The 2 breakpoint regions of an RHCE-D(2-9)-CE allele causing a D phenotype and its RhAG antigen density

Among 195 known RHCE alleles, 10 have evolved to form hybrid RHCE-D-CE alleles encoding aberrant RhCE proteins lacking CE antigen expression. The resulting D-- phenotype predisposes to anti-Hr_o alloimmunization (anti-Rh17). Although hybrid alleles may appear identical at the mRNA level, distinct breakpoint configurations can result in diverse D phenotypes or Rh antigens. We determined the molecular structure of a D-- phenotype. An unrelated RHD allele, DMA had been observed once without serology. We tested Rh antigen densities.

Genomic DNA and cDNA were analyzed using a combination of molecular techniques and commercial red cell genotyping assays targeting the RHD and RHCE genes. A flow cytometric method was developed to quantify RhAG antigen expression. Red cell antigen densities were determined for RhAG and RhD in the D-- and DMA/DAU3 samples serologically.

Nucleotide sequencing of the RHCE gene and its cDNA identified a homozygous CE-D(2-9)-CE hybrid allele in an individual of Afghan origin. The 5' and 3' breakpoint regions included 131 and 4,287 nucleotides. The new allele was linked in a haplotype to the normal RHD allele (RHD*01). Quantitative flow cytometry demonstrated antigen densities of 92,297 RhD and 52,907 RhAG molecules per red cell for the D-- sample (12,465 RhD and 117,871 RhAG for DMA/DAU3).

We describe the breakpoint regions of a Ce-D(2-9)-Ce allele that encoded a D-- phenotype. Our results underscore the importance of breakpoint characterization for identifying clinically relevant RH variants and advancing personalized transfusion medicine. The second DMA observation, in trans to a DAU3 allele, aligned with a weak D phenotype for DMA.