GROWTH PLATE STUDIES IN THE CHONDRODYSTROPHIES

软骨素对骨骼发育、生长和功能至关重要。的 由II、IX和Xi型组成的软骨胶原原纤维 胶原蛋白负责软骨的形成和完整性。相比 其他结缔组织中的胶原纤维，对 软骨胶原纤维。事实上，许多问题仍然没有解决， 它们的组织和组装，小纤维成分的功能， 细胞在调节纤维形成等方面的作用。我们建议使用 结合遗传、细胞和分子生物学方法， 一组天然存在的人类软骨形成突变体， 这些和相关的问题。 过去5年的研究发现， 软骨发育不良，称为显性软骨基质纤维发育不良 （DCMFD）共享显性遗传和定性相似 临床和病理表型。后者包括结构 软骨胶原纤维异常，电泳异常 软骨胶原和粗面内质网扩大。我们假设DCMFD 由编码该成分的基因的杂合突变引起 改变生物合成过程的II、IX和Xi型胶原蛋白链 导致软骨缺损的分子和纤维形成 矩阵该项目继续的目标是描述一个 DCMFD突变谱，并检查其对软骨形成的影响， 阐明这些疾病的分子基础及其表型 变异性和获得洞察正常软骨胶原纤维生物学。 将对40多例DCMFD病例进行逐步分析， 时尚围绕软骨细胞培养系统开发的PI 它提供软骨样组织、新合成的胶原蛋白、DNA和 RNA用于分析。分析胶原蛋白将确定“候选”胶原蛋白 基因进行进一步研究。基因组DNA的Southern和北方印迹分析 然后将mRNA用于筛选总突变的候选基因。 为了检测可能的细微突变，将从 细胞总RNA。然后通过PCR扩增感兴趣的序列， 通过化学裂解分析以检测差异（可能的突变） 候选人和正常人之间的差异“嫌疑人”cDNA将被 测序一旦在基因组DNA中鉴定和确认突变， 突变基因产物将进一步进行生物化学表征; 突变对胶原蛋白生物合成过程的不利影响， 纤维结构和组织将在体外研究， 脉冲追踪、前体定位、免疫电镜和旋转 阴影技术。随着突变变得众所周知，突变：表型 相关性成为可能，基因组DNA的短片段， 其他DCMFD患者的福尔马林固定组织将通过PCR扩增， 确认相关性，并更好地定义分子基础， 表型变异性预计将定义10-15个突变。

Cartilage is critical to skeletal development, growth and function. The cartilage collagen fibril which is comprised of types II, IX and XI collagen is responsible for the form and integrity of cartilage. Compared to collagen fibrils in other connective tissues, much less is known about cartilage collagen fibrils. Indeed, many questions remain unresolved about their organization and assembly, the function of minor fibril constituents, the role of the cell in regulating fibril formation, etc. We propose to use a combined genetic, cellular and molecular biology approach and a unique group of naturally occurring human mutants of chondrogenesis to address these and related questions. Studies over the past 5 years have identified a group of human chondrodysplasias called Dominant Cartilage Matrix Fibril Dysplasias (DCMFDs) which share dominant inheritance and qualitatively similar clinical and pathologic phenotypes. The latter includes structural abnormalities of cartilage collagen fibrils, electrophoretic abnormalities of cartilage collagens and enlargement of RER. We hypothesize that DCMFDs result from heterozygous mutations of the genes encoding the constituent chains of types II, IX and XI collagens which alter biosynthetic processing of the molecules and fibrillogenesis leading to a defective cartilage matrix. The goal of the continuation of this project is to characterize a spectrum of DCMFD mutations and examine their effects on chondrogenesis to elucidate the molecular basis of these disorders and their phenotypic variability and gain insight into normal cartilage collagen fibril biology. The analyses of each of over 40 DCMFD cases will be done in a stepwise fashion centered around a chondrocyte culture system developed by the PI which provides cartilage-like tissue, newly synthesized collagens, DNA and RNA for analysis. Analysis of collagens will identify "candidate" collagen genes for further study. Southern and Northern blot analyses of genomic DNA and mRNA will then be used to screen candidate genes for gross mutations. To detect the mire likely subtle mutations, cDNA will be synthesized from total cellular RNA. Sequences of interest will then be amplified by PCR and analyzed by chemical cleavage to detect differences (possible mutations) between the candidate and normal cDNAs. "Suspect" cDNAs will then be sequenced. Once a mutation is identified and confirmed in genomic DNA, the mutant gene product will be further characterized biochemically; and the adverse effects of the mutation on collagen biosynthetic processing and fibril structure and organization will be investigated in vitro using pulse-chase, precursor localization, immunoelectron microscopic and rotary shadowing techniques. As mutations become known and mutation:phenotype correlations become possible, short segments of genomic DNA from formalin-fixed tissues of other DCMFD patients will be amplified by PCR to confirm the correlations and better define the molecular basis of the phenotypic variability. 10-15 mutations are expected to be defined.