HERITABLE DISORDERS OF CONNECTIVE TISSUE

该科进行研究，以阐明结缔组织遗传性疾病的分子和生化机制，特别是成骨不全(OI)和埃勒斯-丹洛斯(EDS)，并将这些信息应用于这些疾病的治疗。我们为OI的非致命性形式建立了敲入小鼠模型，并将其命名为小鼠脆性(Brtl)。Brt1在其胶原α1(I)链中有一个经典的甘氨酸取代(G348C)，并再现了OI的分子和生化特征。它的骨骼结构松散，矿化不足。我们已经证明，这种突变减少了导致胶原纤维形成的依赖于温度的吸引力。我们还研究了这些小鼠和人类OI中常见的表型的变异性。在有致死和存活结果的小鼠中，我们证明了突变胶原转录本的等量表达和突变蛋白的组织整合。我们还在Brt1突变等位基因中设计了一个核酶切割位点。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。核酶对突变转录物的切割可抑制突变蛋白的表达。我们证明，在成纤维细胞中，突变的胶原转录物受到等位基因特异性的抑制，大约是对照组水平的50%。对于小鼠核酶位点，我们已经在体外证明了特异性和有效性。为了建立核酶的传递系统，我们培育了一只转基因小鼠，表达针对Brt1核酶切割位点的核酶结构。为了确定与正常骨骼功能相容的突变表达水平，我们鉴定了两名马赛克携带者，他们分别是II型和IV型OI患者的母亲。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。这种致命突变的携带者已被证明在白细胞和成纤维细胞中有5%-10%的突变细胞。我们正在启动对载体的成骨细胞研究。我们对培养的OI成骨细胞的研究旨在了解OI作为一种骨病的机制。我们已经证明，与匹配的成纤维细胞相比，成骨细胞在细胞内和分泌到培养液中的电泳性异常胶原的比例更大。脉冲标记研究表明，过度修饰的链在成骨细胞中出现得比成纤维细胞更快，并且有更大的电泳延迟。在我们对EDS患者的研究中，我们发现了导致单倍性功能不全的α1(V)链突变，并证明正常V型胶原缺乏会导致轻度EDS。受影响的个体有剪接缺陷，导致框架外转录带有提前终止密码子。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、Ehler-Danlos综合征、胶原蛋白、核酶-人类受试者和人类受试者：仅采访、问卷或调查

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