title: RHD and RHCE variant and zygosity genotyping via multiplex ligation-dependent probe amplification.
abstract: BACKGROUND
  The presence of a D variant may hamper correct serologic D typing, which may result in D immunization. D variants can be determined via RHD genotyping. However, a convenient single assay to identify D variants is still lacking. We developed and evaluated a multiplex ligation-dependent probe amplification (MLPA) assay to determine clinically relevant RHD and RHCE variant alleles and RHD zygosity.
  
  
  METHODS
  We analyzed 236 cases (73 normal and 163 selected samples) with the RH-MLPA assay, which is able to determine 79 RHD and 17 RHCE variant alleles and RHD zygosity. To confirm the results, mutations were verified by RHD and/or RHCE exon-specific sequencing and RHD zygosity was verified by quantitative real-time polymerase chain reaction (PCR) for 18 cases.
  
  
  RESULTS
  In 99% of the cases, the RH-MLPA assay correctly determined whether a person carried only wild-type RHD and RHCE alleles (n = 69) or (a) variant RHD allele(s) and/or (a) variant RHCE allele(s) (n = 164). In only three cases, including two new RHD variant alleles, the variant allele was not identified, due to lack of detecting probes. These were RHD*DCS2, a new partial RHD allele, RHD*525T (Phe175Leu), and a new D- null allele, RHD*443G (Thr148Arg). All RHD (n = 175) and RHCE variant alleles (n = 79) indicated by the RH-MLPA assay were confirmed by sequencing. RHD zygosity was confirmed by quantitative PCR. Two hematopoietic chimeras were recognized.
  
  
  CONCLUSIONS
  The RH-MLPA genotyping assay is a fast, easy, and reliable method to determine almost all clinically relevant RHD and RHCE variant alleles, RHD zygosity, and RHD+/RHD- chimeras in blood donors, blood recipients, and pregnant women.
authors.affiliation: Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, and MRC-Holland, Amsterdam, the Netherlands. L.Wigman@sanquin.nl
authors.forename: Lonneke
authors.initials: L
authors.lastname: Haer-Wigman
authors.forename: Barbera
authors.initials: B
authors.lastname: Veldhuisen
authors.forename: Remco
authors.initials: R
authors.lastname: Jonkers
authors.forename: Martin
authors.initials: M
authors.lastname: Lodén
authors.forename: Tracey E
authors.initials: TE
authors.lastname: Madgett
authors.forename: Neil D
authors.initials: ND
authors.lastname: Avent
authors.forename: Masja
authors.initials: M
authors.lastname: de Haas
authors.forename: C Ellen
authors.initials: CE
authors.lastname: van der Schoot
mesh_terms: D005091:Exons; D005838:Genotype; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D012204:Rh-Hr Blood-Group System / Q000235:genetics*
chemical_list: C066468:RHCE protein, human; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen