Hereditary Stomatocytosis Associated with a Loss of Function Mutation In Rh-Associated Glycoprotein (RhAG)

Abstract 2040

The erythroid Rh family of proteins includes RhCE and RhD which carry the common Rh antigens, and the related Rh-associated glycoprotein, RhAG. RhAG is required for trafficking of the blood group proteins to the membrane and forms the core of a macro-complex in the membrane which includes glycophorin B, Band 3, CD47, and LW. The Rh proteins are structurally and functionally related to the Amt superfamily of NH₃/NH₄⁺ transport proteins, and RhAG and its nonerythroid paralogs, RhCG and RhBG, have been shown to mediate NH₃/NH₄⁺ transport. RhCG is responsible for part of renal collecting duct epithelial cell NH₃/NH₄⁺ secretion, and Rhcg^-/- mice exhibit incomplete distal renal tubular acidosis due to impaired urinary NH₄⁺ excretion. The Rhag^-/- mouse is grossly normal, and the significance of RhAG-mediated NH₃/NH₄⁺ transport in human erythrocytes remains unclear.

Over-hydrated hereditary stomatocytosis (OHSt) is a rare dominant disorder characterized by moderate hemolytic anemia, increased mean red cell volumes, stomatocytes and echinocytes, and increased red cell permeability to the monovalent cations, Na⁺ and K⁺. Six of the seven OHSt kindred studied by Bruce et al. (Blood. 2009;113:1350) displayed a heterozygous Phe65Ser mutation in RhAG. Expression studies of the mutant 65Ser-RhAG in Xenopus oocytes induced a monovalent cation flux compatible with the cation leak seen in RBCs. The increased Na⁺ and decreased K⁺ contents of mutant RhAG-expressing oocytes suggested that F65S is a gain-of-function mutation that opens a cation leak, likely within the RhAG polypeptide.

In this study the ammonia transport properties of the OHSt mutant 65Ser-RhAG were investigated. Xenopus oocytes were injected with cRNA encoding wild-type RhAG, the OHSt mutant 65Ser-RhAG, and 65Val-RhAG, an engineered mutation with a smaller hydrophobic side chain at position 65. Wild-type and mutant RhAG polypeptides were well-expressed in the oocyte membrane as measured by quantitative immunoblotting. Uptake of the NH₃/NH₄⁺ substrate analog ¹⁴C-methylammonium (MA), was assayed in oocytes previously injected with water (control) or with cRNA. Expression of wild-type RhAG mediated MA uptake at rates 6-fold greater than that of water-injected controls. Uptake of MA by oocytes expressing 65Val-RhAG was equivalent to that of wild type RhAG. However, MA uptake by oocytes expressing OHSt mutant 65Ser-RhAG was greatly reduced to less than 20% that of oocytes expressing wild-type RHAG or 65Val-RhAG, and was only 1.5-fold greater than that of water-injected control oocytes. Co-expression with other, individual Rh complex members glycophorin B, RhD, RhCE, or Band 3 did not alter MA-mediated uptake by RhAG-expressing oocytes.

Importantly, this study reveals that the RhAG mutation Phe65Ser found in patients with type 1 over-hydrated stomatocytosis is a loss of function mutation. Further study is required to define the relationship between loss of NH₃/NH₄⁺ transport and erythrocyte Na⁺ and K⁺ cation content.