Analysis of RHD transcripts from individuals with Asian-type DEL. (A) The schematic diagram of full-length RHD transcript and the primer positions for Sanger sequencing and Nanopore sequencing analyses. Specifically, the primers for fragment amplification of RHD transcripts and Sanger sequencing located on exon 8 to 9 and 3’-UTRs to cover the c.1227A mutation of RHD and the primers for whole coding region amplification of RHD transcripts and Nanopore sequencing located on 5’-UTRs and 3’-UTRs. (B) Schematic representation of RHD transcripts identified in individuals with Asian-type DEL. The RT-PCR products of full-length RHD coding region, which were amplified using cDNA from the cultured erythroblast of individuals with Asian-type DEL, were analyzed using Nanopore sequencing. The top 7 major types of RHD transcripts identified in individuals with Asian-type DEL are presented. The structural schematic diagram and frequency of RHD transcripts are indicated in the right panel. Different types of transcripts are indicated by various colors. (C) Barplot showing the frequencies for major types of RHD transcripts in each individual with Asian-type DEL (n = 8) or D⁺ control sample. The color codes for different types of transcripts are identical to those in panel B. The red bar indicates the full-length transcripts, which was identified in all individuals with Asian-type DEL with lower abundance (mean, 0.18%) but higher abundance in D⁺ control (56.6%). The gray bar indicates other types of transcripts. In the D⁺ control, 3 major transcripts including the full-length RHD transcript in the red bar, the truncated transcripts with exon 7 deletion (19.0% of total, shown in the gray bar accompanied by several other low-frequency transcripts), or exons 7/8/9 deletion (9.0%) in the orange bar, were identified as shown in one previous study.²³ (D) Recognition of RhD antigenic epitopes using anti-D mAbs in vitro. The top 7 types of RHD transcripts identified in Asian-type DEL erythroblasts were cloned into an expression vector and cotransfected into HEK 293T cells with wt RHAG construct. RhD antigen on the transfected cell surface was measured compared with unstained cell negative control and wt RHD-positive control using flow cytometry with 7 different anti-D mAbs.