Cavalli S A, Hirata M H, Hirata R D
Department of Clinical and Toxicological Analysis, Faculty of Pharmaceutical Sciences, Sao Paulo University, Sao Paulo, Brazil.
J Clin Lab Anal. 2001;15(1):35-9. doi: 10.1002/1098-2825(2001)15:1<35::aid-jcla7>3.0.co;2-p.
Several environmental and genetic factors are associated with high levels of cholesterol. Hypercholesterolemia is the main phenotype of Familial Defective Apolipoprotein B and Familial Hypercholesterolemia that are caused by mutations at the apolipoprotein (apo) B and LDL receptor genes, respectively. Identification of the specific genetic alteration associated with hypercholesterolemia is an important issue in clinical diagnosis of high risk for CAD. Apo B gene mutations and polymorphisms are usually screened by SSCP, DGGE, and heteroduplex, which must be confirmed by DNA sequencing or by direct detection using PCR techniques. In this study, we have optimized a PCR-RFLP procedure for identification of 3500Q and 3531 mutations and MspI polymorphism at the apo B gene. The technique can be performed in a single reaction, using the restriction endonuclease MspI for simultaneous detection of 3500Q mutation and MspI polymorphism, and NsiI for detection of 3531 mutation. The procedure was validated by analysis of control DNA samples from individuals carrying these mutations. Screening of 186 Brazilian hypercholesterolemic individuals showed that the frequency of the M-allele (7.8%) of MspI polymorphism was similar to that found in other individuals with CAD. However, neither 3500Q nor 3531 mutations were detected in this group. In conclusion, this procedure is simple and rapid, being easily introduced in clinical laboratories for direct detection of the more frequent mutations at the apo B gene associated with hypercholesterolemia.
多种环境和遗传因素与高胆固醇水平相关。高胆固醇血症是家族性载脂蛋白B缺陷症和家族性高胆固醇血症的主要表型，分别由载脂蛋白（apo）B和低密度脂蛋白受体基因的突变引起。识别与高胆固醇血症相关的特定基因改变是冠心病高危临床诊断中的一个重要问题。载脂蛋白B基因突变和多态性通常通过单链构象多态性（SSCP）、变性梯度凝胶电泳（DGGE）和异源双链分析进行筛查，这些方法必须通过DNA测序或使用PCR技术直接检测来确认。在本研究中，我们优化了一种PCR-限制性片段长度多态性（RFLP）方法，用于识别载脂蛋白B基因的3500Q和3531突变以及MspI多态性。该技术可以在一个反应中进行，使用限制性内切酶MspI同时检测3500Q突变和MspI多态性，使用NsiI检测3531突变。通过对携带这些突变个体的对照DNA样本进行分析，验证了该方法。对186名巴西高胆固醇血症个体的筛查显示，MspI多态性的M等位基因频率（7.8%）与其他冠心病个体中发现的频率相似。然而，在该组中未检测到3500Q和3531突变。总之，该方法简单快速，易于引入临床实验室，用于直接检测与高胆固醇血症相关的载脂蛋白B基因中更常见的突变。
