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Heritable Disorders of Connective Tisue

结缔组织遗传性疾病

基本信息

批准号：
8351215
负责人：
Joan C Marini
金额：
$ 65.32万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/8351215
关键词：
Affect African African American Age Alendronate Alleles Area Binding Biochemical Biological Markers Biomechanics Bone Density Bone remodeling C-telopeptide CCAAT-Enhancer-Binding Proteins COL1A1 gene COL1A2 gene Cartilage Catalytic RNA Cell Line Cells Cellular Stress Cessation of life Chemicals Child Childhood Cleaved cell Clinical Clinical Research Clinical Trials Collagen Collagen Type I Complement Complex Connective Tissue DEXA Data Databases Defect Deposition Development Developmental Bone Diseases Dipeptides Disease Dose Dual-Energy X-Ray Absorptiometry Ehlers-Danlos Syndrome Engraftment Family Female Femur Fracture Genes Genetic Genotype Goals Haplotypes Height Human Image In Vitro Individual International Investigation Knock-in Mouse Knockout Mice Knowledge Laboratories Length Ligand Binding Literature Lower Extremity Minerals Modeling Molecular Molecular Biology Molecular Chaperones Morphology Mus Mutation Organ Osteoblasts Osteogenesis Osteogenesis Imperfecta Osteoporosis Outcome Pain Patients Pattern Peptide Hydrolases Peptides Perinatal Phenotype Physiologic calcification Placebo Effect Prevalence Process Procollagen Property Proteoglycan Proteomics Published Database Randomized Controlled Trials Reporting Role Sampling Site Skeleton Skin Spectroscopy, Fourier Transform Infrared Stem cells Stress Structure Symptoms Testing Thick Tissues Transcript Transgenic Mice Type I Procollagen Variant Weight alanylaspartic acid analog bisphosphonate bone bone cell bone quality bone strength density design experience follow-up heritable connective tissue disorder improved in vivo insight member mineralization mouse model muscle strength mutant novel pamidronate proband programs protein expression protein misfolding pup response skeletal spine bone structure substantia spongiosa treatment strategy

项目摘要

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. A fundamental insight into the mechanism of OI derived from Brtl has involved the role of ER stress. Combined microarray and proteomic investigations of Brtl showed a two fold increase in Gadd153/CHOP expression and protein in lethal pups shortly before death. The increase in Gadd 153 expression was bone specific. Gadd153 is a member of the C/EBP family activated by cell stress, in particular, by ER retention of misfolded protein. These studies suggest that relief of ER stress with chemical chaperones, such as SPB, may have a beneficial effect on the OI skeleton. Using Brtl, we completed a major theraeputic trial of the effect of bisphosphonate, which complements our pediatric trial. We treated Brtl and wild-type littermates with alendonate and compared treated and untreated femora of each genotype. Alendronate treatment increased femoral DXA and cortical volumetric BMD, but did not improve Brtl weight curves or femoral length. Brtl trabecular number and diaphyseal cortical thickness were improved, as was femoral stiffness and load to fracture. However, detrimental changes were also detected in material and cellular parameters of bone. Predicted material strength and elastic modulus of both Brtl and wild-type bone were deceased; brittleness of Brtl femora were unchanged, while that of wild-type was increased. Furthermore, dramatic retention of mineralized cartilage disrupts matrix continuity and may contribute to the weakening of bone material. In addition, the function osteoblasts was impaired, with severe reductions in mineral apposition rate and bone formation rate. Osteoblast morphology was altered, making Brtl Treated osteoblasts flattened, similar to lining cells. These studies contribute to the increased cautionary notes in the literature concerning avoidance of an elevated cummulative bisphosphonate dose. We have collaborated in a study demonstrating that a fluoresecent bisphosphonate analog is an accurate biomarker of deposition and retention in vivo is currently being used for trails of two non-traditional therapies. In a collabaorative study. Brtl was sued for an utero cell tansplantation trailof GFP expression stem cells. Despite low levels of engraftment, the perinatal lethality and femoral geometry and biomechanics of the of the engrafted Brtl mice were improved. The result are encouraging for translational trails. Second, we are modelling a lesson from type I OI to suppress mutantcollagen expression. Specific suprression of transcripts of the mutant collagen allele can biochemically transform individual with severe OI into mild type I OI. We have introduced a R7 target site into the BRTL mutant allele: we have henerated transgenic mice expressing ribozymes tragated to the Brtl mutation. Preliminary dataon Brtl/RZ mice is encouraging for improvement of Brtl biomechanical properties in female mice. We have identified a novel "high bone density" form of OI caused by mutations in the C-proteinase cleavage site of type I procollagen. The Asp-Ala dipeptide between the telopeptide and the C-propeptide of each chain is cleaved by C-proteinase/BMP1 to release mature collagen. We have identified children with substitutions at two of these 4 peptides. They present with fractures and a high DEXA z-score. Interestingly, despite the high DEXA, radiographs and histomorphometry are similar to type I OI and point to matrix deficiency. Pericellular processing of procollagen C-propeptide is delayed, and in vitro cleavage by purified BMP1 is impaired. FTIR imaging of corticicol and trabecular bone confirms elevated mineral/matrix ratios in affected children, compared to normal controls and classical OI samples, as well significantly inreased collagen maturity in trabecular bone BBD (Bone mineralization density distribution) reveals a marked shift toward increased mineralizaton compared to controls but difeerent patterns for each patient, with heterogenity of minerlization density (area of both higher and lower mineralization than normal or classical OI bone)for the COL1A1 mutations and uniformaly high mineralization exceeding even classical OI for the COL1A2 bone. The data not only reveal a novel form of OI but also provide new fundamental on roles of procollagen processing and the mechanism of tissue mineralizations. We are currently generating a murine model for high bone density OI, in order to study the molecular and biochemical mechanism of the mineralization, and its developmental progression. To better understand the relationship of genotype and phenotype in human OI, the BEMB led and international constortium of connective tissue laboratories to assemble an BSEM are being used to study the amount and crystallinity of bone samples from our two probands. These data not only reveal a novel form of OI but also provide new fundamental information on roles of procollagen processing and the mechanism of tissue mineralization. To better understand the relationship of genotype and phenotype in human OI, the BEMB led international consortium of connective tissue laboratories to assemble and analyze a mutation database. The initial database published in 2007 containing over 830 mutations; currently the database under analysis contains over 1300 mutations. Genotype-phenotype modeling revealed different functional relationships for each chain of type I collagen. Lethal mutations in alpha 1 (I) coincide with the Major Ligand Binding Regions. Lethal regions in alpha 2(I) continue to support the Regional Model first proposed by the BEMB, with lethal mutations in regularly-spaced clusters along the chain that coincide with proteoglycan binding regions. This model correctly predicts clinical outcome in 86% of alpha 2(I) mutations. . We are also continuing our clinical studies of children with types III and IV 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 and secondary gains (improved functional level and muscle strength and decreased pain) reported in observational trials. 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. There was no significant change in ambulation level, lower-extremity strength or pain in children with OI treated with pamidronate. Hence the changes previously reported appear to have been a placebo effect in uncontrolled trials. We are recommending that treatment of children with types III and IV OI with pamidronate be limited to at most three years, with subsequent follow-up of bone status. Furthermore, we are currently engaged in a dose comparison trial. We are also focusing on the variability of response to treatment in each group. The improvements in vertebral height and area do not correlate with changes in DXA z-score, nor did the improvement in vertebral height and area correlate for individual children. These differences may be related to important individual variation in ability to synthesize new bone or to remodel bone. They also highlight the inadequacy of DXA as a surrogate for bone strength.

