简介：RNA序列分析可以有效地用于识别异常剪接，考虑到肿瘤抑制基因的功能丧失机制，它们是足够的靶标。桑格测序是RNA序列分析最简单的方法；然而，由于其对无义介导的 mRNA 衰减 (NMD) 的敏感性不足，建议使用具有 NMD 抑制作用的培养样本，这阻碍了其广泛采用。方法：回顾性分析未进行 NMD 抑制的外周血 RNA 的 Sanger 测序结果，对抑癌基因的潜在剪接变异进行检测。对于转录本中未发现变化的阴性病例，通过查阅最新文献来评估 NMD 引起假阴性的可能性。结果：回顾了各种肿瘤抑制基因的十一种潜在剪接变体。根据桑格 RNA 测序确定的无效效应，有 6 种变异被分类为致病性或可能致病性。由于确定了框内变化或两个等位基因的正常表达，四个变体仍然是意义不确定的变体。在回顾了最近的一项研究后，一个变体的结果被怀疑是由 NMD 引起的假阴性，该研究报告了相同的变体对受影响的转录本造成了无效作用。结论：虽然根据规范规则，大多数病例中发现的 RNA 变化预计会经历 NMD，但大多数病例 (10/11) 可以通过 Sanger RNA 测序进行解释，而无需 NMD 抑制，因为尽管 NMD 效率很高，但由于 NMD 效率不完全或等位基因特异性表达.版权所有 © 2023 Ha、Jang、Kim 和 Kim。

Introduction: RNA sequence analysis can be effectively used to identify aberrant splicing, and tumor suppressor genes are adequate targets considering their loss-of-function mechanisms. Sanger sequencing is the simplest method for RNA sequence analysis; however, because of its insufficient sensitivity in cases with nonsense-mediated mRNA decay (NMD), the use of cultured specimens with NMD inhibition has been recommended, hindering its wide adoption. Method: The results of Sanger sequencing of peripheral blood RNA without NMD inhibition performed on potential splicing variants of tumor suppressor genes were retrospectively reviewed. For negative cases, in which no change was identified in the transcript, the possibility of false negativity caused by NMD was assessed through a review of the up-to-date literature. Results: Eleven potential splice variants of various tumor suppressor genes were reviewed. Six variants were classified as pathogenic or likely pathogenic based on the nullifying effect identified by Sanger RNA sequencing. Four variants remained as variants of uncertain significance because of identified in-frame changes or normal expression of both alleles. The result of one variant was suspected to be a false negative caused by NMD after reviewing a recent study that reported the same variant as causing a nullifying effect on the affected transcript. Conclusion: Although RNA changes found in the majority of cases were expected to undergo NMD by canonical rules, most cases (10/11) were interpretable by Sanger RNA sequencing without NMD inhibition due to incomplete NMD efficiency or allele-specific expression despite highly efficient NMD.Copyright © 2023 Ha, Jang, Kim and Kim.