Abstract
Background
As 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.
Material and methods
RH genotypes were determined in 168 DNA samples from SCD patients and 280 DNA samples from African Brazilian donors. Laboratory developed tests, RHD BeadChipTM, RHCE BeadChipTM, cloning and sequencing were used to determine RHD-CE genotypes among patients and African Brazilian blood donors.
Results
The 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.
Discussion
Knowledge 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.
Keywords: RHCE, RHD, RH alleles, RHD-CE haplotypes, sickle cell disease
Introduction
Sickle cell disease (SCD) is the most prevalent hereditary disease in Brazil and affects around 30,000 patients with approximately 3,500 new cases each year. Around 50% of the patients are chronically transfused and alloimmunisation is a serious complication in these patients, with severe clinical consequences, including delay in obtaining matched blood as well as potentially life-threatening delayed haemolytic transfusion reactions, autoantibody formation, and hyperhaemolysis syndrome1. In an effort to reduce alloimmunisation, some programmes have been designed and implemented to provide antigen-matched red blood cell (RBC) transfusions to patients with SCD. Although these programmes are succeeding in reducing alloimmunisation, some chronically transfused patients still become alloimmunised to Rh antigens and develop delayed haemolytic transfusion reactions, indicating the need to transfuse more precisely RH-matched blood2–6.
RH gene variation is frequent in individuals of African descent, generating RHD and RHCE altered alleles predicting partial antigens and lacking high prevalence antigens, such as hrB and hrS, contributing to Rh alloimmunisation in patients with SCD and making it a challenge to find compatible blood7,8. As such, matching programmes used to prevent alloimmunisation in SCD are starting to include matching for relevant RH haplotypes6,9.
Genetic variation in the African population is widespread and complex and multiple RHCE alleles may appear to have the same predicted phenotype10. Different combinations of RhCE variants, such as, ceAR, ceMO, ceEK, ceBI and ceMI proteins, all lacking the high prevalence hrS antigen, have been reported in patients who make anti-Rh1811. However, an antibody made by a patient with one of these variants is not necessarily compatible with RBC from an hrS-negative donor with a different molecular background. In addition, the variant RHCE can be inherited with an altered RHD and therefore in addition to making antibodies to RhCE antigens the patients can make anti-D11.
Some variant RH alleles are due to single nucleotide changes, but the majority are due to gene rearrangements that result in hybrid alleles12. Different methods including polymerase chain reaction (PCR) (restriction fragment length polymorphism [RFLP], allele-specific-PCR, DNA microarrays, sequencing of genomic DNA, cloning and sequencing of complementary DNA) have been used to predict Rh phenotypes and to select the appropriate blood component for the transfusion of patients with SCD.
The purpose of this study was to determine the diversity and frequency of RHD-CE genotypes, predicting partial antigens in patients with SCD and in African Brazilian donors in order to find, through the use of RH genotyping, more closely matched donors for SCD patients who are alloimmunised to Rh antigens.
Materials and methods
Samples
DNA samples were obtained from 280 random African Brazilian donors and from 168 patients with SCD, who were receiving extended antigen-matched RBC units. All donors and patients agreed to participate in this study by signing an Institutional Review Board form giving their approved, informed consent.
DNA preparation
Genomic DNA was extracted from whole blood by manual spin column separation (QIAmp, Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions.
DNA microarray analysis
DNA microarray analysis was performed using wRHCE and wRHD BeadChips from BioArray Solutions (Immucor, Warren, NJ, USA). The chips contained 34 markers associated with RhD altered expression and 25 polymorphisms associated with altered RHCE alleles. Of note, wRHD and wRHCE BeadChips devices are currently for “research use only”.
DNA sequence analysis
DNA sequences were analysed on PCR products amplified from gDNA for all samples that were not characterised by the RHD and RHCE BeadChips, in order to determine the specific allele present using RHD and RHCE specific primers, as previously reported11,13. PCR products were purified by elution from 1% agarose gels using a Qiaex II gel extraction kit (Qiagen, Valencia, CA, USA), and sequenced directly, without subcloning, on an ABI 373XL Perkin Elmer Biosystems (PEB) sequencer, using the PEB Big Dye reagent BD (GenPak, Perkin Elmer Biosystems, Foster City, CA, USA).
