Rhnull Phenotype Research Articles

A novel frameshift mutation in RHAG leads to Rhnull phenotype in a Chinese individual.

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. 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. 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. 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.

A novel nonsense variant in RHAG underlies a Nordic Rhnull phenotype.

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. 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. 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. 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.

Identification of First Patient With Rh Null Phenotype in Southeast Iran

Background and Aims: Rhnull, with an estimated incidence of one per 6,000,000 individuals, is an extremely rare disorder with an autosomal recessive pattern of inheritance that is more common in societies with a high rate of consanguinity.
 Materials and Methods: In this study, we report the first case with Rhnull, a blood group phenotype in southeast Iran, which was diagnosed during pretransfusion testing.
 Results and Conclusions: A 21-year-old woman with a positive parents' consanguineous marriage was found to have an unusual reaction with all packed red blood cell units during routine pretransfusion cross-match testing in the hospital. The patient's serum was reacted with all screening and identification panel cells, suspected to have an alloantibody against a common antigen or multiple alloantibodies against her absence antigens. Further studies revealed negative results for C, c, E, and e, which are highly suspected of Rhnull phenotype. Confirmatory assessments were performed, including adsorption and elution studies and Rh phenotyping of patients, along with known positive and negative controls. Due to the blood requirement of the patient, we performed serological studies on the patient's family members and found that her sister also has a Rhnull phenotype. Blood transfusion from her sister's donated units was performed, and the pregnancy was ended without any complications. Finally, due to the rarity of the Rhnull phenotype, early identification of individuals and autologous or compatible allogeneic blood transfusion should be planned prior to selective or emergency surgeries.

A novel SNV on a previously reported hybrid RHCE background is responsible for an Rh haplotype potentially involved in the generation of an Rh null phenotype.

The authors have disclosed no conflict of interest. Appendix S1 Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Rhnull phenotype: A family study of a novel RHAG allele in Brazilians.

Rhnull phenotype: A family study of a novel RHAG allele in Brazilians.

Blood group systems

Blood group systems

First report of Rhnull individuals in the Indian population and characterization of the underlying molecular mechanisms.

Rhnull phenotype is an extremely rare condition characterized by no expression of Rh antigens at the surface of red blood cells. Although rare, genetic bases of this phenotype are well known and include mutations within either the RH (RHD and RHCE) genes or the RHAG gene. So far Rhnull has been reported in individuals of Caucasian, African, and Asian origin. Here, we report individuals from two families of Indian origin representing such a rare phenotype. Serologic analysis was carried out by testing with anti-D, -C, -c, -E, and -e in Rhnull individuals and their family members. RH genes were analyzed by standard molecular approaches, including Sanger sequencing and quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments. RHAG gene was investigated by exon-specific PCR amplification and Sanger sequencing. In one family, RHAG gene was found to be deleted at the homozygous state in the propositus, suggesting Rhnull of the regulator type. In the other family, a novel splice site variant in RHCE in cis with whole RHD gene deletion was identified at the homozygous state. Further functional analysis by minigene splicing assay showed that this variation, that is, c.801 + 1G>A, completely impairs normal splicing, then inactivating the expression of RhCE protein. Contrary to the former case, these data suggest Rhnull of the amorph type. Overall, we report for the first time the molecular mechanisms responsible for Rhnull phenotype in individuals of Indian origin. This study contributes to extend the molecular spectrum of variations in Rhnull individuals.

A novel nonsense mutation in RHAG gene responsible for Rhnull phenotype in a Chinese individual

BackgroundRhnull is a rare autosomal recessive phenotype, which is characterized by the lack of Rh antigen expression on the red blood cells (RBCs). Rhnull of the regulator type is caused by RHAG mutation. In this study, a novel nonsense mutation in RHAG gene was identified in a Chinese Rhnull individual. Objectives and methodsRh phenotypes of the Rhnull individual and his family members were typed by standard serological methods. DNA sequences of all ten exons of RHAG gene were analyzed using genomic DNA by polymerase chain reaction (PCR) and direct-sequencing. ResultsSerological testing results showed a D-C-c-E-e- phenotype in the proband. Molecular analyses revealed a 540C>A mutation in exon 4 of RHAG gene was present at the homozygous state in the proband. His parents were heterozygous for the mutation, and his brother didn’t carry the mutation. The 540C>A mutation was nonsense mutation, which led to a premature stop codon (Tyr180stop). ConclusionThese results indicated that the 540C>A nonsense mutation in RHAG gene caused the regulator type of Rhnull phenotype in a Chinese individual. Our results contributed to a greater understanding of the genetic mechanisms of Rhnull phenotype.

