BLOOD GROUP GENOMICS
RHD and RHCE variant and zygosity genotyping via multiplex ligation–dependent probe amplification
Article first published online: 9 OCT 2012
© 2012 American Association of Blood Banks
Volume 53, Issue 7, pages 1559–1574, July 2013
How to Cite
Haer-Wigman, L., Veldhuisen, B., Jonkers, R., Lodén, M., Madgett, T. E., Avent, N. D., de Haas, M. and van der Schoot, C. E. (2013), RHD and RHCE variant and zygosity genotyping via multiplex ligation–dependent probe amplification. Transfusion, 53: 1559–1574. doi: 10.1111/j.1537-2995.2012.03919.x
- Issue published online: 11 JUL 2013
- Article first published online: 9 OCT 2012
- Manuscript Accepted: 20 AUG 2012
- Manuscript Revised: 17 JUL 2012
- Manuscript Received: 16 MAR 2012
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.
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.
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.