Molecular Genetics of Familial Tumoral Calcinosis

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DESCRIPTION (provided by applicant): The molecular and cellular pathomechanisms of calcium crystal deposition disorders are complex and focus of intense scientific research due to the steadily growing health problems posed by osteoarthritis and other less common arthropathies and periarthropathies. We propose to characterize the molecular genetics of a rare autosomal recessive disorder, familial tumoral calcinosis (FTC), to identify new key players responsible for periarticular calcium and phosphate homeostasis. FTC, a severely debilitating metabolic disorder is characterized by massive periarticular calcium crystal depositions over large joints and, in a subset of patients, by elevated serum phosphate levels. We have recently demonstrated (Nat Genet 36:579-581, 2004) that this disorder is caused by mutations in GALNT3 encoding ppGalNAc-T3, one of 24 known polypeptide galactosyltransferases. These Golgi-associated biosynthetic enzymes are responsible for mucin type Oglycosylation, a prevalent form of posttranslational modification and glycoprotein synthesis, through transfer of GalNAc from the sugar donor UDP-GalNAc to serine and threonine residues. However, the pathological mechanisms leading to the development of basic calcium crystals and phosphate imbalance remain obscure. We hypothesize that ppGalNAc-T3 deficiency is associated with a decrease in extracellular inorganic pyrophosphate (ePPi), which at normal levels prevents basic calcium phosphate crystal depositions in joints and other tissues. Therefore, we propose to evaluate PPi balance, and expression and function of molecules that control ePPi, such as the ANK protein and the ectoenzyme nucleoside triphosphate pyrophosphate hydrolase PC-1 in primary fibroblast and transformed lymphoblast cultures of FTC patients. To illuminate potential target proteins of ppGalNAc-T3 that are involved in phosphate homeostasis, we will assess changes in the expression and function of phosphatonins, such as fibroblast growth factor 23, and kidney sodiumphosphate transporters. Moreover, there is evidence that FTC is genetically heterogeneous and another gene might be implicated in the etiology of FTC. We propose clinical and genetic linkage studies to identify the second FTC gene and assess the causal role of FTC gene mutations in disorders with overlapping phenotypes. The biological relevance of our in vitro findings in the context of the complex and redundant ppGalNAc T system in vivo will be addressed by developing a transgenic mouse model of FTC using targeted ablation of Galnt3. The phenotypic characterization of ppGalNAc-T3 deficient mice combined with pathophysiological in vitro and in vivo studies will provided new insight in the molecular biology of mucin-type O-glycosylation and its intriguing role in ectopic calcium crystal deposition disorders. The Galnt3-l- mouse model of FTC will be a valuable tool for future research studies exploring the specific targets of individual ppGalNAc transferases and their interactions, for studying the complex mechanisms leading to ectopic crystal deposition and potentially for exploring new therapeutic approaches.