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International audience; 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.

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.

Introduction L’ISBT repertorie plus de 70 alleles RHCE*ce dont 11 alleles nuls. Ces alleles nuls sont le fait d’evenements genetiques varies (insertion, deletion, mutation). Nous presentons ici le cas d’une patiente avec un nouvel RHCE*ce nul et un allele codant un groupe sanguin rare. Methodes Les tests serologiques automatises et manuels pour l’expression des antigenes D, C, E, c, e ont ete realises par des techniques standards d’hemagglutination avec des reactifs Bio-Rad. Apres extraction de l’ADNg, le genotypage RH a ete effectue a l’aide d’analyses de PCR en temps reel et de sequencage des 10 exons du gene RHCE. Etude patiente enceinte de 25 ans de phenotype RH :1,w2,−3,−4,w5 originaire d’Afrique de l’Ouest. Les analyses de PCR en temps reel montrent la presence d’un allele RHCE*CeRN (RHCE*02.10.01) et d’un allele RHCE*ce. Le sequencage confirme ces donnees et permet aussi d’identifier une mutation heterozygote c.48G > C (pTrp16Cys) dans l’exon 1 du gene RHCE et une deletion heterozygote c.366Gdel dans l’exon 3 du gene RHCE (p.Ser122Serfs*2). Cette deletion est associee a un decalage de cadre de lecture dans l’ARNm et a un codon stop premature dans le 3e exon, conduisant a une proteine Rh tronquee et non fonctionnelle. En raison de l’expression affaiblie des antigenes C et e, causee par l’allele RHCE*CeRN et le phenotype c negatif, il est raisonnable de supposer que la nouvelle deletion est sur un allele RHCE*ce. Conclusion Nous decrivons un nouvel allele RHCE*ce nul, RHCE*ce 48 C,366delG, qui a recu par l’ISBT le numero provisoire : RHCE*01N.12. En raison de l’allele rare RHCE*CeRN, le phenotype de la patiente devient alors : RH :1,p2,−3,−4,p5, 32, −46.

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.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.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.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.

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+we+ 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.

Le developpement des techniques de genotypage basees sur l’amplification genique permet de predire la presence ou l’absence d’antigenes de groupes sanguins a la surface des globules rouges. Ces techniques s’averent utiles lorsque les methodes serologiques sont inenvisageables. C’est le cas quand un patient a ete recemment transfuse ou encore si l’on constate une saturation des globules rouges par des autoanticorps. Le genotypage moleculaire avec l’avenement de la PCR en temps reel limite le risque de contamination d’amplicons d’ADN inter echantillons car sans manipulation post-PCR. Au-dela de cet aspect primordial, la nouvelle generation d’automates permet d’obtenir les resultats de genotypage en moins de 45 minutes. Pour repondre a l’urgence transfusionnelle, nous avons developpe au laboratoire IHM de l’EFS IdF un protocole d’urgence de genotypage courant des antigenes de groupes sanguins. Ce protocole est compose de deux etapes : la premiere consiste a extraire l’ADN genomique en moins de 5 minutes a temperature ambiante en mettant en presence 2 μL de sang total avec une solution denaturante ; la deuxieme etape est basee sur l’amplification et la discrimination allelique en point final (PCR en temps reel par sondes d’hydrolyses) des cibles moleculaires FY*1/*2, FY*Fy, JK*1/*2, MNS*3/*4, UvarP2, UvarNY. Nous avons valide ce protocole sur des ADNg extraits a partir de patients atteints de differentes pathologies demontrant ainsi la robustesse et la fiabilite de la technologie PCR en temps reel par sondes d’hydrolyses. Afin d’etre reactifs devant l’urgence, des plaques de 96 puits sont preparees avec le melange reactionnel et conserve a −20 °C.

International audience; 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.

En Ile-de-France, la recherche des haplotypes (C)ce s et RN est systematique depuis 2010 chez les patients drepanocytaires presentant un phenotype C+. Cette preconisation est basee sur une etude ayant demontre la frequence importante de ces haplotypes dans la population afro-antillaise, et le risque d’allo-immunisation anti-C chez les porteurs de ces haplotypes. En 2011, un nouveau variant C partiel, DIVa(C)−, a ete decrit. Dans cette etude, nous avons evalue la frequence de cet haplotype au sein de notre population de patients pour determiner s’il devait etre recherche systematiquement. Trois tests PCR en temps reel developpes au laboratoire, pour determiner la presence des haplotypes (C)ce s , RN et DIVa(C)−, ont ete testes chez 495 patients drepanocytaires C+ adresses au laboratoire IHM de l’EFS IdF entre 2010 et 2014. Les pourcentages de variants (C)ce s et RN, 11 % et 21 % respectivement, correspondent aux frequences deja decrites. Parmi ces 161 patients C partiel de type (C)ce s et RN, 4 sont homozygotes pour l’haplotype (C)ce s (RH:-34) et 2 pour l’haplotype RN (RH:-46). Seulement 3 patients (0,6 %) presentent un haplotype DIVa(C)−. Enfin, pour 11 patients, avec affaiblissement serologique de C, aucune alteration moleculaire connue n’est retrouvee. Cette etude confirme l’interet de rechercher les variants partiels de C chez les patients drepanocytaires. La determination du polymorphisme C (presence ou absence) peut orienter, en premiere intention, vers les variants a rechercher.