Evidence that the RHD(VI) deletion genotype does not exist.
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Gene Deletion↗
Biomedical subjects
B H Faas
Publications and source records attributed to B H Faas.
Molecular background of VS and weak C expression in blacks.
BACKGROUND: The Rh system is complex and consists of as many as 45 different antigens. Red cells of about 25 percent of the black population carry VS an Rh-system antigen (Rh20), but this antigen is very rare in whites. VS positivity is always associated with a weak expression of e, and usually also of C. STUDY DESIGN AND METHODS: The RH genes of 11 black VS-positive donors were studied. Transcripts were sequenced for four VS-positive donors, three of whom had red cells with a weak expression of C. In the other donors, only analysis of genomic DNA was carried out. RESULTS: The occurrence of VS was shown to be related to a single-point mutation in exon 5 of the RHCE gene (cytosine 733 guanine, leading to the Leu245Val substitution). The presence of this polymorphism in exon 5 may explain the simultaneously occurring weak e, because the E/e polymorphism is located in the same exon. Study of VS-positive donors with different Rh phenotypes showed that the polymorphism can occur in different alleles of the RHCE gene. In all three donors whose red cells showed a weak expression of C, a hybrid D-CE-D transcript was found, containing exon 4, 5, 6, 7, and (probably) 8 from the RHCE gene. No transcripts were encountered carrying DNA markers normally associated with C expression. CONCLUSION: It is therefore postulated that the hybrid gene is responsible for the weak expression of C in these individuals. The hybrid gene carried a Leu62Phe substitution, as well as the Leu245Val substitution responsible for VS. The gene most probably cosegregates with a C allele encoding Cys 16 (normally encoded only by the C allele) and Val245 (responsible for VS antigenicity when encoded by the RHCE gene). This explains the combination of weak expression of C and VS positivity that is frequently found in blacks.
Africa, Northern↗
Lower antigen site density and weak D immunogenicity cannot be explained by structural genomic abnormalities or regulatory defects of the RHD gene.
BACKGROUND: The weak D phenotype is characterized serologically by a weak or negative agglutination reaction with polyclonal anti-D in an immediate-spin test. Agglutination is enhanced in the indirect antiglobulin test. Red cells that are typed weak D have a much lower number of apparently complete D antigens at their cell surface and are associated with considerably weaker immunogenicity than are red cells with normal D. In a previous study, the number of D sites per cell was determined in eight unrelated weak D individuals to range from 490 to 1870 D sites per cell, which corresponded to 4 to 14.2 percent of the number of D sites in CcDee samples. STUDY DESIGN AND METHODS: The RHD gene was investigated for structural abnormalities by Southern blot experiments and polymerase chain reaction-based RHD typing in these individuals. In addition, abnormalities in the transcription process were studied by sequence analysis of RH transcripts and by comparing the relative amounts of RHD mRNA in weak D to those in CcDee, CcDEe, and -D- samples by using a semiquantitative reverse transcriptase-polymerase chain reaction analysis. RESULTS: The RHD gene in weak D phenotypes does not show any abnormalities at either the genomic or the transcriptional level when compared to the RHD gene in normal D phenotypes. CONCLUSION: The weaker immunogenicity of weak D is not explained by structural difference in the RHD gene itself. The weaker expression of D might be caused by factors involved in the Rh-related complex or by an as yet unidentified suppressor gene. This study supports the concept that weak D phenotypes carry complete D polypeptides and reflect a quantitative rather than a qualitative variation of D.
Antibodies, Monoclonal↗
The R0Har RH:33 phenotype results from substitution of exon 5 of the RHCE gene by the corresponding exon of the RHD gene.
The highly polymorphic Rh (Rhesus) system is encoded by two homologous genes, one encoding the D polypeptide and the other the CcEe polypeptides. Partial D antigens may be caused by gene rearrangements, deletions or point mutations. In this study the molecular basis of R0Har RH:33, a Rh phenotype of low frequency, is described. The R0Har RH:33 phenotype is characterized by partial expression of D, altered expression of e, absence of G and the presence of two antigens of low frequency: Rh33 and FPTT. Southern blot analysis, RHD typing by PCR and sequence analysis of Rh transcripts revealed that the RHD gene is absent in subjects with this phenotype. Apart from the expected RHCE transcripts, a new Rh transcript, RHc(D)(e), was identified in three unrelated individuals expressing R0Har Rh:33. The RHc(D)(e) transcript showed the same sequence as the RHce transcript, with the exception of exon 5, which was substituted by the corresponding exon of the RHD gene. A method for PCR-based genotyping was developed to determine specifically the c(D)(e) haplotype. The c(D)(e) PCR proved to be a reliable alternative method for R0Har RH:33 typing.