A novel c.790C>T mutation in RHAG gene encoding the Rhnull phenotype in Japanese

Background and ObjectivesMolecular analyses of Rhnull individuals have revealed that abnormalities occur only at the RH or RHAG gene. Rhnull phenotype arises from two distinct genetic mechanisms, the amorph and the regulator type. Here, we genetically analysed a Japanese Rhnull family and identified a novel RHAG mutation.Materials and MethodsRBC samples from two family members were phenotyped using the standard serological techniques and BLOODchip Reference for the predicted phenotype. Scanning electron micrographs of red blood cells (RBCs) were generated. All 10 RHAG exons were subjected to DNA sequencing.ResultsRBCs in 2 Rhnull samples did not contain Rh or RhAG antigens. The screening tests for irregular RBC antibodies identified anti‐Rh29 in 2 samples and anti‐C+e in the older sister of the proband. A molecular base analysis revealed a novel c.790C>T (p.Arg263X) mutation in exon 5 of RHAG gene.ConclusionThe samples with the novel c.790C>T (p.Arg263X) mutation in exon 5 of the RHAG gene were identified as regulator type.

Novel RHAG allele encoding the Rh(null) phenotype in Brazil.

Rhnull is a rare phenotype characterized by the loss of Rh antigen expression. This phenotype can be related to several molecular backgrounds. In this study, we show a novel allele in a Brazilian pregnant woman encoding the Rhnull phenotype due to a change in RHAG exon2 c.310C>T, which leads to a premature stop codon (Gln104Stop).

Molecular background of novel silent RHCE alleles

The absence of expression of C/c and E/e antigens has been associated with rare variant RHCE alleles, referred to as silent RHCE alleles, classically identified among individuals with a rare D- - or Rhnull phenotype. This work reports on different molecular mechanisms identified in three novel silent RHCE alleles. Samples from D- - or Rhnull individuals and their family members, from families for whom Rh phenotype and/or serologic data were unexplained by inheritance of conventional RH alleles, were analyzed. Genomic DNA and transcripts were tested by sequencing analysis. The first silent allele was a RHCE*cE allele carrying an intronic IVS3+5G>A mutation. The second was a RHCE*ce allele carrying an intronic IVS7-2A>G mutation, whereas the third was a silent RHCE*ce allele carrying a 5-bp deletion (Nucleotides 679-683) in Exon 5. In addition to hybrid alleles and nucleotide deletion, intronic mutations may be associated with the nonexpression of RhCE antigens. Regarding the RH system, silent alleles may not be investigated among D- - or Rhnull individuals only. Rh phenotype and/or serologic data unexplained by inheritance of conventional RH alleles should lead to molecular investigations.

Uncommon Rh Phenotypes in a Cross Section of Nigerian Antenatal Women: Implications for Molecular Genotyping of Blood Groups

Background: This study aimed to determine the frequency of rare blood group antigens among pregnant women in Port Harcourt Metropolis. Methods: The frequencies of Rh antigens and phenotypes of the pregnant women in Port Harcourt Metropolis, Nigeria, were determined, using standard serologic techniques. Results: Of the 374 pregnant women studied, the frequencies of the Rh antigens within the population were D (89.0%), c (82.0%), e (54.0%), C (24.3%), E (20.1%). The frequencies of the Rh antithetical antigens were DD/Dd (91.2 %), Cc (19,5%), cc (84.5%), Ee (13.9%), ee (54.3%), CC (25.1%), EE (19.8%) and dd (10.4%). Seven (1.9%) were found to be Rhnull, sixteen(4.3%) were found to be D-- or exalted D. Phenotypes without RhD reactivity were –c- (2.9%), - Cc (0.3%), -C- (0.3%), -Ee (0.5%) and –E- (0.3%). The most frequently occurring Rh phenotype was Dccee with a frequency of (25.8%). RhD-ve was found to be significantly associated with HDN outcome ( 2=6.605 and P=0.01). Conclusion: We concluded that: 1) There is the presence of uncommon Rh phenotypes in our population. 2) Rhnull exists in this study population of which similar studies have not been reported in Nigeria. 3) There is need to conduct studies in other parts of Nigeria in order to compare the frequencies of these rare blood groups in Nigerian population. 4) Molecular studies to establish reasons for deletion and Rhnull phenotypes among Nigerian and Africans in general are hereby advocated.

