扩增片段多态性结合银染法对广西汉族人FⅧ基因内(CA)n的研究

A study on the (CA) n in FⅧ gene in Han ethnic group in Guangxi Zhuang Autonomous Region by amplification polymorphisms combined with silver staining

目的研究广西汉族人群在 FⅧ基因内含子13(CA)n 及内含子22(GT)n(AG)n 两位点的基因频率分布,并用于血友病甲的家系分析。方法采用聚合酶链反应(PCR)扩增 FⅧ基因内含子13(CA)n 及22(GT)n(AG)n 的多态性片段,银染色法显示扩增结果;任选一标本,测其扩增片段长度获得其 i13(CA)n 及 i22(GT)n(AG)n 的具体重复数,从而推算出所有标本两位点的二核苷酸重复数。结果在91名广西汉族人135条 X 染色体中,共检出6种 i13(CA)n 和4种 i22(GT)n(AG)n,i13(cA)n 位点基因频率在 0.0002～0.5408间,多态信息量(polymorphism information content,PIC)为0.5899,实际杂合度为0.6364(28/44);i22(GT)n(AG)n 位点基因频率为0.0444～0.4963,PIC 为0.5359,实际杂合度为0.5227(23/44)。13个有阳性家族史的血友病家系,用该方法进行家系分析,9个家系可作出诊断,可诊断率为69%。结论 (1)i13(CA)n、i22(GT)n(AG)n 在广西汉族人中 PIC 高,是血友病甲家系分析中的有用位点;(2)相比 FⅧ相关的某些单个限制性片段长度多态性位点(restrictive fragment length polymorphism,RFLP),i22(GT)n(AG)n 是一个较为有用的位点。

Objective Hemophilia A is an inherited bleeding disorder caused by defects in factor Ⅷ（FⅧ） gane. In the present study, the frequencies of the microsatellite alleles at introns 13 and 22 in the factor Ⅷ gene were analyzed in the group of Han nationality in Guangxi Zhuang Autonomous Region to explore their diagnostic value for hemophilia A. These two sites were also used to detect the carriers in 13 hemophilia A families. Methods Ninty-one individuals of Han ethnic group in Guangxi Zhuang Autonomous Region （135 X chromosomes） and 13 HA families were subjected to molecular studies. First, these two fragments were PCR amplified simultaneously. Then, silver staining was used later to show their polymorphisms. The investigators selected one sample at random to obtain its lengths of the PCR products at these two sites by ABI310 PCR amplifier. After counting its repeated numbers of （CA） according to the documents concerned, the repeated numbers of the other samples could be counted easily. Results In the 91 individuals, 6 and 4 alleles were detected at these two sites, respectively. At intron 13 the allele frequencies ranged from 0.0002 to 0.5408 and polymorphism information content （PIC） was 0.5899. At intron 22 the allele frequencies ranged from 0.0444 to 0.4963 and its PIC was 0.5359. The actual heterozygosity for intron 13 and intron 22 were 0.6364 （28/44） and 0.5227 （23/44）, respectively. In 13 hemophilia A families with positive history, 9 of them were diagnosed by this method and the diagnosis rate was 69%. Conclusion With high PICs, （CA） nat intron 13 and intron 22 were two valuable sites in the diagnosis of hemophilia A in the population of Han ethnic group in Guangxi Zhuang Autonomous Region. Compared with some other HA restrictive fragment length polymorphisms （RFLP）, intron 22 （GT）n （AG）n was more informative.