Heritable Disorders of Connective Tisue

结缔组织遗传性疾病

• 批准号: 8941517
• 负责人: Joan C Marini
• 金额: $ 65.13万
• 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/8941517
• 关键词: Adult; Affect; African; African American; Age; Alendronate; Alleles; Amish; Antibodies; Area; Back; Binding; Biomechanics; Bone Density; Bone remodeling; C-telopeptide; Cartilage; Catalytic RNA; Cell Line; Cell Transplantation; Child; Childhood; Cleaved cell; Clinical; Clinical Research; Clinical Trials; Collagen; Collagen Type I; Complement; Complex; Connective Tissue; Cuboidal Cell; DEXA; Data; Databases; Defect; Developmental Bone Diseases; Dipeptides; Disease; Dose; Dual-Energy X-Ray Absorptiometry; Ehlers-Danlos Syndrome; Engraftment; Femur; Fracture; Genes; Genetic; Genotype; Geometry; Glycine; Goals; Haplotypes; Height; Human; Individual; International; Knock-in Mouse; Knockout Mice; Knowledge; Laboratories; Length; Ligand Binding; Literature; Lower Extremity; Mechanics; Minerals; Modeling; Molecular Biology; Morphology; Mus; Mutation; Organ; Osteoblasts; Osteocalcin; Osteogenesis; Osteogenesis Imperfecta; Osteoporosis; Outcome; Pain; Peptide Hydrolases; Peptides; Perinatal; Phenotype; Placebo Effect; Prevalence; Process; Procollagen; Property; Proteoglycan; Randomized Controlled Trials; Reporting; Role; Sampling; Serum; Shapes; Site; Skin; Small Interfering RNA; Spectroscopy, Fourier Transform Infrared; Staining method; Stains; Stem cells; Structure; Symptoms; Testing; Thick; Tissues; Transcript; Transgenic Mice; Update; Variant; Weight; alanylaspartic acid; attenuation; bisphosphonate; bone; bone cell; bone geometry; bone mass; bone quality; bone strength; design; experience; follow-up; heritable connective tissue disorder; improved; in utero; light microscopy; long bone; mineralization; mouse model; muscle strength; mutant; novel; pamidronate; proband; programs; response; skeletal; spine bone structure; 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. Using Brtl, we completed a major theraeputic trial of the effect of bisphosphonate, which complements our pediatric trial. In order to examine the effect of bisphosphonate on long bone (femur), 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, the material of the femora was changed by the dramatic retention of mineralized cartilage detected by light microscopy of Masson-stained sections, qCT attenuation and back-scatter EM. Embedded mineralized cartilage disrupts matrix continuity and may contribute to the weakening of bone material. In addition, the function of bone cells, although not their number, was impaired. Histomorphomnetry detected severe reductions in mineral apposition rate and bone formation rate. Osteoblast morphology was altered. Treated wild-type osteoblasts were more irregular in shape than the untreated cuboidal cells; Brtl treated osteoblasts have a flattened morphology, similar to lining cells. These studies contribute to the increased cautionary notes in the literature concerning avoidance of an elevated cummulative bisphosphonate dose. Brtl is also being used as the model for testing an anabolic therapy for OI anti-sclerostin antibody. When growing young Brtl mice were treated with Scl-Ab for 5 weeks, Brtl femora increased cortical bone formation, which improved mechanical strength to WT levels. Administration of Scl-Ab to adult 6 month old Brtl mice resulted in increased bone formation and serum osteocalcin, leading to improved bone mass and mechanical strength. Brtl is currently being used for trials of two non-traditional therapies. 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. These results are encouraging for translational trials. 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 individuals with severe OI into mild type I OI. We have introduced a RZ target site into the Brtl mutant allele; we have also 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 complementary suppression studies, allele specific siRNA was shown to reduce mutant collagen expression by 40%. BEMB has also collaborated in studies of the Old Order Amish mouse (OOA), a knock-in mouse with a glycine substitution in one Col1a1 allele at Gly610 to Cys. The human mutation occurs in a large Amish kinship with variability of expression. To compare the variability of expression inthe mouse, the mutation was crossed into 4 different backgrounds and revealed changes crucial for bone strength and geometry. This approach can be useful for the identification of modifying factors for OI. We have identified a novel "high bone density" form of OI caused by mutation in the C-proteinase cleavage site. The Asp-Ala dipeptide between he 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. FTIR and 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 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. The consortium database now contains over 1300 mutations and the genotype-phenotype analysis is being updated. 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。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。 为了检查双膦酸盐对长骨（股骨）的影响，我们用阿仑糖酸盐处理 Brtl 和野生型同窝小鼠，并比较每种基因型的处理和未处理的股骨。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，通过马森染色切片的光学显微镜、qCT 衰减和背向散射电镜检测到矿化软骨的显着保留，股骨的材料发生了变化。嵌入的矿化软骨会破坏基质的连续性，并可能导致骨材料的弱化。此外，骨细胞的功能虽然数量没有受到损害，但也受到了损害。组织形态测定法检测到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。处理过的野生型成骨细胞的形状比未处理过的立方体细胞更不规则； Brtl 处理的成骨细胞具有扁平的形态，类似于衬里细胞。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。 Brtl 还被用作测试 OI 抗硬化蛋白抗体合成代谢疗法的模型。当正在生长的年轻 Brtl 小鼠接受 Scl-Ab 治疗 5 周时，Brtl 股骨增加了皮质骨形成，从而将机械强度提高到 WT 水平。对 6 个月大的成年 Brtl 小鼠施用 Scl-Ab 会导致骨形成和血清骨钙素增加，从而改善骨量和机械强度。 Brtl 目前正用于两种非传统疗法的试验。在一项合作研究中，Brtl 用于 GFP 表达干细胞的子宫内细胞移植试验。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。这些结果对于转化试验来说是令人鼓舞的。其次，我们正在借鉴 I 型 OI 的经验教训来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。我们在 Brtl 突变等位基因中引入了 RZ 靶位点；我们还培育出了表达针对 Brtl 突变的核酶的转基因小鼠。 Brtl/RZ 小鼠的初步数据对于 Brtl 生物力学特性的改善是令人鼓舞的。在互补抑制研究中，等位基因特异性 siRNA 显示可将突变胶原表达降低 40%。 BEMB 还合作研究了旧秩序阿米什小鼠 (OOA)，这是一种基因敲入小鼠，其中一个 Col1a1 等位基因的 Gly610 处被甘氨酸取代为 Cys。人类突变发生在一个大的阿米什亲属中，具有表达变异性。为了比较小鼠中表达的变异性，将突变交叉到 4 个不同的背景中，揭示了对骨骼强度和几何形状至关重要的变化。这种方法可用于识别成骨不全的改变因素。 我们已经鉴定出一种新的“高骨密度”OI 形式，该形式是由 C 蛋白酶切割位点突变引起的。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。 FTIR 和 BSEM 用于研究我们两个先证者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包含 830 多个突变的突变数据库。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。该联盟数据库现在包含 1300 多个突变，并且基因型-表型分析正在更新。 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，治疗组的椎骨骨密度增量在治疗一到两年后逐渐减少。帕米膦酸钠治疗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。