Denaturing gradient gel electrophoresis: a novel method for determining Rh phenotype from genomic DNA

Abstract

Denaturing gradient gel electrophoresis (DGGE) was carried out on PCR products amplified from exons 2 and 5 of RHD and RHCE. Exon 2 of RHD and exon 2 of the C allele of RHCE have an identical sequence, which differs from that of the c allele of RHCE. One band representing D and/or C, and another representing c, could be distinguished by DGGE of exon 2 amplifications of genomic DNA from individuals with the appropriate Rh phenotype. C and c could only be distinguished in D-negative samples. Exon 5 of RHD and exon 5 of the E and e alleles of RHCE all have different nucleotide sequences. Bands representing D, E and e could be distinguished following DGGE of the products of exon 5 amplification of genomic DNA from individuals with red cells of the appropriate Rh phenotype. In samples from individuals with VS+ red cells (V+ or V-) there was a shift of the band representing e. Sequencing demonstrated that VS is associated with a RHCE e sequence with a single base change predicting a Leu245 --> Val substitution in the Rh polypeptide. This substitution may be responsible for the VS and e5 antigens.

A new blood group system, RHAG: three antigens resulting from amino acid substitutions in the Rh-associated glycoprotein

“…Antigenic changes associated with amino acid substitutions buried in the membrane are well-established in Rh (e.g. VS [31] and DAR [32]), and heterozygosity for an RHAG mutation encoding a Ser65Phe substitution responsible for OHSt may also remove a high-frequency antigenic determinant on RhAG, although that has not been confirmed [6].…”

Weakened expression of ‘e’ owing to concomitant occurrence of Cys16 and Val245 (VS antigen)

“…A plethora of RH variants have been identified at the molecular level [8,12]. Among them, several 'e' variants have been described [5,[14][15][16][17][18][19][20], although the requirements for expression of the e antigen are not fully understood.…”

Two Examples of an Inseparable Antibody That Reacts Equally Well with D^W+ and Rh32+ Red Blood Cells

“…Rh antigens are highly dependent upon conformation and even a single amino acid substitution can dramatically change the antigenicity [12, 14, 17]. For example, the VS antigen is associated with a Leu245 →Val substitution in RHCE and this amino acid is predicted to reside within the lipid bilayer [17, 18]. It is clear that we do not fully understand the effect that an amino acid change(s) can have on the tertiary structure of a protein.…”