家族性载脂蛋白B_(100)缺陷第3500位氨基酸突变检测方法比较及初步应用

为比较和探讨家族性载脂蛋白Ｂ１００缺陷患者ａｐｏＢ－３５００位突变的检测方法，对４个高胆固醇血症家族的１９名成员，采用聚合酶链反应（ＰＣＲ）结合酶切法、等位基因特异寡核苷酸（ＡＳＯ）探针杂交法及ＰＣＲ产物序列分析法检测此点突变，并通过定点突变法构建一人工突变体作为阳性对照，以确保试验方法可靠。结果：４个家族１９名成员通过ＰＣＲ结合酶切及ＡＳＯ探针杂交两种方法皆没有检测到ａｐｏＢ－３５００位突变，与测序结果一致；测序结果验证人工构建突变体的１０６５８位核苷酸为Ａ。结论：比较而言，ＰＣＲ结合酶切法是检测ａｐｏＢ－３５００位突变的一种简便而可靠方法；通过设计适当引物在ＰＣＲ产物中引进酶切位点及构建人工阳性对照的方法，是检测点突变疾病的新思路；ａｐｏＢ－３５００位突变不是引起此４个家族成员胆固醇升高的原因。

Detection of the apoB 3500 mutation in children with familial hypercholesterolemia

To evaluate the methods for detecting apoB 3500 mutation in familial defective apolipoprotein B 100 (FDB), the authors examined 19 members of 4 familial hypercholesterolemic (FH) families by polymerase chain reaction (PCR) combined with endonuclease cleavage, allele specific oligonucleotide (ASO) hybridization and nucleotide sqeuence analysis. In addition, an artificial positive control was constructed by site dirceted mutagenesis to ensure the reliability of the assay. No apoB 3500 mutation was detected in these 19 members by the three methods, and sequencing showed that the 10658 nucleotide in the constructed mutant DNA was A. These results suggested that the method of PCR combined with endonuclease cleavage was easy to perform and reproducible; the idea of introducing an endonuclease cleavage site and constructing artificial mutant into the PCR product proved to be successful, and the hypercholesterolemia of these 4 FH families was not caused by apoB 3500 mutation.