Unitat de Diagnòstic i Teràpia Molecular, Banc de Sang i Teixits, Barcelona, Spain.
J Thromb Haemost. 2011 Apr;9(4):679-88. doi: 10.1111/j.1538-7836.2011.04204.x.
In von Willebrand factor (VWF) the effect of mutations potentially affecting mRNA processing or splicing is less predictable than that of other mutations (e.g. nonsense or missense substitutions). Bioinformatic tools can provide a valuable means to determine the consequences of potential splice site mutations (PSSM), but functional studies are mandatory to elucidate the true effect of the variation detected.
OBJECTIVES, PATIENTS AND METHODS: After identification of PSSM in VWD patients, we began a systematic study of their in vivo effect in RNA extracted from the patients' platelets and leukocytes.
Thirteen pairs of primers were designed for full amplification of VWF mRNA by RT-PCR that, after sequencing of aberrant products, enabled elucidation of the PSSM consequences for mRNA processing. This procedure was used to study seven different PSSM identified in four patients demonstrating diverse molecular mechanisms such as exon skipping (c.533-2A>G and c.8155+3G>C) and the activation of a cryptic splice site (c.7730-1G>C). No visible effect was evident for c.1533+15G>A and c.5170+10C>T and the consequence of c.[546G>A;7082-2A>G] was hidden by nonsense-mediated mRNA decay (NMD). Results were compared with in silico predictions of four splice-site analysis tools. We demonstrate selective degradation of VWF mRNA bearing PSSM by NMD for several mutations, which suggests that NMD represents a general mechanism for truncating mutations in VWF. Furthermore, because NMD efficiency varies between cell types, use of RNA from both platelets and leukocytes for in vivo study of VWF PSSM offers complementary results, particularly in cases in which NMD occurs in the allele carrying the mutation.
在血管性血友病因子 (VWF) 中，潜在影响 mRNA 处理或剪接的突变的影响比其他突变（例如无义或错义取代）更难预测。生物信息学工具可以提供一种有价值的方法来确定潜在剪接位点突变 (PSSM) 的后果，但功能研究是阐明检测到的变异的真实影响所必需的。
目的、患者和方法：在鉴定 VWD 患者的 PSSM 后，我们开始对从患者血小板和白细胞中提取的 RNA 中 PSSM 的体内效应进行系统研究。
设计了 13 对引物，通过 RT-PCR 对 VWF mRNA 进行完全扩增，在对异常产物进行测序后，阐明了 PSSM 对 mRNA 处理的影响。该程序用于研究在四名患者中鉴定的七种不同的 PSSM，这些患者表现出不同的分子机制，如外显子跳跃（c.533-2A>G 和 c.8155+3G>C）和激活隐匿剪接位点（c.7730-1G>C）。c.1533+15G>A 和 c.5170+10C>T 没有明显的影响，c.[546G>A;7082-2A>G] 的后果被无义介导的 mRNA 衰减 (NMD) 隐藏。结果与四种剪接位点分析工具的计算机预测进行了比较。我们证明了 NMD 对几种 PSSM 导致的 VWF mRNA 的选择性降解，这表明 NMD 是 VWF 截断突变的一种普遍机制。此外，由于 NMD 效率在细胞类型之间存在差异，因此使用血小板和白细胞中的 RNA 进行 VWF PSSM 的体内研究可提供互补结果，特别是在 NMD 发生在携带突变的等位基因的情况下。
