Isolation of novel mouse genes associated with ectopic ossification by differential display method using ttw, a mouse model for ectopic ossification.

Mouse mutant ttw (tiptoe walking) is an excellent model for ectopic ossification. This mutant exhibits ossification in various soft tissues, which is histologically similar to human OPLL (ossification of posterior longitudinal ligament of the spine). We previously reported that ttw is caused by a nonsense mutation of the nucleotide pyrophosphatase (ENPP1) gene, and that a polymorphism of the human ENPP1 gene is associated with OPLL. These facts indicate that ENPP1 regulates ectopic ossification in vivo; however, the mechanism is unclear. ENPP1 is an ectoenzyme that generates phosphate (Pi) and pyrophosphate (PPi). PPi is a strong inhibitor of ossification. Abnormal Pi metabolism is observed in patients with OPLL, and diseases with abnormal Pi metabolism such as hypophosphatemic rickets are frequently complicated by ectopic ossification. These lines of evidence suggest Pi-PPi metabolism associated with ENPP1 may play an important role in regulation of ectopic ossification. To clarify the molecular mechanism of ectopic ossification in ttw, we examined the effect of dietary phosphate and calcium on the ttw phenotype and found a high dietary phosphate-accelerated ectopic ossification. Then we examined genes associated with the enhanced ossification in ttw on a high phosphate diet by a differential display method. We identified nine mouse genes; six genes were up-regulated by the high phosphate diet, and three were down-regulated. Six of the nine genes were novel and we cloned and characterized them. Two of the genes were highly specific to cartilage, suggesting their specific role in enchondral ossification. Our identification of the novel genes would give novel insight into the mechanism of ectopic ossification and etiology of OPLL.