Heritable Disorders Of Connective Ti

在一个独特的综合项目的实验室和临床研究，我们研究遗传性结缔组织疾病成骨不全症（OI）和埃勒斯-丹洛斯综合征（EDS）的分子生物学。我们的目标是阐明原发性胶原蛋白缺陷导致骨骼脆弱和其他结缔组织症状的机制，并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。了解突变型胶原分子与细胞外基质的正常和非胶原成分的相互作用，也将增强对正常骨功能的理解，并可能对更常见的骨质疏松症产生见解。我们专注于一个具有经典胶原蛋白突变的非致死动物模型。这种具有a1(I)链甘氨酸取代突变的非致死性敲入小鼠（Brtl）是药物治疗试验、适用于显性疾病的基因治疗方法和OI骨骼基质研究的优秀模型。我们的临床研究包括III/IV型成骨不全的儿童，他们参加了与年龄相适应的治疗方案，形成了一个纵向研究组。a1(I)胶原链的OI/EDS区（Cabral, Letocha, Marini, Leikin） OI/EDS患者是成骨不全患者中一个独特的子集，具有骨骼脆性、EDS、严重关节松弛和早发性脊柱侧弯的特征。在OI/EDS儿童中，我们描述了a1(I)链螺旋区前90个残基的突变。这些突变导致n -前肽加工异常，n -胶原蛋白掺入基质，真皮原纤维直径减小。这一数据提供了EDS症状的机制，而螺旋变化本身是导致骨脆性的原因。由于缺乏a1(I)或a2(I)链上的n -蛋白酶切割位点，它们的EDS机制与EDS VIIA和B的相同。这些检测定义了a1(I)的折叠区域，突变通过改变n蛋白酶切割位点的三螺旋和二级结构导致不同的OI/ED表型。保留n -前肽在胶原链的相当比例限制了纤维的直径。异常原纤维可通过降低对剪切力的抵抗力直接引起关节松弛和棘旁韧带，或通过改变d期重叠区胶原和其他基质组分之间的相互作用间接引起。I型胶原c -前肽突变（Marini, Barnes, Ashok）在一些成骨不全患者中发现了I型胶原c -前肽突变，其表型从致命到中度严重不等。这些突变是有趣的，因为它们位于胶原原纤维组装之前从前胶原中切割的区域。这种突变本身在组织中的胶原原纤维中并不存在。这表明这些突变的病理生理机制不同于纳入基质的α链螺旋区域的突变，并发挥显性负作用。我们在III/IV型成骨不全儿童胶原蛋白的保守残基上发现了4个新的c -前肽突变。所有突变都延迟了α 1链与异源三聚体的结合。细胞周围加工试验表明，c -前肽从含有这些突变的分泌胶原中去除的延迟。在培养中，突变胶原被纳入成纤维细胞基质，形成成熟的交联。我们使用免疫荧光法比较了OI成纤维细胞与正常对照和c端螺旋突变细胞中突变前胶原分子与内质网伴侣蛋白的细胞内相互作用。结果表明，突变的存在和类型与前胶原的亚细胞定位模式有明显的相关性。螺旋结构域羧基端突变的正常前胶原/前胶原表现出明显的网状内质网定位模式，与钙连连蛋白有明显的免疫荧光重叠，而c -前肽突变的前胶原表现出弥漫性内质网定位模式，与内质网伴侣、热休克蛋白-47和蛋白二硫异构酶的IF模式几乎完全重叠。突变沿前胶原链的位置指导内质网伴侣相互作用的性质。双膦酸盐广泛应用于成骨不全症儿童，但对含有异常I型胶原的骨的影响尚未直接研究。IV型OI的Brtl小鼠模型在COL1A1等位基因中敲入了甘氨酸取代（G349C）。我们用阿仑膦酸钠（0.219 mg/kg/周，默克赠送）或生理盐水安慰剂治疗2-14周龄的Brtl和Brtl与CD-1交配的野生型（wt）后代。Brtl小鼠体重和股骨长度明显小于wt，且阿仑膦酸钠未改变。阿仑膦酸钠治疗增加了Brtl和wt患者股骨和腰椎的全骨密度。数据表明差异是由于骨量增加而不是矿化。由于小梁数目增加，治疗后股骨远端骨量占总容积的比例增加了一倍。Brtl和wt型股骨骨干皮质厚度增加。在治疗后的Brtl股骨中，几何形状重塑为更圆的结构。在力学测试中，阿仑膦酸钠治疗增加了股骨刚度，减少了wt的屈服前位移，表明治疗对正常骨不是良性的。治疗后的Brtl股骨的刚度、屈服前位移和屈服载荷不变。阿仑膦酸钠对骨质量有负面影响：1)预测Brtl和wt骨的材料强度和模量降低；2)与未治疗的wt相比，治疗后的Brtl股骨脆性加重；3)治疗后的股骨后侧有较多的矿化软骨残余，可能增加骨折的发生；4)对骨细胞有有害影响。治疗后BFR/BS、MAR、MS/BS均低于治疗前的25%。成骨细胞表面百分率下降。Brtl成骨细胞形态由丰满的立方体成骨细胞转变为支持细胞毒性作用的中间形态。数据表明，有限的治疗可能是获得改善骨几何形状和最小化长期治疗对骨质量不利影响的最佳选择。帕米膦酸盐治疗III/IV型成骨不全症儿童（Letocha, Marini, Gerber, Paul）双膦酸盐治疗成骨不全症儿童的无对照试验报告显示，双膦酸盐可增加椎体骨密度和高度，改善力量/功能水平，减少骨折和骨痛。我们对帕米膦酸钠治疗III/IV型成骨不全症的儿童进行了随机对照试验。第一年是受控的；治疗组患儿给予帕米膦酸钠治疗（10 mg/m2/d，每3个月3天）；治疗组和对照组的儿童都进行了功能、力量和疼痛的PT评估。治疗组再给予帕米膦酸钠6-21个月。在对照期，与对照组相比，治疗组患者的椎体BMD z-score、L1-L4椎体中间高度和总椎面积均显著增加。治疗组长骨骨折发生率未见下降。在延长治疗中，DEXA z-评分、椎体高度和面积在12个月后没有增加。关于最大限度的身体康复，我们没有看到双膦酸盐的额外功能效果。与非对照试验相比，我们发现经帕米膦酸盐治疗的成骨不全症儿童在活动水平、下肢力量或疼痛方面没有显著变化。在治疗组中，一些人在所有测量中都有明显的反应，而另一些人的骨密度增加了，但面积或高度没有增加。DEXA z-score的变化从小于1个SD到大于3个SD。这种反应可变性尚未报道，可能与潜在胶原突变引起的骨基质差异有关。

In a unique 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 elucidating mechanisms by which primary collagen defects cause skeletal fragility and other connective tissue symptoms and applying knowledge gained from our studies to treatment of children with these conditions. Understanding interactions of mutant collagen molecules with normal and non-collagenous components of extracellular matrix will also enhance understanding of normal bone function and may yield insights to more common forms of osteoporosis. We focused on a non-lethal animal model with a classical collagen mutation. This non-lethal knock-in mouse (Brtl), with a glycine substitution mutation in the a1(I) chain, is an excellent model for pharmacological treatment trials, approaches to gene therapy suitable for dominant disorders and studies of OI skeletal matrix. Our clinical studies involve children with types III/IV OI enrolled in age-appropriate