Alternative Splicing of RHD mRNA by the Next-generation Sequencing in RhD-positive Individuals
- VernacularTitle:基于二代测序的RhD阳性个体RHD基因mRNA选择性剪接分析
- Author:
Ya-lin LUO
1
;
Ling WEI
2
;
Yan-li JI
2
;
Guang-ping LUO
2
;
Yong-shui FU
1
;
Ji-zhi WEN
2
;
Cheng-yao LI
1
Author Information
1. Department of Transfusion Medicine，Southern Medical University，Guangzhou 510515，China
2. Institute of Clinical Blood Transfusion， Guangzhou Blood Center， Guangzhou， 510095， China
- Publication Type:Journal Article
- Keywords:
RhD-positive;
next-generation sequencing;
transcripts;
splicing
- From:
Journal of Sun Yat-sen University(Medical Sciences)
2021;42(1):51-56
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveThis study aimed to analyze different transcripts specifically expressed by red blood cells in RhD-positive individuals， and to explore the mechanism of alternative splicing. MethodsErythroblasts from RhD-positive individuals were isolated and cultured in an in vitro erythroblast culture system. The fragment from exon 6 to 3’untranslated coding regions of RHD gene of RhD-positive individuals was amplified by reverse transcription polymerase chain reaction （RT-PCR） method. The PCR products were then sequenced by next-generation sequencing and the mRNA transcripts were analyzed. The maximum entropy values of 5’ splice site （ss） and 3’ ss of all exons of RHD gene were analyzed using bioinformatics. ResultsThe transcripts of the RHD gene were mainly normal full-length mRNA. In addition， eight other abnormal transcripts were also identified. According to the expression frequencies （high to low）， these transcripts were exon 7 deletion， exon 9 deletion， exons 8 and 9 deletion， exons 7-9 deletion， exons 7 and 9 deletion， exons 8-9 deletion and 170 bp insertion between exon 7 and 10， full-length with 170 bp insertion between exon 7 and 8， exon 9 deletion and 170 bp insertion between exon 7 and 8. The last three transcripts were novel. Bioinformatic analysis suggested that the abnormal splicing of exon 7 and exon 9 might be due to the decreased binding ability of 5’ss or 3’ss to the spliceosome. The intron 7 was expressed in mRNA， which might be related to the sequences conserved with 5’ss and 3’ss splicing. ConclusionThe alternative splicing patterns of RHD gene are very complicated， which leads to various transcripts at the mRNA level of the RHD gene from RhD-positive individuals.
