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The ammonia/ammonium (NH3/NH4+, AM) concentration in human erythrocytes (RBCs) is significantly higher than in plasma. Two main possible mechanisms for AM transport, including simple and facilitated diffusion, are described; however, the driving force for AM transport is not yet fully characterized. Since the erythroid ammonium channel RhAG forms a structural unit with anion exchanger 1 (eAE1) within the ankyrin core complex, we hypothesized the involvement of eAE1 in AM transport. To evaluate the functional interaction between eAE1 and RhAG, we used a unique feature of RBCs to swell and lyse in isotonic NH4+ buffer. The kinetics of cell swelling and lysis were analyzed by flow cytometry and an original laser diffraction method, adapted for accurate volume sensing. The eAE1 role was revealed according to (i) the changes in cell swelling and lysis kinetics, and (ii) changes in intracellular pH, triggered by eAE1 inhibition or the modulation of eAE1 main ligand concentrations (Cl− and HCO3−). Additionally, the AM import kinetics was analyzed enzymatically and colorimetrically. In NH4+ buffer, RBCs concentration-dependently swelled and lysed when [NH4+] exceeded 100 mM. Cell swelling and hemolysis were tightly regulated by chloride concentration. The complete substitution of chloride with glutamate prevented NH4+-induced cell swelling and hemolysis, and the restoration of [Cl−] dose-dependently amplified the rates of RBC swelling and lysis and the percentage of hemolyzed cells. Similarly, eAE1 inhibition impeded cell swelling and completely prevented hemolysis. Accordingly, eAE1 inhibition, or a lack of chloride anions in the buffer, significantly decreased NH4+ import. Our data indicate that the eAE1-mediated chloride gradient is required for AM transport. Taken together, our data reveal a new player in AM transport in RBCs. [ABSTRACT FROM AUTHOR]

Background: The Rh system is an extremely important RBC antigen system with over 50 antigens, 5 of which (D, C, E, c and e) are considered most clinically significant. The rare Rhnull phenotype can result from mutations in the RHD and RHCE genes or the RHAG gene that affects their expression. This is a case report of the second type. Case Report: This case reports a multiparous lady who had to be evaluated for a panreactive antibody. The discrepancy was first identified at the centre she reported to. A thorough immunohematological workup was performed at a second reference laboratory. Suspecting Rhnull phenotype, a third referral (molecular typing) was requested at International Blood Group Reference Laboratory (IBGRL), Bristol. Results: A novel RHAG null allele (c.1138+2t>a), causing a Rhnull phenotype was identified. The antibody was most likely an anti‐Rh 29 antibody. Conclusion: The novel c.1138+2 t > a mutation in the RHAG gene causing the Rhnull phenotype and development of a pan reacting antibody(ies) made the patient's pregnancy challenging. Confirmation of the diagnosis, an important step in her management, required use of both serological immunohematology and molecular techniques. [ABSTRACT FROM AUTHOR]

Background: We recently encountered a Rhnull phenotype proband within one family in the Chinese population. Rhnull is a rare autosomal recessive disorder characterized by the absence of the Rh antigens on the erythrocyte membrane, resulting in chronic hemolytic anemia. This study described the serological and molecular analysis of a Chinese Rhnull proband and his immediate family. Methods: Red blood cells antigen phenotyping and antibody screening/identification were conducted. RHD, RHCE, and RHAG were analyzed using genomic DNA by polymerase chain reaction and sequence analysis. Results: Serologic tests showed a D–C–E–c–e– phenotype in the proband associated with the suspicion of anti‐Rh29 (titer 16). Molecular analyses showed a new mutation (c.406dupA) in exon 3 of RHAG. This duplication introduced a reading frameshift (p.Thr136AsnfsTer21). The RHAG mutation was found in the homozygous state for the proband and heterozygous state for his parents. Conclusion: We identified a novel RHAG mutation resulting in the Rhnull phenotype of the regulator type. Inheritance of the novel allele was shown by family study. [ABSTRACT FROM AUTHOR]

