A Bioinformatically Initiated Approach to Evaluate GATA1 Regulatory Regions in Samples with Weak D, Del, or D- Phenotypes Despite Normal Exons.

INTRODUCTION

With over 360 blood group antigens in systems recognized, there are antigens, such as RhD, which demonstrate a quantitative reduction in antigen expression due to nucleotide variants in the non-coding region of the gene that result in aberrant splicing or a regulatory mechanism. This study aimed to evaluate bioinformatically predicted GATA1-binding regulatory motifs in the gene for samples presenting with weak or apparently negative RhD antigen expression but showing normal exons.

METHODS

Publicly available open chromatin region data were overlayed with GATA1 motif candidates in . Genomic DNA from weak D, Del or D- samples with normal exons ( = 13) was used to confirm zygosity by quantitative PCR. Then, promoter, intron 1, and intron 2 regions were amplified for Sanger sequencing to detect potential disruptions in the GATA1 motif candidates. Electrophoretic mobility shift assay (EMSA) was performed to assess GATA1-binding. Luciferase assays were used to assess transcriptional activity.

RESULTS

Bioinformatic analysis identified five of six GATA1 motif candidates in the promoter, intron 1 and intron 2 for investigation in the samples. Luciferase assays showed an enhancement in transcription for GATA1 motifs in intron 1 and for intron 2 only when the haplotype variant (rs675072G>A) was present. GATA1 motifs were intact in 12 of 13 samples. For one sample with a Del phenotype, a novel c.1-110A>C variant disrupted the GATA1 motif in the promoter which was supported by a lack of a GATA1 supershift in the EMSA and 73% transcriptional activity in the luciferase assay. Two samples were D+/D- chimeras.

CONCLUSION

The bioinformatic predictions enabled the identification of a novel allele, c.1-110A>C, which disrupted the GATA1 motif in the proximal promoter. Although the majority of the samples investigated here remain unexplained, we provide GATA1 targets which may benefit future regulatory investigations.