Effects of 14 F9 synonymous codon variants on hemophilia B expression: Alteration of splicing along with protein expression
Effects of 14 F9 synonymous codon variants on hemophilia B expression: Alteration of splicing along with protein expression
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DOI：
10.1002/humu.24377
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发表时间：
2022-04
期刊：
影响因子：
3.9
通讯作者：
Huayang Zhang;Changming Chen;Xi Wu;Can Lou;Q. Liang;Wenman Wu;Xuefeng Wang;Q. Ding
中科院分区：
文献类型：
--
作者：
Huayang Zhang;Changming Chen;Xi Wu;Can Lou;Q. Liang;Wenman Wu;Xuefeng Wang;Q. Ding
越来越多的证据表明，同义密码子变异 (SCV) 可以通过破坏不同的蛋白质生产过程而导致疾病。该研究的目的是调查F9变异数据库中报告的14个SCV是否是B型血友病的致病原因。结合计算机预测、体外小基因剪接测定和细胞表达检测，检测SCV对剪接和蛋白质表达的影响。通过共扩增荧光 PCR 对剪接转录本进行鉴定和定量。通过修改的 pU1snRNA 和 pU7snRNA 方法验证了剪接机制。在八个 SCV 中发现了异常剪接模式。 8 个 SCV 中有 5 个几乎产生了所有异常剪接亚型，预期会截短蛋白质，其中 3 个在剪接和蛋白质分泌方面均出现部分缺陷，总体效果与受影响病例的残留因子 IX 活性一致。其余 6 个 SCV 中的前信使 RNA (mRNA) 剪接过程和蛋白质功能均未受损。总之，我们的研究首先揭示了 14 种 F9 SCV 的致病机制，并强调了在遗传性疾病中进行 SCV mRNA 剪接分析和蛋白表达研究的重要性。
There is growing evidence that synonymous codon variants (SCVs) can cause disease through the disruption of different processes of protein production. The aim of the study is to investigate whether the 14 SCVs reported in the F9 variant database were the pathogenic causes of hemophilia B. The impacts of SCVs on splicing and protein expression were detected using a combination of in silico prediction, in vitro minigene splicing assay and cell expression detection. The splicing transcripts were identified and quantified by co‐amplification fluorescent PCR. The mechanism of splicing was verified by a modified pU1snRNA and pU7snRNA approach. Aberrant splicing patterns were found in eight SCVs. Five of the 8 SCVs produced almost all aberrant splicing isoforms, which were expected to truncate protein, three of them presented a partial defect on both splicing and protein secretion, the overall effects were consistent with the residual Factor IX activity of the affected cases. Neither the pre‐messenger RNA (mRNA) splicing process nor the protein function was impaired in the rest six SCVs. In conclusion, our study firstly revealed the pathogenic mechanism of the 14 F9 SCVs and highlighted the importance of performing mRNA splicing analysis and protein expression studies of SCVs in inherited disorders.