MOLECULAR BIOLOGY OF THE MARFAN SYNDROME

马方综合征的分子生物学

• 批准号: 3161554
• 负责人: Clair A. Francomano
• 金额: $ 27.15万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-15 至 1995-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3161554
• 关键词: Marfan syndrome; autosomal dominant trait; biopsy; blood; chromosome translocation; complementary DNA; fibroblasts; gel electrophoresis; gene mutation; genetic disorder diagnosis; genetic library; human subject; linkage mapping; messenger RNA; molecular cloning; molecular genetics; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; pulsed field gel electrophoresis; restriction mapping; southern blotting

The Marfan syndrome is an autosomal dominant disorder of connective tissue with an estimate prevalence of 1/10,000 people. Dilatation, dissection and rupture of the proximal aorta are the most serious complications of the disorder, other phenotypic features, include ectopia lentis, scoliosis, dural ectasia, and spontaneous pneumothorax. The cause of Marfan syndrome is uncertain, and the molecular basis for these extensive pleiotropic manifestations remains unknown. In the past year, several laboratories have presented immunohistochemical and biochemical evidence pointing to fibrillin, a 350 kD protein found in the microfibrils, as the leading candidate for the mutant molecule in the Marfan syndrome. In the same year, we showed that the Marfan gene is tightly linked to D15S1, an anonymous DNA fragment mapped to human chromosome 15ql5-2l.3. The overall goal of this proposal is the identification of the gene causing the Marfan syndrome and the characterization of mutations in this disorder. The specific aims of this proposal are to (1) develop a long-range restriction map around the Marfan syndrome locus, as best defined currently by D15SI, (2) determine the physical relationship between the Marfan locus, D15SI and the fibrillin gene, (3) assess whether mutations at the fibrillin locus on chromosome 15 cause the Marfan syndrome (4) if the fibrillin locus is not the Marfan locus, identify and characterize the Marfan syndrome locus, and (5) identify and characterize mutations in patients with the Marfan syndrome. Restriction fragments resolved using both pulsed-field gel electrophoresis and "conventional" Southern blot techniques will be analyzed to accomplish specific aims 1 and 2. The physical relationships between D15SI and fibrillin on pulsed-field maps should determine whether the loci are distinct. Mutations will be detected by hybridization of D15S1 and fibrillin cDNA clones to pulsed-field gel blots and Southern blots to look for gross alterations in gene structure. Fibrillin cDNA synthesized from mRNA isolated from Marfan fibroblasts will be amplified by PCR and searched for evidence of mutation using denaturing gradient gel electrophoresis (DGGE) or non-denaturing strand separation techniques. Amplified fragments exhibiting evidence of mutation will be sequenced. If, as expected, substantial genetic heterogeneity is discovered, genotype-phenotype relationships will be explored as one explanation of interfamilial variability of the disorder. Identification of the genetic locus causing the Marfan syndrome and identification and characterization of Marfan mutations are likely to have important clinical implications (e.g. diagnosing presymptomatic persons at risk and isolated cases who may or may not have the full syndrome with its attendant risks), as well as enhancing understanding of the development and pathobiology of the diverse tissues involved in the disorder.

马凡氏综合征是一种常染色体显性遗传的结缔组织疾病 估计患病率为万分之一 扩张、夹层和 近端主动脉破裂是最严重的并发症， 疾病，其他表型特征，包括晶状体异位，脊柱侧凸， 硬脑膜扩张和自发性气胸 马凡氏综合征的病因 是不确定的，这些广泛的多效性的分子基础 其表现尚不清楚。 在过去的一年里，几个实验室 已经提出了免疫组织化学和生化证据， 在微纤维中发现的350 kD的蛋白质，作为主要的 马凡氏综合征突变分子的候选人 在同一 2010年，我们发现Marfan基因与D15 S1紧密相连， 定位于人染色体15 q15 - 21.3的匿名DNA片段。 这项提案的总体目标是确定基因引起的 马凡氏综合征和这种疾病的突变特征。 本提案的具体目标是：（1）制定一项长期的 马凡氏综合征基因座周围的限制性图谱，目前最好的定义 通过D15 SI，（2）确定马凡位点之间的物理关系， D15 SI和D15 S1基因的突变，（3）评估D15 S1基因的突变是否与D15 S1基因的突变有关。 15号染色体上的基因座引起马凡综合征（4），如果该基因座 不是马凡基因座，识别和描述马凡综合征 基因座，和（5）识别和表征突变的患者与 马凡氏综合征 使用两种脉冲场解析限制性片段 凝胶电泳和“常规”Southern印迹技术将在 分析以实现具体目标1和2。的物理关系 脉冲场图上D15 SI和D15 SI之间的差异应确定是否 基因座是不同的。 突变将通过以下杂交检测： 将D15 S1和D15 S2的cDNA克隆进行脉冲场凝胶印迹和Southern杂交， 用印迹法寻找基因结构的重大改变 原纤维蛋白cDNA 从马凡成纤维细胞分离的mRNA合成的DNA将通过以下方法扩增： PCR和使用变性梯度凝胶搜索突变的证据 电泳（DGGE）或非变性链分离技术。 将对显示突变证据的扩增片段进行测序。 如果， 正如所预期的，发现了大量的遗传异质性， 基因型-表型关系将被探索作为一种解释， 疾病的家族间变异性。 马凡氏综合征遗传位点的鉴定及 马凡氏突变的鉴定和表征可能具有 重要的临床意义（例如， 风险和孤立的情况下，谁可能会或可能不会有完整的综合征，其 以及加强对发展的了解， 疾病中涉及的不同组织的病理生物学。