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RHD and RHCE variant and zygosity genotyping via multiplex ligation-dependent probe amplification.
MedLine Citation:
PMID:  23043317     Owner:  NLM     Status:  Publisher    
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. STUDY DESIGN AND 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. CONCLUSION: 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.
Lonneke Haer-Wigman; Barbera Veldhuisen; Remco Jonkers; Martin Lodén; Tracey E Madgett; Neil D Avent; Masja de Haas; C Ellen van der Schoot
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-10-9
Journal Detail:
Title:  Transfusion     Volume:  -     ISSN:  1537-2995     ISO Abbreviation:  Transfusion     Publication Date:  2012 Oct 
Date Detail:
Created Date:  2012-10-9     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0417360     Medline TA:  Transfusion     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
© 2012 American Association of Blood Banks.
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