变性高效液相色谱分析检测人类糖皮质激素受体基因变异

Analysis of glucocorticoid receptor gene polymorphisms by denaturing high-performance liquid chromatography

目的 :以变性高效液相色谱分析 (DHPLC)检测糖皮质激素受体基因 (NR3C1)在中国人群中的变异情况。方法 :提取 6 4例中国人基因组DNA ,参照文献所报道的引物序列 ,PCR方法扩增糖皮质激素受体基因编码区(2～ 9α外显子 )及其邻近的部分内含子序列 ,琼脂糖凝胶电泳鉴定产物后 ,以DHPLC检测PCR产物 ,对洗脱曲线异常者进行DNA测序。结果 :经DHPLC分析和DNA测序证实 ,研究人群的NR3C1基因中存在 8处变异 ,5处为新发现 ;其中有 2组变异呈紧密连锁的单倍型 ,均为首次报道 ,它们在人群中的基因型频率达 3.1%与 14 .1% ;另一处变异出现频率相对较低。结论 :成功地建立了以DHPLC检测NR3C1基因变异的方法及技术参数 ,证实在中国人群中NR3C1基因存在变异 ,其中 2种频率较高的单倍型为新发现。

Objective: To detect polymorphisms of glucocorticoid receptor gene ( NR3C1 ) in Chinese population with the technique of denaturing high performance liquid chromatography (DHPLC). Methods: Genomic DNA was isolated from peripheral blood of 2 healthy volunteer doctors and umbilical cord blood of 62 normal neonates. All the NR3C1 coding exons along with their flanking intron portions were amplified by polymerase chain reaction (PCR). For polymorphism screening, PCR products were analyzed by DHPLC. DNA fragments with aberrant elution profiles revealed by DHPLC were re amplified and sequenced directly. Results: Eight polymorphisms were identified by DHPLC and sequencing analysis. Among them, 5 (1374A>G, IVSG 68IVSG 63delAAAAAA, 2193T>G, IVSH 9C>G, 2382C>T) were novel and 3 (1896C>T, 2166C>T, 2430T>C) were reported previously. Two groups of polymorphisms ([1374A>G +IVSG 68IVSG 63delAAAAAA+IVSH 9C>G+2382C>T] and [1896C>T+2166C>T+2430T>C]) showed to have the complete linkage disequilibrium and were identified as 2 different haplotypes, which was first reported. The frequencies of the 2 novel haplotypes were 3.1% and 14.1%, respectively. Another polymorphism (2193T>G) was relatively rare. Conclusion: DHPLC is a rapid and reliable method for polymorphism screening. Eight polymorphisms in the NR3C1 gene are detected in Chinese population with the technique of DHPLC , of which two haplotypes have been identified for the first time.