Molecular Genetics of Familial Tumoral Calcinosis

描述（由申请人提供）：由于骨关节炎和其他不太常见的关节病和关节周围病造成的健康问题稳步增长，钙晶体沉积疾病的分子和细胞病理机制非常复杂，是密集科学研究的焦点。我们建议描述一种罕见的常染色体隐性遗传病，家族性肿瘤性钙质沉着症（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的关键参与者。FTC是一种严重的使人衰弱的代谢紊乱，其特征在于大关节上的大量关节周围钙晶体沉积，并且在一部分患者中，血清磷酸盐水平升高。我们最近已经证明（Nat Genet 36：579-581，2004），这种病症是由编码ppGalNAc-T3的GALNT 3中的突变引起的，ppGalNAc-T3是24种已知多肽半乳糖基转移酶之一。这些高尔基体相关的生物合成酶通过将GalNAc从糖供体UDP-GalNAc转移到丝氨酸和苏氨酸残基，负责粘蛋白型O糖基化，这是翻译后修饰和糖蛋白合成的普遍形式。然而，导致碱性钙结晶和磷酸盐失衡的病理机制仍然不清楚。 我们假设ppGalNAc-T3缺乏与细胞外无机焦磷酸盐（ePPi）的减少有关，ePPi在正常水平下可防止关节和其他组织中的碱性磷酸钙晶体沉积。因此，我们建议评估PPi平衡，以及控制ePPi的分子的表达和功能，例如ANK蛋白和胞外酶三磷酸核苷焦磷酸水解酶PC-1在FTC患者的原代成纤维细胞和转化的淋巴母细胞培养物中。为了阐明参与磷酸盐稳态的ppGalNAc-T3的潜在靶蛋白，我们将评估磷酸化酶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，另一个基因可能与FTC的病因有关。我们建议临床和遗传连锁研究，以确定第二个FTC基因，并评估FTC基因突变的因果作用，重叠表型的疾病。我们的体外研究结果在体内复杂和冗余的ppGalNAc T系统的背景下的生物学相关性将通过使用Galnt 3的靶向消融开发FTC的转基因小鼠模型来解决。ppGalNAc-T3缺陷小鼠的表型表征结合体外和体内研究的病理生理学将提供新的见解粘蛋白型O-糖基化的分子生物学及其在异位钙晶体沉积疾病中的有趣作用。Galnt 3-l-小鼠模型的FTC将是一个有价值的工具，为未来的研究探索个别ppGalNAc转移酶及其相互作用的具体目标，研究复杂的机制，导致异位晶体沉积和潜在的探索新的治疗方法。

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.