Cheng-Han Huang

The Rh (Rhesus) genes encode a family of conserved proteins that share a structural fold of 12 transmembrane helices with members of the major facilitator superfamily. Interest in this family has arisen from the discovery of Rh factor’s involvement in hemolytic disease in the fetus and newborn, and of its homologs widely expressed in epithelial tissues. The Rh factor and Rh-associated glycoprotein (RhAG), with epithelial cousins RhBG and RhCG, form four subgroups conferring upon vertebrates a genealogical commonality. The past decade has heralded significant advances in understanding the phylogenetics, allelic diversity, crystal structure, and biological function of Rh proteins. This review describes recent progress on this family and the molecular insights gleaned from its gene evolution, membrane biology, and disease association. The focus is on its long evolutionary history and surprising structural conservation from prokaryotes to humans, pointing to the importance of its functional role, related to but distinct from ammonium transport proteins.

Rhesus (Rh) proteins share a conserved 12-transmembrane topology and specify a family of putative CO₂ channels found in diverse species from microbes to human, but their functional essentiality and physiological importance in metazoans is unknown. To address this key issue and analyze Rh-engaged physiologic processes, we sought to explore model organisms with fewer Rh genes yet are tractable to genetic manipulations. In this article, we describe the identification in nematodes of two Rh homologues that are highly conserved and similar to human Rh glycoproteins, and we focus on their characterization in Caenorhabditis elegans. RNA analysis revealed that CeRh1 is abundantly expressed in all developmental stages, with highest levels in adults, whereas CeRh2 shows a differential and much lower expression pattern. In transient expression in human cells, both CeRh1 and CeRh2-GFP fusion proteins were routed to the plasma membrane. Transgenic analysis with GFP or LacZ-fusion reporters showed that CeRh1 is mainly expressed in hypodermal tissue, although it is also in other cell types. Mutagenesis analysis using deletion constructs mapped a minimal promoter region driving CeRh1 gene expression. Although CeRh2 was dispensable, RNA interference with CeRh1 caused a lethal phenotype mainly affecting late stages of C. elegans embryonic development, which could be rescued by the CbRh1 homologue from the worm Caenorhabditis briggsae. Taken together, our data provide direct evidence for the essentiality of the CeRh1 gene in C. elegans, establishing a useful animal model for investigating CO₂ channel function by cross-species complementation.

BACKGROUND:  The rare phenotypes Dc‐ and D‐‐ lack the expression of E/e and CcEe antigens, respectively; their cotransmission in a single family has not been reported.STUDY DESIGN AND METHODS:  Six members of a Chinese family with two exhibiting the Dc‐ phenotype were studied using standard serologic methods. Rh genotypes were analyzed by Southern blot, and RH loci, by exon PCR. Rh transcripts were characterized by gene‐specific RT‐PCR and sequencing.RESULTS:  Although Rh typing detected two members as Dc‐ homozygotes, RFLP analysis and exon PCR showed them to be Dc‐ heterozygotes with a partial deletion of RHCE. cDNA sequencing showed the expression in the family of normal RHD and RHCe as well as hybrid transcripts, RHD(1‐9)/RHCE(10) and RHCE(1‐3)/RHD(4‐10). Thus, the Dc‐ members had the genotype of Dc‐/ D‐‐ and expressed both hybrid genes that were inherited from their parents, respectively.DISCUSSION:  This is the first demonstration in a family that the Dc‐ and D‐‐ complexes neither are linked with a normal RHD or RHCE gene. The segregation of these two different hybrid genes with single break points sug‐gests their independent genetic origin and provides molecular insights into the dynamic nature of genomic rearrangements leading to RH locus contraction.

BACKGROUND: Rh CcEe antigens occur as ce, Ce, cE, or CE alleles in the RBC membrane. Their epitope structures and the location of their cis interacting products remain to be defined.MATERIALS AND METHODS: A rare blood sample from a white male whose parents are first cousins was identified. Hemagglutination was performed using standard methods. RH structure and genotype was assessed by Southern blots. Rh transcripts were obtained by gene‐specific RT‐PCR and sequenced. The mutation was verified by genomic PCR assays.RESULTS: The donor's RBCs typed D+C–c+E–e–f(Rh6)– with a normal c dose, suggesting the Dc– phenotype. Further tests revealed a weak and qualitatively altered e expression. Southern blots indicated a genotype of Dce/dce without other gross changes. RT‐PCR detected a triplet deletion (Δ685AGA687) in the Rhce gene that specifies codon 229 for arginine (Arg229). Sequencing of the region around the mutated exon 5 confirmed the donor to be homozygous for the AGA deletion.DISCUSSION: Arg229 is invariant on external loop 4 and close to the Ala226Pro change specific for e/E polymorphism. The qualitative and quantitative alteration of e antigen defines Arg229 as a crucial component for e/E epitope presentation. Given a normal dose of c antigen, the disruption of f (Rh6) by Arg229 deletion suggests that external loop 4 is a major structural element contributing to the expression of RHCE cis interacting antigenic products.