Background and Objectives: A newborn presented with jaundice in Thailand. The cord red cells tested positive by direct antiglobulin test (DAT) for an unknown maternal red cell antibody. Initial blood group sequencing suggested that the infant carried a novel variant RHAG c.140T>C, responsible for a low‐prevalence antigen in the RHAG blood group system (ISBT 030). We report here on testing of samples from the infant's parents and older sibling to define a new antigen in the RHAG system. Materials and Methods: Massive parallel sequencing (MPS) using a custom‐designed panel was performed on all four family members. Extended serological testing was also performed to determine whether family members with the same variant as the infant showed reactivity with the antibody in the maternal plasma. Results: We identified a novel single nucleotide variant (SNV) (RHAG c.140T>C, p.[Phe47Ser]) in samples from three of the four family members tested (the infant, the older sibling and the father). The variant was not detected in the mother's sample. Maternal plasma showed positive agglutination with all family members tested; however, when tested with routine panel cells, no reactivity was observed. Conclusion: This case study showed that the presence of the novel variant (RHAG c.140T>C), encoding a p.(Phe47Ser) change in the RhAG glycoprotein, was the apparent cause of incompatibility between maternal plasma and that of red cells from the proband, father and older sibling of the proband. We propose this variant to be a new low‐prevalence antigen in the RHAG blood group system. [ABSTRACT FROM AUTHOR]

Objective To study the blood group serology and molecular biology of patients with RhD--, so as to guide clinical blood use. Methods The EDTA-K2 anticoagulant blood of the patient was detected for Rh antigens and antibodies. Meanwhile, DNA was extracted, and the 1-10 exon of RHCE and RHAG gene was sequenced by Sanger sequencing. Results The serological test showed O type RhD--, and all spectral cells were positive. RHCE gene sequencing showed RHCE*02/RHCE*02, RHAG gene sequencing showed mutations at site 808 G > A and site 861 G > A of exon 6. Conclusion When patients were with RhD--, and related immune conditions such as pregnancy and/or transfusion history were present, autologous blood transfusion or plasma exchange could be an option for emergency blood use.

Background and Objectives: The extremely rare Rhnull phenotype is characterized by the absence of all Rh antigens on erythrocytes. It is divided into the regulator and amorph types based on the underlying genetic background. The more common regulator type depends on critical variants silencing RHAG, which encodes RhAG glycoprotein, necessary for RhD/RhCE expression. Rhnull cells have altered expression of glycophorin B and LW glycoprotein. Materials and Methods: Four unrelated Rhnull individuals were investigated. Serological testing was performed according to standard blood bank practice. RHD/RHCE and S/s allele‐specific Polymerase chain reaction (PCR) genotyping was done on genomic DNA using in‐house PCR assays. RHAG, and in some cases also RHD/RHCE, were sequenced. Initial s phenotyping results triggered additional serological investigation. Results: Anti‐Rh29 was identified in all four individuals. Extended typing with anti‐S and anti‐s showed that the three samples predicted to type as s+ failed to react with 2 of 5 anti‐s. Sequence analysis of all 10 RHAG exons and the immediate intron/exon boundaries revealed a single nucleotide variant in the 3′‐end of intron 6, c.946 −2a>g in all samples. RHD/RHCE showed no alterations. Conclusion: A novel Nordic Rhnull allele was identified. In addition, it was shown that s+ Rhnull red blood cells are not only U− but also have qualitative changes in their s antigen expression. [ABSTRACT FROM AUTHOR]

Objective A 77 year old female was admitted with a subdural hematoma requiring 1 unit of apheresis platelets. She was a study subject in the 1960s and was found to be Rhnull, along with another individual who previously served as a directed donor for her. Methods Serologic testing performed by the immunohematology reference laboratory (IRL) confirmed that the patient was Rhnull and expressed anti-Rh29 antibodies. While searching for red blood cells (RBCs) for possible transfusion, it was discovered that the individual from the original study had recently donated an autologous unit. Results The IRL discovered that the donor's antigen typing was r'r'. Testing had been performed using a molecular human erythrocyte antigen BeadChip (HBC). Due to the discrepancy between current and historical testing results, a donor segment was thawed and by tube testing confirmed to be Rhnull. A limitation of HBC is that many null phenotypes will be missed. Conclusion This case demonstrated that Rhnull evaluation of the donor required both serological and molecular methods. [ABSTRACT FROM AUTHOR]

Background: The Rh system is an extremely important RBC antigen system with over 50 antigens, 5 of which (D, C, E, c and e) are considered most clinically significant. The rare Rhnull phenotype can result from mutations in the RHD and RHCE genes or the RHAG gene that affects their expression. This is a case report of the second type., Case Report: This case reports a multiparous lady who had to be evaluated for a panreactive antibody. The discrepancy was first identified at the centre she reported to. A thorough immunohematological workup was performed at a second reference laboratory. Suspecting Rhnull phenotype, a third referral (molecular typing) was requested at International Blood Group Reference Laboratory (IBGRL), Bristol., Results: A novel RHAG null allele (c.1138+2t>a), causing a Rhnull phenotype was identified. The antibody was most likely an anti-Rh 29 antibody., Conclusion: The novel c.1138+2 t > a mutation in the RHAG gene causing the Rhnull phenotype and development of a pan reacting antibody(ies) made the patient's pregnancy challenging. Confirmation of the diagnosis, an important step in her management, required use of both serological immunohematology and molecular techniques., (© 2024 British Blood Transfusion Society.)

Background: We recently encountered a Rh null phenotype proband within one family in the Chinese population. Rh null is a rare autosomal recessive disorder characterized by the absence of the Rh antigens on the erythrocyte membrane, resulting in chronic hemolytic anemia. This study described the serological and molecular analysis of a Chinese Rh null proband and his immediate family., Methods: Red blood cells antigen phenotyping and antibody screening/identification were conducted. RHD, RHCE, and RHAG were analyzed using genomic DNA by polymerase chain reaction and sequence analysis., Results: Serologic tests showed a D-C-E-c-e- phenotype in the proband associated with the suspicion of anti-Rh29 (titer 16). Molecular analyses showed a new mutation (c.406dupA) in exon 3 of RHAG. This duplication introduced a reading frameshift (p.Thr136AsnfsTer21). The RHAG mutation was found in the homozygous state for the proband and heterozygous state for his parents., Conclusion: We identified a novel RHAG mutation resulting in the Rh null phenotype of the regulator type. Inheritance of the novel allele was shown by family study., (© 2024 AABB.)

There are two types of Rh SB null sb phenotype: (1) Amorph type: caused by genetic variations affecting I RHD i and I RHCE i ; or (2) Regulator type: secondary to variants in I RHAG i , which encodes Rh-associated glycoprotein (RhAG).1 A functional RhAG is necessary for the expression of Rh antigens and for the proper assembly of Rh proteins on red blood cell membrane. Keywords: RH; RHAG; RHnull EN RH RHAG RHnull E62 E64 3 09/16/21 20210901 NES 210901 BRIEF BACKGROUND Rh SB null sb is a rare inherited condition characterized by the absence of Rh antigens on red blood cell surface causing abnormalities on erythrocyte morphology, commonly stomatocytosis and mild hemolytic anemia. [Extracted from the article]

