用等位基因特异性PCR／限制性片段长度多态性策略构建原发性青光眼致病基因CYP1B1的单倍型  

Construction of CYP1B1 gene haplotypes predisposing to primary congenital glaucoma through allele-specific PCR/restriction fragment length polymorphism analysis

作　　者：张爱平[1] 李圣杰[1] 欧阳琦[1] 汤荔[1] 王晓蕾[2] 吉建[1] 曹文俊[1] 

机构地区：[1]复旦大学附属眼耳鼻喉科医院检验科,上海200031 [2]复旦大学附属眼耳鼻喉科医院眼科,上海200031

出　　处：《中华医学遗传学杂志》2015年第6期780-784,共5页Chinese Journal of Medical Genetics

基　　金：上海市卫生计生委课题（20124093）

摘　　要：目的建立等位基因特异性PCR／限制性片段长度多态性（allele-specific PCR／restriction fragment length polymorphism，AS-PCR／RFLP）法检测原发性先天性青光眼（primary congenital glaucoma，PCG）致病基因CYP1B1常见单核苷酸多态性（single nucleotide polymorphisms，SNPs）及其单倍型的方法。方法收集20例原发性先天性青光眼患者和20名正常对照为研究对象，首先经测序筛查SNP位点后，再分别以PCR-RFLP和AS-PCR／RFLP策略构建CYP1B1基因rs10012（S1）和rs1056827（S2）及rs1056836（S3）和rs1056837（S4）位点的单倍型，并对这两种策略进行评价。结果测序共发现4个SNP位点，为第2外显子rs10012G／C（s1）及rs1056827T／G（S2）、第3外显子rs1056836C／G（S3）及rs1056837T／C（S4）。这些位点在PCG患者和正常对照中的分布呈现不同特点，同时存在rs10012（S1）和rs1056827（S2）位点的PCG患者和正常对照分别为10例（50％）和5人（25％）；同时存在rs1056836（S3）和rs1056837（S4）位点的PCG患者和正常对照分别为5例（25％）和2人（10％）；均未发现上述SNP位点单独存在。确定各位点的分布特点后，首先用PCR-RFLP策略构建rs10012（S1）和rs1056827（S2）位点单倍型，显示杂合突变型除出现目的条带外还存在底物条带，虽提示同时存在rs10012（S1）和rs1056827（S2），但仍不能证实存在位点间的连锁；AS-PCR／RFLP构建的结果显示，AS-PCR扩增rs10012（S1）位点获得阳性结果的同时，针对rs1056827（S2）位点的特异性RFLP分析也获得阳性结果。AS-PCR／RFLP对位点rs1056836（S3）和rs1056837（S4）的分析获得了类似的结果。应用AS-PCR／RFLP成功构建了C-GErs10012（S1）-rs1056827（S2）]和G-C[rs1056836（S3）～rs1056837（S4）]两种单倍型。结论应用AS-PCR／RFLP策略成功构建PCG致病基因CYP1B1单倍型，该方法准确高效特异可用于构建遗传性疾病基因的单倍型。Objective To develop an allele-specific PCR （AS-PCR）/restriction fragment length polymorphism （RFLP） assay for CYP1B1 gene haplotypes predisposing to primary congenital glaucoma （PCG）. Methods Twenty Chinese PCG patients and 20 healthy controls were recruited. Peripheral blood sample was subjected to direct sequencing for common single nucleotide polymorphisms （SNPs） of the CYP1B1 gene. Based on the results, CYP1B1 gene haplotypes were constructed by PCR-RFLP and AS-PCR combined with RFLP. Results Four SNPs loci were identified by sequencing, which included rsl0012 G〉 C （S1 in exon 2）, rs1056827 T/G （S2 in exon 2）, rs1056836 C/G （S3 in exon 3） and rs1056837T〉C（S4 in exon 3）. The distribution of such loci showed different characteristics between the two groups. 50 % of the PCG patients had rs10012 G〉C and rs1056827 T〉G, while 25% of PCG patients had rs1056836 C2〉 Gand rs1056837T〉C. As for the controls, 25% had rsl0012 G〉C and rs1056827 T〉G, 10% had rs1056836 C〉G and rs1056837T〉C. None of the SNP loci has presented alone. PCR-RFLP was carried out to confirm the results of SNPs typing, but could not confirm the linkage between the SNP loci. By contrast, AS-PCR combined with RFLP has achieved specific amplification for rs10012 G〉C and thorough differentiation of 1056827 T〉G polymorphism. Similar results have been obtained by the same method for rs1056836 C〉G and rs1056837T〉C typing and linkage disequilibrium analysis. Conclusion The AS- PCR/RFLP assay has successfully constructed the haplotypes of the CYPIB1 gene. For its accuracy, efficiency and specificity, the method may be used for constructing haplotypes for hereditary disease studies.

关 键 词：原发性先天性青光眼 CYP1B1基因 单核苷酸多态性 等位基因特异性PCR/限制 性片段长度多态性 单倍型 

分 类 号：R774.63[医药卫生—眼科]