A novel splice-site mutation in RHD gene associated with RhD negative phenotype

Abstract

Introduction: The Rh antigens are encoded by the highly polymorphic RHD and the RHCE genes. Individuals with D variants may make anti-D alloantibodies, which in pregnant women cause severe or fatal hemolytic disease of the fetus and newborn. Accurate D typing using reliable routine methods must be performed. A number of unusual RHD alleles are being currently reported due in large part to the growing implementation of molecular analyses. Case Report: A sample from a Caucasian male blood donor at Northern Italy was serologically classified as RhD negative (Ccee). Instead, the sample was genotyped with all SSP-PCR kits as RhD positive, Dd and the result obtained with Partial D-TYPE (Dva or Va like or Va associated or DBS) was consistent with a suggestion of a new variant. Through a microarray platform analysis (BLOODchip Reference), the sample was genotyped as DV/DBS [RHD-CE(S)-D hybrid], associated with a predicted partial D phenotype for the RhD group, due to the lack of a hybridization signal for RHD exon 5. The PCR analysis of the exon 5 of the RHD gene gave a positive amplification result and the sequencing revealed a polymorphism in hemizygous or homozygous state in position RHD*IVS5+1t. This novel splice-site mutation alters or completely abolishes the specific sequence where the splicing of RHD gene intron 5 takes place. Conclusion: The novel RHD mutation hereby described highlights the significance of using RHD genotyping to confirm and/or clarify D antigen uncertainties in order to prevent the unnecessary immunization of patients.