Background and objectives: The 48 G>C transversion in exon 1 of the RHCE gene leads to Trp16Cys, usually present in the conventional RHCE Ce, while Trp16 is associated with RHCE ce. The presence of Cys16 in RHCE ce is associated with the R(0) (Dce) haplotype in Africans, leading to a weak 'e' antigen expression on red blood cells (RBCs). VS is a common red cell antigen in individuals of African descent and results from a single point mutation in exon 5 of the RHCE (733C>G), leading to Leu245Val substitution; VS positivity is also associated with weak expression of 'e'. This study investigated the association of Cys16 and/or VS with the RHCE ce alleles in a cohort of sickle cell disease (SCD) patients phenotyped as R(0)r or R(0)R(0) and rr.
Materials and methods: DNA samples from 58 SCD patients were tested for the 48 G>C transversion, encoding Cys16, by allele-specific polymerase chain reaction (PCR). We also amplified exon 5 of the RHCE by PCR and subjected the amplified product to restriction fragment length polymorphism analysis, using BfaI, in order to determine the VS status. Further cDNA analysis was performed on three samples to verify whether the mutations were located on the same or on different alleles.
Results: Fifty-six of the 58 SCD patients studied (97%) were heterozygous for 48G/48C (Cys16). Of these, 18 (32%) were also heterozygous for 733C/G (245Val). All of these 18 samples showed weak 'e' expression on RBCs when tested with at least one monoclonal antibody to e antigen. cDNA sequencing of three of 18 patient samples showed that the genes encoding Cys16 and Val245 (VS) were on different alleles.
Conclusions: We found a high incidence of Cys16 associated with the RHCE ce in our SCD cohort. A high percentage of these patients were also found to be heterozygous for VS. cDNA analysis showed that, in at least three samples, the two mutations were on different alleles, with consequent weakening of expression of the e antigen on RBCs.