RHD and RHCE genotyping by next-generation sequencing is an effective strategy to identify molecular variants within sickle cell disease patients

Blood Cells Mol Dis. 2017 Jun;65:8-15. doi: 10.1016/j.bcmd.2017.03.014. Epub 2017 Mar 31.


Background: 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.

Aims: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Anemia, Sickle Cell / blood
  • Anemia, Sickle Cell / diagnosis*
  • Anemia, Sickle Cell / genetics*
  • Anemia, Sickle Cell / therapy
  • Blood Transfusion / methods
  • Disease Management
  • Genetic Testing / methods
  • Genetic Variation*
  • Genotype*
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Isoantibodies / blood
  • Isoantibodies / immunology
  • Phenotype
  • Reproducibility of Results
  • Rh-Hr Blood-Group System / genetics*


  • Isoantibodies
  • RH-antibodies
  • RHCE protein, human
  • Rh-Hr Blood-Group System
  • Rho(D) antigen