SUMMARY Aims/Objectives: We aimed to analyse the molecular backgrounds and red blood cell (RBC) antigen expression of a male blood donor with Rhmod phenotype and his family members. Background: Rh deficiency phenotypes are rarely found worldwide and are characterised by the lack of Rh antigen expression on RBCs. During routine screening, we found a blood donor who seemingly lacked Rh antigens. Therefore, we recruited the donor and his family for further investigation. Methods: RBC serotyping and antibody screening/identification were performed for each sample. A routine blood examination was also conducted. RHD, RHCE and RHAG were sequenced at the genomic DNA or RNA level. Eleven antigens or proteins associated with Rh complex were tested using flow cytometry analysis. Results: The proband and one of his brothers showed extremely weak D antigen and Rh expression levels but did not manifest anaemia. Most of the expressed RBC antigens of the two Rh‐deficient individuals were similar to the previously reported cases but with some exceptions. Molecular analyses demonstrated homozygous expression of a novel RHAG allele, namely, c.[572G>A;707A>C], both in the proband and one of his brothers. Conclusions: To our knowledge, we identified the second double‐variant RHAG allele and the first one related to Rhmod phenotype. The novel allele was also confirmed to be heritable by family analyses. [ABSTRACT FROM AUTHOR]

We report here an instructive case referred at 16 months-old for exploration of hemolysis without anemia (compensated anemia with reticulocytosis). The biology tests confirmed the hemolysis with increased total and indirect bilirubin. The usual hemolysis diagnosis tests were normal (DAT, G6PD, PK, Hb electrophoresis) except cytology and ektacytometry suggesting an association of multiple red blood cell (RBC) membrane disorders. This led us to propose a molecular screening analysis using targeted-Next Generation Sequencing (t-NGS) with a capture technique on 93 genes involved in RBC and erythropoiesis defects. We identified 4 missense heterozygous allelic variations, all of them were described without any significance (VUS) in the SLC4A1 , RhAG , PIEZO1 and SPTB genes. The study of the familial cosegregation and research functional tests allowed to decipher the role of at least two by two genes in the phenotype and the hemolytic disease of this young patient. Specialized t-NGS panel (or virtual exome/genome sequencing) in a disease-referent laboratory and the motivated collaboration of clinicians, biologists and scientists should be the gold standard for improving the diagnosis of the patients affected with RBC diseases or rare inherited anemias. • Red cell membrane disorders as an example of polygenic entity • Interest of targeted-NGS in deciphering the molecular defect association when functional data or phenotype are confusing • Importance and relevance of the functional tests to validate the variants of unknown significance (VUS) [ABSTRACT FROM AUTHOR]

Normal human RBCs have a very low basal permeability (leak) to cations, which is continuously corrected by the Na,K-ATPase. The leak is temperature-dependent, and this temperature dependence has been evaluated in the presence of inhibitors to exclude the activity of the Na,K-ATPase and NaK2Cl transporter. The severity of the RBC cation leak is altered in various conditions, most notably the hereditary stomatocytosis group of conditions. Pedigrees within this group have been classified into distinct phenotypes according to various factors, including the severity and temperature-dependence of the cation leak. As recent breakthroughs have provided more information regarding the molecular basis of hereditary stomatocytosis, it has become clear that these phenotypes elegantly segregate with distinct genetic backgrounds. The cryohydrocytosis phenotype, including South-east Asian Ovalocytosis, results from mutations in SLC4A1, and the very rare condition, stomatin-deficient cryohydrocytosis, is caused by mutations in SLC2A1. Mutations in RHAG cause the very leaky condition over-hydrated stomatocytosis, and mutations in ABCB6 result in familial pseudohyperkalemia. All of the above are large multi-spanning membrane proteins and the mutations may either modify the structure of these proteins, resulting in formation of a cation pore, or otherwise disrupt the membrane to allow unregulated cation movement across the membrane. More recently mutations have been found in two RBC cation channels, PIEZO1 and KCNN4, which result in dehydrated stomatocytosis. These mutations alter the activation and deactivation kinetics of these channels, leading to increased opening and allowing greater cation fluxes than in wild type.

