scholarly journals Rapid Rh D genotyping by polymerase chain reaction-based amplification of DNA

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2975-2980 ◽  
Author(s):  
S Simsek ◽  
BH Faas ◽  
PM Bleeker ◽  
MA Overbeeke ◽  
HT Cuijpers ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Blood Group ◽  
False Positive ◽  
Complete Agreement ◽  
Blood Group System ◽  
Chain Reaction ◽  
Group System ◽  
Allele Specific ◽  
Polymerase Chain ◽  
Rh Blood Group

Rh (rhesus) D is the dominant antigen of the Rh blood group system. Recent advances in characterization of the nucleotide sequence of the cDNA(s) encoding the Rh D polypeptide allow the determination of the Rh D genotype at the DNA level. This can be of help in cases in which red blood cells are not available for phenotyping, eg, when in concerns a fetus. We have tested three independent DNA typing methods based on the polymerase chain reaction (PCR) for their suitability to determine the Rh D genotype. DNA derived from peripheral blood mononuclear cells from 234 Rh-phenotyped healthy donors (178 Rh D positive and 56 Rh D negative) was used in the PCR. The Rh D genotypes, as determined with a method based on the allele-specific amplification of the 3′ noncoding region of the Rh D gene described by Bennett et al (N Engl J Med 329:607, 1993), were not concordant with the serologically established phenotypes in all cases. We have encountered 5 discrepant results, ie, 3 false-positive and 2 false-negative (a father and child). Rh D genotyping with the second method was performed by PCR amplification of exon 7 of the D gene with allele-specific primers. In all donors phenotyped as D positive tested so far (n = 178), the results of molecular genotyping with this method were concordant with the serologic results, whereas a false-positive result was obtained in one of the D-negative donors (also false-positive in the first method). Complete agreement was found between genotypes determined in the third method, based on a 600-bp deletion in intron 4 of the Rh D gene described by Arce et al (Blood 82:651, 1993), and serologically determined phenotypes. The Rh blood group system is complex, and unknown polymorphisms at the DNA level are expected to exist. Therefore, although genotypes determined by the method of Arce et al were in agreement with serotypes, it cannot yet be regarded as the golden standard. More experience with this or other methods is still needed.

scholarly journals Molecular basis of the RhCW (Rh8) and RhCX (Rh9) blood group specificities

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 1196-1201
Author(s):  
I Mouro ◽  
Y Colin ◽  
P Sistonen ◽  
PY Le Pennec ◽  
JP Cartron ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Blood Group ◽  
Point Mutations ◽  
Blood Group Antigens ◽  
Chain Reaction ◽  
Definitive Evidence ◽  
Allele Specific ◽  
The Common ◽  
Polymerase Chain ◽  
Rh Blood Group

The Rh blood group antigens are encoded by two highly related genes, RHD and RHCE, and the sequence of the common alleles (D, Ce, CE, ce, and cE) of these genes has been previously elucidated. In this report, Rh transcripts and gene fragments have been amplified using polymerase chain reaction from the blood of donors with the CW+ andCX+ phenotypes. Sequence analysis indicated that the expression of the CW (Rh8) and CX (Rh9) antigens are associated with point mutations in the RHCE gene, which provides the definitive evidence that the CW and CX specificities are encoded by the same gene as the Cc and Ee antigens. As compared with the common (CW- and CX-) transcripts of the RHCE gene, the CW+ and CX+ cDNAs exhibited A122G and G106A transitions that resulted in Gln41Arg and Ala36Thr amino acid substitutions in the CW+ and CX+ polypeptides, respectively. Therefore, although the CW and CX specificities behave serologically as if they were allelic, they cannot not be considered, stricto sensu, as the products of antithetical allelic forms of the RHCE gene. Based on the CW-/CW+ nucleotide polymorphism, a polymerase chain reaction assay useful for diagnosis purposes has been developed that detects the presence of the CW+ allele by the use of an allele-specific primer.

scholarly journals Molecular basis of the RhCW (Rh8) and RhCX (Rh9) blood group specificities

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 1196-1201 ◽  
Author(s):  
I Mouro ◽  
Y Colin ◽  
P Sistonen ◽  
PY Le Pennec ◽  
JP Cartron ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Blood Group ◽  
Point Mutations ◽  
Blood Group Antigens ◽  
Chain Reaction ◽  
Definitive Evidence ◽  
Allele Specific ◽  
The Common ◽  
Polymerase Chain ◽  
Rh Blood Group

Abstract The Rh blood group antigens are encoded by two highly related genes, RHD and RHCE, and the sequence of the common alleles (D, Ce, CE, ce, and cE) of these genes has been previously elucidated. In this report, Rh transcripts and gene fragments have been amplified using polymerase chain reaction from the blood of donors with the CW+ andCX+ phenotypes. Sequence analysis indicated that the expression of the CW (Rh8) and CX (Rh9) antigens are associated with point mutations in the RHCE gene, which provides the definitive evidence that the CW and CX specificities are encoded by the same gene as the Cc and Ee antigens. As compared with the common (CW- and CX-) transcripts of the RHCE gene, the CW+ and CX+ cDNAs exhibited A122G and G106A transitions that resulted in Gln41Arg and Ala36Thr amino acid substitutions in the CW+ and CX+ polypeptides, respectively. Therefore, although the CW and CX specificities behave serologically as if they were allelic, they cannot not be considered, stricto sensu, as the products of antithetical allelic forms of the RHCE gene. Based on the CW-/CW+ nucleotide polymorphism, a polymerase chain reaction assay useful for diagnosis purposes has been developed that detects the presence of the CW+ allele by the use of an allele-specific primer.

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