Heritable Disorders of Connective Tisue

结缔组织遗传性疾病

• 批准号: 8736903
• 负责人: Joan C Marini
• 金额: $ 56.44万
• 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/8736903
• 关键词: Adult; Affect; African; African American; Age; Alendronate; Alleles; Antibodies; Area; Autophagocytosis; Binding; Biochemical; Biological Markers; Biomechanics; Bone Density; Bone callus; C-telopeptide; Cardiac; Cardiopulmonary; Caring; Cartilage; Catalytic RNA; Cell Transplantation; Child; Childhood; Cleaved cell; Clinical; Clinical Research; Clinical Trials; Coculture Techniques; Collagen; Collagen Type I; Communities; Complement; Complex; Connective Tissue; DEXA; Data; Databases; Defect; Deposition; Development; Developmental Bone Diseases; Dipeptides; Disease; Dose; Dual-Energy X-Ray Absorptiometry; Early Diagnosis; Early treatment; Ehlers-Danlos Syndrome; Engraftment; Female; Femur; Fracture; Fracture Healing; Genes; Genetic; Genotype; Goals; Haplotypes; Height; Human; Image; In Vitro; Individual; International; Knock-in Mouse; Knockout Mice; Knowledge; Laboratories; Ligand Binding; Literature; Lower Extremity; Lung; Lung diseases; Marrow; Mesenchymal Stem Cells; Minerals; Modeling; Molecular; Molecular Biology; Morphology; Mus; Mutation; Organ; Osteoblasts; Osteoclasts; Osteogenesis; Osteogenesis Imperfecta; Osteoporosis; Outcome; Pain; Participant; Pathway interactions; Patients; Peptide Hydrolases; Peptides; Perinatal; Pharmaceutical Preparations; Phase; Phenotype; Physiologic calcification; Placebo Effect; Predisposition; Prevalence; Process; Procollagen; Property; Proteoglycan; Published Database; Randomized Controlled Trials; Reporting; Research; Role; Sampling; Site; Skin; Spectroscopy, Fourier Transform Infrared; Stem cells; Structure; Symptoms; Testing; Therapy Clinical Trials; Thick; Tissues; Transcript; Transgenic Mice; Type I Procollagen; United States National Institutes of Health; Up-Regulation; Western Blotting; alanylaspartic acid; analog; bisphosphonate; bone; bone cell; bone mass; bone quality; bone strength; cohort; density; design; experience; follow-up; heritable connective tissue disorder; improved; in utero; in vivo; insight; mineralization; mortality; mouse model; mutant; nanoindentation; novel; pamidronate; programs; pulmonary function; repaired; research study; response; scoliosis; skeletal; spine bone structure; substantia spongiosa; treatment strategy

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. Brtl mesenchymal stem cells have impaired differentiation, with decreased expression of late osteoblast markers vs WT. Adult MSCs had upregulation of autophagy markers on Western blot. We demonstrated increased osteoclast number and activity. Co-culture experiments that Brtl MSC's secrete a non-RANKL/OPG factor that increases osteoclast number in Brtl and WT marrow precursors. Using Brtl, we completed a major therapeutic trial of bisphosphonate, which complements our pediatric trial. Alendronate treatment increased femoral DXA and cortical volumetric BMD. Brtl trabecular number and diaphyseal cortical thickness were improved, as was femoral stiffness and fracture load. However, detrimental changes