变性高效液相色谱法在α-血红蛋白稳定蛋白基因检测中的应用

Application of denaturing high performance liquid chromatography for detection of α-hemoglobin-stabilizing protein gene

建立了采用变性高效液相色谱（DHPLC）对α-血红蛋白稳定蛋白（AHSP）基因进行基因分型和突变筛查的新方法。将AHSP基因序列分成6个片段,因第一、二、四、六个片段均含有1~2个常见单核苷酸多态性（single nu-cleotide polymorphism,SNP）位点,需单个标本分别进行检测;第三、五个片段不含有常见SNP位点,采用DHPLC结合DNA（脱氧核糖核酸）池的方法进行检测。以基因测序为金标准对所建立的AHSP基因检测方法进行方法学评价,结果显示：40个样品的DHPLC检测结果与测序结果之间完全吻合,说明所建立的检测方法能对AHSP基因6种常见SNP进行准确基因分型。应用DHPLC对365个样品的AHSP基因进行检测,发现2个罕见SNP（11 810G〉A和12 802C〉T）;同时还发现2个错义突变（AHSP D29V和AHSP V56G）,AHSP D29V突变为新突变,AH-SP V56G为罕见突变。结果表明采用DHPLC法可有效地对AHSP基因进行基因分型和突变筛查。

An assay method for α-hemoglobin-stabilizing protein（AHSP） gene was established based on denaturing high performance liquid chromatography（DHPLC）.The AHSP gene sequences are divided into six fragments.Because of one or two common single nucleotide polymorphism（SNPs） in the first,second,fourth and sixth fragments,all samples should be analyzed individually when the fragments were detected.The third and fifth fragments were detected by DHPLC technique combined with DNA pooling for no common SNP in the fragments.The six common SNPs in AHSP gene can be genotyped accurately by the established method.After analyzing AHSP gene of 40 samples by DHPLC detection and gene sequencing,it was found that the results of the two methods were completely consistent.After AHSP gene of 365 samples being analyzed by DHPLC,two rare SNPs（11810 GA and 12802 CT）were found.Two missense mutations（AHSP D29V and AHSP V56G） were also found.AHSP D29V mutation is a novel mutation.AHSP V56G is a rare mutation.It demonstrated that this method is suitable for the detection of α-hemoglobin-stabilizing protein gene.