Molecular basis of the rare gene complex, DIVa(C)-, which encodes four low-prevalence antigens in the Rh blood group system

Background Over 40 years ago, an unusual Rh phenotype denoted DIVa(C)‐ was identified in a case of fatal haemolytic disease of the newborn in the third child of Madame Nou. Her RBCs expressed a partial D, weak C and four low‐prevalence Rh antigens: Go^a (RH30), Rh33 (RH33), Riv (RH45) and FPTT (RH50). The purpose of this study was to determine the molecular basis associated with this rare DIVa(C)‐ complex. Material and Methods Blood samples were from three donors previously identified as carrying the DIVa(C)‐ haplotype. Molecular analyses were performed by standard methods. Results The three donors were heterozygous for RHD and RHD*DIVa.2, and all carried a compound hybrid allele at the RHCE locus. This hybrid RHCE allele contained exons 2 and 3 from RHD*DIVa.2 and exon 5 from RHD [RHCE*CE‐DIVa.2(2‐3)‐CE‐D(5)‐CE] and is in cis to RHD*DIVa.2. The RHCE allele on the in trans chromosome differs between the donors and is RHCE*cE in donor 1, RHCE*ce (254C, 733G) in donor 2 and RHCE*ce in donor 3. Conclusions The RHD*DIVa.2 encodes the Go^a antigen, whereas the compound hybrid allele most likely encodes Rh33, Riv and FPTT. The weakly expressed C antigen on RBCs with the DIVa(C)‐ phenotype could be encoded by exons 2 and 3 from RHD*DIVa.2 in the compound hybrid. This is the first report of RHD*DIVa.2 being involved in a hybrid gene at the RHCE locus. As only one example of anti‐Riv has been described, our molecular analysis and findings provide a tool by which to predict Riv expression.     

On the trail of anti-CDE to unexpected highlights of the RHD*weak 4.3 allele in the Upper Austrian population

Background and Objectives The application of a commercial available microcolumn system for ABO/RH determination lead to irregular results in CDE typing of seemingly D‐ blood samples. In this study, we introduce a comprehensive serological and molecular work‐up of a novel haplotype carrying the RHD*weak 4.3 in combination with an aberrant RHCE*ce. Materials and Methods The molecular background was characterized by RHD and RHCE‐specific DNA sequencing, RHD cDNA sequencing and RHD zygosity testing. Haplotype‐specific extraction and inheritance analysis were initiated to determine the linkage of the polymorphisms. The genetic admixture was studied by whole genome SNP array analysis. Serology was done using commercial available standard techniques and by in‐house sera likewise. Results All samples (n = 29) were shown to harbour an altered RHD(T201R, F223V, P291R) allele known as RHD*weak 4.3 associated with a RHCE*ce(W16C, A36T, L245V) gene formation, expressing C^X and VS. Both anti‐C^X and anti‐V/VS were detected as contaminating antibodies in a commercial available microcolumn system for ABO/RH determination accounting for the positive results in CDE typing. Compared with other population data, the samples were clearly identified as Caucasian. Conclusion The RHD*weak 4.3 allele with markedly reduced antigen D expression was shown to be associated with an altered RHCE gene formation leading to the expression of C^X and VS. Its frequency was estimated 1 in 854 among apparently D‐ Upper Austrian blood donors.     

Frequency and characterization of known and novel RHD variant alleles in 37 782 Dutch D‐negative pregnant women

To guide anti‐D prophylaxis, Dutch D‐ pregnant women are offered a quantitative fetal‐RHD‐genotyping assay to determine the RHD status of their fetus. This allowed us to determine the frequency of different maternal RHD variants in 37 782 serologically D‐ pregnant women. A variant allele is present in at least 0·96% of Dutch D‐ pregnant women The D‐ serology could be confirmed after further serological testing in only 54% of these women, which emphasizes the potential relevance of genotyping of blood donors. 43 different RHD variant alleles were detected, including 15 novel alleles (11 null‐, 2 partial D‐ and 2 DEL‐alleles). Of those novel null alleles, one allele contained a single missense mutation (RHD*443C>G) and one allele had a single amino acid deletion (RHD*424_426del). The D‐ phenotype was confirmed by transduction of human D‐ erythroblasts, consolidating that, for the first time, a single amino acid change or deletion causes the D‐ phenotype. Transduction also confirmed the phenotypes for the two new variant DEL‐alleles (RHD*721A>C and RHD*884T>C) and the novel partial RHD*492C>A allele. Notably, in three additional cases the DEL phenotype was observed but sequencing of the coding sequence, flanking introns and promoter region revealed an apparently wild‐type RHD allele without mutations.     

