incomplete penetrance or extremely mild enamel phenotype, in spite of having the same mutation with the other affected individuals. We believe that these findings will broaden our understanding of the clinical phenotype of AI caused by ENAM mutations.