Normal human RBCs have a very low basal permeability (leak) to cations, which is continuously corrected by the Na,K-ATPase. The leak is temperature-dependent, and this temperature dependence has been evaluated in the presence of inhibitors to exclude the activity of the Na,K-ATPase and NaK2Cl transporter. The severity of the RBC cation leak is altered in various conditions, most notably the hereditary stomatocytosis group of conditions. Pedigrees within this group have been classified into distinct phenotypes according to various factors, including the severity and temperature-dependence of the cation leak. As recent breakthroughs have provided more information regarding the molecular basis of hereditary stomatocytosis, it has become clear that these phenotypes elegantly segregate with distinct genetic backgrounds. The cryohydrocytosis phenotype, including South-east Asian Ovalocytosis, results from mutations in SLC4A1, and the very rare condition, stomatin-deficient cryohydrocytosis, is caused by mutations in SLC2A1. Mutations in RHAG cause the very leaky condition over-hydrated stomatocytosis, and mutations in ABCB6 result in familial pseudohyperkalemia. All of the above are large multi-spanning membrane proteins and the mutations may either modify the structure of these proteins, resulting in formation of a cation pore, or otherwise disrupt the membrane to allow unregulated cation movement across the membrane. More recently mutations have been found in two RBC cation channels, PIEZO1 and KCNN4, which result in dehydrated stomatocytosis. These mutations alter the activation and deactivation kinetics of these channels, leading to increased opening and allowing greater cation fluxes than in wild type. [ABSTRACT FROM AUTHOR]

Background The band 3 macrocomplex (also known as the ankyrin-associated complex) on the red cell membrane comprises two interacting subcomplexes: a band 3/glycophorin A subcomplex, and a Rh/Rh AG subcomplex. Glycophorin B ( GPB) is a component of the Rh/Rh AG subcomplex that is also structurally associated with glycophorin A ( GPA). Expression of glycophorin B-A-B hybrid GP.Mur enhances band 3 expression and is associated with lower levels of Rh-associated glycoprotein (Rh AG) and Rh polypeptides. The goal of this study was to determine whether GP.Mur influenced erythroid Rh/Rh AG expression at the transcript level. Materials and Methods GP.Mur was serologically determined in healthy participants from Taitung County, Taiwan. RNA was extracted from the reticulocyte-enriched fraction of peripheral blood, followed by reverse transcription and quantitative PCR for Rh AG, RhD and RhCcEe. Results Quantification by real-time PCR revealed significantly fewer Rh AG and RhCcEe transcripts in the reticulocytes from subjects with homozygous GYP*Mur. Independent from GYP.Mur, both Rh AG and RhD transcript levels were threefold or higher than that of RhCcEe. Also, in GYP.Mur and the control samples alike, direct quantitative associations were observed between the transcript levels of Rh AG and RhD, but not between that of Rh AG and RhCcEe. Conclusion Erythroid RhD and RhCcEe were differentially expressed at the transcript levels, which could be related to their different degrees of interaction or sensitivity to Rh AG. Further, the reduction or absence of glycophorin B in GYP.Mur erythroid cells affected transcript expressions of Rh AG and RhCcEe. Thus, GPB and GP.Mur differentially influenced Rh/Rh AG expressions prior to protein translation. [ABSTRACT FROM AUTHOR]

