CLONING THE GENE FOR CHILDHOOD-ONSET SMA

脊髓性肌萎缩症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）， II型（中间型）和III型（Kugelberg-Welander）。 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该实验室的最新进展 将SMA基因位点细化到较小的区域（约2- 5cM）， 几个多态侧翼标记。 本文提出了一个研究课题 以克隆疾病基因区域并表征基因突变。 该项目的具体目标是：（1）建设一个远程 SMA基因座上的限制性酶切图谱，以鉴定限制性片段 和两个最接近的侧翼标记内的“CpG岛”;（2）至 构建重叠酵母人工染色体（YAC）和粘粒克隆 通过使用 （3）通过染色体步移技术， 最小遗传区域：通过寻找保守序列， 鉴定CpG岛，并通过筛选从 正常和SMA组织，包括脑、脊髓和肌肉;（4）至 通过检测连锁确定SMA疾病基因突变 不平衡，通过筛选RNA表达的模式， 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（ASO）的候选基因， 大量的SMA和正常DNA样品;（5）表征 疾病基因突变在一个大的受影响的个人和检测 突变模式与其表型之间的相关性 临床表现，如疾病的严重程度，发病年龄和模式， 传承创新的分子遗传技术和战略将是 为实现上述目标而开发。

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.