The highly variable RH locus in nonwhite persons hampers RHD zygosity determination but yields more insight into RH-related evolutionary events

BACKGROUND: Knowledge about paternal RHD hemior homozygosity is of clinical interest in alloinimunized pregnant women. D negativity in white persons is usually caused by deletion of the RHD gene. Recently, the physical structure of the RH locus and the mechanism causing the deletion of the RHD gene have been explored, enabling RHD zygosity determination in white persons by specific detection of a hybrid Rhesus box characteristic for the RHD- locus. STUDY DESIGN AND METHODS: RHD zygosity was determined in 402 samples from five different ethnic groups by polymerase chain reaction (PCR)-restriction fragment length polymorphism and by a newly developed real-time quantitative PCR. The Rhesus boxes of samples showing discrepancies between both tests were cycle sequenced. RESULTS: In nonwhite persons, several mutated Rhesus boxes exist that hamper zygosity determination by detection of the RHD- locus. Such mutated Rhesus boxes in D+ RHD homozygous black persons have a frequency of 0.22. In white persons, no mutated Rhesus boxes were encountered so far. CONCLUSIONS: Owing to the high frequency of the mutated Rhesus boxes, zygosity determination by detection of the RHD- locus is not feasible in nonwhite persons. The cosegregation of variant RHD genes (RHDV and (C)cde(s)) with specific mutated Rhesus boxes yields more insight into the evolutionary events concerning variant RHD genes and mutated Rhesus boxe

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)

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 cluste

Rapid genotyping of blood group antigens by multiplex polymerase chain reaction and DNA microarray hybridization

BACKGROUND: In the Netherlands, 500,000 blood donors are active. Blood of all donors is currently typed serologically for ABO, the Rh phenotype, and K. Only a subset of donors is typed twice for a larger set of red cell (RBC) and/or platelet (PLT) antigens. To increase the direct availability of typed RBCs and PLTs, a high-throughput technique is being developed to genotype the whole donor cohort for all clinically relevant RBC and PLT antigens. STUDY DESIGN AND METHODS: A multiplex polymerase chain reaction was developed to both amplify and fluorescently label 19 gene fragments of RBC and PLT antigens in one reaction. To test the setup of the genotyping method by microarray, a pilot study with human PLT antigen (HPA)-typed donor samples was performed. On each slide, 12 arrays are present containing 20 probes per PLT antigen system (28 for HPA-3). The allele-specific oligohybridization method was used to discriminate between two different alleles. RESULTS: Two blinded panels encompassing 94 donors were genotyped for HPA-1 through -5 and -15; no discrepancies were found compared to their serologic typing (HPA-1, -2, -3, -4, and -5) and genotyping (HPA-15; TaqMan, Applied Biosystems). CONCLUSION: This study shows that the HPA microarray provides a reliable and fast genotyping procedure. With further development an automated throughput for complete typing of large donor cohorts can be obtaine

The Rh complex exports ammonium from human red blood cells

The Rh blood group system represents a major immunodominant protein complex on red blood cells (RBC). Recently, the Rh homologues RhAG and RhCG were shown to promote ammonium ion transport in yeast. In this study, we showed that also in RBC the human Rh complex functions as an exporter of ammonium ions. We measured ammonium import during the incubation of RBC in a solution containing a radiolabelled analogue of NH4Cl (C-14-methyl-NH3Cl). Rh-null cells of the regulator type (expressing no Rh complex proteins) accumulated significantly higher levels (P = 0.05) of radiolabelled methyl-ammonium ions than normal RBC, at room temperature. Rh-null cells of the amorph type (expressing limited amounts of Rh complex proteins) accumulated an intermediate amount of methyl-ammonium ions. To show that decreased ammonium export contributes to its accumulation, the release of intracellular methyl-ammonium from the cells was measured over time. In 30 s, normal RBC released 87% of the intracellular methyl-ammonium ions, whereas Rh-null cells of the regulator type released only 46%. We conclude that the Rh complex is involved in the export of ammonium from RB