PROJECT SUMMARY/ABSTRACT The developmental malformation of dental enamel, known as amelogenesis imperfecta (AI), is a rare inherited condition. Children and their families affected by AI suffer life-long from a disfigured appearance, compromised social interactions, psychological suppression, sensitive teeth, increased risk for caries and bite collapse, inability to masticate. The dental treatment requires full-mouth rehabilitation and frequent, life- long maintenance. All enamel proteins are known to cause AI. Ameloblastin (Ambn) is the second most abundant enamel protein. The Ambn gene belongs to the cluster of secreted calcium-binding phosphoproteins (SCPPs) that originated from the basement membrane gene SPARC. During evolution, genes for mineralization were selected to specialize for enamel, dentin and bone. Consistent with features for SCPPs, Ambn is a disordered, phosphorylated, calcium-binding and proline-rich, acidic protein. The variety of enamel proteins is increased by alternative splicing. Ambn is expressed as full-length and spliced proteins. The spliced segment is expressed by exon 6 and consist of 15 highly conserved residues, including an O-glycosylation site and splice site. Our preliminary data shows that ameloblasts express full-length and spliced Ambn in different concentrations and different developmental stages. During secretory stage, spliced Ambn is expressed higher than full-length Ambn. But at maturation stage, the expression of spliced Ambn reduces and full- length Ambn is expressed higher than spliced. In Ambn null mice, reconstituted with transgenic full-length Ambn, an enamel layer is present, however the enamel layer is not recovered. This finding suggests that full-length Ambn is not sufficient for proper enamel and that spliced Ambn may have an important function in enamel formation. The overall hypothesis is that Ambn splice variants execute distinctly different functions, depending on the enamel stage. In Specific Aim 1 we will determine the stage-specific functions of full- length Ambn compared to spliced Ambn in enamel mineralization. In Specific Aim 2 the stage-specific functions of full-length Ambn vs. spliced Ambn in ameloblasts for cell attachment and basal lamina will be determined. For the proposed studies a team of clinician scientists, experts in Genome Engineering, Next- generation sequencing and bioinformatics was assembled for unique interactions and novel approaches. High resolution imaging and quantification will be applied to study the enamel surface, thickness, density and hardness. Ameloblasts will be analyzed by stage to understand the pathways for full-length and spliced Ambn.