Heritable Disorders Of Connective Tissue

结缔组织遗传性疾病

• 批准号: 7594133
• 负责人: Joan C Marini
• 金额: $ 88.15万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594133
• 关键词: Adipocytes; Adverse effects; Affect; Age; Age of Onset; Apoptosis; Area; Back Pain; Binding; Birth; Bone Density; Bone Tissue; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Proteins; Calvaria; Cell Transplants; Cells; Charge; Child; Childhood; Clinical Protocols; Clinical Research; Clinical Trials; Collagen; Collagen Fibril; Collagen Type I; Condition; Connective Tissue; Data; Data Analyses; Databases; Defect; Developmental Bone Diseases; Dimerization; Discrimination; Disease; Dose; Dysplasia; Ehlers-Danlos Syndrome; Endoplasmic Reticulum; Enrollment; Exons; Extracellular Protein; Family; Fatty acid glycerol esters; Fibroblasts; Fracture; Functional disorder; Genes; Genetic; Genotype; Glycine; Goals; Growth; Heat shock proteins; Height; Helix (Snails); Hour; Human; Individual; Integrins; International; Knock-in Mouse; Knowledge; Laboratories; Lead; Ligand Binding; Ligands; Location; Longitudinal Studies; Lower Extremity; Lung; Mediating; Metabolism; Minority; Modeling; Molecular Biology; Molecular Chaperones; Molecular Genetics; Morbidity - disease rate; Mus; Mutation; Natural History; Object Attachment; Osteoblasts; Osteogenesis Imperfecta; Osteoporosis; Outcome; Pain; Pathogenesis; Patients; Pattern; Phase; Phenotype; Placebo Effect; Positioning Attribute; Procollagen; Proteins; Proteoglycan; Proteomics; RNA Splicing; Randomized Controlled Trials; Range; Reporting; Resistance; Role; Score; Severities; Side; Site; Skeletal system; Skin; Solvents; Somatotropin; Splice-Site Mutation; Stress; Stretching; Survivors; Symptoms; Testing; Time; Tissues; Treatment Protocols; Valine; Western Blotting; Whole Organism; alpha-Crystallin B Chain; base; bisphosphonate; bone; bone cell; bone quality; ear helix; experience; follow-up; hammerhead ribozyme; hearing impairment; heritable connective tissue disorder; improved; long bone; member; monomer; mouse model; muscle strength; mutant; novel; pamidronate; programs; protein folding; pulmonary function; pup; response; scoliosis; spine bone structure; translational study; treatment trial

In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and other connective tissue symptoms and then apply the knowledge gained from our studies to the treatment of children with these conditions. Our Branch has generated a knock-in murine model for OI with a classical collagen mutation, and we are using this model to study disease pathogenesis and the skeletal matrix of OI, the effects of pharmacological therapies, and approaches to gene therapy.We are also continuing our clinical studies of children with types III and IV OI, who form a longitudinal study group enrolled in age-appropriate clinical protocols for treatment of their condition. The Brtl mouse model for OI continues to be investigated to understand the pathological and cellular mechanism of OI. We first approached the secretion, matrix incorporation and interactions of collagen molecules with one and two mutant alpha 1 (I) chains. Collagen with no mutant chains or one mutant chain would be expected to comprise 25 and 50% of matrix collagen content; however we detected a decreased content of molecules with one mutant chain and an increase in the proportion with no mutant chains. We demonstrated selective cellular retention of molecules containing one mutant chain, compared to those with no or two mutant chains. In matrix, collagens were incorporated into matrix proportionately to their presence in media, pointing to the cell as the site of discrimination. Brtl fibroblasts had engorgement of the endoplasmic reticulum, suggesting that these cells were undergoing ER stress. The reactive -SH group in collagen with one mutant chain is exposed to solvent and may form aberrant S-S bonds with other intracellular or extracellular proteins. We also studied the basis for the phenotypic variability in Brtl, since this models the phenotypic variability seen in human alpha 1(I) mutations. Brtl has two discrete phenotypes; about 30% of the pups die within hours of birth, while the survivors have a moderately severe skeletal defect. We used microarray and 2D proteomics for complementary studies of calvarial bone from lethal and surviving Brtl pups. We found that Gadd153 expression was increased 2-3 fold in lethal Brtl mice in comparison to surviving and wt littermates. The increase in Gadd153 expression was detected only in bone tissue and was not found in skin or lung. Furthermore, increased Gadd153 was also present in calvarial protein extracts by Western blot. Gadd153 is a member of the C/EBP family that can pair with C/EBP beta or alpha to inhibit