treatment protocols forming a longitudinal study group. The OI/EDS Region of the a1(I) Collagen Chain (Cabral, Letocha, Marini, Leikin) Patients with OI/EDS form a distinct subset of OI patients having skeletal fragility as well as characteristics of EDS, severe joint laxity and early onset scoliosis. In OI/EDS children we delineated mutations in the first 90 residues of the helical region of a1(I) chain. These mutations cause abnormal N-propeptide processing, incorporation of pN-collagen into matrix, and decreased diameter of dermal fibrils. This data provides a mechanism for EDS symptoms while the helical changes per se are responsible for bone fragility. The mechanism of their EDS is shared with those with EDS VIIA and B due to absence of N-proteinase cleavage site from a1(I) or a2(I) chain. These assays define a folding region of a1(I) where mutations cause a distinct OI/ED phenotype by altering the triple helical and secondary structure of the N-proteinase cleavage site. Retention of N-propeptide in a substantial proportion of collagen chains limits fibril diameter. The abnormal fibrils may cause joint laxity and paraspinal ligaments directly by reduced resistance to shearing forces, or indirectly, by altering interactions between collagen and other matrix components in the overlap zone of the D-periods. Type I Collagen C-propeptide Mutations (Marini, Barnes, Ashok) Mutations in the C-propeptide of type I collagen are found in some OI patients, the phenotype ranging from lethal to moderately severe. These mutations are of interest as they are located in a region that is cleaved from procollagen before collagen fibril assembly. The mutations per se are not expected in collagen fibrils in tissues. This implies that the pathophysiological mechanism of these mutations differ from mutations in the helical region of alpha chains incorporated into matrix and exert a dominant-negative effect. We identified 4 novel C-propeptide mutations at conserved residues in collagen of children with OI III/IV. All mutations delayed incorporation of alpha1 chains into heterotrimers. A pericellular processing assay suggests a delay in C-propeptide removal from secreted collagens containing these mutations. Mutant collagens are incorporated into fibroblast matrix in culture forming mature cross-links. We compared the intracellular interaction of mutant procollagen molecules with ER chaperones in OI fibroblasts to normal controls and cells with a C-terminal helical mutation using immunofluorescence assays. Results show clear correlation between the presence and type of mutation with subcellular localization pattern of procollagen. Normal procollagen/procollagens with mutations in the carboxyl end of the helical domain show distinct reticular pattern of ER localization with significant immunofluorescence overlap with calnexin, while procollagens with C-propeptide mutations have a diffuse ER localization with an almost complete overlap with the IF pattern of ER chaperones, Hsp-47 and protein disulfide isomerase. Location of the mutation along procollagen chains directs the nature of ER chaperone interactions. Alendronate Treatment of Brtl Mouse (Marini, Uveges, Goldstein, Gronowicz) Bisphosphonates are widely administered to OI children however effects on bone containing abnormal type I collagen have not been directly examined. The Brtl mouse model for type IV