Heritable Disorders of Connective Tisue

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突变的OI的敲入小鼠模型。从Brtl衍生出的对成骨不全机制的基本认识涉及内质网应激的作用。联合微阵列和蛋白质组学研究显示，在死亡前不久，致命幼崽的Gadd153/CHOP表达和蛋白质增加了两倍。gadd153表达的增加是骨特异性的。Gadd153是C/EBP家族的一员，可被细胞应激激活，特别是被错误折叠蛋白的内质网保留。这些研究表明，用化学伴侣（如SPB）来缓解内质网应激可能对成骨不全症骨骼有有益的影响。使用Brtl，我们完成了一项关于双膦酸盐效果的主要治疗试验，它补充了我们的儿科试验。我们用阿仑膦酸钠处理Brtl和野生型幼崽，并比较各基因型处理和未处理的股骨。阿仑膦酸钠治疗增加了股骨DXA和皮质体积骨密度，但没有改善Brtl体重曲线或股骨长度。股骨小梁数量和骨干皮质厚度均得到改善，股骨刚度和骨折负荷也得到改善。然而，骨的材料和细胞参数也发生了有害的变化。Brtl和野生型骨的预测材料强度和弹性模量均下降；Brtl股骨脆性基本不变，而野生型股骨脆性增加。此外，矿化软骨的大量保留破坏了基质的连续性，并可能导致骨材料的削弱。此外，成骨细胞功能受损，矿物质附着率和骨形成率严重降低。成骨细胞形态发生改变，使Brtl处理的成骨细胞变平，类似于衬里细胞。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。我们合作的一项研究表明，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，目前被用于两种非传统疗法的试验。在合作研究中。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。这一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型成骨不全的教训，以抑制突变胶原的表达。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R7靶位点：我们产生了表达BRTL突变核酶的转基因小鼠。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。我们发现了一种由I型前胶原c蛋白酶裂解位点突变引起的新型“高骨密度”型成骨不全。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。我们已经鉴定出在这4个肽中的两个具有取代的子代。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。与正常对照和典型成骨不全症样本相比，皮质醇和骨小梁的FTIR成像证实，受影响儿童的矿物质/基质比率升高，骨小梁骨BBD（骨矿化密度分布）的胶原成熟度也显著增加，与对照组相比，矿化程度明显增加，但每个患者的模式不同。COL1A1突变的矿化密度不均匀（矿化面积高于或低于正常或典型成骨不全骨），COL1A2突变的矿化密度均匀高，甚至超过典型成骨不全。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。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的基因型和表型之间的关系，BEMB领导的国际结缔组织实验室联盟组装了一个BSEM，用于研究我们两个先证骨样本的数量和结晶度。这些数据不仅揭示了一种新型的成骨不全，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信息。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领导国际结缔组织实验室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III型和IV型成骨不全儿童的临床研究。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。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）。治疗组椎体参数改善，包括BMD z评分、中央椎体高度和椎体面积。然而，治疗组椎体骨密度的增加在治疗一至两年后逐渐减少。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作为骨强度替代物的不足。

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. <br><br>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. <br><br>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. .<br><br>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.