Background: Unlike the standard RHD+ or RHD- alleles, serologic determination of weak or partial D alleles is often not clear-cut. Most importantly, rare weak D alleles, not typed by serology, are prone to alloimmunization when transfused with D+ blood. Although more than 100 RHD variants have currently been reported, many more rare alleles probably remain to be identified.
Study design and methods: To identify novel unusual RHD alleles, genomic DNA samples were collected from 333 blood donors or recipients in western France. All displayed ambiguity for D phenotype as determined by routinely used serologic reagents and analyzed by means of denaturing high-performance liquid chromatography (DHPLC) analysis in parallel with direct sequencing.
Results: For the first time it has been established that a reliable DHPLC-based approach potentiates the rapid screening of the entire RHD gene-coding sequence. In so doing, a total of 12 novel RHD alleles were identified. Except for the null allele that is in trans with a Weak D type 4 allele, the predicted effects of the other new alleles on gene expression correlated well with the discrepant routine D phenotype results. In particular, the carrier of the p.Leu214Phe missense mutation developed alloanti-D antibodies after transfusion of D+ blood.
Conclusion: The identification of 12 novel RHD alleles represents a significant addition to the known repertoire of unusual RHD variants and, at the same time, serves to deepen our understanding of the molecular basis of weak and partial D. The accurate molecular typing of RHD alleles would allow to reduce the alloimmunization risk.