Comprehensive analysis of RHD splicing transcripts reveals the molecular basis for the weak anti-D reactivity of D_el-red blood cells

Background: Of the Rh blood type, D_el is a rare variant that elicits the weakest anti-D reactivity. In this study, we revisit the genetic changes of D_el allele and characterise the RHD splicing transcripts to realise the molecular basis of D_el formation in the Taiwanese population. Study design and methods: The RHD exons from D_el and D-positive individuals were amplified by polymerase chain reaction (PCR) using different primer pairs followed by sequencing analyses. In addition, full-length RHD transcripts were reversed transcribed and amplified by nested-PCR. The type and frequency of the RHD splicing transcripts were analysed after sequencing the PCR products that were subcloned into a cloning vector. Results: All D_el individuals had a characteristic 1227G>A mutation. No deletion of the exon sequences was found. At least nine types of RHD splicing transcripts including exons 7/8/9 deletion, 7/9 deletion, 8/9 deletion, 9 deletion, 2/3/7/9 deletion, 2/3/7/8/9 deletion, exons 7/8/9 deletion with replacement of exon 3 with RHCE exon 3, exon 9 deletion with cryptic insertion of 170 bp of intron 7 and exons 7/8/9 deletion with cryptic insertion of 117 bp of intron 3 were identified in the D_el-RBC. These aberrant splicing transcripts led to production of frame shift or truncated D antigen. Notably, no full-length RHD transcript was identified in the D_el-RBC. Conclusion: The RHD 1227G>A mutation contributes to the molecular basis of D_el phenotype in the Taiwanese population. The point mutation results in aberrant frame shift or exon deletion transcripts and generates D protein with weak antigen presenting function.