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。 我们的分支已经为OI生成了带有经典胶原蛋白突变的OI敲入鼠模型。使用BRTL，我们完成了对双膦酸盐作用的重大挑战试验，该试验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，我们用艾甘丹酸盐治疗了BRTL和野生型同窝材料，并比较了每种基因型的处理和未经处理的股骨。丙膦酸盐处理增加了股骨DXA和皮质容量BMD，但不能改善BRTL重量曲线或股骨长度。 BRTL小梁数和diaphyseal皮质厚度得到了改善，股骨刚度和骨折负荷也可以提高。然而，在骨骼的材料和细胞参数中也检测到有害的变化。 BRTL和野生型骨的预测材料强度和弹性模量均已死亡。 Brtl股骨的脆性不变，而野生型的肌肉则增加了。此外，通过通过Masson染色切片，QCT衰减和后散布EM检测到的矿化软骨的显着保留，改变了股骨的材料。嵌入的矿化软骨破坏基质连续性，并可能导致骨料的减弱。另外，骨细胞的功能虽然不是数量，但受到了损害。矿物质的矿物质作用率和骨形成率严重降低。成骨细胞的形态发生了变化。所处理的野生型成骨细胞的形状比未处理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。这些研究在文献中有助于提高有关避免双膦酸盐剂量升高的文献中的警告性。 BRTL也被用作测试OI抗骨化蛋白抗体的合成代谢疗法的模型。当生长的年轻BRTL小鼠用SCL-AB处理5周时，BRTL股骨会增加皮质骨形成，从而提高了机械强度到WT水平。对成年6个月大的BRTL小鼠的施用SCL-AB导致骨形成和血清骨钙素增加，从而改善了骨骼质量和机械强度。 BRTL目前正在用于两种非传统疗法的试验。在一项协作研究中，BRTL用于GFP表达干细胞的子宫细胞移植试验。尽管植入水平较低，但植入的BRTL小鼠的围产期致死性和股骨几何形状和生物力学得到了改善。这些结果令人鼓舞。其次，我们正在对I型OI进行建模，以抑制突变胶原蛋白的表达。 突变胶原蛋白等位基因转录本的特定抑制作用可以生化将严重OI的个体转化为轻度的I型OI。我们已经将RZ目标位点引入了BRTL突变等位基因。我们还产生了表达针对BRTL突变的核酶的转基因小鼠。关于BRTL/RZ小鼠的初步数据鼓励改善BRTL生物力学特性。在补充抑制研究中，表现出等位基因特异性siRNA可将突变胶原蛋白表达降低40％。 BEMB还与旧阶Amish小鼠（OOA）进行了合作，这是一种在Gly610的一个COL1A1等位基因中脱甲基替代的小鼠。人类突变发生在大型阿米什人的亲属关系中，表达可变性。为了比较小鼠中表达的变异性，将突变跨入4个不同的背景，并揭示了对骨强度和几何形状至关重要的变化。这种方法可用于识别OI的修改因子。 我们已经确定了由C蛋白酶裂解位点突变引起的一种新型的“高骨密度”形式。 He端肽和每个链的C肽之间的ASP-ALA二肽被C蛋白酶/BMP1裂解以释放成熟的胶原蛋白。我们已经确定了在这4个肽中有两个替代的儿童。他们出现裂缝和高dexa z得分。有趣的是，尽管具有较高的DEXA，X光片和组织形态计量学类似于I型OI，并且指向矩阵缺乏症。 Procollagen C-肽的周围处理延迟。 FTIR和BSEM用于研究我们两个概率的骨样品的数量和结晶度。这些数据不仅揭示了一种新型的OI形式，而且还提供了有关Procollagen加工作用和组织矿化机理的新基本信息。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。联盟数据库现在包含1300多个突变，并且正在更新基因型 - 表型分析。 我们还在继续对III型和IV型儿童的临床研究。 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。目的是在观察性试验中测试主要的骨骼增长和次要收益（提高功能水平和肌肉强度以及疼痛减轻）。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。我们建议使用III型和IV型OI的儿童使用pamiDronate治疗最多三年，随后骨状态随访。此外，我们目前正在进行剂量比较试验。我们还专注于每组对治疗的反应变异性。椎骨高度和面积的改善与DXA Z分数的变化无关，椎骨高度和各个儿童的面积也没有改善。这些差异可能与合成新骨或重塑骨的能力的重要个体变化有关。他们还强调了DXA作为骨骼强度的替代物的不足。