The Impact of R740s Mutation of TCIRG1 on Dental Development in Osteopetrotic Mice

Abstract

Osteopetrosis is a rare disease characterized by sclerotic bone due to impaired osteoclast function. Over 50% of the cases of the most severe, autosomal recessive form of the disease are associated with a defect in the TCIRG1 gene encoding the a3 subunit of vacuolar-type H + -ATPase (V-ATPase). These patients have bone resorption defects, delayed tooth eruption and variable enamel defects; however, little is known about amelogenesis in this condition. We studied an osteopetrotic mouse with a point mutation (R740S) in the V-ATPase a3 subunit, resulting in severe osteopetrosis, hypocalcemia, and dental abnormalities. To elucidate the role of V-ATPases in amelogenesis, we investigated the cellular distribution of a3, spatiotemporal expression of enamel matrix proteins, and measured thickness and mineral content of the enamel in the R740S mouse. Micro CT analysis demonstrated a statistically significant decrease in mineralization and thickness of the enamel in the homozygote relative to the heterozygote and wild type mice. SEM analysis showed that enamel mineralization was slightly delayed in the homozygote, although minimal crystallization was noted in all genotypes. To assess spatiotemporal protein expression of the enamel proteins, mouse mandibles were collected on days 1, 5 and 9 postnatal, fixed, decalcified, and analyzed using immunohistochemistry (IHC). IHC demonstrated that amelogenin and amelotin expression patterns were similar among all genotypes. Expression of the a3 subunit was not detected in the ameloblasts, suggesting that the hypomineralized and hypoplastic enamel found in the R740S mouse may be due to underlying systemic conditions affecting the enamel microenvironment.