PCR-构象敏感凝胶电泳技术在血友病A基因分型诊断及携带者检测中的应用  

Application studies on the gene diagnosis and carrier detection of hemophilia A by using polymerase chain reaction-conformation sensitive gel electrophoresis

作　　者：David Lillicrap 何国平[2] Jayne Leggo 刘雨生[2] 童先宏[2] 周桂香[2] 骆丽华[2] 

机构地区：[1]Department of Pathology and Melecular Medicine, Richardson Laboratory of Queen's University, Canada [2]安徽医科大学附属省立医院生殖医学中心

出　　处：《中华医学遗传学杂志》2009年第4期393-399,共7页Chinese Journal of Medical Genetics

基　　金：安徽省卫生厅医学科研课题（098115）;安徽省教育厅自然科学研究项目（2006KJ066C）;安徽省科技攻关项目（06013125B）

摘　　要：目的建立一种简便、快速的血友病A（hemophilia A，HA）基因突变筛查方法学，并用于直接基因诊断及携带者筛查。方法对于临床确诊的24例血友病A患者和家系女性成员，首先采用倒位PCR及PCR技术，检测是否为F脚基因第22、1内含子倒位或倒位携带者；对非倒位者则用PCR扩增FⅧ基因26个外显子，继而应用构象敏感凝胶电泳（conformation sensitive gel electrophoresis，CSGE）技术对37个扩增片段进行突变筛查，阳性者予以进一步测序验证；据突变类型，对非倒位HA先证者家系女性成员直接测序或行CSGE检测，以判读其是否为携带者。结果24个HA家系中，7例先证者FⅧ基因第22内含子倒位检测为阳性，未发现第1内含子倒位；13个有家族史及3个散发非倒位HA家系中均存在FⅧ基因不同外显子区的单碱基突变，1例未检出突变。本研究中，24个家系中基因分型诊断及携带者筛查阳性率、可诊断率分别为94．12％（16／17），100％（17／17）。结论PCR—CSGE检测HA单碱基突变高度敏感、特异；F脚基因第22、1内含子检测联合PCR—CSGE基因分型诊断技术及核苷酸测序可对所有的HA家系（含散发）进行直接基因诊断，理论上可筛查新突变并明确其突变类型。该法简便、快速、成本低，在HA直接基因诊断及携带者筛查中优势独特，应具重要应用价值。Objective To establish a simple, rapid and easy method for screening the gene mutation in hemophilia A, which was further applied to a direct diagnosis and carrier detection at gene level. Methods Twenty-four clinically diagnosed hemophilia pedigrees, including all the hemophilia patients and female members, were tested for the introns 22 and 1 in factor Ⅷ gene by using inversion polymerase chain reaction （PCR） and regular PCR techniques. All the 26 exons of factor Ⅷ gene were consecutively screened in the 17 patients manifesting non-inverted sequences in intron 22 by using PCR, subsequently all the 37 amplicons resulted from 26 exons were analyzed by conformation sensitive gel electrophoresis （CSGE）, finally the mutated exons were subjected to sequencing verification. According to the mutation results, mothers and twin sisters of the hemophilia prohands were tested by CSGE or subjected to nucleotide sequencing directly, to ascertain if those individuals had the same mutation or were the carriers of disease-causing gene. Results Intron 22inversion was detected in 7 hemophilia probands out of 24 hemophilia pedigrees, intron 1 inversion was not detected in these pedigrees. Single-base mutations distributed in different exons of factor Ⅷ gene were detected in 13 pedigrees with family history and 3 sporadic pedigrees, diagnosed as non-inverted 22 intron patients. By comprehensive usage of PCR-CSGE and nucleotide sequencing, the positive rate and the diagnosahle rate of gene diagnosis or carrier detection in the 24 hemophilia pedigrees was 94.12~ and 100% respectively. Conclusion PCR-CSGE is a highly sensitive and special assay for detecting single base mutation. By integrated utilization of introns 22 and 1 of factor Ⅷ gene detection and PCR-CSGE genotyping, combining with nucleodde sequencing, a direct diagnosis of all hemophilia pedigrees be could nearly make at gene level, including the sporadic families. This method might be used to screen new mutation theoretically and ascertain the mutation type

关 键 词：血友病A 构象敏感凝胶电泳 基因诊断 携带者检测 

分 类 号：R686[医药卫生—骨科学]