Clinically relevant RHD-CE genotypes in patients with sickle cell disease and in African Brazilian donors

BackgroundAs a consequence of the homology and opposite orientation of RHD and RHCE, numerous gene rearrangements have occurred in Africans and resulted in altered RH alleles that predict partial antigens, contributing to the high rate of Rh alloimmunisation among patients with sickle cell disease (SCD). In this study, we characterised variant RH alleles encoding partial antigens and/or lacking high prevalence antigens in patients with SCD and in African Brazilian donors, in order to support antigen-matched blood for transfusion.ResultsThe distributions of RHD and RHCE alleles in donors and patients were similar. We found RHCE variant alleles inherited with altered RHD alleles in 25 out of 168 patients (15%) and in 22 out of 280 (7.8%) African Brazilian donors. The RHD and RHCE allele combinations found in the population studied were: RHD*DAR with RHCE*ceAR; RHD*weak D type 4.2.2 with RHCE*ceAR, RHD*weak D type 4.0 with RHCE*ceVS.01 and RHCE*ceVS.02; RHD*DIIIa with RHCE*ceVS.02. Thirteen patients and six donors had RHD-CE genotypes with homozygous or compound heterozygous alleles predicting partial antigens and/or lacking high prevalence antigens. Eleven patients were alloimmunised to Rh antigens. For six patients with RHD-CE genotypes predicting partial antigens, no donors with similar genotypes were found.DiscussionKnowledge of the distribution and prevalence of RH alleles in patients with SCD and donors of African origin may be important for implementing a programme for RH genotype matching in SCD patients with RH variant alleles and clinically significant Rh antibodies.     

Identification of RHCE and KEL alleles in large cohorts of Afro-Caribbean and Comorian donors by multiplex SNaPshot and fragment assays: a transfusion support for sickle cell disease patients

To lower the alloimmunization risk following transfusion in blacks, we developed two genotyping assays for large-scale screening of Comorian and Afro-Caribbean donors. One was a multiplex SNaPshot assay designed to identify ce(s) (340), ceMO/AR/EK/BI/SM, ce(s) , ce(s) (1006) and KEL*6/*7 alleles. The other was a multiplex fragment assay designed to detect RHD, RHDψ and RHCE*C and 455A>C transversion consistent with (C)ce(s) Type 1 and DIII Type5 ce(s) . Variant RHCE*ce alleles or RH haplotypes were detected in 58·69% of Comorians and 41·23% of Afro-Caribbeans. The ce(s) allele, (C)ce(s) Type 1, and DIII Type 5 ce(s) haplotypes were identified respectively in 39·13%, 14·67% and 4·88% of Comorians and 32·23%, 5·28% and 1·76% of Afro-Caribbeans. Genotypes consistent with partial D, C, c and/or e antigen expression were observed in 26·08% of Comorians and 14·69% of Afro-Caribbeans. No homozygous genotype corresponding to the RH:-18, -34, and -46 phenotypes were found. However, over 50% of genotypes produced low-prevalence antigens at risk for negative recipients, i.e., V, VS, JAL, and/or KEL6. One new variant RHCE*ce(s) (712) allele was identified. This is the first determination of variant RHCE and KEL allele frequencies. Results indicate the most suitable targets for molecular assay screening to optimize use of compatible blood units and lower immunization risk.     

Denaturing gradient gel electrophoresis: a novel method for determining Rh phenotype from genomic DNA

Denaturing gradient gel electrophoresis (DGGE) was carried out on PCR products amplified from exons 2 and 5 of RHD and RHCE . Exon 2 of RHD and exon 2 of the C allele of RHCE have an identical sequence, which differs from that of the c allele of RHCE . One band representing D and/or C, and another representing c, could be distinguished by DGGE of exon 2 amplifications of genomic DNA from individuals with the appropriate Rh phenotype. C and c could only be distinguished in D-negative samples. Exon 5 of RHD and exon 5 of the E and e alleles of RHCE all have different nucleotide sequences. Bands representing D, E and e could be distinguished following DGGE of the products of exon 5 amplification of genomic DNA from individuals with red cells of the appropriate Rh phenotype. In samples from individuals with VS+ red cells (V+ or V−) there was a shift of the band representing e. Sequencing demonstrated that VS is associated with a RHCE e sequence with a single base change predicting a Leu245 → Val substitution in the Rh polypeptide. This substitution may be responsible for the VS and e^s antigens.     

