Insights into anti-D formation in carriers of RhD variants through studies of 3D intraprotein interactions.

BACKGROUND: Many RhD variants associated with anti-D formation (partial D) in carriers exposed to the conventional D antigen carry mutations affecting extracellular loop residues. Surprisingly, some carry mutations affecting transmembrane or intracellular domains, positions not thought likely to have a major impact on D epitopes. STUDY DESIGN AND METHODS: A wild-type Rh trimer (RhD1 RhAG2 ) was modeled by comparative modeling with the human RhCG structure. Taking trimer conformation, residue accessibility, and position relative to the lipid bilayer into account, we redefine the domains of the RhD protein. We generated models for RhD variants carrying one or two amino acid substitutions associated with anti-D formation in published articles (25 variants) or abstracts (12 variants) and for RHD*weak D type 38. We determined the extracellular substitutions and compared the interactions of the variants with those of the standard RhD. RESULTS: The findings of the three-dimensional (3D) analysis were correlated with anti-D formation for 76% of RhD variants: 15 substitutions associated with anti-D formation concerned extracellular residues, and structural differences in intraprotein interactions relative to standard RhD were observed in the others. We discuss the mechanisms by which D epitopes may be modified in variants in which the extracellular residues are identical to those of standard RhD and provide arguments for the benignity of p.T379M (RHD*DAU0) and p.G278D (RHD*weak D type 38) in transfusion medicine. CONCLUSION: The study of RhD intraprotein interactions and the precise redefinition of residue accessibility provide insight into the mechanisms through which RhD point mutations may lead to anti-D formation in carriers.

New molecular basis associated with CD36-negative phenotype in the sub-Saharan African population.

BACKGROUND: CD36 glycoprotein is expressed by various cell types, including platelets (PLTs), monocytes, and erythroid precursors, and is also the receptor for several ligands. However, absence of CD36 expression seems asymptomatic and is poorly described in Caucasians. In contrast, the frequency reaches 7% and 11% in African Caribbean and Asian persons, respectively. Lack of CD36 expression exposes to the risk of immunization in case of pregnancy or PLT transfusion. Two types of deficiency have been described: in Type I, PLTs and monocytes lack CD36 expression and the subjects are homozygous or compound heterozygous for CD36 mutations, whereas in Type II, only PLTs (Type IIa), and rarely also erythroid cells (Type IIb), are affected. Molecular events leading to Type II deficiency are poorly understood. CASE REPORT: An African girl, diagnosed with homozygous sickle cell disease and regularly transfused, was assessed for PLT CD36 expression by immunofluorescence microscopy. The deficiency was then confirmed by monoclonal antibody immobilization of PLT antigen (MAIPA) assay, and the subtype was assessed by flow cytometry. The underlying molecular basis was characterized by DNA sequencing. Furthermore, we tested the serum for possible anti-CD36 immunization. RESULTS AND CONCLUSION: Flow cytometric analysis on the patient's blood samples allowed the diagnosis of Type I CD36 deficiency. CD36 antibodies, probably due to her past history of red blood cell transfusions, were identified by MAIPA and by Luminex technology assay. Interestingly, we identified through sequencing a new molecular basis involved in CD36 deficiency: two adenines were replaced by one guanine in Exon 4 (c.367_368delAAinsG) leading to a stop codon at Position 76.

Defining Blood Group Gene Reference Alleles by Long-Read Sequencing: Proof of Concept in the ACKR1 Gene Encoding the Duffy Antigens.

BACKGROUND: In the novel era of blood group genomics, (re-)defining reference gene/allele sequences of blood group genes has become an important goal to achieve, both for diagnostic and research purposes. As novel potent sequencing technologies are available, we thought to investigate the variability encountered in the three most common alleles of ACKR1, the gene encoding the clinically relevant Duffy antigens, at the haplotype level by a long-read sequencing approach. MATERIALS AND METHODS: After long-range PCR amplification spanning the whole ACKR1 gene locus (∼2.5 kilobases), amplicons generated from 81 samples with known genotypes were sequenced in a single read by using the Pacific Biosciences (PacBio) single molecule, real-time (SMRT) sequencing technology. RESULTS: High-quality sequencing reads were obtained for the 162 alleles (accuracy >0.999). Twenty-two nucleotide variations reported in databases were identified, defining 19 haplotypes: four, eight, and seven haplotypes in 46 ACKR1*01, 63 ACKR1*02, and 53 ACKR1*02N.01 alleles, respectively. DISCUSSION: Overall, we have defined a subset of reference alleles by third-generation (long-read) sequencing. This technology, which provides a "longitudinal" overview of the loci of interest (several thousand base pairs) and is complementary to the second-generation (short-read) next-generation sequencing technology, is of critical interest for resolving novel, rare, and null alleles.

Alloimmunization risk associated with amino acid 223 substitution in the RhD protein: analysis in the light of molecular modeling.