Base Sequence↗
The genetic basis of a new partial D antigen: DDBT.
The Rh system, the most polymorphic system on red cells, is genetically controlled by two different but highly homologous genes on chromosome 1. The RHCE gene encodes different RhCcEe polypeptides and the RHD gene encodes D antigens. It is well established that in D negative individuals the RHD gene is either absent or grossly deleted. The D antigen comprises at least nine serologically defined D epitopes. The D antigen can be divided into different partial D categories, reflecting a different pattern of specific D epitopes. In this study a newly defined partial D antigen, DDBT, was studied. D epitope mapping revealed the presence of D epitopes 6/7 and 8 and the absence of the other D epitopes. The molecular basis of this phenotype was studied by Southern blotting, by RHD typing using the polymerase chain reaction (RHD-PCR) and by sequence analysis of Rh transcripts. The DBT phenotype appeared to be encoded by a hybrid RHD gene, in which exons 5, 6 and 7 (and possibly the identical exon 8) were replaced by the corresponding exons of the RHCE gene. From this study it may be concluded that D epitopes 1, 2, 3, 4, 5 and 9 are dependent on the presence of RHD exons 5, 6, and 7.
Base Sequence↗
Involvement of Ser103 of the Rh polypeptides in G epitope formation.
BACKGROUND: Almost all red cells that carry D and/or C antigens also express the G antigen (Rh12). A study was conducted on the molecular background of the G epitope. STUDY DESIGN AND METHODS: Two unrelated donors with the rare ccDEe, G- phenotype and one donor with the ccEe, G+ phenotype were studied. Genomic DNA and cDNA of these donors were studied with polymerase chain reaction, Southern blot, and sequence analysis, with special focus on exon 2, because it is only in this exon that there are supposed to be similarities between RHD and the RHC allele, but not between RHD and the RHc allele. RESULTS: In both ccDEe, G- donors, a nucleotide substitution was found in exon 2 of RHD; T307 was replaced by C307, which predicted a Ser->Pro substitution at amino acid position 103 of the D polypeptide. The ccEe, G+ donor carried the complete exon 2 of RHD. Moreover, despite the absence of all known D epitopes, this donor also carried RHD characteristics in exons 1 to 3 and exon 9 and further downstream. CONCLUSION: Ser103, encoded by exon 2 of the RH genes, is involved in G epitope formation.
Blotting, Southern↗
Characterization of the hybrid RHD gene leading to the partial D category IIIc phenotype.
BACKGROUND: A D-positive white woman was found to have produced alloanti-D leading to hemolytic disease of the newborn in her third D-positive child. The maternal D was identified as the partial D category IIIc antigen (DIIIc). The molecular basis of this phenotype was studied. STUDY DESIGN AND METHODS: The proposita and her relatives were phenotyped for Rh system antigens with standard reagents. D(IIIc) typing of D-positive red cells was done with serum that contained anti-D from the proposita. Southern blot analysis and RHD-specific polymerase chain reactions were performed with genomic DNA. Rh transcripts were cloned and sequenced. RESULTS: Six relatives of the proposita were found to express the DIIIc phenotype, which traveled with Ce. The DIIIc phenotype was inherited in a Mendelian fashion. Southern blot analysis showed an identical digestion pattern in D(IIIc) individuals and in DD controls. Three different Rh transcripts were found. Two Rh transcripts were derived from RHCE (RHce and RHCe). The RHD-derived Rh transcript was the same as that of the published RHD sequence, apart from exon 3, which appeared to be exon 3 of RHCE. At the genomic level, RHD exon 3 was missing in all individuals expressing D(IIIc). CONCLUSION: This study shows the characteristics of a new hybrid D-CE-D allele encoding D(IIIc). It may be concluded that exon 3 of RHD is not involved in the formation of any of the D epitopes known at present, but rather encodes a new D epitope or D epitopes, as yet undefined by monoclonal anti-D reagents.
Adult↗
Rapid Rh D genotyping by polymerase chain reaction-based amplification of DNA.
