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目的建立凝血因子Ⅷ(FⅧ)基因内含子22的 Xba Ⅰ多态位点高特异的基因连锁分析法,并应用于血友病 A(HA)家系中孕妇携带者检测及产前基因诊断。方法用长距离 PCR 特异性扩增206个无血缘关系个体中 FⅧ基因内含子226.2 kb 片段并进行Ⅹba Ⅰ酶切,计算Ⅹba Ⅰ多态性位点的基因频率和多态信息量;用内含子22倒位检测及 FⅧ基因内Ⅹba Ⅰ,Bcl Ⅰ,Hind Ⅲ和(CA)n 二核苷酸重复序列多态性位点和 FⅧ基因外 DXS52多态性位点对20个 HA 家系进行间接基因诊断。结果ⅩbaⅠ多态性位点的基因频率和多态信息量分别是0.5475和0.4955;20个 HA 家系中,7个为内含子22倒位,13个非倒位家系中有6个家系Ⅹha Ⅰ均提供多态信息,其中2个家系只能用Ⅹba Ⅰ作出分子诊断,诊断率达46.15%;两个或两个以上连锁分析方法均提供多态信息的有8个家系。结论改进的 FⅧ基因内Ⅹba Ⅰ多态位点是一个特异性高,信息量大的分子诊断标志。联合用22内含子倒位及Ⅹba Ⅰ等5种间接检测方法大大地提高了 HA 基因诊断率,是防止患儿出生,阻断致病基因传递的有效措施。
Objective To establish a highly specific genetic linkage analysis of Xba Ⅰ polymorphism of intron 22 of factor Ⅷ (F Ⅷ) gene in pregnant women with hemophilia A (HA) and to diagnose prenatal gene in pregnant women. Methods The 226.2 kb fragment of FⅧ gene was amplified by specific PCR from 206 unrelated individuals and digested with Xba Ⅰ to calculate the gene frequency and polymorphism information of Xba Ⅰ polymorphism. Inversions of intron 22 and 20 HA families were performed using the Xba Ⅰ, Bcl Ⅰ, Hind Ⅲ, and (CA) n dinucleotide repeat polymorphisms in the FⅧ gene and the DXS52 polymorphic site outside the FⅧ gene Indirect genetic diagnosis. Results The frequency and polymorphic information of XbaⅠ locus were 0.5475 and 0.4955 respectively. Among the 20 HA families, 7 were inversions of intron 22 and 6 of 13 non-inversions were Ⅹha Ⅰ All of them provided polymorphic information, of which 2 families could only make molecular diagnosis with Xba Ⅰ, the diagnosis rate was 46.15%. There were 8 pedigrees providing polymorphic information by two or more linkage analysis methods. Conclusion The modified X chromosome Ⅰ polymorphism site of FⅧ gene is a molecular diagnostic marker with high specificity and large amount of information. Combined with 22 intron inversion and Xba Ⅰ and other 5 kinds of indirect detection methods greatly increased the HA gene diagnosis rate is to prevent the birth of children, blocking the transmission of pathogenic genes effective measures.