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首页> 外文期刊>Molecular genetics and metabolism >Two silent substitutions in the PDHA1 gene cause exon 5 skipping by disruption of a putative exonic splicing enhancer.
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Two silent substitutions in the PDHA1 gene cause exon 5 skipping by disruption of a putative exonic splicing enhancer.

机译：PDHA1基因中的两个沉默取代通过推定外显子剪接增强子的破坏导致外显子5跳跃。

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摘要

BACKGROUND: Synonymous mutations within exons may cause aberrant splicing by disrupting exonic splicing enhancer (ESE) motifs in the vicinity of non consensus splice sites. Mutational analysis of PDHA1 revealed only one silent single nucleotide substitution in exon 5 in two unrelated boys and a girl (c.483C>T and c.498C>T variants, respectively). For both patients, pyruvate dehydrogenase complex activity was low and the immunoreactive E1alpha protein was defective in cultured fibroblasts. METHODS AND RESULTS: One of the boys was a somatic mosaic for the c.483C>T variant, as shown by the variable ratio of mutant to normal alleles in fibroblast, lymphocyte and single hair root DNA. Transcript analysis in fibroblasts from the three patients revealed the presence of both normal and truncated cDNAs, with the splicing out of exon 5 predicted to result in a frame shift and premature termination (p.Arg141AlafsX11). The treatment of fibroblasts with emetine before harvesting to prevent nonsense mRNA-mediated decay increased the amount of mutant mRNA. In silico analysis revealed that each variant disrupted a putative SRp55 binding site and that the intron 5 donor splice site (5'ss) contained a weak splicing signal. Transient transfection of COS-7 or Hela cells with hybrid minigene constructs containing wild-type or mutant PDHA1 exon 5, followed by RT-PCR demonstrated that each variant resulted in the incomplete inclusion of PDHA1 exon 5, and that this defect was corrected following the restoration of a perfect consensus sequence for the 5' splice site by site-directed mutagenesis. CONCLUSION: These two synonymous mutations expand the spectrum of rare PDHA1 splicing mutations, all of which are located in non canonical splice sites.
机译：背景：外显子内的同义突变可能会通过破坏非共有剪接位点附近的外显子剪接增强子（ESE）基序来引起异常剪接。 PDHA1的突变分析显示，在两个无关的男孩和一个女孩中，外显子5中仅一个沉默的单核苷酸取代（分别为c.483C> T和c.498C> T变体）。对于这两名患者，丙酮酸脱氢酶复合物活性低，并且在培养的成纤维细胞中免疫反应性E1α蛋白存在缺陷。方法和结果：其中一个男孩是c.483C> T变体的体细胞镶嵌，如成纤维细胞，淋巴细胞和单发根DNA中突变体与正常等位基因的可变比例所显示。在三名患者的成纤维细胞中进行的转录本分析显示正常和截短的cDNA均存在，外显子5的剪接预计会导致移码和过早终止（p.Arg141AlafsX11）。收获前用曲美汀处理成纤维细胞以防止无意义的mRNA介导的衰变增加了突变mRNA的量。在计算机分析中发现，每个变体破坏了一个假定的SRp55结合位点，内含子5供体的剪接位点（5's）包含一个弱的剪接信号。用含有野生型或突变PDHA1外显子5的杂种小基因构建体瞬时转染COS-7或Hela细胞，然后进行RT-PCR证明每种变体均导致PDHA1外显子5的不完全包涵，并且该缺陷在纠正通过定点诱变恢复5'剪接位点的完美共有序列。结论：这两个同义突变扩大了罕见的PDHA1剪接突变的范围，它们均位于非规范剪接位点。

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