THE MOLECULAR BIOLOGY OF ACID MALTASE DEFICIENCY

溶酶体酸性α-葡萄糖苷酶(GAA)缺陷导致 糖原沉积病II型(庞佩氏病)，包括 一系列不同严重程度的疾病，从迅速的 致命的婴儿起病形式到缓慢进展的成人起病 形式。婴儿型的特征是巨大的 糖原在心肌和骨骼肌等组织中的积聚 组织，而在成人发病时，受累仅限于 骨骼肌。研究临床发病的分子基础 GAA缺乏症的异质性，我试图克隆和 分析人类GAA基因。I.基因的分离 为了分离编码序列，我筛选了一个lambda gt11 利用亲和纯化的多克隆抗体构建表达文库 嘎嘎。复测阳性噬菌体对单抗的反应性 抗体鉴定了一个单噬菌体(包含2kb的cDNAs 插入)。该2kb的cDNA特异性地杂交到 包含人类GAA基因的17号染色体(17q21-23) 和3.4kb的mRNA，与大小一致(约105kD) 蛋白质的含量。最后，该cDNAs鉴定出了 GAA基因缺失。因此，在1到2个婴儿GAA中 缺乏的情况下，不能检测到3.4kb的mRNA，而成虫 发现起病缺陷，mRNA的大小和数量减少。 在此基础上，我克隆了一个2kb的cDNAs作为探针， 已经分离出一个较长的cDNA(3.4kb)2)确定 用引物测定cDNA5‘端非翻译区的完整大小 对全长cdna进行延伸和测序 M13亚克隆、缺失亚克隆和GAA的组合 特定的引子。3)分离正常基因组片段， 确定内含子和外显子的数量、限制图和 对接头连接进行排序。二、DNA和RNA的研究 来自NUTANT和遗传多态的正常 分离出的cdna或基因组DNA将用于研究 突变细胞(S)(A)在DNA水平上确定是否存在 在所有外显子中，(B)确定是否存在正常大小和 以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，并发现了与在 前三位病人。(C)确定准确的突变，包括 直接克隆和测序产生和/或产生的信使核糖核酸 缺少信使核糖核酸的基因组DNA。我首先将重点放在 表达信使核糖核酸并表现出异常的 酶蛋白的各种性质，以提供结构- 函数关系。

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.