CLONING OF A CONGENITAL EXTRAOCULAR MUSCLE FIBROSIS GENE

先天性眼外肌纤维化基因的克隆

• 批准号: 2157839
• 负责人: Elizabeth C. Engle
• 金额: $ 7.83万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2157839
• 关键词: artificial chromosomes; autosomal dominant trait; congenital eye disorder; extraocular muscle; family genetics; fibrosis; gene mutation; genetic disorder; genetic library; genetic polymorphism; human subject; linkage mapping; meiosis; messenger RNA; molecular cloning; molecular genetics; muscle disorders; nervous system disorder; nucleic acid repetitive sequence; sequence tagged sites

Human extraocular muscle (EOM) is selectively susceptible to a large number of sporadic and inherited disorders. In the vast majority of these disorders, neither the etiology nor the selectivity for EOM involvement is understood. These questions have been particularly difficult to address in EOM because these muscles are unique and differ from other skeletal muscle both physiologically and structurally. With recent advances in molecular genetics, however, it is now possible to study an inherited disorder by determining the gene and mutation(s) responsible for the disease and studying the defective gene product. This approach has yielded insight into the molecular basis and pathogenesis of disease processes that have otherwise proven difficult to study. The successful application of this approach to a skeletal muscle disease that spares the EOM has been demonstrated with the cloning and characterization of the gene for Duchenne muscular dystrophy. To better understand EOM and its pathophysiology, the applicant is studying an autosomal dominant inherited congenital disorder selective for EOM, referred to as "Congenital Fibrosis of the Extraocular Muscles" (CFEOM). The present proposal aims to isolate and begin the characterization of the gene responsible for CFEOM. A six generation family with CFEOM has been identified by the applicant and DNA has been isolated from 50 family members within five generations. Linkage analysis is being performed using PCR amplified dinucleotide repeat polymorphisms. The specific aims of Phase I of this project are to establish linkage of athe CFEOM disease to a specific chromosomal position and to begin to develop a map of this region. Once linkage has been established within the pedigree, the degree of genetic heterogeneity between families with the same or similar disorders will be determined. If the mutations in other families map to the same locus, these additional meioses will allow further refinement of the localization. YAC clones of this region will be isolated and further CA repeat polymorphisms will be identified and used to construct a detailed linkage map. When the flanking markers are as close as possible, the YAC contig will be the basis for a physical map of the region. In parallel with this molecular genetic approach, the muscle pathology and neuropathology of CFEOM in the pedigree will be more specifically characterized from study of autopsy tissue from affected family members. The specific aims of Phase II are to identify and characterize athe mutated gene. Appropriate cDNA libraries will be constructed. Candidate genes will be identified and new genes will be isolated from the region between the closest flanking markers. These genes will be examined for characteristics consistent with those of the CFEOM gene, and promising candidates will be screened for mutations. The long- range goal is to characterize the function of the CFEOM gene and its protein product and apply this information to the understanding of EOM biology.

人类眼外肌 (EOM) 对大量的选择性敏感 散发性和遗传性疾病。 在这些绝大多数中 疾病，无论是病因学还是 EOM 参与的选择性都不是 明白了。 这些问题特别难以解决 EOM 因为这些肌肉是独特的并且不同于其他骨骼肌 无论是生理上还是结构上。 随着分子生物学的最新进展 然而，遗传学现在可以通过以下方式研究遗传性疾病： 确定导致疾病的基因和突变，以及 研究有缺陷的基因产物。 这种方法产生了洞察力 深入研究疾病过程的分子基础和发病机制 否则证明很难研究。 此方案的成功应用 一种避免 EOM 的骨骼肌疾病方法 通过 Duchenne 基因的克隆和表征得到证明 肌肉萎缩症。 为了更好地了解 EOM 及其病理生理学， 申请人正在研究常染色体显性遗传先天性疾病 选择性 EOM，称为“先天性眼外纤维化” 肌肉”（CFEOM）。目前的提案旨在隔离并开始 负责 CFEOM 的基因的特征。 申请人已确定具有 CFEOM 的六代家庭，并且 已从五代人的 50 名家庭成员中分离出 DNA。 使用 PCR 扩增的二核苷酸重复序列进行连锁分析 多态性。 该项目第一阶段的具体目标是 建立 CFEOM 疾病与特定染色体位置的联系 并开始绘制该地区的地图。 一旦链接完成 系谱内建立的遗传异质性程度 将确定患有相同或相似疾病的家庭之间的情况。 如果 其他家族中的突变映射到相同的基因座，这些额外的 meioses 将允许进一​​步完善本地化。 YAC克隆 该区域将被隔离，进一步的 CA 重复多态性将被 识别并用于构建详细的连锁图。 当侧翼 标记尽可能接近，YAC 重叠群将成为 该地区的物理地图。 与这种分子遗传平行 家系中 CFEOM 的方法、肌肉病理学和神经病理学 尸检组织的研究将更具体地表征 受影响的家庭成员。 第二阶段的具体目标是确定和 表征突变基因。 适当的 cDNA 文库将是 建造的。 候选基因将被识别，新基因将被 与最近的侧翼标记之间的区域隔离。 这些基因 将检查是否具有与 CFEOM 一致的特征 基因，并对有希望的候选者进行突变筛选。 长- 范围目标是表征 CFEOM 基因的功能及其 蛋白质产品并将这些信息应用于 EOM 的理解 生物学。