Rh (rhesus) D is the dominant antigen of the Rh blood group system. Recent advances in characterization of the nucleotide sequence of the cDNA(s) encoding the Rh D polypeptide allow the determination of the Rh D genotype at the DNA level. This can be of help in cases in which red blood cells are not available for phenotyping, eg, when in concerns a fetus. We have tested three independent DNA typing methods based on the polymerase chain reaction (PCR) for their suitability to determine the Rh D genotype. DNA derived from peripheral blood mononuclear cells from 234 Rh-phenotyped healthy donors (178 Rh D positive and 56 Rh D negative) was used in the PCR. The Rh D genotypes, as determined with a method based on the allele-specific amplification of the 3' noncoding region of the Rh D gene described by Bennett et al (N Engl J Med 329:607, 1993), were not concordant with the serologically established phenotypes in all cases. We have encountered 5 discrepant results, ie, 3 false-positive and 2 false-negative (a father and child). Rh D genotyping with the second method was performed by PCR amplification of exon 7 of the D gene with allele-specific primers. In all donors phenotyped as D positive tested so far (n = 178), the results of molecular genotyping with this method were concordant with the serologic results, whereas a false-positive result was obtained in one of the D-negative donors (also false-positive in the first method). Complete agreement was found between genotypes determined in the third method, based on a 600-bp deletion in intron 4 of the Rh D gene described by Arce et al (Blood 82:651, 1993), and serologically determined phenotypes. The Rh blood group system is complex, and unknown polymorphisms at the DNA level are expected to exist. Therefore, although genotypes determined by the method of Arce et al were in agreement with serotypes, it cannot yet be regarded as the golden standard. More experience with this or other methods is still needed.
Alleles↗
Rh E/e genotyping by allele-specific primer amplification.
It has been shown that the Rhesus (Rh) blood group antigens are encoded by two homologous genes: the Rh D gene and the Rh CcEe gene. The Rh CcEe gene encodes different peptides: the Rh C, c, E, and e polypeptides. Only one nucleotide difference has been found between the alleles encoding the Rh E and the Rh e antigen polypeptides. It is a C-->G transition at nucleotide position 676, which leads to an amino acid substitution from proline to alanine in the Rh e-carrying polypeptide. Here we present an allele-specific primer amplification (ASPA) method to determine the Rh E and Rh e genotypes. In one polymerase chain reaction, the sense primer had a 3'-end nucleotide specific for the cytosine at position 676 of the Rh E allele. In another reaction, a sense primer was used with a 3'-end nucleotide specific for the guanine at position 676 of the Rh e allele and the Rh D gene, whereas the antisense primer had a 3'-end nucleotide specific for the adenine at position 787 of the Rh CcEe gene. We tested DNA samples from 158 normal donors (including non-Caucasian donors and donors with rare Rh phenotypes) in these assays. There was full concordance with the results of serologic Rh E/e phenotyping. Thus, we may conclude that the ASPA approach leads to a simple and reliable method to determine the Rh E/e genotype. This can be useful in Rh E/e genotyping of fetuses and/or in cases in which no red blood cells are available for serotyping. Moreover, our results confirm the proposed association between the cytosine/guanine polymorphism at position 676 and the Rh E/e phenotype.
Alleles↗
Involvement of Gly96 in the formation of the Rh26 epitope.
BACKGROUND: The Rh system, a complex blood group system, comprises at least 45 antigens. Red cells expressing c usually express Rh26. Rare cells that are c+ Rh:-26 give variable reactions with anti-c and may have weak expression of f (ce). STUDY DESIGN AND METHODS: Serologic and molecular studies were performed with red cells from persons with the c+ Rh:-26 phenotype occurring in two unrelated Dutch families. Red cells of 11 members of these two families were typed for Rh26, for c (with monoclonal and polyclonal reagents), and for f (ce). The cDNA of three donors was sequenced, while restricted DNA analysis was carried out on material from available members of the two families. RESULTS: Serologic tests showed that the rare c+ Rh:-26 phenotype was associated with a weak expression of c and a normal expression of f. The cDNA analysis of three members of one family revealed a single-point mutation (G286A) in exon 2 of the ce allele. Allele-specific primer amplification, polymerase chain reaction followed by allele-specific restriction analysis, and single-strand conformation polymorphism showed the same polymorphism in all other members of both families, whereas it was absent in 80 control donors. CONCLUSION: The c+ Rh:-26 phenotype, identified in two families, is associated with a single-point mutation at nucleotide 286 (G286A) in the ce allele, which predicts a Gly96Ser amino acid substitution. This substitution also affects c, because all anti-c reagents reacted more weakly. Other polymorphic sites apparently are involved in the formation of the Rh26 epitope as well, because Rh26 is expressed only on the c polypeptide, whereas Gly96 is expressed on all polypeptides.
Alleles↗