GROWTH PLATE STUDIES IN THE CHONDRODYSTROPHIES

软骨对骨骼的发育、生长和功能至关重要。这个 由II、IX和XI三种类型组成的软骨胶原纤维 胶原蛋白对软骨的形态和完整性起着重要作用。相比较 对于其他结缔组织中的胶原纤维，我们知之甚少 软骨胶原纤维。事实上，许多问题仍然没有得到解决。 它们的组织和组装，次要纤维成分的功能， 细胞在调节纤维形成等方面的作用。我们建议使用 一种结合了遗传、细胞和分子生物学的方法和一种独特的 一组自然发生的人类软骨形成突变体要解决 这些和相关的问题。 在过去的5年里，研究发现了一群人类 软骨发育不良称为显性软骨基质纤维发育不良 (DCMFD)具有显性遗传且性质相似 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 软骨胶原染色，粗面粗面增大。我们假设DCMFD 由编码该成分的基因的杂合突变所致 改变生物合成过程的II、IX和XI型胶原链 导致软骨缺陷的分子和纤维形成 矩阵。继续这个项目的目标是描述一个 DCMFD突变谱并检测其对软骨形成的影响 阐明这些疾病的分子基础及其表型 可变性和深入了解正常的软骨胶原原纤维生物学。 对40多个DCMFD病例的分析将分步骤进行 以猪的软骨细胞培养系统为中心的时尚 提供软骨样组织，新合成的胶原蛋白，DNA和 用于分析的RNA。对胶原蛋白的分析将确定“候选”胶原蛋白 用于进一步研究的基因。基因组DNA的Southern和Northern杂交分析 然后，信使核糖核酸将被用于筛选候选基因的大突变。 为了检测可能的微妙突变，将从 细胞总RNA。然后用聚合酶链式反应扩增出感兴趣的序列 通过化学裂解来检测差异(可能的突变) 候选人和正常的DNA之间的差异。然后“可疑”的cDNA将被 已排序。一旦在基因组DNA中发现并确认了突变， 突变的基因产物将进一步进行生化表征；以及 突变对胶原生物合成加工的不利影响 纤维的结构和组织将在体外使用 脉冲追逐、前体定位、免疫电子显微镜和旋转 阴影技术。随着突变变得已知和突变：表型 相关成为可能，基因组DNA的短片段来自 其他DCMFD患者的福尔马林固定组织将通过聚合酶链式反应进行扩增 确认相关关系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.