Reijnen M J, Peerlinck K, Maasdam D, Bertina R M, Reitsma P H
Department of Hematology, University Hospital, Leiden, The Netherlands.
Blood. 1993 Jul 1;82(1):151-8.
Hemophilia B Leyden is an X chromosome-linked bleeding disorder characterized by an altered developmental expression of blood coagulation factor IX. This form of hemophilia B has been found to be associated with a variety of single point mutations in the factor IX promoter region. We now describe a novel point mutation, T-->G at position -21, in two related patients with the hemophilia B Leyden phenotype. This mutation lies within the factor IX promoter region (-40 to -9) that contains overlapping binding sites for hepatocyte nuclear factor 4 (HNF-4) and androgen receptor. Transient transfection assays in HepG2 cells show that the -21 mutation causes a significant reduction in factor IX promoter activity. Gel mobility shift assays and transient cotransfection experiments revealed that the HNF-4-binding site but not the androgen-responsive element is disrupted by the -21 mutation. A comparison of the -21 mutation with the previously described -20 T-->A mutation (associated with the hemophilia B Leyden phenotype) and -26 G-->C mutation (associated with severe hemophilia B throughout life) was made. It shows that the -21 mutation reduced HNF-4 binding and transactivation to a similar level as the -20 mutation, whereas the -26 mutation completely abolished HNF-4 binding and transactivation. Mobility shift experiments indicate that there was no significant difference in binding affinity of recombinant androgen receptor protein for oligonucleotides containing wild-type and -21 or -20 mutated DNA. The binding affinity for the oligonucleotide containing the -26 mutation was twofold lower. The results indicate that the disruption of the HNF-4-binding site by the -21 T-->G mutation is the cause of the bleeding disorder in these two patients. This study adds further support for the notion that the recovery from hemophilia at puberty may not only be related to an intact androgen-responsive element but also to the degree of disruption of the HNF-4-binding site.
莱顿型乙型血友病是一种X染色体连锁的出血性疾病，其特征是凝血因子IX的发育表达发生改变。现已发现这种形式的乙型血友病与因子IX启动子区域的多种单点突变有关。我们现在描述在两名具有莱顿型乙型血友病表型的相关患者中发现的一种新的单点突变，即位于-21位的T→G。该突变位于因子IX启动子区域（-40至-9）内，该区域包含肝细胞核因子4（HNF-4）和雄激素受体的重叠结合位点。在HepG2细胞中进行的瞬时转染试验表明，-21突变导致因子IX启动子活性显著降低。凝胶迁移率变动分析和瞬时共转染实验表明，-21突变破坏了HNF-4结合位点，但未破坏雄激素反应元件。对-21突变与先前描述的-20 T→A突变（与莱顿型乙型血友病表型相关）和-26 G→C突变（与终生严重乙型血友病相关）进行了比较。结果表明，-21突变使HNF-4结合和反式激活降低到与-20突变相似的水平，而-26突变则完全消除了HNF-4结合和反式激活。迁移率变动实验表明，重组雄激素受体蛋白对含有野生型和-21或-20突变DNA的寡核苷酸的结合亲和力没有显著差异。对含有-26突变的寡核苷酸的结合亲和力低两倍。结果表明，-21 T→G突变导致的HNF-4结合位点破坏是这两名患者出血性疾病的原因。这项研究进一步支持了这样一种观点，即青春期血友病的恢复可能不仅与完整的雄激素反应元件有关，还与HNF-4结合位点的破坏程度有关。
