Functional significance of deep intronic mutation in the ATM gene and evidence for an alternative Exon 28a
Functional significance of deep intronic mutation in the ATM gene and evidence for an alternative Exon 28a
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DOI：
10.1002/humu.20170
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发表时间：
2005-01-01
期刊：
影响因子：
3.9
通讯作者：
Gatti, RA
中科院分区：
文献类型：
--
作者：
Coutinho, G;Xie, JY;Gatti, RA
ATM突变的筛查通常是以基因组DNA为模板进行跨外显子区域的PCR扩增，结果是无法分析深层内含子序列。在这里，我们报告了一个新的保留假外显子的深内含子突变(IVS28-159A>；G；G.75117A>；G基于GenBank U82828.1)，以及在来自正常人的淋巴母细胞系(LCL)中表达的ATM基因(外显子28a)中先前未识别的替代外显子的鉴定。用A-T患者的LCL进行的cDNA分析显示，在外显子28和29之间保留了两个异常的内含子片段，即112和190个核苷酸。构建Miniges是为了确定异常剪接区两个基因组变化的功能意义：IVS28-193C>；T(g.75083C>；T)和IVS28-159A>；G，揭示：1)第一个是多态的；2)IVS28-159A>；G削弱了可选外显子28a的5‘剪接位点，并激活了下游隐蔽的5’剪接位点(Ss)83个核苷酸；3)野生型结构还保留了一个29个核苷酸的片段(外显子28a)，作为112和190个核苷酸片段的一部分。对SS强度的最大熵估计证实了cDNA和微型基因的发现。这些突变可能被证明与针对特定剪接异常的计划治疗有关。(C)2005年Wiley-Liss，Inc.
Screening for ATM mutations is usually performed using genomic DNA as a template for PCR amplification across exonic regions, with the consequence that deep intronic sequences are not analyzed. Here we report a novel pseudoexon-retaining deep intronic mutation (IVS28-159A > G; g.75117A > G based on GenBank U82828.1) in a patient with ataxia-telangiectasia (A-T), as well as the identification of a previously unrecognized alternative exon in the ATM gene (exon 28a) expressed in lymphoblastoid cell lines (LCL) derived from normal individuals. cDNA analysis using the A-T patient's LCL showed the retention of two aberrant intronic segments of 112 and 190 nt between exons 28 and 29. Minigenes were constructed to determine the functional significance of two genomic changes in the region of aberrant splicing: IVS28-193C > T (g.75083C > T) and IVS28-159A > G, revealing that: 1) the first is a polymorphism; 2) IVS28-159A > G weakens the 5' splice site of the alternative exon 28a and activates a cryptic 5' splice site (ss) 83 nt downstream; and 3) wild-type constructs also retain a 29-nt segment (exon 28a) as part of both the 112- and 190-nt segments. Maximum entropy estimates of ss strengths corroborate the cDNA and minigene findings. Such mutations may prove relevant in planning therapy that targets specific splicing aberrations. (C) 2005 Wiley-Liss, Inc.