Molecular basis of medium chain acyl-coenzyme A dehydrogenase deficiency. An A to G transition at position 985 that causes a lysine-304 to glutamate substitution in the mature protein is the single prevalent mutation.
Molecular basis of medium chain acyl-coenzyme A dehydrogenase deficiency. An A to G transition at position 985 that causes a lysine-304 to glutamate substitution in the mature protein is the single prevalent mutation.
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中链酰基辅酶 A 脱氢酶缺乏症的分子基础。
DOI：
10.1172/jci114761
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Tanaka,K
中科院分区：
文献类型：
--
作者：
Yokota,I;Indo,Y;Coates,PM;Tanaka,K
我们测序聚合酶链反应（PCR）扩增变异中链酰基辅酶A脱氢酶（MCAD）的cDNAs培养成纤维细胞从三个MCAD缺陷的患者。在所有三名患者中，在编码区的位置985处确定了A到G的转换。由于没有发现合适的限制性位点用于检测该点突变，我们设计了一种PCR方法，从955位扩增87 bp的片段。在包含955至984位的5 ′引物中，A-981被人工地用C取代。在C-981和G-985的存在下，在突变体拷贝中引入Nco I限制性位点。当用这种方法检测来自9名MCAD缺陷患者的成纤维细胞的cDNA或基因组DNA时，所有这些人的拷贝都被Nco I完全切割成两个较短的片段，表明它们的A-G-985转换的纯合性。相比之下，来自所有八个对照的拷贝保持完整。因此，这种A-G-985转换是导致MCAD缺乏症的单一流行突变，这是任何遗传疾病的一个非常不寻常的特征。PCR/Nco I消化法适用于MCAD缺陷症的诊断。图片
We sequenced polymerase chain reaction (PCR)-amplified variant medium chain acyl-CoA dehydrogenase (MCAD) cDNAs in cultured fibroblasts from three MCAD-deficient patients. In all three patients, an A to G transition was identified at position 985 of the coding region. Since no appropriate restriction sites for detecting this point mutation were found, we devised a PCR method that amplifies an 87-bp fragment from position 955. In the 5' primer encompassing positions 955 to 984, A-981 was artificially substituted with C. With the presence of C-981 and G-985, an Nco I restriction site is introduced in the mutant copies. When cDNA or genomic DNA from fibroblasts of nine MCAD-deficient patients were tested with this method, the copies from all of them completely cleaved into two shorter fragments by Nco I, indicating their homozygosity for the A----G-985 transition. In contrast, the copies from all eight controls remained intact. Thus, this A----G-985 transition is the single prevalent mutation causing MCAD deficiency, a highly unusual feature for any genetic disorder. The PCR/Nco I digestion method is suitable for the diagnosis of MCAD deficiency.Images