A Bioinformatically Initiated Approach to Evaluate GATA1 Regulatory Regions in Samples with Weak D, Del, or D– Phenotypes Despite Normal <i>RHD</i> Exons

Abstract

<b><i>Introduction:</i></b> 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 <i>RHD</i> gene for samples presenting with weak or apparently negative RhD antigen expression but showing normal <i>RHD</i> exons. <b><i>Methods:</i></b> Publicly available open chromatin region data were overlayed with GATA1 motif candidates in <i>RHD</i>. Genomic DNA from weak D, Del or D– samples with normal <i>RHD</i> exons (<i>n</i> = 13) was used to confirm <i>RHD</i> zygosity by quantitative PCR. Then, <i>RHD</i> 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. <b><i>Results:</i></b> 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 <i>R</i>^<i>2</i> haplotype variant (rs675072G&gt;A) was present. GATA1 motifs were intact in 12 of 13 samples. For one sample with a Del phenotype, a novel <i>RHD</i> c.1–110A&gt;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. <b><i>Conclusion:</i></b> The bioinformatic predictions enabled the identification of a novel <i>DEL</i> allele, <i>RHD</i> c.1–110A&gt;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 <i>RHD</i> regulatory investigations.