Heritable Disorders Of Connective Tissue

结缔组织遗传性疾病

基本信息

批准号：
7734690
负责人：
Joan C Marini
金额：
$ 90.23万
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/7734690
关键词：
Adipocytes Adverse effects Affect Age Age of Onset Age-Months Apoptosis Area Binding Binding Sites Bone Density Bone Matrix Bone Tissue COL1A1 gene COL1A2 gene Cartilage Cell Communication Cell Transplants Cells Child Childhood Circular Dichroism Clinical Clinical Protocols Clinical Research Clinical Trials Collaborations Collagen Collagen Fibril Collagen Type I Condition Connective Tissue Data Databases Defect Degenerative polyarthritis Developmental Bone Diseases Differential Scanning Calorimetry Disease Distal Dose Dysplasia Ehlers-Danlos Syndrome Enrollment Family Fatty acid glycerol esters Femur Fibroblasts Fracture Functional disorder Genes Genetic Genotype Glycine Goals Growth Height Helix (Snails)Heterogeneity Histology Human Immunohistochemistry Incidence Individual Integrins International Investigation Joints Knee joint Knock-in Mouse Knowledge Laboratories Leg Length Ligand Binding Location Longitudinal Studies Lower Extremity Lung Maps Marrow Measures Mediating Metabolism Modeling Molecular Biology Molecular Chaperones Molecular Genetics Morbidity - disease rate Mus Mutation National Institute of Child Health and Human Development Natural History Numbers Osteoblasts Osteoclasts Osteogenesis Osteogenesis Imperfecta Osteoporosis Outcome Pain Pathogenesis Pathology Patients Pattern Phenotype Placebo Effect Procollagen Production Proteoglycan Puberty Randomized Controlled Trials Range Reporting Research Personnel Reverse Transcriptase Polymerase Chain Reaction Role Score Severities Signal Pathway Site Skeletal system Skin Somatotropin Staining method Stains Stress Surface Symptoms TNFSF11 gene Temperature Testing Time Tissues Treatment Protocols Variant Whole Organism Wild Type Mouse alpha 2 collagen type I base bisphosphonate bone bone cell bone quality calcification ear helix experience follow-up hammerhead ribozyme hearing impairment heritable connective tissue disorder improved insight melting mouse model muscle strength mutant novel pamidronate programs pulmonary function response scoliosis spine bone structure substantia spongiosa tibia translational study treatment trial triple helix type I collagen alpha 1

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, 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 have investigated the cellular contribution to Brtl bone properties.Brtl cortical and trabecular bone are reduced before and after puberty, with BV/TV decreased 40-45%. Brtl osteoblast surface is comparable to wild-type, and Brt and wild-type marrow generate equivalent numbers of osteoblast precursors at both ages. Interestingly, the osteoclast surface is increased in Brtl at both ages (36-45%), as are the number of TRAP positive cells (57-47%). After puberty, Brtl osteoblast matrix production decreases to half of wild-type values. In contrast, osteoclasts remain significantly elevated compared to wild-type. The combination of osteoblast and osteoclast changes results in a decline in BFR from normal to half of wild-type values at 6 months. We investigated whether the RANKL/OPG signaling pathway is responsible for the uncoupled cell numbers.Both immunohistochemnistry and real-time RT-PCR show increased RANK, RANKL and OPG levels in Brtl, but a normal RANKL/OPG ratio. Surprisingly, we found that TRAP+ precursors are markedly elevated in Brtl marrow cultures and form more osteoclasts,suggesting that osteoclast increases arise from increased numbers of precursors. This exciting data suggest that OI pathology is due to acombination of deficient bone formation by osteoblasts and increased numbers of osteoclasts,triggered independently by abnormal bone matrix. We have also completed a collaborative study which shows that the Brtl mouse develops an early and rapidly progressive osteoarthritis. The knee joint OA was detected by micro-MRI and micro-CT and confirmed by histology and immunohistochemistry. Brtl mice show thinning of subchondral bone by 2 months of age; by 12 months Brtl has severe OA with joint space narrowing.Safranin O staining showed reduced cartilage proteoglycans compared to wild type mice of the same age. We demonstrated that this cartilage degeneration was not due abnormal spacial expression of collagen by immunohistochemistry. These studies are important for the common condition, osteoarthristis, as well as for the rare dysplasia OI. They demonstrate that abnormalities in subchondral bone can cause OA in mice with initially normal cartilage. To better understand the relationship of genotype and phenotype in human OI, the BEMB led and international consortium of connective tissue laboratories to assemble and analyze a mutation database containing over 830 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 have participated in several collaborations which have extended our understanding of genotype-phenotype relationships. Our long and continuing collaboration with the laboratory of Sergey Leikin at NICHD has yielded insight into structural heterogeneity in the the type I collagen triple helix and its role in osteogenesis imperfecta. Using differential scanning calorimetry and circular dichroism, changes in collagen melting temperature were measured for 41 different glycine substitutions. There was no correlation of melting temperature with substituting residue. Instead, the variations in Tm defined regions along the triple helix which were further confirmed with an activation energy map. We deduced two large, flexible regions important for collagen fibril assembly and ligand binding, one of which aligned with a lethal region for glycine substitutions in the alpha 1(I) chain. The mapping of collagen functional domains was also extended in terms of cell and matrix interaction domains on the collagen fibril. This effort utilized a database of hundreds of type I collagen ligand binding sites and mutations on a 2D model of the fibril. This fibril based model supports important roles for the interaction with integrins in a cell interaction domain, as well as a matrix interaction domain important for fibril connections with proteoglycans. Complementary to the larger mapping enterprise,was our collaboration with investigators mapping the osteonectinbinding sites on type I collagen, using OI mutations to validate the importance of a major and secondary binding site. 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. 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 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. We have also done a clinical radiographic investigation which parallels our studies of recessive OI. We have demonstrated that popcorn calcifications occur in recessive OI; now we delineate its incidence and progression in dominant OI. We retrospectively examined serial lower limb radiographs of 45 children with known dominant mutations in type I collagen. The mean age of onset of popcorn calcifications was 7 years, with a range of 4-14 years. Those children with popcorn always had this finding in their distal femora, and most also had it in proximal tibiae. Unilateral popcorn calcifications were shown to contribute to leg length discrepancy, but not to the severe linear growth deficiency of OI. The type I collagen mutations associated with popcorn calcification occur equally in both COL1A1 and COL1A2 and have no preferential location along the chains.

