Service d'Hématologie Biologique, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, Lyon, France.
EA 4609 Hémostase et Cancer, Université Claude Bernard Lyon 1, University Lyon, Lyon, France.
Haemophilia. 2019 Mar;25(2):306-315. doi: 10.1111/hae.13687. Epub 2019 Jan 28.
Classically, the study of splicing impact of variation located near the splice site is performed by both in silico and mRNA analysis. However, RNA sample was rarely available.
To characterize a panel of putative haemophilia A splicing variations.
Twenty-six F8 variations identified from a cohort of 2075 haemophilia A families were studied using both bioinformatic tools and in vitro minigene assays in HeLa and Huh7 cells.
An aberrant splicing was demonstrated for 21/26 tested sequence variations. A good correlation between in silico and in vitro analysis was obtained for variations affecting donor splice site (12/14) and for the synonymous variations located inside an exon (6/6). Conversely, no concordant results were observed for the six variations affecting acceptor splice sites. The variations resulted more frequently in exon skipping (n = 13) than in activation of nearby cryptic splice sites (n = 5), in use of a de novo splice site (n = 2) or in insertion of large intronic sequences (n = 1). This study allowed to reclassify 5 synonymous substitutions c.1167A>G (p.Gln389Gln), c.1569G>T (p.Leu523Leu), c.1752G>A (p.Gln584Gln), c.5586G>A (p.Leu1862Leu) and c.6066C>T (p.Gly2022Gly) as splicing variations. The pathological significance of five variations remained unclear (c.222G>A [p.Thr74Thr], c.237C>T [p.Asn79Asn], c.240C>T [p.Ile80Ile], c.2113+5_2113+8del and c.2113+5G>A).
The minigene assay herein gave additional evidences for the clinical significance of 21/26 F8 putative splice site mutations. Such investigation should be performed for each F8 putative splice site variation for which no mRNA sample is available, notably to greatly improve the genetic counselling given to female carriers.
经典的研究表明，位于剪接位点附近的变异对剪接的影响是通过计算机分析和 mRNA 分析来进行的。然而，很少有 RNA 样本可供使用。
描述一组可能的血友病 A 剪接变异。
从 2075 个血友病 A 家系的队列中鉴定出 26 个 F8 变异，使用生物信息学工具和 HeLa 和 Huh7 细胞中的体外小基因试验进行研究。
在 26 个测试序列变异中，有 21 个显示出异常剪接。对供体位点剪接（12/14）和内含子内同义变异（6/6）的分析，计算机分析与体外分析具有良好的相关性。相反，对于影响受体剪接位点的 6 个变异，没有观察到一致的结果。变异更频繁地导致外显子跳跃（n=13），而不是激活附近的隐蔽剪接位点（n=5），使用新的剪接位点（n=2）或插入大的内含子序列（n=1）。本研究重新分类了 5 个同义突变 c.1167A>G(p.Gln389Gln)、c.1569G>T(p.Leu523Leu)、c.1752G>A(p.Gln584Gln)、c.5586G>A(p.Leu1862Leu)和 c.6066C>T(p.Gly2022Gly)为剪接变异。五种变异的病理学意义仍不清楚（c.222G>A[p.Thr74Thr]、c.237C>T[p.Asn79Asn]、c.240C>T[p.Ile80Ile]、c.2113+5_2113+8del 和 c.2113+5G>A）。
本文中的小基因试验为 26 个 F8 假定剪接位点突变中的 21 个提供了额外的临床意义证据。对于没有 mRNA 样本的每个 F8 假定剪接位点变异，都应该进行这种研究，这对于提高对女性携带者的遗传咨询非常重要。
