title: RHD and RHCE genotyping by next-generation sequencing is an effective strategy to identify molecular variants within sickle cell disease patients.
abstract: The complexity of Rh genetic variation among sickle cell disease (SCD) patients is high. Conventional molecular assays cannot identify all genetic variants already described for the RH locus as well as foresee novel alleles. Sequencing RHD and RHCE is indicated to broaden the search for Rh genetic variants.
  
  
  
  To standardize the Next Generation Sequencing (NGS) strategy to assertively identify Rh genetic variants among SCD patients with serologic suspicion of Rh variants and evaluate if it can improve the transfusion support.
  
  
  
  Thirty-five SCD patients with unexplained Rh antibodies were enrolled. A NGS-based strategy was developed to genotype RHD and RHCE using gene-specific primers. Genotype and serological data were compared.
  
  
  
  Data obtained from the NGS-based assay were gene-specific. Ten and 25 variant RHD and RHCE alleles were identified, respectively. Among all cases of unexplained Rh antibodies, 62% had been inaccurately classified by serological analysis and, of these, 73.1% were considered as relevant, as were associated with increased risk of hemolytic reactions and shortage of units suitable for transfusion.
  
  
  
  The NGS assay designed to genotype RH coding regions was effective and accurate in identifying variants. The proposed strategy clarified the Rh phenotype of most patients, improving transfusion support.
authors.affiliation: Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil.
authors.forename: Marcia R
authors.initials: MR
authors.lastname: Dezan
authors.affiliation: Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil.
authors.forename: Ingrid Helena
authors.initials: IH
authors.lastname: Ribeiro
authors.affiliation: Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil.
authors.forename: Valéria B
authors.initials: VB
authors.lastname: Oliveira
authors.affiliation: Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil.
authors.forename: Juliana B
authors.initials: JB
authors.lastname: Vieira
authors.affiliation: Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil.
authors.forename: Francisco C
authors.initials: FC
authors.lastname: Gomes
authors.affiliation: Institute of Tropical Medicine, Department of Infectious Disease, University of São Paulo, São Paulo, Brazil.
authors.forename: Lucas A M
authors.initials: LAM
authors.lastname: Franco
authors.affiliation: Institute of Tropical Medicine, Department of Infectious Disease, University of São Paulo, São Paulo, Brazil.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Varuzza
authors.affiliation: Institute of Tropical Medicine, Department of Infectious Disease, University of São Paulo, São Paulo, Brazil.
authors.forename: Roberto
authors.initials: R
authors.lastname: Ribeiro
authors.affiliation: Discipline of Hematology, University of São Paulo School of Medicine, São Paulo, Brazil.
authors.forename: Karen Ziza
authors.initials: KZ
authors.lastname: Chinoca
authors.affiliation: Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil.
authors.forename: José Eduardo
authors.initials: JE
authors.lastname: Levi
authors.affiliation: Discipline of Hematology, University of São Paulo School of Medicine, São Paulo, Brazil.
authors.forename: José Eduardo
authors.initials: JE
authors.lastname: Krieger
authors.affiliation: Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of São Paulo school of Medicine.
authors.forename: Alexandre Costa
authors.initials: AC
authors.lastname: Pereira
authors.affiliation: Discipline of Hematology, University of São Paulo School of Medicine, São Paulo, Brazil.
authors.forename: Sandra F M
authors.initials: SFM
authors.lastname: Gualandro
authors.affiliation: Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil; Discipline of Hematology, University of São Paulo School of Medicine, São Paulo, Brazil.
authors.forename: Vanderson G
authors.initials: VG
authors.lastname: Rocha
authors.affiliation: Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Mendrone-Junior
authors.affiliation: Institute of Tropical Medicine, Department of Infectious Disease, University of São Paulo, São Paulo, Brazil.
authors.forename: Ester Cerdeira
authors.initials: EC
authors.lastname: Sabino
authors.affiliation: Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil; Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of São Paulo school of Medicine. Electronic address: cdinardo@sp.gov.br.
authors.forename: Carla Luana
authors.initials: CL
authors.lastname: Dinardo
mesh_terms: D000483:Alleles; D000755:Anemia, Sickle Cell / Q000097:blood / Q000175:diagnosis* / Q000235:genetics* / Q000628:therapy; D001803:Blood Transfusion / Q000379:methods; D019468:Disease Management; D005820:Genetic Testing / Q000379:methods; D014644:Genetic Variation; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007518:Isoantibodies / Q000097:blood / Q000276:immunology; D010641:Phenotype; D015203:Reproducibility of Results; D012204:Rh-Hr Blood-Group System / Q000235:genetics*
chemical_list: D007518:Isoantibodies; C033242:RH-antibodies; C066468:RHCE protein, human; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen