MOLECULAR CHARACTERIZATION OF SEVERE HEMOPHILIA-A SUGGESTS THAT ABOUT HALF THE MUTATIONS ARE NOT WITHIN THE CODING REGIONS AND SPLICE JUNCTIONS OF THE FACTOR-VIII GENE
MOLECULAR CHARACTERIZATION OF SEVERE HEMOPHILIA-A SUGGESTS THAT ABOUT HALF THE MUTATIONS ARE NOT WITHIN THE CODING REGIONS AND SPLICE JUNCTIONS OF THE FACTOR-VIII GENE
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DOI：
10.1073/pnas.88.16.7405
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发表时间：
1991-08-01
影响因子：
11.1
通讯作者：
ANTONARAKIS, SE
中科院分区：
文献类型：
--
作者：
HIGUCHI, M;KAZAZIAN, HH;ANTONARAKIS, SE
A 型血友病是一种 X 染色体连锁疾病，由凝血因子 VIII 缺乏引起，凝血因子 VIII 是凝血中的重要蛋白质。据报道，因子 VIII 基因中存在大量致病突变。然而，由于基因体积大、外显子多且分散，而且新生突变频率高，对突变进行全面分析一直很困难。最近，我们发现几乎所有导致轻中度甲型血友病的突变都可以通过 PCR 和变性梯度凝胶电泳 (DGGE) 进行检测。 In this study, we attempted to discover the mutations causing severe hemophilia A by analyzing 47 unselected patients, 30 of whom had severe hemophilia and 17 of whom had mild-to-moderate disease.使用DGGE作为筛选方法，我们分析了该基因99%的编码区、94%的剪接点、启动子区和多聚腺苷酸化位点。我们在 17 名轻度至中度疾病患者中的 16 名 (94%) 中发现了突变，但在 30 名重度血友病 A 患者中仅发现了 16 名 (53%)。由于计算机分析后的 DGGE 似乎检测到了给定片段中的所有突变，因此重度疾病患者中突变率低于预期可能不是由于检测方法失败；而是由于检测方法失败。这可能是由于研究区域之外的 DNA 序列中存在突变。 Such sequences may include locus-controlling regions, other sequences within introns or outside the gene that are important for its expression, or another gene involved in factor VIII expression that is very closely linked to the factor VIII gene.
Hemophilia A is an X chromosome-linked disorder resulting from deficiency of factor VIII, an important protein in blood coagulation. A large number of disease-producing mutations have been reported in the factor VIII gene. However, a comprehensive analysis of the mutations has been difficult because of the large gene size, its many scattered exons, and the high frequency of de novo mutations. Recently, we have shown that nearly all mutations resulting in mild-to-moderate hemophilia A can be detected by PCR and denaturing gradient gel electrophoresis (DGGE). In this study, we attempted to discover the mutations causing severe hemophilia A by analyzing 47 unselected patients, 30 of whom had severe hemophilia and 17 of whom had mild-to-moderate disease. Using DGGE as a screening method, we analyzed 99% of the coding region, 94% of the splice junctions, the promoter region, and the polyadenylylation site of the gene. We found the mutation in 16 of 17 (94%) patients with mild-to-moderate disease but in only 16 of 30 (53%) patients with severe hemophilia A. Since DGGE after computer analysis appears to detect all mutations in a given fragment, the lower-than-expected yield of mutations in patients with severe disease is likely not due to failure of the detection method; it is probably due to the presence of mutations in DNA sequences outside the regions studied. Such sequences may include locus-controlling regions, other sequences within introns or outside the gene that are important for its expression, or another gene involved in factor VIII expression that is very closely linked to the factor VIII gene.