BACKGROUND: Partial D status is a major concern for transfusion and pregnancy, due to the possibility of carriers becoming immunized. When known carriers of a D variant have never been exposed to complete D, they are assumed to have D partial status based on the position of the amino acid substituted. New approaches for predicting immunization risk are required. We built a three-dimensional (3D) structural model to investigate the consequences of substitutions of Amino Acid 223 involved in a large number of D variants. STUDY DESIGN AND METHODS: Homology modeling was performed with multiple templates. The model was evaluated by comparing the interactions of the known p.Phe223Val variant (RHD*08.01) and a new p.Phe223Ser variant (RHD*52) to RhD reference allele (p.Phe223). The consequences predicted by modeling the variants were compared with serologic data. RESULTS: The 3D structural model was generated from two related protein structures and assessed with state-of-the-art approaches. An analysis of the interactions of the variant Residue 223 in the proposed 3D model highlighted the importance of this position. Modeling predictions were consistent with the serologic and clinical data obtained for the D antigen with a substitution of Amino Acid 223. CONCLUSION: We used a 3D structural model to evaluate the effect of the p.Phe223 substitution on the conformation of the RhD protein. This model shed light on the influence of substitutions on the structure of the RhD protein and the associated alloimmunization risk. These initial findings indicate that the p.Phe223Ser variant can be considered partial.

High immunogenicity of red blood cell antigens restricted to the population of African descent in a cohort of sickle cell disease patients.

BACKGROUND: Sickle cell disease (SCD) patients undergo multiple red blood cell (RBC) transfusions and are regularly exposed to low-prevalence (LP) antigens specific to individuals of African descent. This study evaluated the prevalence of antibodies against LP antigens in SCD patients and the need to identify these antibodies in everyday practice. STUDY DESIGN AND METHODS: Plasma from 211 SCD patients was tested with RBCs expressing the following LP antigens: RH10 (V), RH20 (VS), RH23 (DW ), RH30 (Goa ), KEL6 (Jsa ), and MNS6 (He). RESULTS: Nine LP antibodies were found in eight patients (3.8%): five anti-RH23, two anti-RH30, and two anti-MNS6. The exposure risk, calculated for each LP antigen, was below 3% per RBC unit, for all antigens tested. Thus, in this cohort of transfused SCD patients, the prevalence of LP antibodies was similar to that of antibodies against antigens of the FY, JK, and MNS blood group systems. These findings also reveal the occurrence of anti-RH23 in SCD patients. No anti-RH20 or anti-KEL6 were found, despite the high frequency of mismatch situations. CONCLUSION: These results highlight the immunogenicity of these LP antigens, and the evanescence of antibodies against LP antigens. They also highlight the importance of appropriate pretransfusion testing for patients frequently transfused, who are likely to be exposed to multiple types of blood group antigens.

Genotyping in Sickle Cell Disease Patients: The French Strategy.

This review presents the French strategy for blood group genotyping in high-responder and newly diagnosed sickle cell disease (SCD) patients. In addition to FY, JK, and MNS genotyping, the RH blood group system is now explored in SCD patients in France. Molecular typing has been used for the deduction of partial RH2 (C) antigens since 2010, and the gradual implementation of systematic RHD and RHCE genotyping nationwide was initiated in late 2014. In our laboratory, 962 RH:2 (C-positive) SCD patients have been tested since 2010, and 1,148 SCD patients of all RH phenotypes have been genotyped for clinically relevant alleles of RHD and RHCE since late 2014.

Molecular dynamics of the human RhD and RhAG blood group proteins.

Introduction: Blood group antigens of the RH system (formerly known as "Rhesus") play an important role in transfusion medicine because of the severe haemolytic consequences of antibodies to these antigens. No crystal structure is available for RhD proteins with its partner RhAG, and the precise stoichiometry of the trimer complex remains unknown. Methods: To analyse their structural properties, the trimers formed by RhD and/or RhAG subunits were generated by protein modelling and molecular dynamics simulations were performed. Results: No major differences in structural behaviour were found between trimers of different compositions. The conformation of the subunits is relatively constant during molecular dynamics simulations, except for three large disordered loops. Discussion: This work makes it possible to propose a reasonable stoichiometry and demonstrates the potential of studying the structural behaviour of these proteins to investigate the hundreds of genetic variants relevant to transfusion medicine.

RHCE*cE734C allele encodes an altered c antigen and a suppressed E antigen not detected with standard reagents.

BACKGROUND: The RH blood group system has many RHCE variant alleles that have arisen through gene conversion or nucleotide changes. Two probands, with red blood cells (RBCs) that were D+C+E-c+(w) e+ were sent to our laboratories to resolve the weak c expression. STUDY DESIGN AND METHODS: Hemagglutination tests were performed by automated and manual procedures. Genomic DNA analysis was performed by sequencing of Exons 1 to 10 of RHCE and RHD. RESULTS: The probands' RBCs did not react with standard monoclonal anti-E reagents from Bio-Rad, Diagast, DiaMed, Immucor, Ortho, and Quotient. The RBCs reacted variably with anti-c reagents from Diagast, DiaMed, Immucor, or Ortho and did not react with the Quotient anti-c reagent. Surprisingly, sequencing results of RHCE showed the presence of C/G at Position 676 (E/e polymorphism) and the association of the E polymorphism with a 734T>C transition in Exon 5 of the RHCE, encoding a Leu245Pro amino acid substitution in the mature RhcE polypeptide. Replacement of leucine 245 by proline in the eighth transmembrane domain of the RhcE protein may have a steric effect on the protein such that most anti-E reagents do not bind and the interaction between anti-c and c antigen is also affected. CONCLUSION: We report a novel RHCE*cE allele, RHCE*cE734C, which was assigned the provisional ISBT allele name RHCE*cE.14 or RHCE*03.14. It was found in two probands whose RBCs had weakened c expression and typed E- with conventional anti-E reagents. These data, once again, highlight the fact that the genotype does not always reflect the phenotype.

