Molecular Genetics of Familial Tumoral Calcinosis

描述（由申请人提供）：由于骨关节炎和其他不常见的关节病和关节周围病引起的健康问题不断增加，钙晶体沉积紊乱的分子和细胞病理机制是复杂的，也是科学研究的重点。我们建议描述一种罕见的常染色体隐性遗传病，家族性肿瘤钙质沉着症（FTC）的分子遗传学特征，以确定负责关节周围钙和磷酸盐稳态的新关键参与者。FTC是一种严重使人衰弱的代谢紊乱，其特征是大关节周围大量钙晶体沉积，并且在一部分患者中，血清磷酸盐水平升高。我们最近证明（Nat Genet 36:579-581, 2004）这种疾病是由编码ppGalNAc-T3的GALNT3突变引起的，ppGalNAc-T3是已知的24种多肽半乳糖转移酶之一。这些高尔基相关的生物合成酶负责粘蛋白型糖基化，这是翻译后修饰和糖蛋白合成的一种普遍形式，通过糖供体甘氨酸-甘氨酸将GalNAc转移到丝氨酸和苏氨酸残基上。然而，导致碱性钙晶体和磷酸盐失衡的病理机制尚不清楚。我们假设ppGalNAc-T3缺乏与细胞外无机焦磷酸盐（ePPi）的减少有关，正常水平的ePPi可以防止关节和其他组织中的碱性磷酸钙晶体沉积。因此，我们建议在FTC患者的原代成纤维细胞和转化淋巴细胞培养中评估PPi平衡以及控制epi的分子，如ANK蛋白和外切酶核苷三磷酸焦磷酸水解酶PC-1的表达和功能。为了阐明ppGalNAc-T3参与磷酸盐稳态的潜在靶蛋白，我们将评估磷酸素的表达和功能的变化，如成纤维细胞生长因子23和肾磷酸钠转运蛋白。此外，有证据表明FTC是遗传异质性的，另一种基因可能与FTC的病因有关。我们提出临床和遗传连锁研究，以确定第二个FTC基因，并评估FTC基因突变在重叠表型疾病中的因果作用。我们的体外研究结果在体内复杂和冗余的ppGalNAc T系统背景下的生物学相关性将通过开发一种靶向消融Galnt3的FTC转基因小鼠模型来解决。ppGalNAc-T3缺陷小鼠的表型特征结合体外和体内病理生理研究将为粘蛋白型o糖基化的分子生物学及其在异位钙晶体沉积紊乱中的有趣作用提供新的见解。FTC的galnt3 - 1小鼠模型将为未来的研究探索单个ppGalNAc转移酶的特定靶点及其相互作用，研究导致异位晶体沉积的复杂机制以及潜在的探索新的治疗方法提供有价值的工具。

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.