The proximal cis-regulatory region of the RHD/RHCE promoter is 105 bp and contains a 55-bp core devoid of known binding motifs but necessary for transcription.

BACKGROUND: The RhD and RhCE polypeptides are erythroid-specific members of the RH gene family. Little is known about the promoter cis-regulatory proximal region responsible for transcription. STUDY DESIGN AND METHODS: The 1246-bp 5'-flanking regions of the RHD and RHCE promoter were amplified and ligated to a luciferase reporter vector and erythroid-specific transcription was evaluated in K562, HEL, U937, and HeLa cell lines. Deletion and substitution promoter-reporter constructs were generated to define the minimal cis-regulatory region. RESULTS: Deletion analysis in K562 cells revealed that the cis-regulatory region extended to a position between -78 and -120 relative to the site of initiation of transcription. Electrophoretic mobility shift assays confirmed binding motifs for Sp1, GATA-1, and E2F transcription factors. The use of 15-bp substitution mutagenesis showed that the minimal region required for transcription extended to -105 bp, and 6-bp substitution mutants from -35 to -90 identified a region necessary for transcription yet devoid of known cis-regulatory binding motifs. CONCLUSION: The proximal RHD/RHCE promoter regions contain cis-regulatory binding motifs and an internal sequence-dependent region that together regulate transcription. The results suggest that this region may be relevant in the weak expression of RhD.

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. STUDY DESIGN AND 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. CONCLUSION: 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.