Complementary DNA cloning and sequencing
In order to determine RH allelic combinations on samples identified with RHCE variants, we performed Rh-cDNA cloning and sequencing. RNA was isolated from reticulocytes with TriZol (Invitrogen, Carlsbad, CA, USA). Reverse transcription was carried out with Superscript First Strand Synthesis (Invitrogen, Carlsbad, CA, USA) using gene-specific primers. PCR products, amplified from cDNA, were purified with ExoSAP-IT (USB, Cleveland, OH, USA), cloned into a TA vector (Invitrogen) and sequenced using primers, as previously reported14.
Determination of RHD zygosity
For specific detection of the RHD gene deletion, we used PCR-RFLP (amplification of the downstream and hybrid Rhesus box as well as digestion of the PCR products with the restriction enzyme Pst I), as previously reported15. We also used a quantitative PCR approach16, complemented by the specific detection of RHDΨ17.
Serological testing
Serological testing for specific Rh antigens in all patients and donors with RH variant alleles was performed by haemagglutination in tubes or on gel cards. Antibodies were screened for and identified by an indirect antiglobulin test in gel. A direct antiglobulin test and autologous control were performed in gel for all samples that were antigen-positive for the corresponding Rh antibody specificity. Eluates were obtained from all samples with a positive direct antiglobulin test using an acid elution method (Diacidel, Bio-Rad, Cressier-sur-Morat, Switzerland). Adsorption onto autologous RBC was also performed to aid the differentiation of autoantibodies and alloantibodies.
Results
Population studied
One hundred and sixty-eight patients with SCD (homozygous for haemoglobin S) were enrolled in the present study. The median age of the patients was 33 years old and they were receiving an average of 83 blood units each year (range: 3–386). Two-hundred and eighty blood donors who were self-identified as African Brazilians were also included in the study.
RHCE alleles
Clinically relevant RHCE variant alleles were found in 73/168 (43%) patients and in 35/280 (12.5%) donors. The distribution of the alleles is shown in Table I. RHCE genotypes encoding partial antigens were found in 15/168 (9%) of patients and in 4/280 (1.4%) donors as represented in Table II.
Table I.
Clinically relevant RHCE variant alleles in patients with SCD and in African Brazilian donors.
| Patients n (%) | Donors n (%) | RHCE alleles | Predicted phenotypes |
|---|---|---|---|
| 19 (11.3) | 11 (3.9) | RHCE*ceVS.01 (733G) | partial c, partial e, hrB+w/− |
| 21 (12.5) | 14 (5) | RHCE*ceVS.02 (48C,733G) | partial c, partial e, hrB− |
| 7 (4.2) | 4 (1.4) | RHCE*ceVS.03 (48C, 733G, 1006T) | partial c, partial e, hrB− |
| 4 (2.4) | 2 (0.7) | RHCE*ceVS.04 (48G, 733G, 1025T) | partial e, hrB− |
| 11 (6.5) | 3 (1.1) | RHCE*ceAR | partial c, partial e, hrS− |
| 5 (3) | 1 (0.4) | RHCE*ceMO | partial c, partial e, hrB−, hrS− |
| 1 (0.6) | 0 | RHCE*ceEK | partial c, partial e, hrS− |
| 1 (0.6) | 0 | RHCE*ceTI | partial e |
| 2 (1.2) | 0 | RHCE*ceBI | hrS− |
| 1 (0.6) | 0 | RHCE*ceAG | partial e, hrB− |
| 1 (0.6) | 0 | RHCE*ceJAL | partial e |
SCD: sickle cell disease.
Table II.
Clinically relevant RHCE genotypes in patients with SCD and in African Brazilian donors.
| Patients n (%) | Donors n (%) | RHCE genotypes |
|---|---|---|
| 4 (2.4) | 2 (0.7) | RHCE*ceVS.02/RHCE*ceVS.02 |
| 3 (1.8) | 1 (0.4) | RHCE*ceVS.01/RHCE*ceVS.01 |
| 2 (1.2) | 0 | RHCE*ceAR/RHCE*ceAR |
| 2 (1.2) | 0 | RHCE*ceAR/RHCE*ceVS.02 |
| 1 (0.6) | 0 | RHCE*VS.01/RHCE*VS.03 |
| 1 (0.6) | 1 (0.4) | RHCE*ceAR/RHCE*ceVS.01 |
| 1 (0.6) | 0 | RHCE*ceMO/RHCE*ceMO |
| 1 (0.6) | 0 | RHCE*ceEK/RHCE*ceEK |
SCD: sickle cell disease.