A novel c.166A>T (p.Thr56Ser) mutation in GYPB*S accounting for unusual S antigen expression

We found an individual with weakened S antigen expression on red blood cells (RBCs) during routine blood grouping. The proband was typed S+s+ by polyclonal antibodies, but the RBCs demonstrated different reactivity with three monoclonal anti‐S. The proband did not have alloanti‐S. Cloning and Sanger sequencing revealed that the proband had a c.166A>T (p.Thr56Ser) mutation in exon 4 of GYPB*S. When antibody screening of 60 455 blood donors was performed using the proband RBCs, no antibodies were detected. GYPB*S with c.166T should encode an unusual S antigen but the creation of a novel antigen remains to be investigated.     

Anti-T haemolysins: the effects of sialic acid removal and 2-aminoethylisothiouronium bromide treatment of erythrocytes on immune lysis

Background and Objectives Intravascular haemolytic reactions are reported in red‐cell T‐activated patients after blood transfusion. The relationship between T antigen antibodies present in normal plasma and these reactions remains unclear. In this study, we assessed the haemolytic activity of T antibodies in vitro in comparison with anti‐A/B antibodies. Materials and Methods We established a haemolysis assay based on treating target red‐blood‐cells (RBCs) with 2‐aminoethylisothiouronium bromide (AET). Two hundred and seven blood donor sera were analysed for anti‐T, anti‐A/B haemolysins and anti‐T agglutinins. Results Anti‐T haemolysins were found in 4 (1·9%) blood donor sera using a standard haemolysis method and in 174 (84%) samples using AET‐treated RBCs. Haemolysis correlated with agglutination titres (P < 10^?7). With both methods, anti‐T haemolysins were much weaker than anti‐A and anti‐B haemolysins. Gradual desialylation of RBCs showed a correlation between sialic acid level as indicated by agglutination with Sambucus nigra lectin and anti‐T mediated haemolysis that was significantly increased (fold 2·4) independently of T antigen expression. Conclusion These data indicate that, in vitro, anti‐T‐mediated haemolysis depends primarily on the degree of desialylation of target RBCs. They suggest that the haemolytic activity of T antibodies‐containing human sera is usually weak and may only become significant in the very rare setting of a profound desialylation of RBCs.     

Specificity and sensitivity of RHD genotyping methods by PCR-based DNA amplification

We have compared the sensitivity and specificity of four PCR methods of RHD gene detection using different sets of primers located in the regions of highest divergence between the RHD and RHCE genes, notably exon 10 (method I), exon 7 (method II), exon 4 (method III) and intron 4 (method IV). Methods I–III were the most sensitive and gave a detectable signal with D-pos/D-neg mixtures containing only 0.001% D-positive cells. Moreover, method II could detect the equivalent DNA amount present in only three nucleated cells in the assay without hybridization of PCR products, whereas the sensitivity of the other methods was 10–50 times less. Investigation of D variants indicated that false-negative results were obtained with method II (D^IVb variant), method III (D^VI and DFR variants) and method IV (D^VI variants), but not method I. Weak D (D^u) was correctly detected as D-positive by all methods, but most cases of Rh_null appeared as false-positives, as they carry normal RH genes that are not phenotypically expressed. Some false-positive results were obtained with method I in a few Caucasian DNA samples serotyped as RhD-neg but carrying a C - or E -allele, whereas a high incidence of false-positives was found among non-Caucasian Rh-negative samples by all methods. In the Caucasian population, however, we found a full correlation between the predicted genotype and observed phenotype at birth of 92 infants. Although we routinely use the four methods for RHD genotyping, a PCR strategy based on at least two methods is recommended.     

Prevalence of RHD alleles in Japanese individuals with weak D phenotype: Identification of 20 new RHD alleles

We identified 46 different RHD alleles from 226 Japanese individuals with weak D phenotype, 26 of which had been previously described and 20 that were novel. Among these weak D individuals, the alleles with c.960G>A, c.845G>A (RHD*15) or c.1013T>C (RHD*01W.24) mutations were most prevalent with relative occurrences of 36·7%, 15·9% and 9·7%, respectively. These findings demonstrate that the prevalence of common weak D alleles in the Japanese population significantly differs from that of Caucasian populations.     

Rare and transient anti‐D antibody response in D− liver transplant recipients transfused with D+ red blood cells

A retrospective analysis was conducted on 20 D^? liver transplant (LT) recipients transfused with D⁺ RBCs perioperatively and screened for RBC antibodies between 2 and 6 months later. None developed anti‐D detectable by the indirect antiglobulin test. Two patients produced weak anti‐D that reacted only with papain‐treated RBCs at 10 and 11 days without any sign of immune haemolysis. Antibodies became quickly undetectable. These data suggest an unusual pattern of alloimmunization in LT recipients with rapid, weak and transient antibody response and support the safety of transfusing D⁺ RBCs in most of D^? patients during LT surgery.     

