Nucleotide deletion in RHCE*cE (907delC) is responsible for a D- - haplotype in Hispanics.

BACKGROUND: Lack of Cc and Ee expression is associated with either hybrid alleles in which regions of RHCE are replaced by RHD or nucleotide deletion(s) in RHCE. The former have been found as D- - phenotypes, and the latter as Rh(null) when accompanied by deletion of RHD. We investigated RH in eight samples, three presenting as D- -, whose c-E- red blood cell (RBC) typing was discordant with the RHCE genotype that predicted c+E+. STUDY DESIGN AND METHODS: Serologic and molecular testing was performed by standard methods. CASES AND RESULTS: RBCs from Patient 1 were D+C-E-c+e+(w) but DNA testing predicted E+. RBCs from Patients 2, 3, and 4 typed as D+C-E-c-e- but DNA testing predicted c+E+. All had alloantibodies strongly reactive with all RBCs tested except D- - and Rh(null). Patient 5 had anti-c and anti-E but DNA testing predicted she was c+E+. RBCs from three donors typed D+C+E-c-e+ with DNA testing predicting c+E+. All had RHCE*cE with deletion of nucleotide 907C in Exon 6 predicted to cause a premature stop codon at Amino Acid 303 (Leu303Stop). HphI polymerase chain reaction-restriction fragment length polymorphism was used to confirm the deletion and to screen 100 Hispanic, 100 Caucasian, and 100 African American donor samples. One additional example was found. CONCLUSIONS: A novel allele, RHCE*cE 907delC (ISBT provisional designation RHCE*03N.02), silences c and E and in the homozygous state resulted in a D- - phenotype and production of anti-Rh17. All eight probands were Hispanic. The allele is associated with discrepant molecular typing, with an approximate frequency of 0.005 in Hispanics.

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).

The JAL antigen (RH48) is the result of a change in RHCE that encodes Arg114Trp.

BACKGROUND: The JAL antigen (Rh48) was discovered more than 30 years ago when it caused hemolytic disease of the fetus and newborn in an African American family. A decade later it was found to cause hemolytic disease of the fetus and newborn in a Caucasian family. The presence of the same low-prevalence antigen in two different ethnic groups is rare, but additional JAL+ in both groups was subsequently identified. This study was undertaken to investigate the RH gene(s) responsible for expression of JAL and to determine the structural relationship between JAL and other Rh antigens. STUDY DESIGN AND METHODS: Samples from 17 JAL+ people were included: 2 Caucasian, 6 African American, 7 African Brazilian, 1 Caribbean, and 1 Puerto Rican. RHCE and RHD were investigated at the genomic level, and Rh cDNAs were cloned and sequenced for some samples. RESULTS: Caucasian JAL+ probands had RHCE*Ce, while JAL+ probands with African ancestry had RHCE*ce, but all had a nucleotide 340C>T change in Exon 3 of RHCE predicted to encode Arg114Trp. The JAL-encoding RHCE*ce also had 733C>G (Leu245Val) and was linked to conventional RHD or to RHD*DAU0. CONCLUSIONS: JAL+ results from a nucleotide 340C>T (Arg114Trp) on either a Ce or ce background. Homology modeling of the JAL+ RhCE protein suggests that the Arg-->Trp change eliminates a critical loop-stabilizing H-bond between the side chain of Arg114 and the e-specific amino acid Ala226. Additionally, accommodation of the bulky tryptophan would disrupt the conformation of the extracellular loops containing C/c- and e-specific amino acids, providing a structural hypothesis for the simultaneous altered expression of C/c, e, and V/VS antigens.

A DOB allele encoding an amino acid substitution (Phe62Ser) resulting in a Dombrock null phenotype.

BACKGROUND: The gene polymorphisms responsible for the antigens Doa, Dob, Hy, and Joa in the Dombrock (Do) blood group system have been identified. Four different mutations have been reported to cause the Dombrock null [Gy(a-)] phenotype. These include splice mutations, an eight-nucleotide deletion, and insertion of a stop codon. Here a Dombrock null caused by a single-amino-acid substitution in the full-length protein is reported. STUDY DESIGN AND METHODS: DOA and DOB were determined by polymerase chain reaction-restriction fragment length polymorphism, and DO (ART4) exons and flanking regions were sequenced from genomic DNA. Expression analysis was performed by transfection of wild-type and mutant cDNAs into HEK 293T cells followed by flow cytometry and immunoblotting. Homology modeling was used to map the mutation on the protein structure. RESULTS: The patient's sample carried nt 793G/G, indicating a DOB/DOB background. Exon 2 sequencing showed the sample carried a new mutation, nt 185T>C, causing a Phe62Ser substitution. This variant Do was not expressed on the surface of transfected HEK 293T cells. The mutation maps to a highly conserved FDDQY motif located between the beta1-strand and alpha1-helix near the COOH terminus in the native molecule. CONCLUSIONS: The Dombrock null reported here is due to a single Phe62Ser mutation. The expression data confirmed that 62Ser is responsible for lack of cell surface Do, and protein modeling suggests the mutation disrupts important aromatic side chain interactions between Phe62 and His160. Production of an antibody to a high prevalence Dombrock antigen (anti-Gya) in this patient was consistent with complete absence of Dombrock/ART4 protein.