Rh proteins: Key structural and functional components of the red cell membrane

Rh (Rhesus) proteins (D, CcEe) are expressed in red cells (RBC) in association with other membrane proteins (RhAG, LW, CD47 and GPB). By interacting with the spectrin-based skeleton through protein 4.2 and ankyrin, the Rh complex contributes to the maintenance of the mechanical properties of the erythrocyte membrane. The RH system is one of the most immunogenic and polymorphic human blood group system. Molecular basis of most Rh phenotypes, including the Rh null phenotype associated with hemolytic anemia, have been determined. The demonstration that the RHD -positive locus is composed of the RHD and RHCE genes, whereas the RHD gene is deleted in most RhD-negative individuals, allowed fetal RhD genotyping by non-invasive PCR assays for antenatal diagnosis of pregnancy at risk for Rh hemolytic disease of the newborn. In mammals, the Rh protein family includes two non-erythroid members, RhBG and RhCG, mainly expressed in liver and kidney, two organs specialized in ammonia genesis and excretion. Functional analyses in heterologous systems revealed that RhAG, RhBG and RhCG can mediate ammonium (NH 3 and/or NH 4 + ) transport across the cell membrane and might represent mammalian specific ammonium transporters. Furthermore, recent studies performed in human and murine red blood cells (RBC) indicate that RhAG facilitates CH 3NH 2/NH 3 movement across the membrane and represents a potential example of gas channel. The crystallographic structure of the bacterial ammonia channel AmtB and functional studies showing that AmtB conducts NH 3 into reconstituted vesicles is fully consistent with these latter studies. In RBCs , RhAG may transport NH 3 to detoxifying organs like kidney and liver and with non-erythroid tissues orthologs may contribute to regulation of the acid-base balance.

A hypothesis of the stomatocytosis in individuals with the phenotype Rhnull

A hypothesis of the stomatocytosis in individuals with the Rhnull phenotype is proposed on the assumptions that the RhAG polypeptide of the Rh antigenic complex has the function of transporting ammonium and that a previously proposed mechanism of erythrocyte shape control is valid.

Blood groups and diseases associated with inherited abnormalities of the red blood cell membrane

HE UNIQUE design of the red blood cell (RBC), and of the RBC membrane that encapsulates hemoglobin within it, ensures that the RBC is able to accomplish the major function of transporting respiratory gases throughout the body. The RBC membrane is thus a flexible, deformable, semifluid slructure that allows the cell passage through small capillaries. Because there are no internal Olganelles to repair damage in the mature RBC, the membrane must be resilient. These unique mechanical properties are attributable to a meshwork of membrane skeleton proteins that interacts with the lipid bilayer through the integral membrane proteins, some of which carry blood group antigens (Fig 1). The blood group antigens are inherited, polymorphlc, structural characteristics, defined by specific amino acid sequences on proteins or by carbohydrates attached to either proteins or lipids. More than 250 blood group antigens have been described and placed into 25 blood group systems. 1 The study of individuals who have a null phenotype for a certain blood group system and thus have RBCs that lack or have altered membrane protein(s) has provided a key to the function of that protein. For example, RBCs with the rare Rhnull phenotype were found to lack several proteins. That these proteins have a role in maintaining RBC morphology and integrity is indicated by the finding that Rhnull RBCs have stomatocytic morphology and reduced in vivo survival. In recent years, the characterization of the molecular genetics of most of the 25 blood group systems, combined with the study of null phenotypes, has expanded our understanding of the structure and function of the membrane components carrying blood group antigens. The functions attributed to, or proposed for, these membrane components are diverse and include

Molecular genetic analysis of the Japanese amorph Rhnull phenotype

Molecular genetic analysis of the Japanese amorph Rhnull phenotype

A splicing mutation of the RHAG gene associated with the Rhnull phenotype.

