Rhnull Disease Research Articles

Rhnull Syndrome: Identification of a Novel Mutation in RHce Gene.

Background: Rhnull syndrome, which includes the amorph and regulator types, is a rare genetic disorder characterized by stomatocytosis and chronic mild hemolytic anemia. The suppression of Rh antigen expression for regulator types is attributed to mutations of the RHAG gene. The deficiency of Rh proteins on the red blood cells (RBCs) from the rare individuals of the Rhnull amorph type may be the result of homozygosity for a silent RHCE allele at the RH locus in which the RHD is absent. We studied a Brazilian family transmitting an amorph Rhnull disease gene on a consanguineous background and identified a novel mutation in RHce gene causing the loss of function phenotype.Methods: RBCs from two Rhnull sisters (G1 and G2) of the amorph type and from family members were analyzed by serology and flow cytometry with monoclonal antibodies specific for Rh (D, C, c, E and e), and LW antigens, for RhAG and CD47 as well as alloantibodies directed against GPB (S, s and U) antigens. Genomic DNA samples and transcripts were tested by PCR and sequence analysis.Results: RBCs from G1 and G2 did not react with the anti-Rh and anti-LW reagents and reacted weakly with anti-RhAG, anti-CD47, anti-s, and anti-U reagents. RBCs from G1 were S+ and from G2 S-. Molecular analyses showed a deletion of the nucleotide at position 963 in exon 7 of the RHce gene [GGG(Glu321→GG)] in addition to a deletion of the RHD gene. This nucleotide deletion in exon 7 introduced a frameshift after Glu321 and a premature stop codon, resulting in a shorter predicted protein with 359 amino acids, including a new C-terminal sequence that is likely to alter the protein conformation and impair the Rh complex assembly.Conclusion: We describe a novel mutation of an amorph Rhnull disease gene leading to a loss of function phenotype. Our findings reinforce previous studies suggesting that protein folding and/or protein-protein interaction mediated by the C-terminal domain of the Rh proteins plays a role in the stability of the Rh membrane complex.

Rh null disease: The amorph type results from a novel double mutation in RhCe gene on D-negative back-ground : Huang C-H, Chen Y, Reid ME, et al. Blood 92:664–671, 1998

Rh null disease: The amorph type results from a novel double mutation in RhCe gene on D-negative back-ground : Huang C-H, Chen Y, Reid ME, et al. Blood 92:664–671, 1998

Rh mod Syndrome: A Family Study of the Translation-Initiator Mutation in the Rh50 Glycoprotein Gene

Rhmod syndrome is a rare genetic disorder thought to result from mutations at a "modifier" but not at the suppressor underlying the regulator type of Rhnull disease. We studied this disorder in a Jewish family with a consanguineous background and analyzed RH and RHAG, the two loci that control Rh-antigen expression and Rh-complex assembly. Despite the presence of a d (D-negative) haplotype, no other gross alteration was found at RH, and cDNA sequencing showed a normal structure for D, Ce, and ce Rh transcripts in family members. However, analysis of RHAG transcript, which encodes Rh50 glycoprotein, identified a single G-->T transversion in the initiation codon, causing a missense amino acid change (ATG[Met]-->ATT[Ile]). This point mutation also occurred in the genomic region spanning exon 1 of RHAG, and its genotypic status in the mother and two children was confirmed by analysis of single-strand conformation polymorphism. Although blood typing showed a very weak expression of Rh antigens, immunoblotting barely detected the Rh proteins in the Rhmod membrane. In vitro transcription-coupled translation assays showed that the initiator mutants of Rhmod-but not those of the wild type-could be translated from ATG codons downstream. Our findings point to incomplete penetrance of the Rhmod mutation, in the form of "leaky" translation, leading to some posttranslational defects affecting the structure, interaction, and processing of Rh50 glycoprotein.