RHD-CE genotypes
The distribution of clinically relevant RHD and RHCE variant alleles in donors and patients was similar. As shown in Table III, we found RHCE variant alleles inherited with altered RHD alleles in 25/168 patients (15%) and in 22/280 (7.8%) African Brazilian donors. The RHD and RHCE allele combinations found in the studied population 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 and RHD*DIIIa-CE(4–7)-D with RHCE*ceVS.02 and RHCE*ceVS.03.
Table III.
RHCE variant alleles inherited with altered RHD alleles in patients with SCD and in African Brazilian donors and RHD-CE genotypes in patients with SCD alloimmunised to Rh antigens and in African Brazilian donors.
| Patients n (%) | Donors n (%) | Combinations of RHD and RHCE variant alleles | Patients n (%) | Donors n (%) | RHD-CE genotypes | Predicted phenotypes | Rh antibodies |
|---|---|---|---|---|---|---|---|
| 2 (1.2) | 3 (1.1) | RHD*weak D type 4.0/RHCE*ceVS.01 | 3 (1.8) | 0 | RHD*weak D type 4.0-RHD*Ψ/RHCE*ceVS.01-VS.01 | partial D, partial c, partial e | anti-D, anti-e |
| 4 (2.4) | 7 (2.5) | RHD*weak D type 4.0/RHCE*ceVS.02 | 2 (1.2) | 2 (0.7) | RHD*weak D type 4.0-RHDΨ/RHCE*ceVS.02-VS.02 | partial D, partial c, partial e, hrB− | anti-e |
| 7 (4.2) | 6 (2.1) | RHD*weak D type 4.2.2/RHCE*ceAR | 2 (1.2) | 1 (0.4) | RHD*weak D type 4.2.2-deleted D/RHCE*ceAR-cE | partial D, partial e, hrS− | anti-e |
| 4 (2.4) | 2 (0.7) | RHD*DAR/RHCE*ceAR | 1 (0.6) | 1 (0.4) | RHD*DAR-DAR/RHCE*ceAR-ceAR | partial D, partial c, partial e, hrS− | anti-D, anti-hrS |
| 2 (1.2) | 1 (0.4) | RHD*DAR/RHCE*ceVS.02 | 1 (0.6) | 1 (0.4) | RHD*DAR-deleted D/RHCE*ceAR-ceVS.02 | partial D, partial c, partial e | ------ |
| 1 (0.6) | 1 (0.4) | RHD*DAU-0/RHCE*ceMO | 1 (0.6) | 1 | RHD*DAR-RHD*Ψ/RHCE*ceAR-ceVS.02 | partial D, partial c, partial e | ------ |
| 1 (0.6) | 1 (0.4) | RHD*DIIIa/RHCE*ceVS.02 | 1 (0.6) | 0 | RHD*DIIIa-DIIIa/RHCE*ceVS.02-ceVS.02 | partial D, partial c, partial e, hrB− | anti-D, anti-e |
| 2 (1.2) | 1 (0.4) | RHD*DIIIa/RHCE*ce VS.03 | 2 (1.2) | 0 | RHD*DIIIa-CE(4–7)-D/ RHCE*ce-VS.02-VS.03 | partial C, partial c, partial e, hrB− | Anti-C, anti-hrB |
| 1 (0.6) | 0 | RHD*DOL-2/RHCE*ceBI | |||||
| 1 (0.6) | 0 | RHD*DIVa/RHCE*ceTI |
SCD: sickle cell disease.
RHD-CE genotypes predicting partial antigens and/or lacking hrB or hrS antigens
Thirteen patients and six donors had RHD-CE genotypes with homozygous or compound heterozygous alleles predicting partial antigens and/or lacking high prevalence antigens such as hrB and hrS. The frequencies of RHD-CE genotypes in the patients and donors are shown in Table III.
Rh alloimmunisation in patients with clinically relevant RHD-CE genotypes
Antibody screening and autologous adsorption results showed that 11 of 13 patients with altered RHD-CE genotypes had Rh alloantibodies. RHD-CE genotypes found in patients and the Rh antibodies produced are displayed in Table III.
