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.

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.

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.

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.

Osteoblastic differentiation of human mesenchymal stem cells with platelet lysate.

Culture of expanded mesenchymal stem cells (MSCs) seeded on biomaterials may represent a clinical alternative to autologous bone graft in bone regeneration. Foetal bovine serum (FBS) is currently used for MSC expansion, despite risks of infectious disease transmission and immunological reaction due to its xenogenic origin. This study aimed to compare the osteogenic capacities of clinical-grade human MSCs cultured with FBS or allogenic human platelet lysate (PL). In vitro, MSCs cultured in PL both accelerate the expansion rate over serial passages and spontaneously induce osteoblastic gene expression such as alkaline phosphatase (ALP), bone sialoprotein (BSP), osteopontin (Op) and bone morphogenetic protein-2 (BMP-2). In vivo, ectopic bone formation is only observed on ceramics seeded with MSCs grown in PL medium implanted under the skin of immunodeficient mice for 7 weeks. In conclusion, allogenic human PL accelerates MSC proliferation and enhances MSC osteogenic differentiation.