Rhnull is a syndrome serologically characterized by the deficiency of all Rh antigens on human red blood cells. Rhnull is divided into two types: regulator and amorph. Recently, Cherif-Zahar et al. proposed that the RHAG gene encoding the Rh50 glycoprotein is a candidate for inducing regulator type Rhnull. We investigated both the RH and RHAG genes in an Rhnull individual. The reticulocytes from the propositus had RHD, RHcE, and RHCe transcripts without any mutation. However, the sequence analysis of RHAG cDNA showed a deletion of 122 bp from nucleotide 946 to 1067. This deletion was revealed to be due to a homozygous splicing mutation, which is a single base substitution at the consensus sequence of the splicing acceptor site (AG-->AT). The mutation appeared to break the 'GT-AG' splicing rule and to cause 122 bp exon skipping accompanied by a frameshift. This study confirms that the RHAG gene is the most likely candidate for the 'regulator' gene of Rhnull cases.

Candidate gene acting as a suppressor of the RH locus in most cases of Rh-deficiency.

The Rh antigen is a multi-subunit complex composed of Rh polypeptides and associated glycoproteins (Rh50, CD47, LW and glycophorin B); these interact in the red cell membrane and are lacking or severely reduced in Rhnull cells. As a result, individuals with Rhnull suffer chronic haemolytic anaemia known as the Rh-deficiency syndrome. Most frequently, Rhnull phenotypes are caused by homozygosity of an autosomal suppressor gene unlinked to the RH locus (Rhnull regulator or Rhmod types). We have analysed the genes and transcripts encoding Rh, CD47 and Rh50 proteins in five such unrelated Rhnull cases. In all patients, we identified alteration of Rh50--frameshift, nucleotide mutations, or failure of amplification--which correlated with Rhnull phenotype. We propose that mutant alleles of Rh50, which map to chromosome 6p11-21.1, are likely candidates for suppressors of the RH locus accounting for most cases of Rh-deficiency.

Normal transport of glutathione S-conjugates in erythrocytes of a patient with the Rh null phenotype

Normal transport of glutathione S-conjugates in erythrocytes of a patient with the Rh null phenotype

Defining the Rh blood group antigens: Biochemistry and molecular genetics

The Rh blood group antigens (D, Cc and Ee series) are carried by a family of non glycosylated hydrophobic transmembrane proteins of 30–32 kDa which are missing from the red cells of rare Rh null individuals that express several membrane defects. The structure of these proteins has been deduced from cDNA cloning and studies have shown that the Rh proteins are erythroid specific and share no sequence homology with any known protein. The RhD and non-D proteins exhibit 92% sequence identity and their predicted membrane topology is similar as most of the molecules appear to reside between the leaflets of the phospholipid bilayer with only short hydrophilic loops connecting the twelve putative transmembrane helices. The RHD and RHCE genes encoding the Rh proteins (D and Cc/Ee, respectively) are organized in tandem on chromosome 1p34-p36 and most likely derived by duplication of a common ancestral gene. This concept is supported by the identification of RH-like genes in non human primates. The human RH locus is best described as a two-gene model in which all RhD-positive and most RhD-negative haplotypes are composed of two ( RHD and RHCE) or only one ( RHCE) structural genes, respectively. The RHD gene encodes the D protein and the RHCE gene encodes the C/c and E/e proteins presumably by alternative splicing of a pre messenger RNA. The correlation between the blood group D epitopes and the amino acid polymorphism of the Rh proteins is not yet established, but amino acid polymorphisms at positions 103 and 226 determine the molecular basis for the C/c (Ser → Pro) and E/e (Pro → Ala) specificities, respectively. Most variants analyzed so far are caused by gene conversion which appears as the principal mechanism responsible for polymorphism and gene diversity in the RH system. However, gene deletions have also been found in some occasions. To date, all Rh null phenotypes investigated most likely result from transcriptional regulatory mechanisms that are not yet understood. Rh null individuals suffer a clinical syndrome of varying severity and their red cells are characterized by morphological and functional abnormalities of cation transport and phospholipid asymmetry. In addition, several membrane components including the Rh proteins and other glycoproteins recently characterized (Rh50 glycoprotein, CD47, glycophorin B, Duffy, LW) are absent or severely decreased on these cells. These findings suggest that the Rh proteins are assembled into a multimeric complex with these glycoproteins and further studies should clarify the role in biosynthesis and the potential function of each component in this complex.