Molecular mechanisms that lead to reduced expression of Duffy antigens

BACKGROUND: in the Duffy blood group system, the null phenotype Fy(a-b-) ha s been classically associated with a mutated GATA box, while the Fy(x) phen otype weak Fy(b) is associated with Arg89Cys and Ala100Thr mutations. This report assesses the prevalence of the Duffy GATA box and the Fy(x)-associat ed mutations in white and African American (black) donors and investigates the molecular mechanism underlying the Fy(x) phenotype. STUDY DESIGN AND METHODS: PCR RFLP Duffy genotyping was performed on blood samples from blacks and whites. Duffy antigen expression (Fy(a), Fy(b), Fy6 , Fy3) on RBCs was measured by flow cytometry. By site-directed mutagenesis , the relevance of each F-x-associated mutation to Duffy (mRNA, antigen, an d protein) expression was analyzed in transfectants by Northern blotting, f low cytometry, and immunoblotting. RESULTS: The mutated GATA box occurred at a high allele frequency (0.8) in blacks and was rare among whites. Conversely, the Fy(x)-associated mutation s were absent in blacks, but present in 3.5 percent of whites. By flow cyto metry, Duffy antigens (Fy(a) or Fy(b), Fy6 and Fy3) showed a dosage effect in RBC samples that were transcriptionally silenced by the GATA box mutatio n in one allele. By contrast, the reduced (10%) Duffy protein in Fy(x) RBCs was shown by heterologous expression analysis not to be due to reduced RNA levels, but to protein instability caused by Arg89Cys. CONCLUSIONS: Reduced Duffy expression can result from mutations affecting t ranscription (mutated GATA box in one allele) or instability of the transla ted protein (Arg89Cys). The frequencies of these mutations vary among popul ations.