Hereditary Dentin Defects (HDD) affect 1 in 8,000 people. The genetic causes of most HDD correlate with the dysfunction of dentin proteins: type I collagen and dentin sialophosphoprotein (DSPP). All DSPP mutations reported to date show a dominant pattern of inheritance. This is because DSPP mutations manifest their phenotype through a dominant negative or gain of function mechanism?not by haplo- insufficiency. Reducing the normal amount of DSPP by half, as in Dspp heterozygous mice, does not cause dentin malformations. Dspp-/- null mice show a severe phenotype due to the absence of DSPP?not by the autosomal dominant pathological mechanism that causes HDD in humans. This distinction is important. Therapeutically, HDD in the absence of Dspp-/- could be reversed by restoring DSPP expression, whereas human HDD caused by DSPP mutations could not be restored in this way because the condition is not due to a lack of DSPP protein, but rather, is due to the pathological effects of aberrant DSPP in odontoblasts. This proposal ?DSPP Function, Pathophysiology, and Genetic Diagnosis? seeks to improve our under- standings of 1) DSPP-derived proteins during normal dentinogenesis, 2) the pathological mechanism of Dspp -1 frameshift mutations, and 3) to develop a practical approach for HDD genetic testing to specifically identify the causative mutation and establish a definitive diagnosis.
Three Specific Aims are proposed: SA1: Determine the role of DSPP-derived proteins during initial dentin mineral formation and coalescence by characterizing early dentin mineralization in Dspp+/+, Dspp-1fs/-1fs, Dspp-2fs/-2fs and Dspp-/- mice. SA2: Localize the DSPP -1 frameshift protein in vivo to determine where it accumulates and causes odontoblast cell pathology. SA3: Improve the diagnosis and management of HDDs by establishing an efficient genetic testing algorithm (sequence of actions that identifies the exact genetic cause of HDD in a given individual). Strategy: We hypothesize that DSPP helps initiate the mineralization of dentin calcospherites and promotes their growth and coalescence into a continuous mineral layer. By characterizing and comparing early dentin mineralization in Dspp+/+, Dspp-/-, and Dspp-2fs/-2fs mice using Focus Ion Beam Scanning Electron Microscopy (FIB-SEM), we can determine if dentin sialoprotein (DSP) or dentin phosphoprotein (DPP) is promoting the initiation and/or coalescence of dentin. We hypothesize that DSPP -1 frameshift mutations cause odontoblast cell pathology, possibly through ER stress. We test this hypothesis using Dspp -1 frameshift knockin mice that closely mimic human disease. Odontoblast pathology is assessed by FIB-SEM and TEM double immunogold labeling for the mutant protein and organelle markers in vivo. To improve the diagnosis and management of HDD, we apply a genetic testing algorithm to recruited HDD families to optimize its reproducibility and efficiency in identifying the underlying disease-causing mutations.
The long-term objectives of this research are to advance our understanding of dentin formation and mineralization and to develop an efficient genetic testing protocol to diagnosis, manage, and eventually treat patients with hereditary dentin defects (HDD). Achieving our objectives will allow us to distinguish between persons with dentin defects and those with dentin defects and fragile bones, which is important because the tooth defects are often the only clinical symptom at the time of diagnosis, and knowing there is a risk of bone fractures calls for extra precautions during treatment and in everyday life. This research also leads to a better understanding of the disease process in persons with dentin defects in the absence of bone defects and leads to treatments that can cure this problem.