Elucidation of genetic abnormality and molecular pathogenesis of hereditary hemorrhagic telangiectasia

遗传性出血性毛细血管扩张症（HHT）是一种常染色体显性遗传疾病，其特征是外周血管发育不良和这些病变的复发性出血。最近的研究已经将HHT的负责基因之一定位于染色体9 q33-q34 ;随后，在编码转化生长因子β（TGF-β）结合蛋白的内皮糖蛋白基因中已经鉴定出9种不同的突变。我们发现了一个日本HHT家族，并鉴定了外显子4的C至A突变，该突变将Ala^<160>密码子（GCT）变为Asp^<160>密码子（GAT）。为了揭示在具有该突变的患者中引起血管发育不良的机制，使用在MCF 7细胞中表达的重组正常和突变内皮糖蛋白分析TGF-β信号传导功能。我们证实，正常和突变的endoglin在COS-1细胞的表面膜上表达在相似的程度，通过免疫细胞化学。还在MCF 7细胞中建立了表达正常或突变内皮糖蛋白的稳定细胞系。当TGF-β加入到这些稳定的转化体中时，这些转化体预先用Smad 2-flag质粒转染，在表达正常内皮素的MCF 7细胞中通过免疫荧光分析检测到Smad 2-flag蛋白的核转位。而在表达突变型endoglin的MCF 7细胞中未观察到免疫荧光信号。这些结果提示了几种可能性：（1）突变的endoglin缺乏与TGF-β的结合活性，（2）不能将TGF-β提呈给TGF-β的II型受体或（3）不能组装成同源二聚体结构。目前正在进行这些分析。

Hereditary hemorrhagic telangiectasia(HHT) is an autosomal dominant disorder characterized by peripheral vascular dysplasia and recurrent hemorrhage from those lesions. Recent investigation has mapped one of the responsible genes for HHT to chromosome 9q33-q34 ; subsequently, nine different mutations have been identified in the endoglin gene, which encodes a transforming growth factor beta (TGF- beta) binding protein. We found a Japanese HHT family and identified a C to A mutation in exon 4 which changed an Ala^<160> codon(GCT) to an Asp^<160> codon(GAT). In order to reveal the mechanism by which vascular dysplasia was elicited in patients with having this mutation, TGF- beta signaling function was analyzed using recombinant normal and mutant endoglin proteins expressed in MCF7 cells. We confirmed that both normal and mutant endoglins were expressed on surface membranes of COS-1 cells at similar degrees by immunocytochemistry. Stable cell lines expressing normal or mutant endoglin were also established in MCF7 cells. When TGF- beta was added to these stable transformants, which were previously transfected with Smad2-flag plasmid, nuclear translocation of Smad2-flag protein was detected by immunofluoresense analysis in MCF7 cells expressing normal endoglin. Whereas, no immunofluoresense signal was observed in MCF7 cells expressing mutant endoglin. These results indicated several posibilities that mutant endoglin (1) lacks the binding actibity with TGF- beta, (2) can not present TGF- beta to type II receptor of TGF- beta or (3) fails to assemble to homodimer structure. These analyses are being undertaken now.