Discussion
We transfuse about 19,500 RBC units per year to all 368 patients with SCD registered in our institution. Extended genotyping is performed on patients with a recent transfusion history (chronically transfused patients), patients with a positive direct antiglobulin test and those with RH variants. Although we have a large inventory of phenotyped/genotyped RBC units in our hospital, only 5% are genotyped for RH variants. Consequently, we do not have an ongoing programme for RH genotype matching in SCD patients with RH variant alleles and clinically significant Rh antibodies. This situation is of concern as prophylaxis to maintain higher haemoglobin levels in patients is restricted; therefore, the number of transfusions in such patients is lower and, due to the clinical risk, the least incompatible blood is being transfused. This concern prompted us to determine the diversity and frequency of RHD-CE genotypes to predict partial antigens and/or those high prevalence antigens that are lacking in patients with SCD and in African Brazilian donors in order to find more closely matched donors for patients with SCD who are alloimmunised to Rh antigens.
Our results show that RHCE*ceVS.01 and RHCE*ceVS.02, predicting partial c, partial e and lack of the high prevalence hrB antigen, are the most prevalent variant alleles in patients and donors, followed by RHCE*ceAR predicting partial c, partial e and the lack of the high prevalence hrS antigen. For the most common RHCE alleles, we found at least one compatible donor but when we analysed the homozygous and compound heterozygous RHCE genotypes, we verified that, although rare, they are more prevalent in patients (9%) than in blood donors (1.4%). Our donors were found to have more RHCE variant alleles in trans to a conventional RHCE allele than our patients and those cases demonstrating expression of partial RhCE antigens were dependent on the second RHCE allele. As the Brazilian population is heterogeneous in ethnic origin, having undergone an intense process of admixture between descendants of Europeans, Africans and Native Americans18, we believe that this difference between patients and donors with regards to homozygous and compound heterozygous RHCE genotypes is due to a higher degree of admixture in donors than in patients.
When we analysed variant RHCE alleles inherited with altered RHD alleles, we found the same combinations in patients and donors, although the RHD-CE genotypes predicting partial antigens and/or lacking high prevalence antigens were less frequent in donors. Using this donor pool, we evaluated the feasibility of considering patient and African Brazilian donor RH genotypes to provide perfectly RH-matched blood to the alloimmunised patients with SCD and verified that, for six patients with confirmed antibodies, we would not have donors with the same RHD-CE genotypes. In addition, we noted that even having donors with the same RH genotypes, we need to screen many more African donors to meet the needs of patients with variant alleles requiring long-term transfusion support.
Eleven of 13 patients with RHD-CE genotypes predicting partial antigens and/or lacking high prevalence antigens, who received an average of 83 RBC units per year, developed Rh antibodies (Table III). This result reinforces our previous finding that patients with RH variants alleles can make clinically significant Rh antibodies19.
It is well established that transfusion recipients with the most common weak D type 1, 2 and 3 are not at risk of forming alloanti-D when exposed to conventional RhD-positive RBC20–22, but there are few reports of anti-D in patients with weak D type 4.023. In our study, three patients with the RHD*weak D type 4.0 allele in trans to RHDΨ developed anti-D, therefore providing new evidence that patients with this weak D type can make anti-D.
The possibility of identifying RhD and RhCE variants and discriminating partial antigens using molecular methods improved the practice of transfusion, especially for patients with SCD. The provision of RH-genotyped matched units may decrease the rate of Rh alloimmunisation and delayed haemolytic transfusion reactions in such patients and even without a perfect match, we could select the units based on RH alleles. As the number of RH genotyped donors available is limited at present to meet the needs of patients with variants and confirmed alloantibodies, it is important to develop an approach to identify donors with RH variants to ensure safe transfusions for patients
Conclusions
Knowledge of clinically relevant RH genotypes in patients with SCD and in donors is essential to assess the potential risk of alloimmunisation and is necessary to determine the number of donors required to support patients with SCD with RH-genotype matched units.
Acknowledgement
The Authors thank Nicola Conran for her careful English review of the manuscript.
Footnotes
Authorship contributions
LC designed the study and wrote the manuscript; ACG performed laboratory assays and wrote the manuscript; EAS and MDdM collected the samples and data, and JPJ and FFC revised the manuscript.
The Authors declare no conflicts of interest.
Funding and resources
This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grant n. 2015/07559-9 (LC) and 2014/00984-3 (FFC).
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