Background and Objectives: The extremely rare Rh null phenotype is characterized by the absence of all Rh antigens on erythrocytes. It is divided into the regulator and amorph types based on the underlying genetic background. The more common regulator type depends on critical variants silencing RHAG, which encodes RhAG glycoprotein, necessary for RhD/RhCE expression. Rh null cells have altered expression of glycophorin B and LW glycoprotein., Materials and Methods: Four unrelated Rh null individuals were investigated. Serological testing was performed according to standard blood bank practice. RHD/RHCE and S/s allele-specific Polymerase chain reaction (PCR) genotyping was done on genomic DNA using in-house PCR assays. RHAG, and in some cases also RHD/RHCE, were sequenced. Initial s phenotyping results triggered additional serological investigation., Results: Anti-Rh29 was identified in all four individuals. Extended typing with anti-S and anti-s showed that the three samples predicted to type as s+ failed to react with 2 of 5 anti-s. Sequence analysis of all 10 RHAG exons and the immediate intron/exon boundaries revealed a single nucleotide variant in the 3'-end of intron 6, c.946 -2a>g in all samples. RHD/RHCE showed no alterations., Conclusion: A novel Nordic Rh null allele was identified. In addition, it was shown that s+ Rh null red blood cells are not only U- but also have qualitative changes in their s antigen expression., (© 2023 The Authors. Vox Sanguinis published by John Wiley & Sons Ltd on behalf of International Society of Blood Transfusion.)

Objectives: Concentrations of O 2 and CO 2 in the fetal circulation differ to that in maternal blood. Previous studies done in algae demonstrate the functional role of Rh antigen as CO 2 transporter. As a preliminary study, it was the aim of this project to compare the expression of Rh polypeptides on cord and adult red blood cell progenitors during ex vivo proliferation and differentiation of CD34 + cells during erythropoeisis. Materials and Methods: CD34 positive hematopoeitic progenitor cells were isolated from umbilical cord blood and adult peripheral blood using an immunomagnetic system and cultured in serum free medium containing erythropoietin in order to compel them along the erythroid lineage. Cultured cells were analyzed for cell surface marker expression by flow cytometry, using monoclonal antibodies to RhAG, Glycophorin A, Rh polypeptides, CD47 and Band 3. Cytospin analysis was also done to study the morphology of cultured cells. Results: The appearance of cell surface markers analyzed on different days of culture varied slightly between samples. There was no evidence to suggest that RhAG, GPA, CD47 and Band 3 expression was any different between adult and cord derived cells. Nevertheless, the results of Rh antigenic expression suggest a reasonable difference between the two groups with adult sample derived cells showing higher and earlier expression than cord blood derived cells. These preliminary findings require further investigation. Conclusion: Comparing the expression of cell surface markers especially Rh polypeptides between adult and cord blood derived erythroid progenitors might assist in discerning their functions and could be valuable in the study of erythropoeisis.

The ammonia/ammonium (NH/NH) influx into red blood cells (RBCs) is mediated by surface glycoprotein RhAG that forms a structural complex with anion exchanger 1 (AE1, band 3). Owing to the activity of this complex, RBCs exposed to the isosmotic ammonium buffer swell and finally lyse. Isoosmotic NH-containing media alters the pH gradient in RBCs (intracellular alkalosis in response to NH/NH influx) and triggers the AE1 activity resulting in redundant chloride and water influx and finally in cell swelling. Here we demonstrate that the ammonia/ammonium transport in human RBCs depends on the pH (pH optimum 7.4 ± 0.1), temperature (Q 2.6 ± 0.3), HCO concentration (EC 4.7 ± 0.3 mM), and AE1 function. The data confirm functional interactions between AE1 and RhAG. The initial velocity of cell swelling increased almost 50-fold in the isosmotic ammonium buffer containing 25 mM HCO (37°C) in comparison to the reaction in the same buffer without HCO. This indicates that the reaction is facilitated mostly by the carrier proteins, not just owing to the simple diffusion of NH across the erythrocyte membrane. We demonstrate that pH reaches its maximum value much faster than the volume increase does. These data suggest that there is no direct correlation between pH changes and the influx of NH/NH. Taken together, our data show that the RhAG and AE1 complex activity enables erythrocytes to be ammonia/ammonium storage sites in order to maintain the physiological blood ammonia/ammonium equilibrium. [ABSTRACT FROM AUTHOR]

