载脂蛋白M启动子区的一个新单核苷酸多态性位点及其与冠心病易感性的关系

Association between a new SNP in proximal promoter region of apolipoprotein M and susceptibility of coronary artery diseases in HaM Chinese population

目的检测载脂蛋白M（apoM）基因启动子区域的单核苷酸多态性（SNP），探讨多态性位点对转录活性的影响以及与冠心病易感性的关系。方法将受试者分为2组：冠心病组，经冠状动脉造影证实1或1支以上血管直径狭窄50％以上者206例，男165例，女41例，平均年龄（61．9±9．2）岁；对照组，冠状动脉造影无病变者209例，男157例，女性52例，平均年龄（60．4±9．1）岁。设计2对引物用以扩增apoM启动子区序列，采用PCR产物直接测序法进行序列分析。比较冠心病组和正常组间各基因型及等位基因频率分布的差异。进一步利用基因重组和定点突变技术体外观察SNP对apoM基因启动子转录活性的影响。结果在apoM启动子区一724位发现1个新的胞嘧啶核苷酸缺失突变（一724delC）。冠心病组-724del等位基因频率高于对照组（8．0％比4．1％，OR：2．054，95％CI1．125～3．749，P：0．017），-724del等位基因携带者血浆apoM的表达水平降低．总胆固醇水平明显高于C／C基因型[（6．04±0．90）mmol／L比（4．95-4-1．00）mmol／L，P〈0．01]。与apoM野生型启动子比较，一724C位点发生缺失突变后，apoM启动子活性明显下降（相对荧光素酶活性比值：1．13±0．25比2．11±0．15，P：0．009）。结论新发现的-724delC缺失突变可降低apoM启动子活性，下调apoM蛋白表达水平，增加冠心病的发病风险。

Objective To investigate the association between genetic polymorphisms of proximal promoter region of apolipoprotein M （apoM） gene and susceptibility of coronary artery diseases （CAD） in HaM Chinese population. Methods Two pairs of primers were designed according to the sequence （ GenBank accession nos. EU030444. 1 ） and the PCR products of apoM proximal promoter region were directly sequenced. Two hundred and six patients [ 165 males, mean age （61.9 ＋ 9. 2 ） years old] diagnosed with CAD according to the results of angiography （ a lesion was classed as being significant when stenosis was more than 50% ） were enrolled in the present study, 209 age- and gender-matched patients [ 157 males, mean age （60. 4 ± 9. 1 ） years old ] without CAD according to the results of angiography were selected as the control group. The allelic frequencies and genotype distributions of polymorphism in CAD and non- CAD patients were analyzed. Furthermore the wide-type and mutant promoter region of apoM were cloned into the luciferase expression vector pGL3, respectively. Luciferase reporter assay was used to detect the activity of apoM promoter. Results A new deletion mutation -724delC in apoM promoter was found. The frequency of Del C allele was 8.0% in CAD patients and only 4. 1% in the non-CAD controls （ OR = 2. 054, 95% CI 1. 125 - 3. 749, P = 0. 017）. The mean TC level was lower in groups with wide-type homozygotes compared to the mutant allele carriers [ （ 6.04± 0. 90） mmol/L vs. （4. 95±1.00 ） mmol/L, P 〈 0. 01 ]. -724delC mutant showed obvious decreased luciferase activities （ 1.13 ±0. 25 vs. 2. 11 ±0. 15 ,P =0. 009）. Conclusion It is reasonable to speculate that -724delC could affect the activity of the apoM promoter anddownregulate apoM expressions, therefore, influence the susceptibility of CAD in this patient cohort.