Premium
RHD and RHCE variant and zygosity genotyping via multiplex ligation–dependent probe amplification
Author(s) -
HaerWigman Lonneke,
Veldhuisen Barbera,
Jonkers Remco,
Lodén Martin,
Madgett Tracey E.,
Avent Neil D.,
Haas Masja,
Schoot C. Ellen
Publication year - 2013
Publication title -
transfusion
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.045
H-Index - 132
eISSN - 1537-2995
pISSN - 0041-1132
DOI - 10.1111/j.1537-2995.2012.03919.x
Subject(s) - zygosity , genotyping , multiplex ligation dependent probe amplification , allele , multiplex , biology , genetics , typing , multiplex polymerase chain reaction , microbiology and biotechnology , polymerase chain reaction , genotype , exon , gene
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 * DCS 2 , a new partial RHD allele, RHD *525T (Phe175Leu), and a new D – null allele, RHD *443 G ( T hr148 A rg). 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.