In this study we characterized ammonia and ammonium (NH3/NH4+) transport by the rhesus-associated (Rh) glycoproteins RhAG, Rhbg, and Rhcg expressed in Xenopus oocytes. We used ionselective microelectrodes and two-electrode voltage clamp to measure changes in intracellular pH, surface pH, and whole cell currents induced by NH3/NH4+ and methyl amine/ammonium (MA/MA+). These measurements allowed us to define signal-specific signatures to distinguish NH3 from NH4+ transport and to determine how transport of NH3 and NH4+ differs among RhAG, Rhbg, and Rhcg. Our data indicate that expression of Rh glycoproteins in oocytes generally enhanced NH3/NH4+ transport and that cellular changes induced by transport of MA/MA+ by Rh proteins were different from those induced by transport of NH3/NH4+. Our results support the following conclusions: 1) RhAG and Rhbg transport both the ionic NH4+ and neutral NH3 species; 2) transport of NH4+ is electrogenic; 3) like Rhbg, RhAG transport of NH4+ masks NH3 transport; and 4) Rhcg is likely to be a predominantly NH3 transporter, with no evidence of enhanced NH4+ transport by this transporter. The dual role of Rh proteins as NH3 and NH4+ transporters is a unique property and may be critical in understanding how transepithelial secretion of NH3/NH4+ occurs in the renal collecting duct. [ABSTRACT FROM AUTHOR]

Background: Flower development is central to angiosperm reproduction and is regulated by a broad range of endogenous and exogenous stimuli. It has been well documented that ambient temperature plays a key role in controlling flowering time; however, the mechanisms by which temperature regulates floral organ differentiation remain largely unknown. Results: In this study, we show that low temperature treatment significantly increases petal number in rose (Rosa hybrida) through the promotion of stamen petaloidy. Quantitative RT-PCR analysis revealed that the expression pattern of RhAG, a rose homolog of the Arabidopsis thaliana AGAMOUS C-function gene, is associated with low temperature regulated flower development. Silencing of RhAG mimicked the impact of low temperature treatments on petal development by significantly increasing petal number through an increased production of petaloid stamens. In situ hybridization studies further revealed that low temperature restricts its spatial expression area. Analysis of DNA methylation level showed that low temperature treatment enhances the methylation level of the RhAG promoter, and a specific promoter region that was hypermethylated at CHH loci under low temperature conditions, was identified by bisulfite sequencing. This suggests that epigenetic DNA methylation contributes to the ambient temperature modulation of RhAG expression. Discussion: Our results provide highlights in the role of RhAG gene in petal number determination and add a new layer of complexity in the regulation of floral organ development. Conclusions: We propose that RhAG plays an essential role in rose flower patterning by regulating petal development, and that low temperatures increase petal number, at least in part, by suppressing RhAG expression via enhancing DNA CHH hypermethylation of the RhAG promoter. [ABSTRACT FROM AUTHOR]

Inherited disorders of erythrocyte volume homeostasis are a heterogeneous group of rare disorders with phenotypes ranging from dehydrated to overhydrated erythrocytes. Clinical, laboratory, physiologic, and genetic heterogeneities characterize this group of disorders. A series of recent reports have provided novel insights into our understanding of the genetic bases underlying some of these disorders of red cell volume regulation. This report reviews this progress in understanding determinants that influence erythrocyte hydration and how they have yielded a better understanding of the pathways that influence cellular water and solute homeostasis. [ABSTRACT FROM AUTHOR]

Abstract: The mammalian Rh glycoproteins belong to the solute transporter family SLC42 and include RhAG, present in red blood cells, and two non-erythroid members RhBG and RhCG that are expressed in various tissues, including kidney, liver, skin and the GI tract. The Rh proteins in the red blood cell form an “Rh complex” made up of one D-subunit, one CE-subunit and two RhAG subunits. The Rh complex has a well-known antigenic effect but also contributes to the stability of the red cell membrane. RhBG and RhCG are related to the transporters of the yeast and bacteria but their exact function is yet to be determined. This review describes the expression and molecular properties of these membrane proteins and their potential role as NH3/ and CO2 transporters. The likelihood that these proteins transport gases such as CO2 or NH3 is novel and significant. The review also describes the physiological importance of these proteins and their relevance to human disease. [Copyright &y& Elsevier]

