基于三代测序技术的RHD、RHCE基因mRNA研究  

Study on RHD and RHCE gene mRNA based on three generation sequencing technology

作　　者：官笑羽 纪欣[2,3] 李玲 徐海霞[2,3] 田力 任琪 何知航 王珏 刘忠 GUAN Xiaoyu;JI Xin;LI Ling;XU Haixia;TIAN Li;REN Qi;HE Zhihang;WANG Jue;LIU Zhong(School of Public Health,Anhui Medical University,Hefei 230032,China;Institute of Blood Transfusion,Chinese Academy of Medical Sciences and Peking Union Medical College,Chengdu 610052,China;Key Laboratory of Transfusion Adverse Reactions,Chinese Academy of Medical Sciences)

机构地区：[1]安徽医科大学公共卫生学院,安徽合肥230032 [2]中国医学科学院北京协和医学院输血研究所 [3]中国医学科学院输血不良反应重点实验室

出　　处：《中国输血杂志》2023年第2期115-120,共6页Chinese Journal of Blood Transfusion

基　　金：中国医学科学院医学与健康科技创新工程(2021-1-I2M-060);四川省国际科技创新合作/港澳台科技创新合作项目(2020YFH0024)。

摘　　要：目的建立基于Nanopore的RH基因mRNA三代测序方法,探讨D阳性和D^(el)表型中RHD、RHCE基因mRNA转录本的种类和表达水平差异。方法选取2021年3月~2022年5月成都市各医院送检至本实验室的经RhD血清学初筛及确证试验阴性的RhD阳性标本5例、D^(el)型标本5例。分离有核红细胞和白膜层,提取DNA和RNA,采用PCR-SSP对RhD阳性、D^(el)型基因分型,mRNA逆转录为cDNA后,采用1对引物同时对RHD、RHCE基因的全长mRNA扩增。分别采用Sanger测序和基于纳米孔的三代测序技术对扩增产物进行测序,并对RHD、RHCE基因mRNA转录本的不同剪接体种类和表达量进行分析。结果本研究建立的方法可同时扩增出RHD和RHCE的全长转录本,在10例标本中检测到了10种不同RHD基因mRNA转录本、9种RHCE基因mRNA转录本。D^(el)型中可检测到RHD的全长转录本(RHD-201),但表达量显著低于RhD阳性标本,D^(el)标本中转录本RHD-207(D^(el)789)表达量显著高于RhD阳性标本,转录本RHD-208(D^(el)8910+213)仅在D^(el)型个体中检出,其他RHD转录本以及所有RHCE转录本在2种个体中未发现显著差异。结论本研究成功建立了RHD、RHCE基因mRNA三代测序全长转录本异构体的分析方法,发现RHD、RHCE基因存在复杂的选择性剪接模式,为血型基因变异体mRNA选择性剪接研究提供了新方法。Objective To establish RH gene mRNA sequencing method based on nanopores sequencing and to explore the RHD and RHCE mRNA transcripts in D positive and D^(el)individuals.Methods From March 2021 to May 2022,5 RhD positive samples and 5 D^(el)samples screened out by hospitals in Chengdu were sent to our laboratory for futher examination.The erythrocytes and buff coat were isolated,then DNA and RNA were extracted.All 10 samples were genotyped by PCR-SSP.After the mRNA was reversely transcribed into cDNA,the full-length mRNA of RHD and RHCE genes were simultaneously amplified by a pair of primers.Sanger sequencing and third-generation sequencing technology based on Nanopore were used to sequence the amplified products,and the types and expressions of different splices of RHD and RHCE gene mRNA transcripts were analyzed.Results The method established in this study can simultaneously amplify the full length transcripts of RHD and RHCE.Ten different RHD gene mRNA transcripts and nine RHCE gene mRNA transcripts were detected in 10 samples.RHD full-length transcript(RHD-201)can be detected in RhD D^(el)type,but the expression amount was significantly lower than that in RhD positive samples.The expression amount of transcript RHD-207(D^(el)789)in D^(el)samples was significantly higher than that in RhD positive samples.The transcript RHD-208(D^(el)8910+213)was only detected in RhD D^(el)type individuals,and no significant difference was found between other RHD transcripts and all RHCE transcripts in the two phenotypes.Conclusion In this study,an analytical method for sequencing full-length transcript isomers of RHD and RHCE mRNA via the third generation was successfully established,and complex alternative splicing patterns were found in RHD and RHCE genes,providing a new method for the study of alternative splicing of blood group gene variants mRNA.

关 键 词：RH基因 D^(el)表型 三代测序技术 转录本 选择性剪接 

分 类 号：Q344.13[生物学—遗传学]