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The Duffy (FY) blood group antigens are carried by the DARC glycoprotein, a widely expressed chemokine receptor. The molecular basis of the Fya/Fyb and Fy(a-b-) polymorphisms has been clarified, but little is known about the Fyx antigen and the FY*X allele associated with weak expression of Fyb, Fy3, Fy5, and Fy6 antigens. We analyzed here the structure and expression of the FY gene in 4 Fy(a-bweak) individuals. As compared with Fy(a-b+) controls, the Fy(a-bweak) red blood cell membranes contained residual amount of DARC polypeptide and these cells were poorly bound by anti-Fy antibodies and chemokines. The FY gene from Fy(a-b+) and Fy(a-bweak) individuals differed by one substitution, C286T. The resulting Arg89Cys amino acid change reduced the binding of anti-Fy antibodies and chemokines to DARC transfectants. We concluded that the Fybweak donors carried the FY*X allele at the FY locus and that the Fyx antigen corresponds to highly reduced expression of a grossly normal Fyb polypeptide caused by the Arg89Cys substitution. Because FY is a single copy gene, this defect should also affect DARC expression in nonerythroid cells. Because the Fyx phenotype is not associated with apparent clinical consequences, we discussed these findings in the light of the putative roles of DARC in various tissues. Finally, we developed a Fyx DNA typing assay that should be useful for genetic studies and clinical transfusion medicine., (Copyright 1998 by The American Society of Hematology.)

The RH blood group locus is composed of two sequence-related genes, RHD and RHCE, encoding the D, Cc, and Ee antigens in common Rh-positive phenotypes. In this report, we have analyzed the molecular basis of Rh antigens expression in weak D (Du) and RN donors, in whom there is a severe reduction of the D and C/e antigens, respectively. Genomic and transcript analysis of three unrelated low-grade weak D (Du) variants indicated that the very low expression of the D antigen is not the result of rearrangement or mutation in the coding sequence of the RHO gone. Accordingly, weak D (Du) erythrocytes should carry a normal RhD polypeptide, which is in agreement with the observation that these variants never produce anti-D antibodies. Comparative polymerase chain reaction analysis showed a lower steady-state level of RhD transcripts in weak D (Du) reticulocytes, as compared with normal RhD-positive controls, thus providing direct evidence that the difference between the D antigen of D-positive and weak D (Du) red blood cells is quantitative only. Conversely, analysis of the molecular genetic basis of the RN phenotype Indicated that the severely decreased expression of the RhC and Rhe antigens in three variants is associated with a qualitative alteration identified as a segmental DNA exchange between the RHCE and RHD genes. These genomic rearrangements, which resulted in hybrid RhCe-D-Ce proteins expressing the low frequency Rh32 but not the high incidence Rh46 antigens, involved either axon 4 alone or both exons 3 and 4. These findings show that an identical phenotypical alteration of Rh antigens (reduced expression) may result either from a quantitative or a qualitative alteration of the RH genes expression.

The LW blood group is carried by a 42-kD glycoprotein that belongs to the family of intercellular adhesion molecules. The LW gene is organized into three exons spanning an HindIII fragment of approximately 2.65 kb. The exon/intron architecture correlates to the structural domains of the protein and resembles that of other Ig superfamily members except that the signal peptide and the first Ig-like domain are encoded by the first exon. The 5'UT region (nucleotides -289 to +9) includes potential binding sites for various transcription factors (Ets, CACC, SP1, GATA-1, AP2) and exhibited a significant transcriptional activity after transfection in the erythroleukemic K562 cells. No obvious abnormality of the LW gene, including the 5'UT region, has been detected by sequencing polymerase chain reaction-amplified genomic DNA from RhD+ or RhD- donors and from an Rhnull variant that lacks the Rh and LW proteins on red blood cells. However, a deletion of 10 bp in exon 1 of the LW gene was identified in the genome of an LW (a- b-) individual (Big) deficient for LW antigens but carrying a normal Rh phenotype. The 10-bp deletion generates a premature stop codon and encodes a truncated protein without transmembrane and cytoplasmic domain. No detectable abnormality of the LW gene or transcript could be detected in another LW(a- b-) individual (Nic), suggesting the heterogeneity of these phenotypes.

The Rh blood group antigens are encoded by two highly related genes, RHD and RHCE, and the sequence of the common alleles (D, Ce, CE, ce, and cE) of these genes has been previously elucidated. In this report, Rh transcripts and gene fragments have been amplified using polymerase chain reaction from the blood of donors with the CW+ andCX+ phenotypes. Sequence analysis indicated that the expression of the CW (Rh8) and CX (Rh9) antigens are associated with point mutations in the RHCE gene, which provides the definitive evidence that the CW and CX specificities are encoded by the same gene as the Cc and Ee antigens. As compared with the common (CW- and CX-) transcripts of the RHCE gene, the CW+ and CX+ cDNAs exhibited A122G and G106A transitions that resulted in Gln41Arg and Ala36Thr amino acid substitutions in the CW+ and CX+ polypeptides, respectively. Therefore, although the CW and CX specificities behave serologically as if they were allelic, they cannot not be considered, stricto sensu, as the products of antithetical allelic forms of the RHCE gene. Based on the CW-/CW+ nucleotide polymorphism, a polymerase chain reaction assay useful for diagnosis purposes has been developed that detects the presence of the CW+ allele by the use of an allele-specific primer.

The Rh (Rhesus) blood group antigens, D, Cc, and Ee, are carried by three unglycosylated membrane proteins of the human erythrocytes encoded by two highly related genes, D and CcEe. The major antigen, D, is a mosaic composed of at least nine determinants (epD1 through epD9). The lack of expression of some of these D epitopes at the surface of variant red blood cells defines the so-called D category phenotypes. In this report, we have determined the molecular basis of the DVI category phenotype characterized by the lack of epitopes D1, D2, D5, D6/7, and D8. Southern blot analysis and mRNA sequencing showed that the DVI phenotype is associated with two types of rearrangement of the D gene. Of 10 DVI genomes investigated, 8 exhibited a segmental DNA replacement (gene conversion) between the D fragment encompassing exons 4, 5, and 6 and the equivalent region of the CcEe gene. In the two other variants, these three exons are deleted. In both cases, the genomic rearrangement did not alter the reading frame of the variant RhD transcripts that are translated in 417 and 266 amino acid polypeptides, respectively. A heterogeneity of category DVI samples based on variable reactivity of the red blood cells with anti-D antibodies was previously found to be associated with the CDVIe or cDVIE haplotypes. Interestingly, our present results indicated that this serologic subdivision of the DVI category is correlated to two types of genomic rearrangements of the D gene.