摘要
目的通过mRNA异常剪接体外验证实验,即体外微基因剪接系统(minigene splicing assay,MSA),研究中国人群中发现的RHAG突变对于RHAG基因mRNA剪接的影响。方法通过对KMxD数据库中RHAG基因数据的分析,选择10种中国人群中分布频率相对较高的位于RHAG基因剪接位点附近的突变或编码区域的同义突变,构建相应的RHAG外显子野生型及突变型pSplicePOLR2G微基因表达质粒。转染HEK-293T细胞,48 h后收集细胞提取总RNA,反转录后进行PCR扩增,然后进行琼脂糖凝胶电泳检测和毛细管电泳检测,根据实验结果判断RHAG基因突变是否会影响相应外显子的正常剪接。结果2种RHAG基因剪接位点附近突变(c.158-5delT,c.807+3A>C)轻微减弱了原剪接位点的剪接效率,其余8种突变(c.312G>A,c.341+3G>A,c.609C>T,c.681G>A,c.861G>A,c.957T>A,c.984T>C和c.1139-7G>A)均不影响RHAG基因mRNA的剪接。结论RHAG基因在剪接方面比较保守,剪接位点附近突变及编码区同义突变较少会引起RHAG基因异常剪接。
Objective To study the effect of RHAG variants identified in Chinese population on mRNA splicing by minigene splicing assay(MSA)in vitro.Methods The pSplicePOLR2G minigene expression plasmids were constructed for 10 RHAG mutations with relatively high distribution frequency in Chinese population near splicing sites or synonymous mutations by analyzing the RHAG gene data in the KMxD database.Then,the wild-type and mutant plasmids were transfected into HEK 293T cells,and RNA was extracted 48 hours after transfection.After reverse transcription,specific primers were used for PCR amplification,and then agarose gel electrophoresis and capillary electrophoresis were performed to determine whether the mutations will affect the normal splicing of exons.Results MSA in vitro showed that 2 mutations(c.158-5delT,c.807+3A>C)near the splicing site reduced the amount of normal transcripts slightly.The remaining 8 synonymous mutations(c.312G>A,c.341+3G>A,c.609C>T,c.681G>A,c.861G>A,c.957T>A,c.984T>C and c.1139-7G>A)had no impact on the splicing of RHAG mRNA.Conclusion This study showed that RHAG gene was conservative in terms of splicing,and the mutations near splicing sites and synonymous mutations were less likely to cause abnormal splicing of RHAG gene.
作者
贾双双
孙明明
温机智
魏玲
罗广平
姬艳丽
JIA Shuangshuang;SUN Mingming;WEN Jizhi;WEI Ling;LUO Guangping;JI Yanli(Institute of Clinical Transfusion,Guangzhou Blood Center,Guangzhou 510095,China;KingMed Diagnostics Group Co.,Ltd.)
出处
《中国输血杂志》
CAS
2023年第11期986-990,共5页
Chinese Journal of Blood Transfusion
基金
广东省基础与应用基础研究基金项目(2021A1515012139)
广州市卫生健康科技项目(20231A010047)
广州市科技计划项目(2023A03 J0552)
广州市医学重点学科(2021-2023)项目。