Abstract: Autosomal dominant overhydrated cation-leak stomatocytosis in humans has been associated with missense mutations in the erythroid membrane transport genes AE1, RhAG, and GLUT1. Syndromic stomatocytosis has been reported in three dog breeds, but stomatocytosis in Standard Schnauzers is usually asymptomatic, and is accompanied by minimal if any anemia. We have extended the evaluation of a cohort of schnauzers. We found that low-level stomatocytosis was accompanied by increased MCV and increased red cell Na content, and minimal or no reticulocytosis. Red cells from two affected dogs exhibited increased currents in on-cell patches measured in symmetrical NaCl solutions, but Na,K-ATPase and NKCC-mediated cation flux was minimal. Three novel coding polymorphisms found in canine RhAG cDNA and three novel polymorphisms found in canine SLC4A1 cDNA did not cosegregate with MCV or Na content. The GLUT1 cDNA sequence was normal. We conclude that unlike human overhydrated cation-leak stomatocytosis, stomatocytosis in this cohort of Standard Schnauzers is not caused by mutations in the genes encoding RhAG, SLC4A1, or GLUT1. [Copyright &y& Elsevier]

Abstract: The hereditary stomatocytoses (HSt) are a diverse group of conditions. Common features include hemolytic anemia, a red cell cation leak and morphological changes, but the severity of the condition can vary enormously. We have previously shown that one form of HSt (cryohydrocytosis), where the monovalent cation leak is increased at low temperature, results from amino acid substitutions in the membrane domain of band 3 (anion exchanger 1, SLC4A1). These substitutions appear to convert band 3 from an anion exchanger into a cation channel. More recently we found that over-hydrated hereditary stomatocytosis (OHSt) results from amino acid substitutions in Rh-associated glycoprotein (RhAG), a putative gas channel protein. Both band 3 and RhAG associate in the red cell membrane to form a macrocomplex that is thought to be involved in red cell gas exchange. In this paper I will review the data that has been published so far on the molecular basis of HSt. I will mention other similar conditions that cause either a cation leak or stomatocytosis or both, and consider the mechanisms of red cell shape change and permeability. [Copyright &y& Elsevier]

Objectives: Concentrations of O2 and CO2 in the fetal circulation differ to that in maternal blood. Previous studies done in algae demonstrate the functional role of Rh antigen as CO2 transporter. As a preliminary study, it was the aim of this project to compare the expression of Rh polypeptides on cord and adult red blood cell progenitors during ex vivo proliferation and differentiation of CD34+ cells during erythropoeisis. Materials and Methods: CD34 positive hematopoeitic progenitor cells were isolated from umbilical cord blood and adult peripheral blood using an immunomagnetic system and cultured in serum free medium containing erythropoietin in order to compel them along the erythroid lineage. Cultured cells were analyzed for cell surface marker expression by flow cytometry, using monoclonal antibodies to RhAG, Glycophorin A, Rh polypeptides, CD47 and Band 3. Cytospin analysis was also done to study the morphology of cultured cells. Results: The appearance of cell surface markers analyzed on different days of culture varied slightly between samples. There was no evidence to suggest that RhAG, GPA, CD47 and Band 3 expression was any different between adult and cord derived cells. Nevertheless, the results of Rh antigenic expression suggest a reasonable difference between the two groups with adult sample derived cells showing higher and earlier expression than cord blood derived cells. These preliminary findings require further investigation. Conclusion: Comparing the expression of cell surface markers especially Rh polypeptides between adult and cord blood derived erythroid progenitors might assist in discerning their functions and could be valuable in the study of erythropoeisis. [ABSTRACT FROM AUTHOR]