osteoblast differentiation or to promote cellular apoptosis, respectively. Conversely, the alpha B-crystallin chain was determined to be relatively increased in both expression (1.6 fold) and protein (doubled) in the non-lethal Brtl pups. alpha B-crystallin has a role opposite to that of Gadd153. It is a small heat shock protein, which is known to confer resistance to apoptosis. This data suggests that apoptosis is the key factor in the phenotypic variability of Brtl mice. We are currently exploring the dimerization partners of Gadd153 in Brtl osteoblasts in culture. To better understand the relationship of phenotype to genotype in human OI cases, the BEMB led an international consortium of connective tissue laboratories to assemble and analyze a database of structural mutations in type I collagen causing OI. The consortium assembled over 830 mutations, including 682 glycine substitutions and 150 splice site defects. Genotype-phenotype modelling revealed different functional relationships for each chain of type I collagen. Glycine substitutions in the alpha 1 (I) chain have a generally more severe outcome, with 36 percent of substitutioons resulting in a lethal phenotype. Substitutions by residues with charged or branched side chains have a lethal outcome in the majority of occurrences. Mutations in the amino quarter of the chain are non-lethal; even those involving residues with charged or branched side chains. We observed two stretches of exclusively lethal mutations in the carboxyl quarter of the chain. The two regions coincide with the Major Ligand Binding Regions (MLBR) of several ligands on the collagen monomer, including integrins and fibronection. Glycine substitutions in the alpha 2 (I)chain have a more moderate outcome on average, with less than 20 percent of occurrences resulting in lethality. Substitutions by residues with charged side chains are predominantly lethal, as in alpha 1 (I), but valine, with a branched side chain, is lethal in only 17 percent of occurrences in alpha 2 (I)(as compared with 73% in alpha 1 (I)). As for alpha 1 (I), occurrences in the amino third of the chain are non-lethal. Thereafter, the lethal mutations occur in eight clusters that are regularly spaced along the chain. The distribution of lethal mutations in alpha 2 (I) continues to follow the pattern we previously described in the Regional Model for this chain and correctly predict the phenotype of 86% of cases in alpha 2 (I). The lethal regions coincide with the binding regions for matrix proteoglycans on the collagen fibril. Finally, splice site mutations lead to mild OI in a minority of cases. Most splice site mutations, even those in invariant positions, lead to significant dysplasia, suggesting that use of an alternative donor or acceptor generates translatable products that can be incorporated into matrix. Most mutations that lead to simple exon skipping have a severe or lethal outcome. This modelling provides testable hypothesis for the mechanisms of OI. The BEMB undertook the first randomized controlled trial of bisphosphonate in children with types III and IV OI. The aim was to test both the primary skeletal gains (increased bone density and decreased fractures) and secondary gains (improved functional level and muscle strength and decreased pain) reported in observational trials. Children in the treatment group received pamidronate for 18-23 months. The treatment group experienced improvement in vertebral parameters, including BMD z-scores, central vertebral height and vertebral area. However, the increment in vertebral BMD in the treatment group tapered off after one to two years of treatment. Furthermore, the treatment group did not experience a decrease in long-bone fractures. No functional effect was seen from bisphosphonate treatment. There was no significant change in ambulation level, lower-extremity strength or pain in children with OI treated with pamidronate. Some patients reported increased endurance or decreased back pain, but most reported no perceptible changes. Hence, the changes previously reported in these parameters appear to have been a placebo effect in the uncontrolled trials. The data from our controlled trial were in accord with three other controlled trials that were conducted in the same time frame. We are now recommending that treatment of children with types III and IV OI with pamidronate be limited to one to two (or at most three) years, with subsequent follow-up of bone status. Furthermore, we are currently engaged in a dose comparison trial, using the dose from our first trial and a lower dose. This trial has now entered the data analysis phase; our hypothesis is that the children will gain a comparable benefit from the lower dose with decreased detrimental effects.

