Connie M. Westhoff

BACKGROUND: The partial D phenotype DIIIa was originally reported to be associated with 455A>C in Exon 3, 602C>G in Exon 4, and 667T>G in Exon 5. Other alleles with these changes were subsequently identified and designated DIII Types 5, 6, and 7, as they had additional alterations. The observation that DNA samples associated with the DIIIa phenotype had more changes than those originally reported motivated us to reanalyze the DIIIa probands (BP and DJ) from the original study. We also studied additional DIIIa samples to clarify the RHD background and establish the associated RHCE.STUDY DESIGN AND METHODS: Hemagglutination testing was performed by standard methods. RHD and RHCE were analyzed by combinations of polymerase chain reaction–restriction fragment length polymorphism, exon‐specific sequencing, cloning, or direct sequencing of Rh‐cDNAs.RESULTS: The RHD alleles from BP, DJ, and 58 additional DIIIa samples had the three reported nucleotide changes as well as 186G>T, 410C>T, and 819G>A. The DIIIa allele was associated with several altered RHCE*ce‐alleles, the prominent one being ceS (48C, 733G, 1006T).CONCLUSION: The DIIIa phenotype is associated with six RHD changes, five of which encode amino acid changes, and partial DIIIa and DIII Type 5 are encoded by the same RHD allele. In all samples, RHD*DIIIa was inherited with altered RHCE*ce. Patients with partial DIIIa are at risk for production of alloanti‐D, but they are also at risk for alloanti‐e, ‐c, or antibodies to high‐prevalence Rh antigens if there is no conventional RHCE*ce in trans. Among 39 patients studied, 16 had alloanti‐D and 27 had alloanti‐e or anti‐hrB.

The erythrocyte Rh-associated glycoprotein (RhAG) was recently found to mediate transport of ammonia/ammonium when expressed in Xenopus laevis oocytes and yeast Saccharomyces cerevisiae. Nonerythroid homologs, RhBG and RhCG, are expressed in the mammalian kidney connecting segment and the collecting duct, major sites of urinary ammonia secretion. This study characterizes the transport properties of murine RhBG and RhCG by ammonium analog [¹⁴C]methylamine (MA) uptake and two-electrode voltage clamping of X. laevis oocytes. Both RhBG and RhCG mediated transport of ammonia, but differed in affinity for substrate ( K_m= 2.5 and 10 mM, respectively). The rates of RhBG- and RhCG-mediated transport were sensitive to the concentration of the protonated MA species and were stimulated by extracellular alkalosis and inhibited by acidosis, suggesting a role for H⁺in the transport process. Whereas expression of RhBG or RhCG caused a small increase in plasma membrane conductance, [¹⁴C]MA uptake was not affected by depolarization of oocytes with 100 mM extracellular K⁺or by clamping the membrane potential between 0 and −100 mV, indicating that RhBG- and RhCG-mediated transport was independent of the membrane potential. These results strongly suggest that RhBG and RhCG transport ammonia by an electroneutral process that involves NH₄⁺/H⁺exchange resulting in net NH₃translocation. The polarized localization of RhBG and RhCG in kidney tubules and the different substrate affinities may enable these proteins to participate in transepithelial ammonia secretion and to therefore play an important role in whole animal acid-base regulation.

Serological observations have suggested that numerous D, many e (especially in Blacks), several E, and rare c variants exist within the Rh blood group system. The molecular basis for expression of many of these variants has been elucidated. This study describes five unrelated Caucasians whose red blood cells reacted with polyclonal anti‐e but did not react with some monoclonal anti‐e, which suggested that they carried a variant e antigen. Molecular investigation revealed the presence of a 48G→C change (encoding cysteine instead of tryptophan at amino acid 16) in their RHce gene. No other differences were found, which suggests that amino acid residues located in the first transmembrane region can affect expression of the e antigen, whose critical residues are on the predicted fourth external loop of the protein. This polymorphism has not previously been observed because polyclonal anti‐e does not distinguish this variant from wild type. This position is polymorphic in RHce alleles and the presence of the 48C nucleotide is often found in the R₀ (Dce) haplotype.

To investigate the evolution of the Rh blood-group system in anthropoid apes, New and Old World monkeys, and nonprimate animals, serologic typing of erythrocytes from these species with antibodies specific for the human Rh blood-group antigens was performed. In addition, genomic DNA from these animals was analyzed on Southern blots with a human Rh-specific cDNA. Consistent with earlier reports, serologic results showed that gorilla and chimpanzee erythrocytes had epitopes recognized by human Rh D and c antisera, and gibbon erythrocytes were recognized by the c antisera. Surprisingly, some Old and New World monkeys also expressed a Rh c epitope on their erythrocytes. No erythrocytes from the nonprimate animals reacted specifically with any of the human Rh antisera. Southern blot analysis with a human Rh-specific cDNA probe detected Rh-related sequences in anthropoid apes, all New and Old World monkeys, and in most nonprimate animals tested. Although some Rh-related restriction fragments were conserved across species lines in primates, the Rh locus was more polymorphic in chimpanzees and gorillas than in humans. In addition, restriction fragments segregating with the presence of the D antigen in humans were present in the primate species that expressed the D antigen.