were 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 wild-type femora was increased. Furthermore, dramatic retention of mineralized cartilage may contribute to the weakening of bone material. Also, the function osteoblasts was impaired, with reductions in mineral apposition rate and bone formation rate. Osteoblast morphology was altered, making Brtl treated osteoblasts flattened. These studies contribute to the increased cautionary notes in the literature concerning avoidance of an elevated cummulative bisphosphonate dose. Further, when we examined fracture healing in Brtl mice treated with bisphosphonate, we found that the drug localized in the callus, where it decreased crystallinity and delayed late-phase fracture healing. We have collaborated in a study demonstrating that a fluoresecent bisphosphonate analog is an accurate biomarker of deposition and retention in vivo. In addition, Brtl bone itself was demonstrated to have inherently increased susceptibility to microdamage using an ulnar loading model. This property makes OI bone more likely to become increasingly fragile from inhibition of microdamage repair by bisphosphonates. Much excitement in the osteoporosis research community focuses on the novel anabolic drugs that inhibit sclerostin. Brtl has also been used to model sclerostin antibody treatment (Scl-AB). Sost stimulates osteoblasts via the cannonical Wnt pathway and its antibody is an anabolic drug. Only 2 weeks of treatment resulted in improved bone mass and reduced fragility. Nanoindentation studies indicated unchanged mineralization, showing that the hypermineralization of bisphosphonate treatment did not occur. Anti-sclerostin antibody represents a potential new therapy for pediatric OI patients. In a collaborative study, Brtl was used for an in utero cell transplantation trial of GFP expression stem cells. Despite low levels of engraftment, the perinatal lethality and femoral geometry and biomechanics of the engrafted Brtl mice were improved. The results are encouraging for translational trails. Second, we are modelling a lesson from type I OI to suppress mutant collagen expression. Specific suppression of transcripts of the mutant collagen allele can biochemically transform individual with severe OI into mild type I OI. We have introduced a Ribozyme target site into the BRTL mutant allele: we have generated transgenic mice expressing ribozymes targeted to the Brtl mutation. Preliminary data on 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 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 cortical and trabecular bone confirms elevated mineral/matrix ratios in affected children, compared to normal controls and classical OI samples, as well as significantly increased collagen maturity in trabecular bone BBD (Bone mineralization density distribution) reveals a marked shift toward increased mineralization compared to controls. The data not only reveal a novel form of OI but also provide new fundamental insight on roles of procollagen processing and the mechanism of tissue mineralization. 