Molecular Genetics Of Human Skeletal Dysplasias

该项目的工作重点是三种不同类型的骨骼发育不良： (1)骨骼发育不良的软骨发育不全家族，（2）骨骼发育不良的常染色体隐性形式，腕毛发育不全（CHH），和（3）Schwarz-Jampel综合征。骨骼发育不良的软骨发育不全家族包括三种以前公认的诊断和一种在本项目下定义的诊断。三种公认的条件是软骨发育不全，软骨发育不良和致死性发育不良（TD）。我们实验室在过去发表的工作描述了一种新的综合征，严重软骨发育不全伴发育迟缓和黑色素变性（SADDAN）。该家族中的所有四种疾病都是由编码成纤维细胞生长因子受体3（FGFR 3）的基因突变引起的。使用敲入策略，产生了K644 E和K644 M突变杂合的小鼠。正在进行的工作集中在创建具有K644 N突变的小鼠，以模拟人类疾病软骨发育不良。在过去的一年中，K644 E突变的组织特异性表达已经使用携带cre重组酶的小鼠在组织特异性启动子的控制下完成。这些小鼠正在协助评估突变型FGFR 3在发育中的中枢神经系统中的表达对这些疾病的发病率和死亡率的贡献。一份描述这些观察结果的手稿现在正在《人类分子遗传学》上出版。我们预计在未来的一年里，使用微阵列研究来剖析与这三种疾病发病机制相关的下游途径。 最近发现腕毛发育不全（CHH）是由RNMP基因突变引起的，RNMP基因编码一种参与线粒体核糖核蛋白复合物的RNA。一篇关于阿米什人CHH突变的论文今年发表在《美国医学遗传学杂志》上。在与道格拉斯华莱士博士的合作中，我们计划在未来一年内探索突变对线粒体功能的影响。此外，在未来的一年里，Shepherd Schurman博士将致力于创建一个小鼠模型，用于治疗毛囊发育不全 我们与Yoshi Yamada博士及其同事在NINDR的合作工作表明，Schwarz-Jampel综合征是由串珠素基因突变引起的。 除了我们在特定骨骼发育不良方面的工作外，ZJ Zhang博士还一直在使用体外方法来创建和增强用于软骨发育的器官培养方法。希望这些研究将导致孟德尔和软骨生长和发育的复杂疾病的治疗策略的发展。Zhang博士已经证明，脉冲低强度超声对基质产生和软骨细胞肥大产生明显的合成代谢作用。Zhang博士的工作描述了脉冲低强度超声的影响，今年发表在Ultrasound Mol Biol上。Zhang博士还一直在进行旨在研究颗粒培养在软骨工程中的效用的研究，分离的软骨细胞在颗粒培养中生长2周。聚集蛋白聚糖染色和II型胶原免疫组织化学显示，软骨细胞表型在这个专门设计的培养系统中稳定。在培养期间，软骨细胞保持IX型胶原的表达，其功能是将胶原纤维与聚集蛋白聚糖连接。电镜观察细胞外基质的重建。到2周时，工程化软骨中的纤维网络在纤维直径和密度方面与天然软骨相似，但纤维取向较不有序。描述这一努力的手稿已提交出版，目前正在审查中。 人类基因组信息管理系统还继续维护骨骼系统基因数据库，这是一个公开的电子资源，收录了骨骼组织中发现的所有基因和表达序列，以及基因与影响骨骼系统的疾病状态或特征之间的所有已知关联。

Work on this project has focused on three different types of skeletal dysplasia: (1) the achondroplasia family of skeletal dysplasias, (2) an autosomal recessive form of skeletal dysplasia, Cartilage Hair Hypoplasia (CHH), and (3) the Schwarz-Jampel syndrome. The achondroplasia family of skeletal dysplasias includes three previously recognized diagnoses and one that has been defined under this project. The three well-established conditions are achondroplasia, hypochondroplasia and thanatophoric dysplasia (TD). Work published by our lab in the past described a new syndrome, Severe Achondroplasia with Developmental Delay and Acanthosis Nigricans (SADDAN). All four disorders in this family of conditions are caused by mutations in the gene encoding fibroblast growth factor receptor 3 (FGFR3). Using a knock-in strategy, mice heterozygous for K644E and K644M mutations have been generated. Ongoing work is focused on creation of mice with K644N mutations, to model the human disorder hypochondroplasia. Over the past year, tissue-specific expression of the K644E mutation has been accomplished using mice carrying the cre recombinase under the control of tissue-specific promoters. These mice are assisting in assessing the contribution of mutant FGFR3 expression in the developing central nervous system to morbidity and mortality in these disorders. A manuscript describing these observations is now in press in Human Molecular Genetics. We anticipate using microarray studies to dissect the downstream pathways associated with pathogenesis of these three disorders over the coming year. Cartilage hair hypoplasia (CHH) has recently been found to be caused by mutations in the RNMP gene, which encodes an RNA that participates in the Mitochondrial Ribonuclear Protein complex. A paper reporting on the Amish CHH mutation was published this year in the American Journal of Medical Genetics. In collaboration with Dr. Douglas Wallace, we plan to explore the effects of the mutation on mitochondrial function during the coming year. In addition, over the coming year Dr. Shepherd Schurman will be working to create a mouse model for Cartilage Hair Hypoplasia Our collaborative work with Dr. Yoshi Yamada and his colleagues at the NINDR has demonstrated that the Schwarz-Jampel syndrome is caused by mutations in the perlecan gene. In addition to our work on specific skeletal dysplasias, Dr. ZJ Zhang has been using in vitro methods to create and enhance an organ culture method for the development of cartilage. It is hoped that these studies will lead to the development of treatment strategies for mendelian and complex disorders of cartilage growth and development. Dr. Zhang has shown that pulsed low intensity ultrasound produces a demonstrable anabolic effect on matrix production and chondrocyte hypertrophy. Dr. Zhang's work describing the effects of pulsed low intensity ultrasound was published this year in Ultrasound Mol Biol. Dr. Zhang has also been pursing studies designed to investigate the utility of pellet-culture for cartilage engineering, isolated chondrocytes were grown in pellet culture for 2 weeks. The chondrocyte phenotype was stabilized in this specially designed culture system as show by aggrecan staining and immunohistochemistry for type II collagen. During the culture period, chondrocytes maintained expression of type IX collagen, which functions to link collagen fiber with aggrecan. The reconstruction of extracellular matrix was demonstrated by electron microscopy. By 2 weeks, the fiber network in the engineered cartilage was similar to the native cartilage at fiber diameter and density, but the fiber orientation was less organized. A manuscript describing this effort has been submitted for publication and is currently under review. The HGIMS is also continuing to maintain the Skeletal Gene Database, a publicly available electronic resource cataloguing all genes and expressed sequences found in skeletal tissue, and all known associations between genes and disease states or traits affecting the skeletal system.