Multiple sequence variants of BRCA2 exon 7 alter splicing regulation
Multiple sequence variants of BRCA2 exon 7 alter splicing regulation
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DOI：
10.1136/jmedgenet-2012-100965
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发表时间：
2012-10-01
影响因子：
4
通讯作者：
Martins, Alexandra
中科院分区：
文献类型：
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作者：
Gaildrat, Pascaline;Krieger, Sophie;Martins, Alexandra
背景在患者中鉴定的未知生物学意义的外显子变体（VUS）可以通过改变50或30个剪接位点或通过修饰剪接调控元件来影响mRNA剪接。这些元素的生物信息学预测仍然是不准确的，只有少数这样的元素已在BRCA 2中的功能映射。我们研究了从法国UMD-BRCA 2突变database.Methods中选择的8个外显子7 VUS对剪接的影响，我们进行了剪接小基因测定和患者RNA分析。我们还开发了一种基于焦磷酸测序的定量分析，以测量患者RNA中每个等位基因对含外显子7转录物产生的相对贡献。此外，外显子剪接增强子（ESE）依赖的小基因分析被用来评估野生型和突变片段的剪接调控特性。在minigene测定中，c.517G > T和c. 631 G> A改变了天然剪接位点，c.572 A> G产生了新的50剪接位点，c.520 C> T、c.587 G> A和c.617 C> G诱导了外显子7的跳跃（分别为66%、25%和46%）。患者RNA的焦磷酸测序证实了c.520C > T和c.617C > G的这些外显子跳跃水平。结论BRCA 2外显子7剪接受多种外显子调控，对疾病相关序列变异敏感。测量患者来源的RNA中的等位基因不平衡和/或使用微基因测定的定量分析提供了对部分剪接缺陷程度的有价值的估计。对具有部分剪接效应的变异体的致病性的评估等待更多的证据，特别是分离分析的完成。
Background Exonic variants of unknown biological significance (VUS) identified in patients can affect mRNA splicing, either by changing 50 or 30 splice sites or by modifying splicing regulatory elements. Bioinformatic predictions of these elements are still inaccurate and only few such elements have been functionally mapped in BRCA2. We studied the effect on splicing of eight exon 7 VUS, selected from the French UMD-BRCA2 mutation database.Methods We performed splicing minigene assays and analyses of patient RNA. We also developed a pyrosequencing-based quantitative assay, to measure, in patient RNA, the relative contribution of each allele to the production of exon 7-containing transcripts. Moreover, an exonic splicing enhancer (ESE)-dependent minigene assay was used to evaluate the splicing regulatory properties of wild-type and mutant segments.Results Six out of the eight variants induced splicing defects. In the minigene assay, c.517G > T and c. 631G > A altered the natural splice sites, c.572A > G created a new 50 splice site, and c.520C > T, c.587G > A and c.617C > G induced exon 7 skipping (66%, 25% and 46%, respectively). Pyrosequencing of patient RNA confirmed these levels of exon skipping for c.520C > T and c.617C > G. Results from the ESE-dependent minigene assay indicated that c.520C > T and c.587G > A disturb splicing regulatory elements.Conclusions BRCA2 exon 7 splicing is regulated by multiple exonic elements and is sensitive to disease-associated sequence variations. Measurements of allelic imbalance in patient-derived RNA and/or quantitative analyses using minigene assays provide valuable estimates of the extent of partial splicing defects. Assessment of pathogenicity of variants with partial splicing effect awaits additional evidence and especially the completion of segregation analyses.