21-羟化酶缺乏症全基因测序诊断方法的建立  

Molecular diagnosis of 21-hydroxylase deficiency based on directly sequencing

作　　者：孙菲[1] 陈霞[2] 刘炳丽[1] 乔洁[3] 宋怀东[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院医学基因组学国家重点实验室,上海市内分泌代谢病研究所,200025 [2]江苏省人民医院老年科,南京 [3]上海市第九人民医院内分泌科

出　　处：《中华内分泌代谢杂志》2010年第4期325-329,共5页Chinese Journal of Endocrinology and Metabolism

摘　　要：目的建立一种特异、可靠的21-羟化酶缺乏症的分子诊断方法。方法通过基因序列比对，寻找CYP21A2基因及其假基因CYP21A1P中碱基不同的位点，在差异明显处设计真基因的巢式PCR引物，通过对PCR扩增产物直接测序，评估该基因诊断方法的特异性和准确性。用11名正常人测序以证实该方法的可靠性，并用该方法对1例21．羟化酶缺乏症病人的父母进行基因突变的诊断以验证其可行性。结果扩增出3．8kb的CYP21A2全长基因，其中包含CYP21A2和CYP21A1P基因之间86个碱基差异位点。通过PCR产物直接测序，发现在86个差异碱基中，有97．4％（167／172）的位点的碱基和真基因CYP21A2序列相同。表明该方法可以特异性地扩增CYP21A2基因序列。在1例病人分子生物学诊断为21-羟化酶缺乏症的病人父母中，均检出有CYP21A2基因的1174N杂合突变。结论建立了一种基于PCR产物直接测序的可靠的CYP21A2全基因测序的方法，该方法能够避免假基因的干扰，特异性地扩增CYP21A2基因序列，可用于21-羟化酶缺乏症的分子诊断。Objective To establish a method based on the directly sequencing for identifying 21-hydroxylase gene mutations. Methods The nest PCR primers were designed based on the discrepancy between CYP21 A2 gene and CYP21A1P pseudogene via comparison of gene sequence. The PCR products were directly sequenced in identifying the mutation in CYP21A2 gene. Eleven healthy subjects and parents of a patient of 21-hydroxylase deficiency （I172N） were included to evaluate the specificity and accuracy of the method. Results The total length of CYP21A2 gene, containing 3. 8 kb, was amplified successfully and sequenced. Among 86 different nucleotides between CYP21A2 gene and CYP21AIP pseudogene, 97.4% （167/172） bases was identified as the CYP21 A2 gene. The parents, of a patient molecular biologically diagnosed as 21-hydroxylase deficiency, were both I172N heterozygous mutation carriers. Conclnsion A new method based on the directly sequencing for molecular diagnosis of 21-hydroxylase deficiency has been established. The method can identify CYP21A2 gene from CYP21A1P pseudogene with high specificity.

关 键 词：21-羟化酶缺乏症 CYP21A2基因 CYP21A1P基因 假基因 

分 类 号：R586[医药卫生—内分泌]