The human Rh50 glycoprotein gene—Structural organization and associated splicing defect resulting in Rh null disease : Huang C-H. J Biol Chem 273:2207–2213, 1998

The human Rh50 glycoprotein gene—Structural organization and associated splicing defect resulting in Rh null disease : Huang C-H. J Biol Chem 273:2207–2213, 1998

Rh50 Glycoprotein Gene and Rhnull Disease: A Silent Splice Donor Is trans to a Gly279→Glu Missense Mutation in the Conserved Transmembrane Segment

Rhnull disease includes the amorph and regulator types that are thought to result from homozygous mutations at theRH30 and RH50 loci, respectively. Here we report an unusual regulator Rhnull where two G→A nucleotide (nt) transitions occurred in trans, targeting different regions of the two copies of Rh50 gene. The nt 836 G→A mutation was a missense change located in exon 6; it converted Gly into Glu at position 279, a central amino acid of the transmembrane segment 9 (TM9). While cDNA analysis showed expression of the 836A(Glu279) allele only, genomic studies showed the presence of both 836A(Glu279) and 836G(Gly279) alleles. A detailed analysis of gene organization led to the identification in the Rh50(836G) allele of a defective donor splice site, caused by a G→A mutation in the invariant GT element of intron 1. This is the first known example of such mutations that has apparently abolished the functional splicing of a pre-mRNA encoding a multipass integral membrane protein. With a silent phenotypic copy intrans, the negatively charged Glu279 residue may disrupt TM9 and impair the interaction of the missense protein with Rh30 polypeptides. To evaluate the significance of the mutation, we took a comparative genomic approach and identified Rh50 homologues in different species. We found that Gly279 is a conserved residue and its adjacent amino acid sequence is identical fromCaenorhabditis elegans to human. These findings provide new insight into the diversity of Rhnull disease and suggest that the C-terminal region of Rh50 may also participate in protein-protein interactions involving Rh complex formation.© 1998 by The American Society of Hematology.

Rh50 Glycoprotein Gene and Rhnull Disease: A Silent Splice Donor Is trans to a Gly279→Glu Missense Mutation in the Conserved Transmembrane Segment

Rhnull disease includes the amorph and regulator types that are thought to result from homozygous mutations at theRH30 and RH50 loci, respectively. Here we report an unusual regulator Rhnull where two G→A nucleotide (nt) transitions occurred in trans, targeting different regions of the two copies of Rh50 gene. The nt 836 G→A mutation was a missense change located in exon 6; it converted Gly into Glu at position 279, a central amino acid of the transmembrane segment 9 (TM9). While cDNA analysis showed expression of the 836A(Glu279) allele only, genomic studies showed the presence of both 836A(Glu279) and 836G(Gly279) alleles. A detailed analysis of gene organization led to the identification in the Rh50(836G) allele of a defective donor splice site, caused by a G→A mutation in the invariant GT element of intron 1. This is the first known example of such mutations that has apparently abolished the functional splicing of a pre-mRNA encoding a multipass integral membrane protein. With a silent phenotypic copy intrans, the negatively charged Glu279 residue may disrupt TM9 and impair the interaction of the missense protein with Rh30 polypeptides. To evaluate the significance of the mutation, we took a comparative genomic approach and identified Rh50 homologues in different species. We found that Gly279 is a conserved residue and its adjacent amino acid sequence is identical fromCaenorhabditis elegans to human. These findings provide new insight into the diversity of Rhnull disease and suggest that the C-terminal region of Rh50 may also participate in protein-protein interactions involving Rh complex formation.© 1998 by The American Society of Hematology.

Rhnull Disease: The Amorph Type Results From a Novel Double Mutation in RhCe Gene on D-Negative Background

Rhnull disease, which includes the amorph and regulator types, is a rare genetic disorder characterized by stomatocytosis and chronic hemolytic anemia. We studied here a German family transmitting a putative amorph Rhnull disease gene and identified a rare mutation causing the loss-of-function phenotype. We analyzed the genomic and transcript structure of RH30, RH50, andCD47, the three loci thought to be most critical for expression of the Rh complex in the red blood cell membrane. We showed that in this family the Rh50 and CD47 transcripts were normal in primary sequence. However, the RH30 locus contained an unusual double mutation in exon 7 of the RhCe gene, in addition to a deletion of the RhD gene. The mutation targeted two adjacent codons in multiple arrangements probably via the mechanism of microgene conversion. One scheme entails a noncontiguous deletion of two nucleotides, [ATT(Ile322)→AT] and [CAC(His323)→CC], whereas the other involves a T→C transition [ATT(Ile322)→ATC] and a dinucleotide deletion [CAC(His323)→C]. They caused the same shift in open reading frame predicted to encode a shortened protein with 398 amino acids. The loss of two transmembrane domains and gain of a new C-terminal sequence are likely to alter the protein conformation and impair the Rh complex assembly. Our findings establish the molecular identity of an amorph Rhnull disease gene, showing that Rh30 and Rh50 are both essential for the functioning of the Rh structures as a multisubunit complex in the plasma membrane.

