Alteration of RH gene structure and expression in human dCCee and DCW-red blood cells: Phenotypic homozygosity versus genotypic heterozygosity

This report describes a comparative study on the dCCee and DCW- red blood cells devoid of RhD and CcEe antigens, respectively. Southern blots showed that the two variants carried opposite deletions in the D and non-D (CcEe) genes, Rh haplotyping and exon polymerase chain reaction (PCR) assay indicated that the deletions did not extend beyond the 5' region upstream from exon 1 or the 3' region downstream from exon 10 of the respective genes, This was confirmed by finding intact promoters and 3' untranslated regions in both D and non-D genes in each variant, Reverse transcriptase-PCR and cDNA sequencing showed the expression of two transcripts in each cell type, In dCCee cells, one transcript was the regular Ce form and the other occurred as a D-Ce-D hybrid whose Ce sequence spanned exons 2 through 9. In DCW- cells, the two transcripts were derived from reversely arranged hybrid genes, ie, the CW-D gene was formed by fusion of C-W axon 1 with D exons 2 through 10, whereas the reverse product was formed by fusion of D exons 1 through 9 with non-D exon 10, These results indicated that DNA deletion and recombination had occurred in either cis or trans configuration and involved both RH loci in the dCCee or DCW- genome, Identification of such compound alterations correlates the genotypes with phenotypes and explains the lost Rh antigenic expression. A reinvestigation of gene organization also led to the reassignment of several 5' and 3' splice sites, Together, this study not only shows the complexity of Rh phenotypic diversity, but also points to the importance of concurrent analysis of genomic structure and transcript expression in deciphering the underlying genetic mechanisms. (C) 1996 by The American Society of Hematology.