OI has a glycine substitution (G349C) knocked-into one COL1A1 allele. We treated Brtl and wild type (wt) offspring of Brtl x CD-1 matings from 2-14 weeks of age with alendronate (0.219 mg/kg/wk, gift of Merck) or saline placebo. Brtl mouse weight and femor length were significantly smaller than wt and unchanged by alendronate. Alendronate treatment increased whole bone density of femurs and lumbar vertebrae in Brtl and wt. Data suggest differences are due to increased bone volume rather than mineralization. Distal femoral bone volume per total volume doubled with treatment due to increased trabecular number. Diaphyseal cortical thickness increased in Brtl and wt femurs. In treated Brtl femurs, geometry reshaped to a more rounded structure. In mechanical testing, alendronate treatment increased femoral stiffness and decreased pre-yield displacement in wt, indicating that treatment is not benign for normal bone. Stiffness, pre-yield displacement, and yield load were unchanged in treated Brtl femora. Alendronate negatively impacts bone quality: 1) predicted material strength and modulus of Brtl and wt bone is decreased; 2) brittleness of treated Brtl femurs was worsened compared to untreated wt; 3) metaphyses of treated Brtl femurs have increased remnants of mineralized cartilage which may increase fracture initiation; and 4) there was a detrimental effect on bone cells. After treatment, BFR/BS, MAR and MS/BS was less than 25% of pretreatment. Percent osteoblast surface decreased. Morphology of Brtl osteoblasts changed from plump cuboidal osteoblasts to an intermediate morphology supporting a toxic effect on the cells. Data suggest limited treatment may be optimal for obtaining improved bone geometry and minimizing detrimental effect of extended treatment on bone quality. Pamidronate Treatment of Children with Types III/IV OI (Letocha, Marini, Gerber, Paul) Uncontrolled trials of bisphosphonates in OI children report increased vertebral bone density and height, improved strength/functional level and decreased fractures and bone pain. We did randomized controlled trial of pamidronate in children with Types III/IV OI. Year 1 was controlled; children in treatment group received pamidronate (10 mg/m2/day for 3 days every 3 months); Children in both treatment and control had PT assessments measuring function, strength and pain. The treatment group received pamidronate for an additional 6-21 months. In the controlled phase, treated patients had significant increase in vertebral BMD z-scores, L1-L4 mid-vertebral height and total vertebral area compared to controls. The treatment group did not have decreased long bone fractures. In extended treatment, DEXA z-scores, vertebral heights and areas did not increase beyond 12-month value. Regarding maximized physical rehabilitation we did not see an additional functional effect from bisphosphonates. In contrast to uncontrolled trials we found no significant changes in ambulation level, lower extremity strength or pain in OI children treated with pamidronate. Within the treatment group, some had a robust response in all measurements while others had increased bone density but not increased area or height. Changes in DEXA z-scores were less than 1 SD to more than 3 SD. This response variability has not been reported and is presumably related to differences in bone matrix caused by underlying collagen mutations.