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。我们已经研究了细胞对BRTL骨特性的贡献。BRTL皮质和小梁骨在青春期之前和之后降低，BV/TV降低了40-45％。 BRTL成骨细胞表面与野生型相媲美，并且BRT和野生型骨髓在两个时代都会产生等效的成骨细胞前体。有趣的是，破骨细胞表面增加在两个年龄段的BRTL（36-45％）中，陷阱阳性细胞的数量也是如此（57-47％）。青春期后，BRTL成骨细胞基质产生降至野生型值的一半。相比之下，与野生型相比，破骨细胞仍然显着升高。成骨细胞和破骨细胞的组合变化导致BFR从6个月时BFR下降到野生型值的一半。我们调查了RANKL/OPG信号通路是否负责未偶联的单元格数。正常的RANKL/OPG比率。令人惊讶的是，我们发现陷阱+前体在BRTL骨髓培养物中明显升高并形成更多的破骨细胞，这表明破骨细胞的增加是由前体数量增加引起的。这些令人兴奋的数据表明，OI病理学是由于成骨细胞对缺乏骨骼形成的结合，并增加了破骨细胞的数量，并由异常的骨基质独立触发。我们还完成了一项协作研究，该研究表明，BRTL小鼠患有早期和快速进行性骨关节炎。通过微MRI和微CT检测膝关节OA，并通过组织学和免疫组织化学确认。 BRTL小鼠表现出2 几个月；到12个月之前，BRTL的关节空间狭窄。染色显示软骨蛋白聚糖与相同年龄的野生型小鼠相比降低。我们证明了这种软骨变性不是由于免疫组织化学对胶原蛋白异常的空间表达。这些研究对于共同条件，骨关节炎以及罕见的发育不良。他们表明，软骨下骨异常可能会导致小鼠的OA 最初是正常的软骨。为了更好地了解人OI中基因型和表型的关系，BEMB LED和国际结缔组织实验室联盟组装和分析包含超过830个突变的突变数据库。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。 α1（i）中的致命突变与主要配体结合区域一致。 Alpha 2（i）中的致命区域继续支持BEMB首先提出的区域模型，并在沿链条的常规间隔簇中具有致命突变，与蛋白聚糖结合区域重合。该模型正确预测了86％的α2（i）突变的临床结果。我们参加了几项合作，这些合作扩展了我们对基因型 - 表型关系的理解。我们与NICHD的Sergey Leikin实验室的漫长而持续的合作，已经深入了解了I型胶原蛋白三型螺旋中的结构异质性及其在成骨剂Imperfecta中的作用。使用差异扫描量热法和圆形二色性，测量了41种不同的甘氨酸取代的胶原蛋白熔化温度的变化。熔化温度与取代残基没有相关性。取而代之的是，TM沿三螺旋中定义的区域的变化通过激活能图进一步证实。我们推导了两个对胶原原纤维组件和配体结合至关重要的大型柔性区域，其中一个与致命区域对齐α的甘氨酸取代 1（i）链。胶原功能结构域的映射也扩展了胶原原纤维上的细胞和基质相互作用域。这项工作利用了数百个I型胶原配体结合位点的数据库和原纤维2D模型上的突变。这种基于原纤维的模型支持与整联蛋白在细胞相互作用域中相互作用的重要作用，以及对于与蛋白聚糖的原纤维连接重要的矩阵相互作用域。与较大的映射企业的互补是我们与研究人员合作在I型胶原蛋白上绘制ostocecotinbinding位点的合作，并使用OI突变来验证主要和次要结合位点的重要性。 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）。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。然而，治疗组的椎骨BMD的增加在一到两年后逐渐减弱。在使用pamidronate治疗的OI儿童中，行动水平，较低的肢体强度或疼痛没有显着变化。因此，先前报道的变化似乎是不受控制的试验中的安慰剂作用。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。我们还进行了一项临床放射学研究，与我们对隐性OI的研究相似。我们已经证明了爆米花钙化发生在隐性OI中。现在，我们描述了其在主要OI中的发病率和进展。我们回顾性地检查了45名在I型胶原蛋白中已知显性突变的儿童的串行下肢X光片。爆米花钙化的平均发作年龄为7年，范围为4 - 14年。那些患有爆米花的孩子总是在股骨远端有这一发现，大多数人也都在胫骨近端。单侧爆米花钙化显示出有助于腿长差异，但不能导致OI的严重线性生长缺乏。与爆米花钙化相关的I型胶原突变同样发生在COL1A1和COL1A2中，并且沿链中没有优先位置。