MOLECULAR BIOLOGY OF ACID MALTASE DEFICIENCY

溶酶体酸性α葡糖苷酶（GAA）缺乏导致 糖原累积病II型（庞普病），包括 一系列不同严重程度的疾病， 致死性婴儿期发作至缓慢进行性成人期发作 form. 婴儿型的特点是 糖原在心肌和骨骼肌及其他组织中的积累 组织，而在成人发病形式的参与仅限于 骨骼肌 为了研究临床应用的分子基础， GAA缺乏症的异质性，我试图克隆， 分析人类GAA的基因 I.基因的分离 为了分离编码序列，我筛选了一个λ gt 11 使用亲和纯化的多克隆抗体构建表达文库， GAA。 复检阳性噬菌体对单克隆抗体的反应性 抗体鉴定了单个噬菌体（含有2Kb cDNA 插入）。 2Kb的cDNA特异性地杂交到 17号染色体（17 q21 -23），含有人GAA基因座 和一个3.4 Kb的mRNA，与大小一致（约。105 Kd） 蛋白质。 最后，cDNA鉴定出了 GAA缺陷者的mRNA。 因此，在1或2个婴儿GAA中， 缺陷，3.4 Kb mRNA是不可检测的，而成人 发现起始缺陷和大小和数量减少的mRNA。 在此基础上，我克隆了一个2Kb的cDNA作为探针， 已经分离出了一个更长的cDNA（3.4 Kb）2）确定了 通过引物扩增cDNA的5'非翻译区的完整大小 延伸并使用一个引物对全长cDNA进行完全测序。 M13亚克隆、缺失亚克隆和GAA的组合 特异引物 3)分离正常基因组片段， 确定内含子和外显子的数量，限制性酶切图谱，和 对剪接点进行测序。 二. DNA和RNA的研究 从坚果和遗传多态性正常 细胞分离的cDNA或基因组DNA将用于研究 突变细胞（A），以在DNA水平上确定 （B）确定是否存在正常大小， mRNA的量，并通过以下方法检测剪接错误和缺失： S1核酸酶分析。 我还分析了另外11个 患者，并发现相同程度的异质性， 前三位患者 (C)以确定精确的突变， 直接克隆和测序产生的mRNA和/或 基因组DNA，mRNA不存在。 我将首先关注 那些表达mRNA并表现出异常的个体， 酶蛋白的各种性质，以提供结构- 功能关系。

Deficiency of lysosomal acid alpha glucosidase (GAA) results in glycogen storage diseasae type II (Pompe's disease), encompassing a spectrum of disorders of varying severity ranging from a rapidly fatal infantile onset form to a slowly progressive adult onset form. The infantile form is characterized by massive accumulation of glycogen in cardiac and skeletal muscle and other tissues, while in the adult onset form involvement is limited to skeletal muscle. To study the molecular basis for the clinical heterogeneity of GAA deficiency disease, I sought to clone and analyze the gene for human GAA. I. ISOLATION OF THE GENE To isolate the coding sequences, I screened a lambda gt11 expression library using an affinity purified polyclonal antibody to GAA. Retesting positive phage for reactivity to monoclonal antibodies identified a single phage (containing a 2 Kb cDNA insert). The 2 Kb cDNA hybridized specifically to the portion of chromosome 17 (17q21-23) containing the locus for human GAA and to a 3.4 Kb mRNA, consistent with the size (approx. 105 Kd) of the protein. Finally, the cDNA identified differences in the mRNA of GAA deficients. Thus, in 1 or 2 infantile GAA deficients, the 3.4 Kb mRNA was not detectable, while an adult onset deficient and mRNA of reduced size and amount was found. Based upon the above, I have cloned a 2 Kb cDNA as probe, and have already isolated a longer cDNA (3.4 Kb) 2) determine the complete size of the 5' untranslated region of the cDNA by primer extension and complete sequencing the full length cDNA using a combination of M13 subcloning, deletion subcloning and GAA specific primers. 3) isolate the normal genomic fragment, determine the number of introns and exons, a restriction map, and sequence the splice junctions. II. STUDIES OF DNA AND RNA FROM NUTANT AND GENETICALLY POLYMORPHIC NORMAL CELLS The isolated cDNA or genomic DNA will be used to study mutant cell(s) (A) to determine at the level of DNA the presence of all exons, (B) to determine the presence of normal size and amounts of mRNA and to detect splicing errors and deletions by S1 nuclease analysis. I have also analyzed mRNA from another 11 patients and found the same degree of heterogeneity as in the first three patients. (C) to determine precise mutations including direct cloning and sequencing of mRNA where produced and/or genomic DNA where mRNA is absent. I will initially focus on individuals who express mRNA and who exhibit abnormalities in various properties of the enzyme protein, to provide structure- function relationships.