Fojo S S, Beisiegel U, Beil U, Higuchi K, Bojanovski M, Gregg R E, Greten H, Brewer H B
Molecular Disease Branch, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892.
J Clin Invest. 1988 Nov;82(5):1489-94. doi: 10.1172/JCI113756.
The DNA, RNA, and protein of apo C-II have been analyzed in a patient with apo C-II deficiency (apo C-IIHamburg). Markedly reduced levels of plasma and intrahepatic C-II apolipoprotein were demonstrated by immunoblotting and immunohistochemical analysis. Northern, slot blot, and in situ hybridization studies revealed low levels of a normal-sized apo C-II mRNA. No major rearrangement of the apo C-II gene was detected by Southern blotting. Sequence analysis of apo C-II genomic clones revealed a G-to-C substitution within the donor splice site of intron II. This base substitution resulted in the formation of a new Dde I and loss of a Hph I restriction enzyme cleavage site. Amplification of the mutant sequence by the polymerase chain reaction and digestion with Dde I and Hph I restriction enzymes established that the patient was homozygous for the G-to-C mutation. This is the initial report of the DNA sequence of an abnormal apo C-II gene from a patient with deficiency of apo C-II. We propose that this donor splice site mutation is the primary genetic defect that leads to defective splicing and ultimately to an apo C-II deficiency in this kindred.
对一名载脂蛋白C-II缺乏症患者（载脂蛋白C-II汉堡型）的DNA、RNA和蛋白质进行了分析。免疫印迹和免疫组织化学分析显示，血浆和肝内载脂蛋白C-II水平显著降低。Northern印迹、斑点印迹和原位杂交研究显示，正常大小的载脂蛋白C-II mRNA水平较低。Southern印迹未检测到载脂蛋白C-II基因的主要重排。载脂蛋白C-II基因组克隆的序列分析显示，内含子II供体剪接位点内发生了G到C的替换。这种碱基替换导致形成了一个新的Dde I位点，并失去了一个Hph I限制性内切酶切割位点。通过聚合酶链反应扩增突变序列并用Dde I和Hph I限制性内切酶消化，确定该患者为G到C突变的纯合子。这是关于一名载脂蛋白C-II缺乏症患者异常载脂蛋白C-II基因DNA序列的首次报告。我们认为，这种供体剪接位点突变是导致该家族剪接缺陷并最终导致载脂蛋白C-II缺乏的主要遗传缺陷。
