CLONING THE GENE FOR CHILDHOOD-ONSET SMA

脊髓性肌萎缩症(SMA)是一种进行性神经退行性疾病。 有三种不同的临床表现：I型(Wernig-Hoffmann)， 第二类(中级)和第三类(库格伯格-韦兰德)。SMA基因 最近在CIDA计划中定位于染色体5q11.2-13.3 赞助乔里安博士的实验室。这个实验室的最新进展是 将SMA基因座细化到较小的区域(约2-5 cM) 几个多态的侧翼制造者。这里提出了一个研究项目。 克隆致病基因区域，并对基因突变进行鉴定。 本项目的具体目标是：(1)建设一个远距离的 跨越SMA基因座的限制图以识别限制片段 和“CpG岛”位于最接近的两个侧翼标志内； 重叠酵母人工染色体(YAC)和粘粒克隆的构建 跨SMA基因座，通过使用 “染色体行走”技术；(3)识别基因编码片段 最小遗传区：通过搜索保守序列，通过 鉴定CpG岛，并筛选构建的CpG岛 正常和SMA组织，包括脑、脊髓和肌肉； 利用连锁检测鉴定SMA病基因突变 不平衡，通过筛选来自于 正常和SMA等位基因，通过直接测序和PCR扩增 构建等位基因特异性寡聚体(ASOS)的候选基因 大量SMA和正常DNA样本；(5)表征 在一大批受影响的个体中检测疾病基因突变 突变模式与其表型的相关性 疾病的严重程度、发病年龄和模式等表现 继承。创新的分子基因技术和战略将是 为实现上述目标而开发的。

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease with three different clinical manifestations: type I (Wernig-Hoffmann), type II (intermediate), and type III (Kugelberg-Welander). The SMA gene has recently been localized to chromosome 5q11.2-13.3 in this CIDA program sponsor Dr. Gilliam's laboratory. Recent progress in this laboratory has refined the SMA gene locus to a smaller region (approximately 2-5 cM) with several polymorphic flanking makers. A research project is proposed here to clone the disease gene region and to characterize the gene mutation. The specific aims of this project are: (1) to construct a long-range restriction map across the SMA locus to identify the restriction fragments and "CpG islands" within the two closest flanking markers; (2) to construct overlapping yeast artificial chromosome (YAC) and cosmid clones across the SMA locus by screening both YAC and cosmid libraries using the "chromosomal walk" techniques; (3) to identify gene-coding segments within the "minimal genetic region: by searching for conserved sequences, by identifying CpG islands, and by screening cDNA libraries constructed from normal and SMA tissues including brain, spinal cord, and muscle; (4) to identify the SMA disease gene mutation by detecting linkage disequilibrium, by screening for the pattern of RNA expression from the normal and SMA alleles, by direct sequencing and PCR amplification of the candidate gene to construct allele specific oligomers (ASOs) to assay large number of SMA and normal DNA samples; (5) to characterize the disease gene mutation in a large set of affected individuals and detect the correlation between the patterns of mutation and their phenotypic manifestations such as disease severity, age of onset, and pattern of inheritance. Innovative molecular genetic technology and strategy will be developed to achieve the above goals.