在实验室和临床研究的综合项目中，我们研究遗传性结缔组织疾病成骨不全症 (OI) 和埃勒斯-当洛斯综合征 (EDS) 的分子生物学。我们的目标是阐明主要基因缺陷导致骨骼脆弱和其他结缔组织症状的机制，然后将我们研究中获得的知识应用于治疗患有这些疾病的儿童。 我们的分支机构已经生成了具有经典胶原蛋白突变的成骨不全症敲入小鼠模型。从 Brtl 衍生的对 OI 机制的基本了解涉及 ER 应激的作用。 Brtl 的微阵列和蛋白质组学综合研究显示，致死幼崽在死亡前不久，Gadd153/CHOP 表达和蛋白质增加了两倍。 Gadd 153 表达的增加是骨特异性的。 Gadd153 是 C/EBP 家族的成员，可被细胞应激激活，特别是通过错误折叠蛋白的内质网滞留激活。这些研究表明，用 SPB 等化学伴侣缓解内质网应激可能对 OI 骨骼产生有益影响。使用 Brtl，我们完成了一项关于双膦酸盐作用的主要治疗试验，这补充了我们的儿科试验。我们用阿仑糖酸治疗 Brtl 和野生型同窝小鼠，并比较每种基因型的治疗和未治疗的股骨。阿仑膦酸钠治疗增加了股骨 DXA 和皮质体积 BMD，但没有改善 Brtl 体重曲线或股骨长度。 Brtl 小梁数量和骨干皮质厚度得到改善，股骨刚度和骨折负荷也得到改善。然而，在骨的材料和细胞参数中也检测到了有害的变化。 Brtl和野生型骨的预测材料强度和弹性模量均下降； Brtl股骨的脆性没有变化，而野生型的脆性有所增加。此外，矿化软骨的显着保留会破坏基质的连续性，并可能导致骨材料的弱化。此外，成骨细胞功能受损，矿物质沉积率和骨形成率严重降低。成骨细胞形态发生改变，使得经过 Brtl 处理的成骨细胞变平，与衬里细胞类似。这些研究有助于增加文献中有关避免双膦酸盐累积剂量升高的警告。我们合作进行的一项研究表明，荧光双膦酸盐类似物是体内沉积和保留的准确生物标志物，目前正用于两种非传统疗法的试验。在一项合作研究中。 Brtl 因 GFP 表达干细胞的子宫细胞移植试验而被起诉。尽管植入水平较低，但植入的 Brtl 小鼠的围产期致死率、股骨几何形状和生物力学均得到改善。结果对于转化试验来说是令人鼓舞的。其次，我们正在模拟 I 型 OI 的教训，以抑制突变胶原蛋白的表达。特异性抑制突变胶原等位基因的转录本可以通过生化将严重 OI 患者转变为轻度 I 型 OI。我们已将 R7 靶位点引入 BRTL 突变等位基因中：我们已培育出表达 Brtl 突变核酶的转基因小鼠。 Brtl/RZ 小鼠的初步数据对于雌性小鼠 Brtl 生物力学特性的改善是令人鼓舞的。 我们发现了一种新的“高骨密度”OI 形式，由 I 型原胶原 C 蛋白酶切割位点突变引起。每条链的端肽和C-前肽之间的Asp-Ala二肽被C-蛋白酶/BMP1裂解，释放出成熟的胶原蛋白。我们已经鉴定出儿童在这 4 种肽中的 2 种上有替换。他们出现骨折且 DEXA z 得分较高。有趣的是，尽管 DEXA 较高，但 X 光片和组织形态学与 I 型 OI 相似，表明基质缺乏。前胶原 C 前肽的细胞周加工被延迟，并且纯化的 BMP1 的体外切割受到损害。皮质醇和小梁骨的 FTIR 成像证实，与正常对照和经典 OI 样本相比，受影响儿童的矿物质/基质比率升高，并且小梁骨 BBD（骨矿化密度分布）中的胶原成熟度显着增加，揭示了与对照相比，矿化明显增加，但每个患者的模式不同，矿化密度存在异质性（比正常或经典 OI 骨矿化度更高和更低的区域）对于 COL1A1 突变和 COL1A2 骨的均匀高矿化度甚至超过经典 OI。这些数据不仅揭示了成骨不全的一种新形式，而且还为前胶原加工的作用和组织矿化机制提供了新的基础。我们目前正在建立高骨密度成骨不全症小鼠模型，以研究矿化的分子和生化机制及其发育进程。为了更好地了解人类成骨不全症中基因型和表型的关系，BEMB 领导的国际结缔组织实验室联盟正在使用 BSEM 来研究我们两个先证者的骨样本的数量和结晶度。这些数据不仅揭示了成骨不全的一种新形式，而且还提供了关于前胶原加工的作用和组织矿化机制的新的基本信息。为了更好地了解人类成骨不全症中基因型和表型的关系，BEMB 领导国际结缔组织实验室联盟收集并分析了突变数据库。 2007 年发布的初始数据库包含超过 830 个突变；目前正在分析的数据库包含 1300 多个突变。基因型-表型模型揭示了 I 型胶原每条链的不同功能关系。 α 1 (I) 的致死突变与主要配体结合区域一致。 α 2(I) 中的致死区域继续支持 BEMB 首次提出的区域模型，其中沿链规则间隔的簇中存在致死突变，与蛋白多糖结合区域一致。该模型正确预测了 86% 的 alpha 2(I) 突变的临床结果。 . 我们还在继续对 III 型和 IV 型 OI 儿童进行临床研究。 BEMB 开展了第一个针对 III 型和 IV 型 OI 儿童的双磷酸盐随机对照试验。目的是测试观察性试验中报告的主要骨骼增益和次要增益（改善功能水平和肌肉力量以及减轻疼痛）。治疗组的椎骨参数有所改善，包括 BMD z 评分、椎骨中心高度和椎骨面积。然而，治疗组的椎骨 BMD 增量在治疗一到两年后逐渐减少。接受帕米膦酸钠治疗的成骨不全儿童的步行水平、下肢力量或疼痛没有显着变化。因此，之前报道的变化似乎是非对照试验中的安慰剂效应。我们建议帕米膦酸钠治疗 III 型和 IV 型成骨不全儿童的期限最多为三年，并随后随访骨状况。此外，我们目前正在进行剂量比较试验。我们还关注每组治疗反应的差异。椎体高度和面积的改善与 DXA z 分数的变化不相关，对于个体儿童来说，椎体高度和面积的改善也不相关。这些差异可能与合成新骨或重塑骨的能力的重要个体差异有关。他们还强调了 DXA 作为骨强度替代指标的不足。