RHCE*ceCF encodes partial c and partial e but not CELO, an antigen antithetical to Crawford.

BACKGROUND: RH43 (Crawford) is encoded by RHCE*ce with nucleotide changes 48G>C, 697C>G, and 733C>G (RHCE*ceCF). We investigated the Rh antigen expression and antibody specificities in four patients with this allele. STUDY DESIGN AND METHODS: Hemagglutination tests, DNA extraction, polymerase chain reaction (PCR)-restriction fragment length polymorphism, allele-specific PCR, reticulocyte RNA isolation, reverse transcription-PCR cDNA analyses, cloning, and sequencing were performed by standard procedures. RESULTS: Red blood cells (RBCs) from two patients typed D+C-E-c+e+/-, hrS-/+W, hrB- and their serum was reactive (3+) with all RBC samples of common Rh phenotype tested, but nonreactive with Rhnull or D-- RBCs (apparent alloanti-Rh17). At the RHCE locus, Patient 1 was homozygous for RHCE*ceCF, and Patient 2 inherited RHCE*ceCF in trans to a silenced RHCE*cE. Cross-testing of serum and RBCs from these two samples showed mutual compatibility, indicating that both antibodies define the same novel high-prevalence antigen on Rhce. Two additional patients, one whose serum contained alloanti-c but the RBCs typed C+c+ and one whose serum contained anti-e but the RBCs typed E+e+, also had RHCE*ceCF. RHCE*Ce was present in trans in the former and RHCE*cE in the latter patient. CONCLUSION: We report that amino acid changes on RhceCF (Trp16Cys, Gln233Glu, and Leu245Val) alter the protein to the extent that c and e antigens are partial, and a high-prevalence antigen, we have named CELO (provisional ISBT Number 004058; RH58) is not expressed. CELO is antithetical to RH43 (Crawford).

Two MER2-negative individuals with the same novel CD151 mutation and evidence for clinical significance of anti-MER2.

BACKGROUND: MER2 (RAPH1), the only antigen of the RAPH blood group system, is located on the tetraspanin CD151. Only four examples of alloanti-MER2 are known. We report here two new examples of alloanti-MER2, in women of Pakistani and Turkish origin, one of whom showed signs of a hemolytic transfusion reaction (HTR) after transfusion of 3 units of red cells (RBCs). STUDY DESIGN AND METHODS: Standard serologic methods were used. A monocyte monolayer assay (MMA) was used to assess the potential clinical significance of one of the antibodies. All exons and flanking intronic sequences of CD151 were amplified and sequenced. A homology model for CD151 second extracellular loop (EC2) was constructed based on the crystal structure of CD81. RESULTS: RBCs of both patients did not react with alloanti-MER2, and neither of their antibodies reacted with MER2-negative RBCs. The MMA results suggested that the antibody that appeared to have caused an HTR had the potential to be clinically significant. Both patients were homozygous for a 511C>T mutation in CD151 encoding an Arg171Cys change. This change did not result in any significant structural rearrangement in the protein model. CONCLUSIONS: Two MER2-negative patients with anti-MER2 are homozygous for the same novel mutation encoding an amino acid substitution in the EC2 of CD151. One of the antibodies may have been responsible for an HTR, and crossmatch-compatible RBCs should be recommended for transfusion to patients with anti-MER2.

The molecular diversity of Sema7A, the semaphorin that carries the JMH blood group antigens.

BACKGROUND: Semaphorin 7A (Sema7A), the protein that carries the JMH blood group antigen, is involved in immune responses and plays an important role in axon growth and guidance. Because previous serologic studies on red blood cells (RBCs) suggested a considerable diversity of Sema7A, the present study was designed to elucidate the complex picture of the molecular diversity of this protein. STUDY DESIGN AND METHODS: The JMH antigen status was determined by serology, flow cytometry, and Western blot. Genomic and transcript analysis of SEMA7A was performed by nucleotide sequencing. Recombinant Sema7A proteins were used for genotype-phenotype correlation. A three-dimensional model of Sema7A was generated for topologic analyses. RESULTS: Our studies on 44 individuals with unusual JMH phenotypes and their family members revealed that aberrant Sema7A expression can be an inherited or an acquired phenomenon and is based on reduced surface expression or qualitative changes in Sema7A. These different phenotypes are caused by variations of the SEMA7A gene or seem to be generated by autoimmune-related or RBC lineage-specific mechanisms. The variant JMH phenotypes were related to the presence of missense mutations in SEMA7A, predicting amino acid changes in the semaphorin domain of Sema7A. Sequence analysis of the variant SEMA7A alleles revealed mutations affecting codons 207 and 460/461. Topologic analyses showed that Sema7A polymorphisms were prominently located on the top and bottom of the semaphorin domain, suggesting a functional relevance of these sites. CONCLUSION: These findings provide a basis with which to delineate the various ligand-binding surfaces of Sema7A.

Molecular basis of the LOCR (Rh55) antigen.

BACKGROUND: In 1994 during the investigation of a case of hemolytic disease of the newborn, a new low-incidence red cell (RBC) antigen, LOCR, was described. Although the presence of LOCR was associated with altered expression of Rh antigens, its formal assignment to the Rh blood group system did not occur until haplotype and linkage analysis conducted in 2003 provided the necessary proof. The current study was undertaken in an attempt to define the underlying RH mutation in LOCR+ individuals. STUDY DESIGN AND METHODS: Genomic DNA from five unrelated LOCR+ individuals and three Rh-matched control individuals was amplified by polymerase chain reaction with intronic primers flanking all 10 exons of RH. Amplified products were separated on 1 percent agarose gels and isolated for DNA sequence analysis in both the forward and the reverse directions. RESULTS: DNA sequence analysis of the three LOCR+ D- individuals revealed a single heterozygous 286G>A nucleotide substitution resulting in a predicted Gly>Ser substitution at amino acid 96. DNA sequence analysis from the two LOCR+ D+ individuals revealed the identical mutation, as well as all of the changes associated with the common RHD gene. CONCLUSIONS: Based on our results, a Gly96Ser substitution in the Rhce polypeptide defines the low-incidence RBC antigen known as LOCR. This same amino acid change has previously been shown to be involved in the Rh:-26 phenotype, which suggests that LOCR and Rh26 are antithetical. Serologic investigations with various Rh:-26 cells and serum samples, however, reveal that only some c+ Rh:-26 phenotypes are LOCR+.

The RHCE allele ceCF: the molecular basis of Crawford (RH43).

BACKGROUND: The Crawford antigen (RH43) was described in 1980. It occurred in African American people, as a low-prevalence Rhesus antigen, who were also VS+. STUDY DESIGN AND METHODS: Twelve blood samples were analyzed because of inquiries into discrepant reactions in routine anti-D typing. The RHCE alleles were determined by nucleotide sequencing from genomic DNA. The D epitope profile was determined with 60 monoclonal anti-D. The population frequency was estimated in four major US regional blood centers. RESULTS: The novel RHce(W16C, Q233E, L245V) allele, dubbed ceCF, was found to be occurring in the cde haplotype as cause of the reactivity with the immunoglobulin M anti-D GAMA401. The ceCF phenotype expressed few D epitopes resembling but not matching the reaction patterns observed with other RhCE variants, like R0 (Har), ceRT, and ceSL. The frequency of the ceCF phenotype was 0.056 percent among African American persons and 0.007 percent in the general US population. CONCLUSION: The novel RHce(W16C, Q233E, L245V) allele, which is a variant of the known ce(s) allele, RHce(W16C, L245V), occurs in a haplotype with the RHD deletion and represents the molecular basis of the Crawford antigen.

SCER and SCAN: two novel high-prevalence antigens in the Scianna blood group system.

BACKGROUND: More than 20 years ago, two probands were described whose red blood cells (RBCs) typed Sc:1,-2,3. Their serum samples contained alloantibodies reactive with all RBCs tested except those of the Sc:-1,-2,-3 phenotype. Cloning of the Scianna gene allowed us to determine the molecular bases of these samples. STUDY DESIGN AND METHODS: In a collaborative effort, the two probands' samples and also two Sc:-1,-2,-3 samples were obtained from frozen storage. All 11 SC (ERMAP) exons and their flanking regions were sequenced. RESULTS: The two probands with antibodies to Scianna-related antigens were homozygous, respectively, for an ERMAP(R81Q) allele caused by a G to A substitution at nucleotide 242 in the ERMAP gene and for an ERMAP(H26Y,G35S) allele, in which the G35S substitution was caused by a G to A substitution at nucleotide 103. Two patients with the Sc:-1,-2,-3 phenotype both carried ERMAP(R332X) alleles caused by a C to T substitution at nucleotide 994 that differed at one nucleotide position in the noncoding region of exon 11. In eight samples carrying orphan low-prevalence antigens, no ERMAP variants were detected that could be implicated in Scianna antigen expression. CONCLUSION: SCER and SCAN expanded the Scianna blood group system to seven antigens, have been assigned the ISBT numbers 013.006 (Sc6) and 013.007 (Sc7), and were associated with ERMAP(R81Q) and ERMAP(G35S) proteins, respectively. ERMAP(R332X) is a second molecular basis for the Sc(null) phenotype. The eight low-prevalence antigens By, To(a), Pt(a), Re(a), Je(a), Li(a), SARA, and Sk(a) do not belong to the Scianna blood group system.