Heterogeneity of alleles encoding high‐ and low‐prevalence red blood cell antigens across Africa: useful data to facilitate transfusion in African patients

Ethnic variations in red blood cell (RBC) antigens can be a source of alloimmunization, especially in migrant populations. To improve transfusion safety in continental Africa and countries with African migrants, we performed RBC genotyping to determine allele frequencies coding for high‐ and low‐prevalence antigens. A total of 481 blood samples were collected in ethnic groups from West, Central and East Africa. Molecular typing was performed using a polymerase chain reaction – reverse sequence specific oligonucleotide method. Results demonstrated no DI*1, DI*3, YT*2, SC*2, LW*7, KN*2 alleles in any sample and the CO*2 allele was rare. The frequency of LU*1 was comparable to that of European‐Caucasians (2%) except in Biaka pygmies (8%). The frequency of CROM*?1 was high in Mbuti pygmies (13%). High frequency of KN*7 and KN*6 may reflect selection pressure in the countries investigated. Analysis of Dombrock allele patterns confirmed uneven distribution of the DO*1 and DO*2 alleles with high frequencies of DO*?4 and DO*?5 in all groups. Altogether, findings demonstrated extensive allele‐frequency heterogeneity across Africa and suggested that knowledge of patient ethnicity gives information about the high‐prevalence antigens that may be lacking. These data are medically useful to support transfusion care of African migrants living in countries where the majority of the population is from a different ethnical background.     

Molecular characterization of a new D‐ ‐ haplotype in a Comorian man

The D‐ ‐ phenotype is a genetic variant of the Rh blood group system. It expresses D antigen but lacks C, c, E and e antigens. In D‐ ‐ phenotype, the RHCE coding region is extensively modified by RHD sequence replacement, nucleotide deletion or splice‐site changes. This article reports the identification of a new D‐ ‐ haplotype in a Comorian man. It exhibits a hybrid gene in which RHCE gene exons 3–8 have been replaced by RHD sequences on the RHCE * C allele background. This allele is associated with no expression of c/C and e/E antigens and overexpression of RhD antigen.     

Helicobacter pylori infection in the Australian community: current prevalence and lack of association with ABO blood groups

Aims: To assess the current prevalence of Helicobacter pylori infection in an Australian urban population sample and to relate this to age, gender and ABO and Rhesus blood groups. Methods: We performed a prospective epidemio­logical survey of H. pylori serological status in 500 consecutive voluntary blood donors who presented for the purpose of blood donation at the central ­Melbourne branch of the Australian Red Cross Blood Service, Victoria, Australia, and gave a Melbourne suburban home address. Results: The overall prevalence of specific anti‐H. pylori IgG antibodies in this cohort was 32% (95% confidence interval = 28?36%) and H. pylori sero­positivity increased with age. The rate of H. pylori infection was not significantly different in men and women, with anti‐H. pylori IgG anti­bodies detected in 35% (97/277) of men compared with 28% (63/233) of women (P = 0.12). Similarly, H. pylori serological status was not significantly different between subjects of different ABO (P = 0.18) or Rhesus blood groups (P = 0.55). Conclusion: This study showed that, contrary to expectation, the updated prevalence of H. pylori seropositivity (32%) in this Melbourne sample is at least as high as that found in previous Australian studies over the past 19 years. Seropositivity increased with age, and was not related to gender, confirming the infection pattern seen in other developed nations. Despite epidemiological evidence of increased peptic ulcer disease in ABO blood group O subjects, and recent evidence that H. pylori adhesion to gastric epithelial cells is mediated by blood group epitopes, no significant association between blood groups and H. pylori serological status was detected. (Intern Med J 2003; 33: 163?167)     

Blood group genotype analysis for the quality improvement of reagent test red blood cells

