PCR-RFLP检测载脂蛋白CⅢ基因T-455C多态性方法的建立及应用

Establishment of PCR-RFLP for detection of apolipoprotein CⅢ T-455C polymorphism and its application

目的建立聚合酶链反应-限制性片段长度多态性技术(PCR-RFLP)检测载脂蛋白CⅢ(apoCⅢ)T-455C多态性的方法,探讨南通汉族人群中apoCⅢ多态性与高三酰甘油血症(HTG)的相关性。方法用PCR扩增apoCⅢ基因启动子区包含T-455C多态性在内的DNA片段,扩增产物用限制性内切酶BseGⅠ酶切后电泳,分析apoCⅢ基因型,同时采用生化方法检测研究对象的血脂和载脂蛋白水平。结果检测到3种基因型,分别为-455TT、-455TC和-455CC。HTG患者组和健康对照组apoCⅢ-455C等位基因频率(50.44%比33.11%)和apoCⅢ-455CC基因型频率(26.55%比12.16%)均有显著差异(P<0.01);C等位基因携带者患HTG的风险是非携带者的2.06倍(95%CI:1.31～3.24);-455C等位基因携带者有较高的三酰甘油(TG)和apoCⅢ蛋白水平(P<0.05)。结论PCR-RFLP是一种快速、敏感的分析apoCⅢ基因启动子区T-455C多态性的方法;apoCⅢ-455C等位基因与HTG的发生相关。

Objective To establish a method of polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） to analyze the polymorphism of apolipoprotein C Ⅲ （ apoC Ⅲ） T-455C and to investigate the association of apo C Ⅲ T-455C polymorphism with hypertriglyceridemia（HTG） susceptibility. Methods Polymerase chain reaction （PCR） was performed to amplify apoC Ⅲ including T-455C polymorphism. PCR products were digested with restriction endonuclease, apoC Ⅲ genotype was analyzed after electrophoresis： The levels of serum lipid and apolipoprotein （apo） profiles were also studied by biochemical methods. Results The three kinds of genotypes at apoCⅢ -455 locus detected were -455TT, -455TC and -455CC. The frequencies of -455C allele （50.44% vs 33.11% in HTG patients and healthy subjects group respectively） and the -455CC genotype （26.55% vs 12.16% ） of apoCⅢ were significantly differ- ent in statistics. Compared with the T allele, the C allele had a significantly increased HTG risk （ OR = 2.06, 95% CI ： 1.31 to 3.24）：. The carriers with minor allele of -455C bad higher levels of serum TG and apoC Ⅲ. Conclusions PCR-RFLP is a sensitive and rapid method to investigate apo C Ⅲ gene promoter T-455C polymorphism, The apoCⅢ -455C allele might contribute to an increased risk of HTG among Chinese population.