Rh50 Glycoprotein Gene and Rhnull Disease: A Silent Splice Donor Is trans to a Gly279→Glu Missense Mutation in the Conserved Transmembrane Segment

Rh null disease includes the amorph and regulator types that are thought to result from homozygous mutations at the RH30 and RH50 loci, respectively. Here we report an unusual regulator Rh null where two G→A nucleotide (nt) transitions occurred in trans , targeting different regions of the two copies of Rh50 gene. The nt 836 G→A mutation was a missense change located in exon 6; it converted Gly into Glu at position 279, a central amino acid of the transmembrane segment 9 (TM9). While cDNA analysis showed expression of the 836A(Glu 279 ) allele only, genomic studies showed the presence of both 836A(Glu 279 ) and 836G(Gly 279 ) alleles. A detailed analysis of gene organization led to the identification in the Rh50(836G) allele of a defective donor splice site, caused by a G→A mutation in the invariant GT element of intron 1. This is the first known example of such mutations that has apparently abolished the functional splicing of a pre-mRNA encoding a multipass integral membrane protein. With a silent phenotypic copy in trans , the negatively charged Glu 279 residue may disrupt TM9 and impair the interaction of the missense protein with Rh30 polypeptides. To evaluate the significance of the mutation, we took a comparative genomic approach and identified Rh50 homologues in different species. We found that Gly 279 is a conserved residue and its adjacent amino acid sequence is identical from Caenorhabditis elegans to human. These findings provide new insight into the diversity of Rh null disease and suggest that the C-terminal region of Rh50 may also participate in protein-protein interactions involving Rh complex formation. © 1998 by The American Society of Hematology.