A Review of the Literature Organized Into a New Database: RHeference.

Hundreds of articles containing heterogeneous data describe D variants or add to the knowledge of known alleles. Data can be difficult to find despite existing online blood group resources and genetic and literature databases. We have developed a modern, elaborate database for D variants, thanks to an extensive literature search with meticulous curation of 387 peer-reviewed articles and 80 abstracts from major conferences and other sources. RHeference contains entries for 710 RHD alleles, 11 RHCE alleles, 30 phenotype descriptions (preventing data loss from historical sources), 35 partly characterized alleles, 3 haplotypes, and 16 miscellaneous entries. The entries include molecular, phenotypic, serological, alloimmunization, haplotype, geographical, and other data, detailed for each source. The main characteristics are summarized for each entry. The sources for all information are included and easily accessible through doi and PMID links. Overall, the database contains more than 10,000 individual pieces of data. We have set up the database architecture based on our previous expertise on database setup and biocuration for other topics, using modern technologies such as the Django framework, BioPython, Bootstrap, and Jquery. This architecture allows an easy access to data and enables simple and complex queries: combining multiple mutations, keywords, or any of the characteristics included in the database. RHeference provides a complement to existing resources and will continue to grow as our knowledge expands and new articles are published. The database url is http://www.rheference.org/.

Partial C antigen in sickle cell disease patients: clinical relevance and prevention of alloimmunization.

BACKGROUND: Partial Rh antigens have been widely described in black individuals. Carriers are prone to immunization when exposed to the normal antigens. In sickle cell disease (SCD), patient alloimmunization is a major cause of transfusion failure. The potential of individuals with partial C antigen to make anti-C has not been investigated. We sought partial C status and anti-C production in a cohort of SCD patients with the C+ phenotype, to determine whether exposure to normal C antigen should be avoided. STUDY DESIGN AND METHODS: We constituted a cohort of 177 randomly selected SCD patients expressing C antigen. We screened for (C)ce(s) and R(N) haplotypes, presumably associated with partial C antigen in Afro-Caribbeans, and we recorded the number of transfused C+ red blood cell (RBC) units, immunization status, and extended phenotype. RESULTS: Forty-nine patients carried abnormal C antigen, deduced from the presence of (C)ce(s) and/or R(N), not compensated by a normal RHC allele in trans. Among patients with partial C phenotype exposed repeatedly to C+ RBCs, 30% produced anti-C. Two patients experienced hemolysis. In our hospital, with 22% of SCD patients expressing C, prevention of anti-C immunization for all individuals with partial C antigen would require a 7% increase in the use of C- RBC units. These RBCs are already in short supply for SCD patients who are C-. CONCLUSION: This study demonstrates the need to detect partial C within C+ SCD patients and to prevent immunization. A larger number of Afro-Caribbeans donors is needed to provide these patients with C- RBCs.

Weak D phenotypes and transfusion safety: where do we stand in daily practice?

BACKGROUND: Weak D Types 1, 2, and 3 recipients cannot be immunized when exposed to D antigen. Molecular biology is very efficient to type weak D variants but rarely implemented in daily practice. The serologic typing practice of weak D in a Caucasian patient population was analyzed and a transfusion strategy is proposed. STUDY DESIGN AND METHODS: Samples typed either ddCcee or ddccEe in routine laboratories were tested with the indirect antiglobulin test (D(u) test). D(u)-positive samples were screened for weak D alleles Types 1, 2, and 3 and further tested with immunoglobulin M (IgM) anti-D reagents, used in a fully automated device. RESULTS: A total of 468 of 55,162 samples were found to be ddCcee or ddccEe. Ninety-three expressed weak D after the D(u) test leading to D+ assignment for transfusion. Seventy-three percent of D(u)-positive samples were weak D alleles Type 1, 2, or 3. Almost all weak D Types 1, 2, and 3 were positive with IgM reagents in gel matrix with an automated device. Other variants that could be potentially associated with anti-D alloimmunization, however, were also positive. CONCLUSION: Serology is very sensitive to detect weak D Types 1, 2, and 3, but there is no cutoff to distinguish variants of clinical significance. When molecular analysis is not available, it is proposed that a D+ status for blood recipients found to be weak D with a sensitive method be assigned, except for women of childbearing age or younger, because of the remaining possibility to be partial D or other rare weak D who can be immunized.