Akeson A L, Wiginton D A, States J C, Perme C M, Dusing M R, Hutton J J
Proc Natl Acad Sci U S A. 1987 Aug;84(16):5947-51. doi: 10.1073/pnas.84.16.5947.
Adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, we synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNAs showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These changes do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency.
腺苷脱氨酶(ADA;腺苷氨基水解酶,EC 3.5.4.4)缺乏是遗传性疾病严重联合免疫缺陷的一个病因。为了鉴定导致ADA缺乏的突变,我们从两名ADA缺乏的免疫缺陷患者来源的GM2756和GM2825A这两个细胞系中合成了ADA mRNA的cDNA。对GM2756 cDNA克隆进行序列分析发现,每个等位基因都有一个不同的点突变,导致丙氨酸变为缬氨酸以及精氨酸变为组氨酸的氨基酸变化。GM2825A的一个等位基因也有一个导致丙氨酸被缬氨酸取代的点突变。发现GM2825A的另一个等位基因产生的mRNA中第4外显子已被剪接掉,但没有其他有害突变。对GM2825A mRNA进行S1核酸酶作图显示,全长ADA mRNA和缺失第4外显子的ADA mRNA丰度相等。几个ADA cDNA克隆延伸至主要起始位点的5'端,表明ADA转录有多个起始位点。GM2756和GM2825A中的点突变以及GM2825A中第4外显子的缺失似乎直接导致了ADA缺乏。对多个正常和突变的ADA cDNA序列进行比较,发现密码子第三个碱基有一些变化。这些变化不影响氨基酸序列。对来自不同细胞系的ADA cDNA分析检测到异常RNA种类,这些异常RNA种类要么包含第7内含子,要么排除第7外显子。它们的存在是前体mRNA异常剪接的结果,与导致ADA缺乏的突变无关。