Novel 3'Rhesus box sequences confound RHD zygosity assignment.
BACKGROUND
Paternal RHD zygosity is required for genetic counseling and management of HDN caused by anti-D. The most common D- haplotype is due to the deletion of RHD, which results in the formation of a hybrid Rhesus box, theoretically through the recombination of 5' and 3'Rhesus boxes.


METHODS
The validity of Rhesus box PstI analysis was assessed to determine RHD zygosity by correlating D phenotype, most probable genotype, and Rhesus box PCR-RFLP. RHD exons were examined, and a 501-bp Rhesus box fragment was sequenced that flanked the polymorphic PstI site.


RESULTS
Rhesus box analysis and the most probable genotype differed for 60 of 200 of the samples (30%). The incorrect zygosity assignment by the most probable genotype method is the likely reason for the difference. However, 8 of 328 samples showed Rhesus box copy numbers that were inconsistent with the D phenotype. Two D- samples with one hybrid Rhesus box had a nonfunctional RHD. Six D+ samples appeared to have two copies of the hybrid Rhesus box due to novel 3'Rhesus boxes that contained nucleotide polymorphisms previously assigned to the 5' and hybrid Rhesus boxes. All eight samples were from people of black descent, as determined by the GATA-1 silencing mutation at the FY locus.


CONCLUSIONS
Rhesus box PCR-RFLP analysis for RHD zygosity assignment is confounded by the presence of nonfunctional RHD+ (2.3% of D-) and novel, low frequency (0.9% of all alleles) 3'Rhesus box sequences.
Matheson
Kimberly A
KA
Canadian Blood Services, Research and Development; Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.
Denomme
Gregory A
GA
D020413:3' Untranslated Regions / Q000235:genetics*; D001483:Base Sequence; D044383:Black People / Q000235:genetics; D004247:DNA / Q000235:genetics; D005091:Exons / Q000235:genetics; D005838:Genotype; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D011544:Pseudogenes; D011993:Recombinant Fusion Proteins; D012204:Rh-Hr Blood-Group System / Q000235:genetics*; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid
D020413:3' Untranslated Regions; D015514:Oncogene Proteins, Fusion; D011993:Recombinant Fusion Proteins; D012204:Rh-Hr Blood-Group System; C117760:RhD fusion protein, human; C026410:Rho(D) antigen; D004247:DNA