Overcoming methodical limits of standard RHD genotyping by next-generation sequencing.
OBJECTIVE
Molecular variations of the RHD gene may result in the reduced expression of the D antigen and altered Rh phenotypes. In many occasions, they cannot be typed reliably by standard serological methods. Sequence-based typing is the gold standard to determine rare and unknown RHD genotypes. For this pilot study, sequence-based typing by standard Sanger sequencing was compared to a newly established next-generation sequencing approach based on pyrosequencing.


METHODS
Twenty-six DNA samples were selected after primary serological testing exhibiting a weak reaction in Rh phenotype. Parallel sequence analysis of the complete coding sequence including adjacent intronic sequences allowed a comparison of the methodical potency in mutation detection of Sanger with next-generation sequencing.


RESULTS
Sanger sequencing revealed 39 RHD polymorphisms in 21 of 26 samples in the RHD coding region, while pyrosequencing detected all but two alterations resulting in a concordance rate of 94·9% and clearly revealed a heterozygous compound mutation in one sample with RHDψ and Weak D type 4 alleles. The resolution of cis/trans linkage of polymorphisms and exact characterization of a 37 bp duplication was achieved by next-generation sequencing.


CONCLUSIONS
Our data suggest that next-generation sequencing offers a new development for high-throughput and clonal sequencing for molecular RHD genotyping. However, further attempts in the methodical set-up have to be undertaken prior to validation and introduction as a routine service.
Red Cross Transfusion Service for Upper Austria, Krankenhausstrasse 7, Linz, Austria. stephanie.stabentheiner@o.roteskreuz.at
S
S
Stabentheiner
M
M
Danzer
N
N
Niklas
S
S
Atzmüller
J
J
Pröll
C
C
Hackl
H
H
Polin
K
K
Hofer
C
C
Gabriel
D001788:Blood Grouping and Crossmatching / Q000379:methods* / Q000592:standards; D005260:Female; D006801:Humans; D008297:Male; D011110:Polymorphism, Genetic; D012204:Rh-Hr Blood-Group System / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards
D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen