MOLECULAR BIOLOGY OF THE MARFAN SYNDROME

马凡综合征是一种常染色体显性结缔组织遗传病。 估计患病率为十万分之一。扩张、解剖和 近端主动脉破裂是最严重的并发症。 疾病，其他表型特征，包括异位晶状体，脊柱侧弯， 硬脑膜扩张和自发性气胸。马凡综合征的病因 是不确定的，而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 已经提交了免疫组织化学和生化证据，表明 纤维蛋白，一种在微纤维中发现的350kD蛋白质，是主要的 马凡综合征突变分子的候选者。在相同的 年，我们发现马凡基因与D15S1紧密连锁，这是一种 匿名DNA片段定位于人类染色体15q15-2l.3。 这项提案的总体目标是确定导致 马凡综合征和这种疾病的突变特征。 这项建议的具体目的是(1)发展一种长期的 马凡综合征基因座周围的限制图，目前最好的定义 通过D15SI，(2)确定马凡轨迹之间的物理关系， D15SI和纤维蛋白基因，(3)评估纤维蛋白基因的突变 15号染色体上的基因座导致马凡综合征(4)，如果纤维蛋白原基因座 不是马凡基因，识别和描述马凡综合征 基因座，以及(5)确定和表征患者的突变。 马凡综合征。使用两个脉冲场解析的限制性片段 凝胶电泳法和“常规”的Southern印迹技术 分析以实现具体目标1和2。物理关系 脉冲场图上D15SI和纤维蛋白之间的关系应该决定 这些轨迹是截然不同的。突变将通过杂交检测到 脉冲场凝胶印迹杂交和Southern杂交的D15S1和纤维蛋白基因克隆 印迹法以寻找基因结构的严重变化。纤维素基因 从马凡成纤维细胞中分离出的mRNA合成将通过 用变性梯度凝胶寻找突变证据 电泳法(DGGE)或非变性链分离技术。 显示突变证据的扩增片段将被测序。如果， 正如预期的那样，发现了实质性的遗传异质性， 我们将探讨基因-表型之间的关系，作为对 这种疾病的家族间变异性。 马凡综合征和马凡氏综合征遗传位点的鉴定 马凡突变的鉴定和特征可能具有 重要的临床意义(例如，在 风险和个别病例，可能有或可能没有其全部综合征 随之而来的风险)，以及加强对发展和 与这种疾病有关的各种组织的病理生物学。

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.