606 TRANSFUSION Volume 46, April 2006 Blackwell Publishing IncMalden, USATRFTransfusion0041-11322006 American Association of Blood BanksApril 2006464606615Original Article RHD(T201R, F223V) CLUSTER ANALYSISGROOTKERK-TAX ET AL. ABBREVIATIONS: MPX = multiplex; PCR-SSP = sequence- specific polymerase chain reaction. From Sanquin Research, Amsterdam; Landsteiner Laboratory and the Department of Hematology, Academic Medical Center, University of Amsterdam, Amsterdam; the Department of Research and Development, Sanquin Blood Bank Southwest Region, Rotterdam; Sanquin Diagnostics, Amsterdam; and the Department of Hematology, Erasmus Medical Center, Erasmus University, Rotterdam. Address reprint request to: Martine G.H.M. Grootkerk-Tax, Sanquin Blood Bank Southwest Region, Wytemaweg 10, 3015 CN Rotterdam, the Netherlands; E-mail: Martine.Grootkerk@ bloodrtd.nl. This research was supported by the Netherlands Organization for Health, Research and Development, Grant 21000031. Received for publication May 25, 2005; revision received August 16, 2005, and accepted August 16, 2005. doi: 10.1111/j.1537-2995.2006.00759.x TRANSFUSION 2006;46:606-615. IMMUNOHEMATOLOGY RHD(T201R, F223V) cluster analysis in five different ethnic groups and serologic characterization of a new Ethiopian variant DARE, the DIII type 6, and the RHD(F223V ) Martine G.H.M. Grootkerk-Tax, Joyce D. van Wintershoven, Peter C. Ligthart, Dick J. van Rhenen, C. Ellen van der Schoot, and Petra A. Maaskant-van Wijk BACKGROUND: The RHD phylogeny in humans shows four main clusters of which three are predominantly observed in (African) black persons. Each of the African clusters is characterized by specific amino acid substitutions relative to the Eurasian RHD allele. RH phylogeny defines the framework for identification of clinically relevant aberrant alleles. This study focuses on the weak D type 4 cluster (characterized by RHD(T201R, F223V) (602C>G 667T>G)) in five ethnic groups. STUDY DESIGN AND METHODS: A total of 1702 samples were screened for the presence of 602C>G and 667T>G by sequence-specific polymerase chain reaction (PCR-SSP). Eighty samples were assigned to the weak D type 4 cluster and were molecularly characterized by PCR-SSP and RHD sequencing. Antigens of aberrant alleles were characterized with monoclonal anti-D according to the 37-epitope model when possible. RESULTS: Five new aberrant alleles, DIII type 6, DIII type 7, DARE, RHD(T201R, F223V) (without 819G>A), and RHD(F223V), were identified and DIII type 6, DARE, and RHD(F223V) were serologically characterized with monoclonal anti-D. Both the DARE and RHD(F223V) showed epitope loss. It is postulated that the 1136C>T nucleotide substitution (characteristic for the DAU allele cluster) is present on the DVa(KOU) allele. CONCLUSION: Identification of the new variant alleles refines the phylogeny of RHD in humans. The proposed DVa(KOU) allele with 1136C>T (DVa(KOU)T379M) is probably caused by conversion of the DAU0 allele and the DVa(KOU) allele, forming a phylogenetic link between the DV allele and the DAU cluster. By describing the RHD(F223V) (602C>G) and RHD(T201R, F223V) (602C>G and 667T>G) alleles formal proof is given for the origin of the non-Eurasian cluster. he RhD blood group antigen is a highly immu- nogenic polypeptide present on the red blood cell (RBC) membrane in D + individuals. In transfusion medicine, D mismatch may cause hemolytic transfusion reactions and hemolytic disease of the fetus and newborn and Rh antibodies are often involved in autoimmune hemolytic anemia. The RhD protein is encoded by the RHD gene (chromosome 1, p34.3-p36.1). D negativity in Caucasian persons is mostly caused by a deletion of the RHD gene. 1 In black persons, D negativity is frequently caused by RHD ψ and (C)cde s alleles (allele frequencies of 0.0714 and 0.036, respectively). 2-4 Both alleles carry RHD-specific sequences and therefore hamper RHD genotyping strat- egies. Variant D antigens encoded by aberrant RHD alle- les have been found with high frequencies in black persons and, with much lower frequencies, in white per- sons. 5-7 These aberrant RHD alleles are mainly caused by T