Comparative in vitro and in silico analyses of variants in splicing regions of BRCA1 and BRCA2 genes and characterization of novel pathogenic mutations.
Comparative in vitro and in silico analyses of variants in splicing regions of BRCA1 and BRCA2 genes and characterization of novel pathogenic mutations.
复制标题
DOI：
10.1371/journal.pone.0057173
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Radice P
中科院分区：
文献类型：
--
作者：
Colombo M;De Vecchi G;Caleca L;Foglia C;Ripamonti CB;Ficarazzi F;Barile M;Varesco L;Peissel B;Manoukian S;Radice P
在疾病相关基因的剪接区域已经发现了几种未分类的变异（UVs），它们作为致病性突变或良性多态性的特征对于理解它们在疾病发展中的作用至关重要。在本研究中，通过转录本分析鉴定了位于BRCA1和BRCA2剪接位点的24个uv。这些结果被用来评估9个生物信息学程序在预测导致异常剪接的遗传变异（剪接变异体）和异常转录本性质方面的能力。BRCA1的11个变异和BRCA2的8个变异，包括8个以前未在转录水平上表征的变异，被确定影响mRNA剪接。其中，16个导致含有过早终止密码子（ptc）的异常转录本的合成，2个导致含有ptc的自然发生的替代转录本的上调，1个导致BRCA2 DNA结合域编码区域的框内缺失，导致失去结合伴侣蛋白DSS1和ssDNA的能力。对于每个计算程序，我们评估了非信息性分析的比率，即那些不能识别野生型序列中自然剪接位点的分析，以及假阳性预测的比率，即错误分类为剪接的变异，作为其特异性的衡量标准，在预测灵敏度设置为100%的条件下。表现较好的程序是Human Splicing Finder和Automated Splice Site Analyses，两者都表现出100%的信息性和特异性。对于10个突变，我们观察到隐剪接位点的激活，但我们无法推导出简单的标准来选择，在生物信息学分析预测的不同隐剪接位点中，实际使用的那些。与之前的报道一致，我们的研究提供了证据，证明硅工具可以用于选择剪接位点变异进行体外分析。然而，后者仍然是强制性的表征异常转录本的性质。
Several unclassified variants (UVs) have been identified in splicing regions of disease-associated genes and their characterization as pathogenic mutations or benign polymorphisms is crucial for the understanding of their role in disease development. In this study, 24 UVs located at BRCA1 and BRCA2 splice sites were characterized by transcripts analysis. These results were used to evaluate the ability of nine bioinformatics programs in predicting genetic variants causing aberrant splicing (spliceogenic variants) and the nature of aberrant transcripts. Eleven variants in BRCA1 and 8 in BRCA2, including 8 not previously characterized at transcript level, were ascertained to affect mRNA splicing. Of these, 16 led to the synthesis of aberrant transcripts containing premature termination codons (PTCs), 2 to the up-regulation of naturally occurring alternative transcripts containing PTCs, and one to an in-frame deletion within the region coding for the DNA binding domain of BRCA2, causing the loss of the ability to bind the partner protein DSS1 and ssDNA. For each computational program, we evaluated the rate of non-informative analyses, i.e. those that did not recognize the natural splice sites in the wild-type sequence, and the rate of false positive predictions, i.e., variants incorrectly classified as spliceogenic, as a measure of their specificity, under conditions setting sensitivity of predictions to 100%. The programs that performed better were Human Splicing Finder and Automated Splice Site Analyses, both exhibiting 100% informativeness and specificity. For 10 mutations the activation of cryptic splice sites was observed, but we were unable to derive simple criteria to select, among the different cryptic sites predicted by the bioinformatics analyses, those actually used. Consistent with previous reports, our study provides evidences that in silico tools can be used for selecting splice site variants for in vitro analyses. However, the latter remain mandatory for the characterization of the nature of aberrant transcripts.
登录
查看更多内容
影响因子：
9.8
作者：
Easton, Douglas F.;Deffenbaugh, Amie M.;Goldgar, David E.
通讯作者：
Goldgar, David E.
影响因子：
4
作者：
Gaildrat, Pascaline;Krieger, Sophie;Martins, Alexandra
通讯作者：
Martins, Alexandra
影响因子：
14.9
作者：
Desmet FO;Hamroun D;Lalande M;Collod-Béroud G;Claustres M;Béroud C
通讯作者：
Béroud C
影响因子：
14.9
作者：
Hebsgaard, SM;Korning, PG;Brunak, S
通讯作者：
Brunak, S
影响因子：
3.9
作者：
Houdayer, Claude;Caux-Moncoutier, Virginie;Stoppa-Lyonnet, Dominique
通讯作者：
Stoppa-Lyonnet, Dominique