Rhnull Disease: The Amorph Type Results From a Novel Double Mutation in RhCe Gene on D-Negative Background

AbstractRhnull disease, which includes the amorph and regulator types, is a rare genetic disorder characterized by stomatocytosis and chronic hemolytic anemia. We studied here a German family transmitting a putative amorph Rhnull disease gene and identified a rare mutation causing the loss-of-function phenotype. We analyzed the genomic and transcript structure of RH30, RH50, andCD47, the three loci thought to be most critical for expression of the Rh complex in the red blood cell membrane. We showed that in this family the Rh50 and CD47 transcripts were normal in primary sequence. However, the RH30 locus contained an unusual double mutation in exon 7 of the RhCe gene, in addition to a deletion of the RhD gene. The mutation targeted two adjacent codons in multiple arrangements probably via the mechanism of microgene conversion. One scheme entails a noncontiguous deletion of two nucleotides, [ATT(Ile322)→AT] and [CAC(His323)→CC], whereas the other involves a T→C transition [ATT(Ile322)→ATC] and a dinucleotide deletion [CAC(His323)→C]. They caused the same shift in open reading frame predicted to encode a shortened protein with 398 amino acids. The loss of two transmembrane domains and gain of a new C-terminal sequence are likely to alter the protein conformation and impair the Rh complex assembly. Our findings establish the molecular identity of an amorph Rhnull disease gene, showing that Rh30 and Rh50 are both essential for the functioning of the Rh structures as a multisubunit complex in the plasma membrane.

The Human Rh50 Glycoprotein Gene: STRUCTURAL ORGANIZATION AND ASSOCIATED SPLICING DEFECT RESULTING IN Rhnull DISEASE

The Rh (Rhesus) protein family comprises Rh50 glycoprotein and Rh30 polypeptides, which form a complex essential for Rh antigen expression and erythrocyte membrane integrity. This article describes the structural organization of Rh50 gene and identification of its associated splicing defect causing Rhnull disease. The Rh50 gene, which maps at chromosome 6p11-21.1, has an exon/intron structure nearly identical to Rh30 genes, which map at 1p34-36. Of the 10 exons assigned, conservation of size and sequence is confined mainly to the region from exons 2 to 9, suggesting that RH50 and RH30 were formed as two separate genetic loci from a common ancestor via a transchromosomal insertion event. The available information on the structure of RH50 facilitated search for candidate mutations underlying the Rh deficiency syndrome, an autosomal recessive disorder characterized by mild to moderate chronic hemolytic anemia and spherostomatocytosis. In one patient with the Rhnull disease of regulator type, a shortened Rh50 transcript lacking the sequence of exon 7 was detected, while no abnormality was found in transcripts encoding Rh30 polypeptides and Rh-related CD47 glycoprotein. Amplification and sequencing of the genomic region spanning exon 7 revealed a G-->A transition in the invariant GT motif of the donor splice site in both Rh50 alleles. This splicing mutation caused not only a total skipping of exon 7 but also a frameshift and premature chain termination. Thus, the deduced translation product contained 351 instead of 409 amino acids, with an entirely different C-terminal sequence following Thr315. These results identify the donor splicing defect, for the first time, as a loss-of-function mutation at the RH50 locus and pinpoint the importance of the C-terminal region of Rh50 in Rh complex formation via protein-protein interactions.

A case of Rh Null disease with haemolytic anaemia and pelger-huet anomaly

An incidental finding in the blood of a 31 -yr-old Caucasian female was the presence of red cell spherocytosis and stomatocytosis. (Pelger-Huët phenomenon was also present in her neutrophils.) She is symptomatic, with mild splenomegaly and a haemoglobin level of about 11.5 g/dl, with reticulocytosis of 6-10%. The degree of red cell atypia has been quite variable on films taken at different times. Incubated osmotic fragility and autohaemolysis are increased. No immune haemolysis or enzyme defect is demonstrable. Rh phenotyping discloses that the patient has no demonstrable Rh antigens and is thus Rh Null. Serological investigation of the family shows that both parents of the patient have suppressed Rh antigens; the remainder of the family possess varying amounts of Rh antigen including one child who appears to have increased amounts compared with the normal population. The probable genotype of the patient's 3 children is R₁r while the remainder of the family appears to be R₁R₁ This family demonstrates the effect of suppressor genes on the expression of Rh antigens and also the quantitative variation of the antigens between members of the family. This and other blood group deletions should be considered in patients with obscure haemolytic anaemia.