血友病甲患儿及其家系凝血因子Ⅷ基因内含子重复序列多态性研究  

Polymorphism of intron13(CA)n repeat within FⅧ gene in hemophilia A children and kindred

作　　者：刘方[1] 滕智平[2] 孙金英[3] 王彬[3] 王勇玲[3] 邓焕霞[3] 刘桂兰[3] 

机构地区：[1]北京大学人民医院外科监护室,100044 [2]北京大学人民医院血液病研究所,100044 [3]北京大学人民医院儿科,100044

出　　处：《中华儿科杂志》2001年第11期686-688,共3页Chinese Journal of Pediatrics

摘　　要：目的　探索一种更简便 ,更特异的方法 ,用于血友病甲的基因诊断及其家系的遗传咨询。方法　应用聚合酶链反应 (PCR)和双链异源泳动分析 (HMA)对我国 10个血友病甲患儿及其家系成员中FⅧ基因内 13内含子二核苷酸重复序列 (CA)n多态性和等位基因进行分析。结果　 10个家系中 ,男性 2 0例 ,女性 10例 ,共 4 0条X染色体 ,经PCR扩增、琼脂糖电泳均可显示出 14 0～ 15 0bpDNA条带 ,男性呈现单一的DNA条带 ,女性则根据等位基因的不同 ,出现两条DNA条带 (杂合子 )或者一条DNA条带 (纯合子 )。结论　二核苷酸重复序列广泛分布于中国人群FⅧ基因中 ,重复序列的多态性作为第二代遗传标志 ,结合等位基因分析和HMA杂交电泳泳动分析 ,可明确鉴别出血友病甲基因的携带者 。Objective Hemophilia A is the most common severe inherited and X linked bleeding disorder, and caused by a defect in factor VIII gene, FVIII. The intragenic and entigenic restriction fragment length polymorphism (RFLP) has been known as an important technique for detection of carrier and prenatal diagnosis of haemophilia A,while RFLP can be used in certain types of mutations. With the development and application of the polymerase chain reaction (PCR),it is possible to detect the polymophism of dinucleotide repeat sequence in intron 13 of FVIII. This technique has been successfully applied to gene tracking in families segregating hemophilia A. This study aimed at evaluating the usage of this technique as a simple and specific method for genetic diagnosis of hemophilia A and identification of carriers. Methods The genomic DNA was isolated from the peripheral blood of 10 hemophilic patients and their family members, including 20 males and 10 females. The intron 13 of FVIII gene was analyzed to detect the (CA)n repeat sequence by using PCR and heteroduplex mobility analysis (HMA) techniques. Results The (CA)n repeat sequence of intron 13 was detected. The number of the (CA)n repeat sequence was from 20 to 30. A DNA band of 140～150 bp was revealed by PCR amplification and agarose gel electrophoresis. In males a single band (homozygote) was observed and in females two bands (beterozygote) were obseved. Conclusion Dinucleotide repeat sequence (CA)n showed extensive esistence in FVIII gene of hemophilic related Chinese patients. The polymorphism of the repeat sequence may be used as a genetic marker along with the allelic gene analysis for detecting the next generation and screening the carriers of hemophilia A gene.

关 键 词：聚合酶链反应 异源双链分析 核酸重复序列 儿童 基因多态性 血友病A 凝血因子Ⅷ 

分 类 号：R725.5[医药卫生—儿科]