BIOCHEMICAL GENETICS OF CHILDHOOD IMMUNODEFICIENCY

遗传性缺乏腺苷脱氨酶（ADA）是最常见的原因， 严重的联合免疫缺陷病，一种致命的儿童疾病。 受影响儿童的细胞ADA缺乏率低于1%。 有3 长期目标：1）克隆、定位和测序正常人ADA 基因，2）比较核苷酸序列和组织模式， 来自ADA缺陷儿童的突变ADA基因与正常儿童，以及3） 用分子术语解释细胞ADA缺乏， ADA基因的结构和表达与结构和/或 ADA mRNA和蛋白质的量。 细胞系可从19个不同的ADA缺陷儿童获得。 的 品系在体外表达酶缺乏， 突变ADA蛋白、mRNA和基因组DNA的可用来源。 研究 突变的ADA蛋白和mRNA已经证明了这些线代表了一种 ADA基因中存在大量不同的突变。 全长cDNA 与正常人ADA mRNA的同源性已被克隆并测序。 正常和 选择的突变ADA基因将克隆到噬菌体载体中， 通过与ADA cDNA探针杂交鉴定。 正常的ADA基因 将通过桑格程序和外显子的组织进行测序， 内含子和侧翼序列。 一组限制 代表单独内含子、外显子和侧翼区的片段 将被准备和克隆。 这些将作为序列的探针 代表正常基因的不同区域， 用于表征mRNA前体和突变基因的试剂。 ADA 来自ADA缺陷细胞的mRNA将通过斑点印迹定量，大小通过 北方凝胶电泳，并通过S1核酸酶技术作图。 利用在mRNA中鉴定的缺陷作为可能的突变位点的指导， 将克隆的突变基因组DNA或克隆的cDNA的适当区域 测序并与正常人进行比较。 各种技术，包括在 体外转录、翻译和转染将提供额外的 正常和突变ADA基因的功能表征。 有一个 这些研究很有可能会发现 一系列导致致命遗传病的突变等位基因 的 信息也与ADA缺乏症的产前诊断有关 并涉及用于将正常ADA基因转移到 缺陷细胞

Hereditary lack of adenosine deaminase (ADA) is the most common cause of severe combined immunodeficiency disease, a lethal childhood disorder. Cells from affected children have less than 1% ADA deficiency. There are 3 long-term objectives: 1) to clone, map, and sequence the normal human ADA gene, 2) to compare nucleotide sequences and patterns of organization of mutant ADA genes from ADA deficient children with the normal, and 3) to explain cellular ADA deficiency in molecular terms which link changes in the structure and expression of the ADA gene to changes in structure and/or amounts of ADA mRNA and protein. Cell lines are available from 19 different ADA deficient children. The lines express the enzyme deficiency in vitro and represent readily available soures of mutant ADA proteins, mRNAs, and genomic DNAs. Studies of the mutant ADA proteins and mRNAs have proved that the lines represent a large number of different mutations in the ADA gene. The full length cDNA to normal human ADA mRNA has been cloned and sequenced. Normal and selected mutant ADA genes will be cloned in phage vectors and will be identified by hybridization to the ADA cDNA probe. The normal ADA gene will be sequenced by the Sanger procedure and the organization of exons, introns, and flanking sequences will be defined. A set of restriction fragments representing the separate introns, exons, and flanking regions will be prepared and cloned. These will serve as probes for sequences representing different regions of the normal gene and will be useful reagents for characterization of mRNA precursors and mutant genes. ADA mRNAs from ADA deficient cells will be quantitated by dot blot, sized by Northern gel electrophoresis, and mapped by the S1 nuclease technique. Using defects identified in mRNAs as a guide to probable sites of mutation, appropriate regions of cloned mutant genomic DNAs or cloned cDNAs will be sequenced and compared to normal. A variety of techniques including in vitro transcription, translation, and transfection will provide additional functional characterization of normal and mutant ADA genes. There is a high probability that these studies will identify the molecular defects in a series of mutant alleles responsible for a fatal hereditary disease. The information is also relevant to the prenatal diagnosis of ADA deficiency and to the design of procedures for transfer of normal ADA genes to defective cells.