HERITABLE DISORDERS OF CONNECTIVE TISSUE

该科进行研究，以阐明遗传性结缔组织疾病的分子和生物化学机制，特别是骨生成障碍（OI）和Ehlers-Danlos（EDS），并将这些信息应用于治疗这些疾病。我们产生了非致死形式的OI的敲入小鼠模型，并将该小鼠命名为脆性（brtl）。Brt1在其胶原α 1（I）链之一中具有经典的甘氨酸取代（G348C），并再现了OI的分子和生物化学特征。它的骨骼结构紊乱，矿化不足。我们已经表明，这种突变降低了负责胶原纤维形成的温度依赖性吸引力。我们还研究了这些小鼠中存在的表型的变异性，以及在人类OI中常见的表型的变异性。在具有致死和存活结果的小鼠中，我们证明了突变胶原转录物的等效表达和突变蛋白的组织掺入。我们还将核酶切割位点工程化到Brtl突变等位基因中。我们一直在开发锤头状核酶作为OI和其他显性疾病的治疗剂。核酶切割突变体转录本可以抑制突变体蛋白的表达。我们证明了等位基因特异性抑制成纤维细胞中的突变型胶原蛋白转录到其在对照中的水平的约50%。对于鼠核酶位点，我们已经证明了体外特异性和效率。为了创建核酶的递送系统，我们已经产生了表达对Brt1核酶切割位点特异性的核酶构建体的转基因小鼠。为了确定与功能正常骨相容的突变表达水平，我们已经确定了两个嵌合体携带者，他们分别是II型和IV型OI受影响儿童的母亲。我们证明了IV型携带者在她的白细胞和成纤维细胞中有很高比例的突变细胞。已证明致死突变的携带者在白细胞和成纤维细胞中具有5 - 10%的突变细胞。我们开始对携带者进行成骨细胞研究。我们对成骨细胞的培养研究旨在了解成骨不全作为一种骨疾病的机制。我们已经证明，与匹配的成纤维细胞相比，成骨细胞在细胞内检测到的和分泌到培养基中的胶原蛋白比例更大。脉冲标记研究表明，过度修饰的链出现更迅速地在成骨细胞比成纤维细胞，并有更大的电泳延迟。在我们对EDS患者的研究中，我们已经确定了α 1（V）链中的突变，该突变导致单倍不足，并证明正常V型胶原蛋白的缺乏会导致轻度EDS。受影响的个体具有剪接缺陷，其导致具有提前终止密码子的框外转录物。扫描电子显微镜显示真皮I型胶原原纤维大小和花椰菜原纤维的变化。- 成骨不全，ehlers-danlos综合征，胶原蛋白，核酶-人类受试者和人类受试者：访谈，实习医生，或调查仅

The Section conducts studies to elucidate the molecular and biochemical mechanisms of heritable disorders of connective tissue, specifically osteogenesis imperfecta (OI) and Ehlers- Danlos(EDS), and to apply this information to the treatment of these disorders. We generated a knock-in mouse model for the non-lethal form of OI and have designated this mouse Brittle (brtl). Brtl has a classic glycine substitution (G348C) in one of its collagen alpha1(I) chains and reproduces the molecular and biochemical features of OI. Its bone structure is disorganized and undermineralized. We have shown that this mutation reduces the temperature-dependent attractive forces responsible for collagen fibrillogenesis. We also investigated the variability of phenotype present in these mice and frequently seen in human OI. In mice with lethal and surviving outcomes, we demonstrated both equivalent expression of mutant collagen transcripts and tissue incorporation of mutant protein. We also engineered a ribozyme cleavage site into the Brtl mutant allele. We have been developing hammerhead ribozymes as a theraputic agent for OI and other dominant disorders. Cleavage of the mutant transcript by ribozyme can suppress expression of the mutant protein. We demonstrated allele-specific suppression of the mutant collagen transcript in fibroblasts to about 50% of its level in controls. For the murine ribozyme site, we have demonstrated in vitro specificity and efficiency. To create a delivery system for ribozymes, we have generated a transgenic mouse expressing a ribozyme construct specific for the Brtl ribozyme cleavage site. To determine the level of mutation expresssion that is compatible with functionally normal bone, we have identified two mosaic carriers who are the mothers of type II and IV OI affected children, respectively. We demonstrated the type IV carrier has a high percent of mutant cells in her leukocytes and fibroblasts. The carrier of the lethal mutation has been demonstrated to have 5-10% mutant cells in leukocytes and fibroblasts. We are initiating osteoblast studies on the carriers. Our studies of cultured OI osteoblasts are aimed at understanding the mechanism of OI as a bone disease. We have demonstrated that a greater proportion of electrophoretically abnormal collagen is detected both intracellularly and secreted into media by osteoblasts than by matched fibroblasts. Pulse labelling studies showed that overmodified chains appear more rapidly in osteoblasts than fibroblasts and have greater electrophoretic delay. In our studies of patients with EDS, we have identified a mutation in an alpha1(V) chain which causes haploinsufficiency and demonstrates that deficiency of normal type V collagen causes mild EDS. Affected individuals have a splicing defect which results in an out-of- frame transcript with a premature termination codon. Scanning electron microscopy demonstrates variation in dermal type I collagen fibril size and cauliflower fibrils. - osteogenesis imperfecta, ehlers-danlos syndrome, collagen, ribozymes - Human Subjects & Human Subjects: Interview, Questionaires, or Surveys Only