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 an international consortium of connective tissue laboratories to assemble and analyze a mutation database. The initial database published in 2007 contained 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 treatment group experienced improvement in vertebral parameters, which leveled 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 highlight the inadequacy of DXA as a surrogate for bone strength. We have also focused on the cardiopulmonary aspects of OI. Findings in the NIH pediatric OI cohort revealed a significant progressive decline in pulmonary function and increased restrictive pulmonary disease independent of scoliosis. Most participants in the study showed mild cardiac valvular regurgitation, independent of pulmonary and skeletal findings. This data will change the standard of anticipatory care for OI and promote early detection and treatment of the greatest cause of mortality in OI.

在一个综合的实验室和临床研究项目中，我们研究了遗传性结缔组织疾病成骨不全症（OI）和ehers - danlos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，然后将从我们的研究中获得的知识应用于患有这些疾病的儿童的治疗。我们的分支已经建立了一个具有经典胶原蛋白突变的成骨不全小鼠敲入模型。Brtl间充质干细胞分化受损，与WT相比，晚期成骨细胞标志物的表达降低。Western blot结果显示，成年MSCs自噬标志物上调。我们发现破骨细胞数量和活性增加。共培养实验表明，Brtl MSC分泌一种非rankl /OPG因子，可增加Brtl和WT骨髓前体细胞中的破骨细胞数量。使用Brtl，我们完成了一项主要的双膦酸盐治疗试验，补充了我们的儿科试验。阿仑膦酸钠治疗增加股骨DXA和皮质体积骨密度。股骨小梁数量和骨干皮质厚度以及股骨刚度和骨折负荷均得到改善。然而，在骨的材料和细胞参数检测到有害的变化。Brtl和野生型骨的预测材料强度和弹性模量均下降；野生型股骨脆性增加。此外，矿化软骨的显著保留可能会导致骨材料的削弱。同时，成骨细胞功能受损，矿物质附着率和骨形成率降低。成骨细胞形态发生改变，使Brtl处理的成骨细胞变平。这些研究增加了文献中关于避免升高的双膦酸盐累积剂量的警告说明。此外，当我们检查用双膦酸盐治疗的Brtl小鼠的骨折愈合时，我们发现该药物局限于愈伤组织，在那里它降低了结晶度并延迟了后期骨折愈合。我们合作进行了一项研究，证明荧光双膦酸盐类似物是体内沉积和滞留的准确生物标志物。此外，使用尺骨加载模型证明Brtl骨本身对微损伤具有固有的易感性。这种特性使得成骨不全骨更容易因双膦酸盐抑制微损伤修复而变得越来越脆弱。许多令人兴奋的骨质疏松研究集中在新的合成代谢药物，抑制硬化蛋白。Brtl也被用于模拟硬化蛋白抗体治疗（scll - ab）。Sost通过典型的Wnt途径刺激成骨细胞，其抗体是一种合成代谢药物。仅2周的治疗就改善了骨量，降低了脆性。纳米压痕研究表明矿化没有改变，表明双膦酸盐处理的高矿化没有发生。抗硬化蛋白抗体为儿童成骨不全症患者提供了一种潜在的新疗法。在一项合作研究中，Brtl被用于GFP表达干细胞的子宫细胞移植试验。尽管移植水平较低，但移植的Brtl小鼠的围产期死亡率和股骨几何形状和生物力学均得到改善。这些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型成骨不全以抑制突变型胶原表达的经验。特异性抑制突变型胶原等位基因的转录本可生化地将重度成骨不全个体转化为轻度I型成骨不全。我们在BRTL突变等位基因中引入了一个核酶靶点：我们产生了表达针对BRTL突变的核酶的转基因小鼠。Brtl/RZ小鼠的初步数据对改善雌性小鼠Brtl生物力学性能具有鼓舞作用。我们已经确定了一种新的“高骨密度”形式的OI，由I型前胶原的c蛋白酶裂解位点突变引起。每条链的端肽和c前肽之间的Asp-Ala二肽被c蛋白酶/BMP1切割，释放成熟胶原。我们在这4个多肽中鉴定出两个有取代的子代。他们有骨折和高DEXA z评分。有趣的是，尽管DEXA很高，但x线片和组织形态测量与I型成骨不全相似，并指向基质缺乏。前胶原c -前肽的细胞周围加工被延迟，纯化的BMP1体外切割受损。与正常对照和典型OI样本相比，皮质骨和小梁骨的FTIR成像证实，受影响儿童的矿物质/基质比率升高，小梁骨BBD（骨矿化密度分布）的胶原成熟度显著增加，与对照组相比，矿化程度明显增加。这些数据不仅揭示了一种新的成骨不全形式，而且为前胶原加工的作用和组织矿化机制提供了新的基础见解。我们目前正在建立高骨密度成骨不全小鼠模型，以研究矿化的分子生化机制及其发育过程。为了更好地理解人类成骨不全症中基因型和表型的关系，BEMB领导了一个由结缔组织实验室组成的国际联盟，组装并分析了一个突变数据库。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型胶原蛋白各链的不同功能关系。α 1 (I)的致死突变与主要配体结合区一致。α 2(I)中的致死区域继续支持BEMB首先提出的区域模型，在与蛋白聚糖结合区域一致的链上有规则间隔的簇中存在致死突变。该模型正确预测了86%的α 2(I)突变的临床结果。我们还在继续对III型和IV型成骨不全儿童进行临床研究。BEMB进行了第一个双膦酸盐治疗III型和IV型成骨不全症儿童的随机对照试验。治疗组的椎体参数有所改善，治疗一至两年后趋于平稳。经帕米膦酸盐治疗的成骨不全症患儿的活动水平、下肢力量或疼痛无显著变化。因此，先前报道的变化在非受控试验中似乎是一种安慰剂效应。我们建议使用帕米膦酸盐治疗III型和IV型成骨不全症的儿童时间限制在最多3年，随后随访骨骼状况。此外，我们目前正在进行剂量比较试验。我们还关注每组患者对治疗反应的可变性。椎体高度和面积的改善与DXA z-score的变化不相关，个体儿童椎体高度和面积的改善也不相关。这些差异突出了DXA作为骨强度替代指标的不足。我们也关注成骨不全的心肺方面。NIH儿童成骨不全队列研究结果显示肺功能显著进行性下降，与脊柱侧凸无关的限制性肺部疾病增加。大多数参与者在研究中显示轻微的心脏瓣膜反流，独立于肺部和骨骼的发现。这一数据将改变成骨不全的预期治疗标准，促进早期发现和治疗导致成骨不全死亡的最大原因。