BACKGROUND AND OBJECTIVES: Reagent red blood cells (RBCs) for antibody detection should express certain important antigens as a double dose, that is, the donors must be homozygous for the corresponding alleles. Traditionally, dose is determined by serological typing and known allele frequencies. However, RHD zygosity cannot be predicted serologically owing to the absence of an antithetical antigen, and FY zygosity is confounded by two variant haplotypes, FY*0 and FY*X. Furthermore, lack of reagents hampers our ability to type for some clinically important antigen pairs such as Do(a)/Do(b). MATERIALS AND METHODS: Genomic DNA was isolated from reagent RBC samples. Established, validated methods were used to determine the RHD, FY, and DO genotypes. RESULTS: Three of 52 D+ samples gave results that differed from the predicted genotype: two presumed R(1)R(1) samples and an R(2)R(2) sample were shown to be R(1)r' and R(2)r', respectively. Five of 59 samples that were from presumed homozygotes for either FY*A or FY*B were heterozygous, together with either FY*X (three samples) or FY*0 (two samples). Seventy-five samples tested for DO were DO*A/A (n = 14), DO*A/B (n = 39), or DO*B/B (n = 22). CONCLUSIONS: The results show that serologically determined RhD and Duffy phenotypes of reagent RBCs are unreliable and that antigens we thought were represented as a double dose were single dose. The addition of Dombrock genotyping provides information which is useful in antibody identification. We conclude that selected genotype analyses are a valuable quality assurance measure to ensure that reagent RBCs comply with national and international recommendations for test sensitivity.     

Lack of G blood group antigen in DIIIb erythrocytes is associated with segmental DNA exchange between RH genes

Summary. The Rh blood group antigens D, Cc and Ee are encoded by two highly related genes, RHD and RHCE. Almost all red cells which carry D and all cells which carry C also express the G (Rh12) antigen. In this report we have determined the molecular basis of the D^IIIb category phenotype which represents a very rare condition characterized by the presence of most of the D epitopes and the total absence of the antigen G. mRNA sequencing and Southern blot analysis of two unrelated samples indicated that the D^IIIb category phenotype is associated with a segmental DNA exchange between exon 2 of the RHD and RHCE genes resulting in three D c amino acid substitutions (Ile60Leu, Ser68Asn Ser103Pro).     

Weakened expression of 'e' owing to concomitant occurrence of Cys16 and Val245 (VS antigen)

BACKGROUND AND OBJECTIVES: The 48 G>C transversion in exon 1 of the RHCE gene leads to Trp16Cys, usually present in the conventional RHCE Ce, while Trp16 is associated with RHCE ce. The presence of Cys16 in RHCE ce is associated with the R(0) (Dce) haplotype in Africans, leading to a weak 'e' antigen expression on red blood cells (RBCs). VS is a common red cell antigen in individuals of African descent and results from a single point mutation in exon 5 of the RHCE (733C>G), leading to Leu245Val substitution; VS positivity is also associated with weak expression of 'e'. This study investigated the association of Cys16 and/or VS with the RHCE ce alleles in a cohort of sickle cell disease (SCD) patients phenotyped as R(0)r or R(0)R(0) and rr. MATERIALS AND METHODS: DNA samples from 58 SCD patients were tested for the 48 G>C transversion, encoding Cys16, by allele-specific polymerase chain reaction (PCR). We also amplified exon 5 of the RHCE by PCR and subjected the amplified product to restriction fragment length polymorphism analysis, using BfaI, in order to determine the VS status. Further cDNA analysis was performed on three samples to verify whether the mutations were located on the same or on different alleles. RESULTS: Fifty-six of the 58 SCD patients studied (97%) were heterozygous for 48G/48C (Cys16). Of these, 18 (32%) were also heterozygous for 733C/G (245Val). All of these 18 samples showed weak 'e' expression on RBCs when tested with at least one monoclonal antibody to e antigen. cDNA sequencing of three of 18 patient samples showed that the genes encoding Cys16 and Val245 (VS) were on different alleles. CONCLUSIONS: We found a high incidence of Cys16 associated with the RHCE ce in our SCD cohort. A high percentage of these patients were also found to be heterozygous for VS. cDNA analysis showed that, in at least three samples, the two mutations were on different alleles, with consequent weakening of expression of the e antigen on RBCs.     

Molecular analysis of blood group Rh transcripts from a rGr variant

The Rh blood group antigens D, Cc and Ee are encoded by two related genes, RHD and RHCE . The RhG antigen (Rh12) is associated with the expression of RhC and/or RhD, except in rare variant red cells. Here we have determined the molecular basis of G expression in the absence of D and C in the r^Gr phenotype. Nucleotide sequence analysis revealed that the r  ^G allele resulted either from a segmental DNA exchange between part of exon 2 of the RHce gene and the equivalent region of the RHCE or RHD genes or from a crossing over between positions nt150 and nt178 of the RHce and RHCe genes. The predicted protein encoded by the hybrid r  ^G gene ( c-C-e or c-D-e ) carries Ile60, Ser68 and Ser103 (as C and D polypeptides); any of these positions appear to be critical in the formation of the G antigen. In addition, Cys16 was found to be important in the phenotypic expression of C.