Aflac Cancer and Blood Disorders Center, Emory University/Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
The Children's Hospital of Philadelphia, The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Philadelphia, Pennsylvania, USA.
J Thromb Haemost. 2021 Jul;19(7):1653-1665. doi: 10.1111/jth.15314. Epub 2021 May 20.
Elucidating the molecular pathogenesis underlying East Texas bleeding disorder (ET) led to the discovery of alternatively spliced F5 transcripts harboring large deletions within exon 13. These alternatively spliced transcripts produce a shortened form of coagulation factor V (FV) in which a large portion of its B-domain is deleted. These FV isoforms bind tissue factor pathway inhibitor alpha (TFPIα) with high affinity, prolonging its circulatory half-life and enhancing its anticoagulant effects. While two missense pathogenic variants highlighted this alternative splicing event, similar internally deleted FV proteins are found in healthy controls.
We identified a novel heterozygous 832 base pair deletion within F5 exon 13, termed F5-Atlanta (F5-ATL), in a patient with severe bleeding. Our objective is to investigate the effect of this deletion on F5 and FV expression.
METHODS & RESULTS: Assessment of patient plasma revealed markedly elevated levels of total and free TFPI and a FV isoform similar in size to the FV-short described in ET. Sequencing analyses of cDNA revealed the presence of a transcript alternatively spliced using the ET splice sites, thereby removing the F5-ATL deletion. This alternative splicing pattern was recapitulated by heterologous expression in mammalian cells.
These findings support a mechanistic model consisting of cis-acting regulatory sequences encoded within F5 exon 13 that control alternative splicing at the ET splice sites and thereby regulate circulating FV-short and TFPIα levels.
阐明导致东德克萨斯出血性疾病（ET）的分子发病机制，发现了 F5 转录本的选择性剪接，该转录本在exon13 内含有大片段缺失。这些选择性剪接的转录本产生凝血因子 V（FV）的缩短形式，其中其 B 结构域的大部分被缺失。这些 FV 同工型与组织因子途径抑制剂 α（TFPIα）具有高亲和力结合，延长其循环半衰期并增强其抗凝作用。虽然两种错义致病性变异突出了这种选择性剪接事件，但在健康对照中也发现了类似的内部缺失 FV 蛋白。
我们在一名严重出血的患者中鉴定出 F5exon13 内的一个新的杂合 832 碱基对缺失，称为 F5-Atlanta（F5-ATL）。我们的目的是研究该缺失对 F5 和 FV 表达的影响。
对患者血浆的评估显示总 TFPI 和游离 TFPI 水平显著升高，FV 同工型大小与 ET 中描述的 FV-short 相似。cDNA 的测序分析显示存在使用 ET 剪接位点进行选择性剪接的转录本，从而去除了 F5-ATL 缺失。这种选择性剪接模式通过在哺乳动物细胞中的异源表达得到重现。
这些发现支持一个机制模型，该模型由 F5exon13 内编码的顺式作用调节序列组成，控制 ET 剪接位点的选择性剪接，从而调节循环 FV-short 和 TFPIα 水平。