在实验室和临床研究的综合计划中，我们研究了可遗传的结缔组织疾病的分子生物学成骨肌发生Imperfecta（OI）和Ehlers-Danlos综合征（EDS）。我们的目标是阐明主要基因缺陷会导致骨骼脆弱性和其他结缔组织症状，然后将我们从研究中获得的知识应用于患有这些条件的儿童的知识。我们的分支机构为OI产生了一个具有经典胶原蛋白突变的OI敲击鼠模型，我们正在使用该模型研究疾病的发病机理和OI的骨骼基质，药理学疗法的影响，药物治疗的作用以及基因疗法的方法。我们还继续对儿童进行III和III和IV OI类型的临床研究，以进行长期研究，以培训他们的临床研究。 继续研究了OI的BRTL小鼠模型，以了解OI的病理和细胞机制。我们首先接近胶原蛋白分子与一个和两个突变α1（i）链的分泌，基质结合和相互作用。没有突变链或一个突变链的胶原蛋白将占基质胶原蛋白含量的25％和50％；但是，我们检测到一个突变链的分子含量降低，没有突变链的比例增加。与没有或两个突变链的分子相比，我们证明了包含一个突变链的分子的选择性细胞保留。在基质中，将胶原蛋白与其在培养基中的存在成比例地合并到基质中，将细胞作为歧视部位。 BRTL成纤维细胞具有内质网状的体积，表明这些细胞正在经历ER应激。一个突变链的胶原蛋白中的反应性-SH组暴露于溶剂中，可能与其他细胞内或细胞外蛋白形成异常的S -S键。 我们还研究了BRTL表型变异性的基础，因为这对人α1（i）突变中的表型变异性进行了模型。 BRTL具有两个离散表型。大约30％的幼崽在出生后数小时内死亡，而幸存者的骨骼缺陷中等。我们使用微阵列和2D蛋白质组学进行了致命和幸存的BRTL幼犬的钙骨骨的互补研究。我们发现，与存活的和WT的同窝仔相比，致命的BRTL小鼠的GADD153表达增加了2-3倍。仅在骨组织中检测到GADD153表达的增加，在皮肤或肺中未发现。 此外，通过Western印迹中钙化蛋白提取物中也存在增加的GADD153。 GADD153是C/EBP家族的成员，可以与C/EBPβ或α配对以抑制成骨细胞分化或促进细胞凋亡。相反，在非致命性BRTL幼犬中，确定αB-晶状蛋白链在表达（1.6倍）和蛋白质（加倍）中相对增加。 Alpha b-crystallin与GADD153的作用相反。这是一种小的热休克蛋白，已知可以赋予凋亡的耐药性。该数据表明凋亡是BRTL小鼠表型变异性的关键因素。我们目前正在培养文化中BRTL成骨细胞中GADD153的二聚化伙伴。 为了更好地理解表型与基因型在人OI病例中的关系，BEMB领导了一个国际结缔组织实验室联盟，以组装和分析I型胶原蛋白中的结构突变数据库，从而导致OI。 该财团组装了830多个突变，包括682个甘氨酸取代和150个剪接部位缺陷。基因型 - 表型建模揭示了I型胶原链的每个链的不同功能关系。 Alpha 1（I）链中的甘氨酸取代的结果通常更严重，替代品的36％导致致命的表型。在大多数发生的情况下，用带电或分支侧链的残留物取代具有致命的结果。链的氨基四分之一的突变是非致命的。甚至那些涉及带电或分支侧链的残留物的人。我们观察到了链的羧基四分之一的两次伸展致死突变。这两个区域与胶原蛋白单体的几种配体的主要配体结合区（MLBR）一致，包括整联蛋白和纤维纤维。 Alpha 2（i）链中的甘氨酸取代平均结果更为中等，不到20％的发生导致致死性。带有带电侧链的残基的取代主要是致命的，如在α1（i）中，但是带有侧链链的瓦林在α2（i）中仅17％发生在alpha 2（i）中（与Alpha 1（i）中的73％相比，althal是17％。至于alpha 1（i），氨基三分之一的链中的发生是非致命的。此后，致命突变发生在沿链条规定间隔的八个簇中。 Alpha 2（i）中致死突变的分布继续遵循我们先前在该链区域模型中描述的模式，并正确预测了Alpha 2（i）中86％的病例的表型。致命区域与胶原原纤维上基质蛋白聚糖的结合区域一致。最后，在少数情况下，剪接部位突变导致温和的OI。大多数剪接部位突变，甚至是处于不变位置的突变，都会导致明显的发育不良，这表明使用替代供体或受体会生成可翻译的产品，这些产品可以掺入矩阵中。导致简单外显子跳过的大多数突变都会产生严重或致命的结果。该建模为OI机制提供了可检验的假设。 BEMB在III型和IV型OI的儿童中进行了双膦酸盐的首次随机对照试验。目的是在观察试验中测试主要的骨骼增长（增加骨密度和减少骨折）和继发性收益（提高功能水平和肌肉强度和疼痛）。治疗组的儿童接受了18-23个月的pamidronate。该治疗组的椎骨参数有所改善，包括BMD Z得分，中央椎骨高度和椎骨区域。但是，治疗一到两年后，治疗组中椎BMD的增加逐渐减弱。此外，治疗组没有长骨骨折的减少。从双膦酸盐处理中未观察到功能效应。用pamidronate治疗的OI儿童的行动水平，较低的高度强度或疼痛没有显着变化。一些患者报告说耐力增加或减轻了背痛，但大多数报告没有明显的变化。因此，这些参数中先前报道的变化似乎是不受控制的试验中的安慰剂效应。我们对照试验的数据与在同一时间范围内进行的其他三个对照试验一致。现在，我们建议对III型和IV型儿童进行pamiDronate的治疗，仅限于一到两年（或最多三年），随后进行骨状态随访。此外，我们目前使用第一次试验和较低剂量的剂量进行了剂量比较试验。该试验现在进入了数据分析阶段。我们的假设是，儿童将从较低剂量中获得可比的益处，并减少有害影响。