Functional investigation of the RHD gene promoter: Molecular changes are rarely responsible for variant D phenotype...

While modern technology is increasingly available to identify efficiently the molecular alterations responsible for variant phenotype, a subset of samples still escapes those analyses. In addition to the promoter region, two intronic regions containing GATA1 binding sites within the RHD gene locus were recently shown to modulate the transcriptional activity of the gene. We then thought to explore these regions in a subset of samples with unresolved genotypes.

Thirty-three Thai donor samples (weak D, n = 10; D-negative, 23) were selected for molecular analysis by conventional Sanger sequencing. In parallel, a novel functional assay was designed for investigating the potential effect of variants within the RHD gene promoter. Three variants previously reported, that is, c.1-115A>C, c.1-110A>C, and c.1-83G>T, were tested by this approach.

No variant was identified in the 33 samples, suggesting that the molecular basis of the respective phenotypes remains to be resolved. Interestingly, beyond the ~200 base pair (bp) promoter region commonly described, another ~300 bp region located upstream appears to contain elements that positively regulate the transcriptional activity of the RHD gene. Also, the deleterious effect of the c.1-110A>C variant was confirmed, conversely to the two other variants, which did not show any functional alteration.

Regulation of RH gene expression is likely to involve other levels of complexity that remain to be identified. Future investigations are necessary to characterize those critical genomic regions that potentially carry rare variants responsible for weakening or abolishment of D antigen expression.