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

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.

Rhnull phenotype in an Indian patient due to a novel c.1138 + 2 t > a mutation in the RHAG gene.

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.

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

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.

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

BACKGROUND: Rhnull 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 METHODS: Rh 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. RESULTS: Serological 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). CONCLUSION: These 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.

ABO gene editing for the conversion of blood type A to universal type O in Rhnull donor-derived human-induced pluripotent stem cells.

The limited availability of red cells with extremely rare blood group phenotypes is one of the global challenges in transfusion medicine that has prompted the search for alternative self-renewable pluripotent cell sources for the in vitro generation of red cells with rare blood group types. One such phenotype is the Rhnull , which lacks all the Rh antigens on the red cell membrane and represents one of the rarest blood types in the world with only a few active blood donors available worldwide. Rhnull red cells are critical for the transfusion of immunized patients carrying the same phenotype, besides its utility in the diagnosis of Rh alloimmunization when a high-prevalence Rh specificity is suspected in a patient or a pregnant woman. In both scenarios, the potential use of human-induced pluripotent stem cell (hiPSC)-derived Rhnull red cells is also dependent on ABO compatibility. Here, we present a CRISPR/Cas9-mediated ABO gene edition strategy for the conversion of blood type A to universal type O, which we have applied to an Rhnull donor-derived hiPSC line, originally carrying blood group A. This work provides a paradigmatic example of an approach potentially applicable to other hiPSC lines derived from rare blood donors not carrying blood type O.

First Report of Known Rare Rhnull Phenotype Individuals in Iran.

Rhnull phenotype is a rare blood group with a frequency of approximately 1 in 6 million individuals, transmitted via an autosomal recessive mode. It is characterized by the weak (Rhmod) or lack (Rhnull) of expression of all Rh antigens on the red cells. The clinical significance of its assessment is that such patients with Rhnull syndrome are associated with chronic hemolytic anemia of varying degrees. Another clinical importance is that such subjects readily form alloantibodies when exposed to Rh antigens.We report herein a rare Rhnull phenotype in a sibling, which was detected as a part of the difficult sample work-up for red cell antibody screening and identification.