Functional study of a novel RHD variant IVS4+2delT leading to RhD-negative phenotype
10.13303/j.cjbt.issn.1004-549x.2026.06.014
- VernacularTitle:一种新型RHD变异IVS4+2delT导致RhD抗原阴性表型的功能研究
- Author:
Xiao HAO
1
;
Ruirui LI
1
;
Lu ZHANG
1
;
Yongchun YANG
1
Author Information
1. Jinan Blood Center, Jinan 250000, China
- Publication Type:Journal Article
- Keywords:
novel RHD variant;
splicing;
PacBio sequencing;
minigene assay
- From:
Chinese Journal of Blood Transfusion
2026;39(6):795-798
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To investigate the effect of a novel RHD genotype (RHD
01N.01/RHD
01.01 with IVS4+ 2delT mutation)on the RhD phenotype through in vitro experiments in a case with a serologically RhD-negative phenotype. Methods: Serological screening was performed using saline method, and confirmed by indirect antiglobulin test (IAT). RhCE phenotyping was determined by RhCE typing cards. The full-length RHD gene was sequenced using PacBio long-read sequencing technology. Bioinformatics analysis and Minigene splicing variant analysis technology were used to elucidate the abnormal splicing mechanism of novel splice site variations. Results: The serological presentation was RhD negative. The PacBio sequencing revealed a compound heterozygote RHD
01N.01 and a new RHD
01.01 allele, which carried a new mutation at the 5′ splice site (IVS4+2delT) of intron 4. Bioinformatics predicted that the mutation disrupts the donor splice site and activates downstream recessive splice sites. The minigene experiment confirmed that this mutation leads to abnormal splicing, producing two types of mRNA: one with a 10 bp insertion and the other with a 15 bp insertion. Conclusion: A novel allele of the RHD gene IVS4+2delT causing an RhD-negative phenotype was identified, revealing that it affects D expression through an abnormal splicing mechanism.
