RT-PCR splicing analysis of the NF1 open reading frame
RT-PCR splicing analysis of the NF1 open reading frame
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DOI：
10.1007/s00439-002-0714-6
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发表时间：
2002-05-01
期刊：
影响因子：
5.3
通讯作者：
Wallace, MR
中科院分区：
文献类型：
--
作者：
Thomson, SAM;Wallace, MR
神经纤维瘤病1（NF 1）是一种常染色体显性遗传疾病，由于基因的大小和大量独特的NF 1基因突变，其分子诊断具有挑战性。一些剪接和无义突变已被证明会导致外显子跳跃。最近，在离体组织中发现了温度诱导的NF 1异常剪接。这促使我们研究整个NF 1转录本的这种异常剪接。我们发现了几个新的外显子跳跃，出现从头或最初存在，并增加在老化/冷却血液：外显子20，外显子20和21组合，外显子33，外显子34，外显子37，外显子40，外显子45，外显子43和45组合，外显子43的一部分，和外显子12 b的第一个密码子。当血液被抽入Qiagen PAXgene管而不是EDTA真空采血管时，一些异常剪接形式是检测不到的，我们证明了这些异常剪接事件是如何成为基于RNA的NF 1突变表征的潜在陷阱。使用相同的逆转录/聚合酶链反应策略来筛选施万细胞和其他七种组织中新型NF 1选择性剪接。尽管没有发现Schwann特异性的选择性外显子，但我们发现了微小的新型剪接异构体差异表达，如外显子37和外显子40的跳跃。在所有分析的组织中发现外显子43、外显子43的部分和外显子12 b的第一个密码子的跳跃。这些形式表明NF 1信息中基于组织的变异性比以前认为的更大，并且可能表明蛋白质水平上的少量异质性。
Neurofibromatosis 1 (NF1) is an autosomal dominant condition whose molecular diagnosis is challenging because of the large size of the gene and the vast number of unique NF1 gene mutations. Some splicing and nonsense mutations have been shown to cause exon skipping. Recently, temperature-induced abnormal splicing has been found in NF1 in ex-vivo tissues. This prompted us to investigate the entire NF1 transcript for such aberrant splicing. We found several novel exon skips that appeared de novo or were present initially and increased in aged/cooled blood: exon 20, exons 20 and 21 combined, exon 33, exon 34, exon 37, exon 40, exon 45, exons 43 and 45 combined, part of exon 43, and the first codon of exon 12b. Some aberrant splice forms were undetectable when blood was drawn into Qiagen PAXgene tubes, rather than EDTA vacutainers, and we demonstrate how these aberrant splicing events are a potential pitfall for RNA-based NF1 mutation characterization. The same reverse transcription/polymerase chain reaction strategy was used to screen for novel NF1 alternative splicing in Schwann cells and seven other tissues. Even though no Schwann-specific alternative exons were identified, we found minor novel splicing isoforms differentially expressed such as skips of exon 37 and exon 40. Skipping of exon 43, part of exon 43, and the first codon of exon 12b were found in all tissues analyzed. These forms suggest greater tissue-based variability in the NF1 message than was previously thought and may indicate minor amounts of heterogeneity at the protein level.