一种新型RHD变异IVS4+2delT导致RhD抗原阴性表型的功能研究OA

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 no-vel allele of the RHD gene IVS4+2delT causing an RhD-negative phenotype was identified,revealing that it affects D ex-pression through an abnormal splicing mechanism.