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

Area of Science:

• Molecular Biology
• Immunogenetics
• Blood Group Serology

Background:

• The Rh blood group system, crucial in transfusion medicine and diagnostics, involves complex RHD and RHCE gene variations.
• Accurate identification of Rh antigens is vital for preventing hemolytic disease of the fetus and newborn and transfusion reactions.
• Previous methods for Rh typing faced limitations in distinguishing certain closely related alleles.

Purpose of the Study:

• To develop and validate a molecular method for differentiating key Rh antigens (D, C, c, E, e) using PCR-DGGE.
• To investigate the genetic basis of the VS antigen and its association with RHCE gene sequences.
• To explore the potential link between a specific Rh polypeptide substitution and the VS/e5 antigen expression.

Main Methods:

• Polymerase chain reaction (PCR) amplification of RHD exon 2, RHCE exon 2, RHD exon 5, and RHCE exon 5.
• Denaturing gradient gel electrophoresis (DGGE) to analyze PCR products and differentiate DNA sequence variations.
• DNA sequencing of specific PCR products to confirm sequence identity and identify novel variations.

Main Results:

• DGGE successfully distinguished between RHD D/C alleles and RHCE c alleles using exon 2 amplifications.
• DGGE differentiated RHD D, RHCE E, and RHCE e alleles using exon 5 amplifications.
• A distinct band shift in DGGE for VS+ red blood cells indicated a sequence variation in RHCE exon 5, identified by sequencing as a Leu245Val substitution.

Conclusions:

• PCR-DGGE is a reliable method for molecular Rh phenotyping, distinguishing critical D, C/c, and E/e antigens.
• The VS antigen is associated with a specific RHCE sequence variant in exon 5, involving a Leu245Val substitution.
• This identified amino acid substitution is a likely cause of the VS and e5 antigen expression on red blood cells.