Volume 44, March 2004 TRANSFUSION 391 Blackwell Science, LtdOxford, UKTRFTransfusion0041-11322004 American Association of Blood BanksMarch 2004443Original Article ALTERED E ANTIGEN EXPRESSION BY ARG229 DELETIONCHEN ET AL. ABBREVIATIONS: Arg 229 = codon 229 for arginine; BP = Brazilian propositus; TM = transmembrane. From the Biochemistry and Molecular Genetics Laboratory and the Immunohematology Laboratory, New York Blood Center, New York, New York; Fundazao Pro, Sangue/Hemocentro de Sao Paulo, Sao Paulo, Brazil. Address reprint requests to: Cheng-Han Huang, MD, PhD, Laboratory of Biochemistry and Molecular Genetics, New York Blood Center, 310 East 67th Street, New York, NY 10021; e-mail: chuang@nybloodcenter.org. The work was funded by HL66274 and DK62704 (to C-HH) and HL54459 (to MER) from the National Institutes of Health. Received for publication June 26, 2003; revision received September 26, 2003, and accepted October 2, 2003. TRANSFUSION 2004;44:391-398. IMMUNOHEMATOLOGY Deletion of arginine codon 229 in the Rhce gene alters e and f but not c antigen expression Y.X. Chen, J. Peng, M. Novaretti, M.E. Reid, and C.-H. Huang BACKGROUND: Rh CcEe antigens occur as ce, Ce, cE, or CE alleles in the RBC membrane. Their epitope structures and the location of their cis interacting products remain to be defined. MATERIALS AND METHODS: A rare blood sample from a white male whose parents are first cousins was identified. Hemagglutination was performed using standard methods. RH structure and genotype was assessed by Southern blots. Rh transcripts were obtained by gene-specific RT-PCR and sequenced. The mutation was verified by genomic PCR assays. RESULTS: The donor’s RBCs typed D+C–c+E–e– f(Rh6)– with a normal c dose, suggesting the Dc– phenotype. Further tests revealed a weak and qualitatively altered e expression. Southern blots indicated a genotype of Dce/dce without other gross changes. RT-PCR detected a triplet deletion (D685AGA687) in the Rhce gene that specifies codon 229 for arginine (Arg229). Sequencing of the region around the mutated exon 5 confirmed the donor to be homozygous for the AGA deletion. DISCUSSION: Arg229 is invariant on external loop 4 and close to the Ala226Pro change specific for e/E polymorphism. The qualitative and quantitative alteration of e antigen defines Arg229 as a crucial component for e/E epitope presentation. Given a normal dose of c antigen, the disruption of f (Rh6) by Arg229 deletion suggests that external loop 4 is a major structural element contributing to the expression of RHCE cis interacting antigenic products. and CcEe are the common antigens of the human Rh blood group system but are diver- sified with a large number of genetic polymor- phisms at the level of populations. 1,2 They can stimulate allo- or autoantibody production, which causes HDN, transfusion reaction or autoimmune hemolytic anemia. 3 As highly similar polytopic transmembrane (TM) proteins of the RBC, RhD and RhCE differ only by 35 amino acids and are encoded respectively by RHD and RHCE, a linked locus on the short arm of chromosome 1. 4-6 Although RHD appears as a more recent duplicate, RHCE occurs in four allelic forms as ce, cE, Ce, or CE. 7 The surface presentation of Rh antigens as a whole also requires a coordinate expression and physical association with RhAG, the product of a homologous gene, on chro- mosome 6. 8 Defining the sequence context and spatial orga- nization of Rh epitope structures (epitope mapping) is fundamental to understanding their nature as potent immunogens causing hemolytic reactions. Approaches including molecular analysis of natural Rh variant phe- notypes, 1,2 ex vivo expression of site-directed mutants, 9 and phage display 10 have been used to gain insight into D