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Journal article
A Bioinformatically Initiated Approach to Evaluate GATA1 Regulatory Regions in Samples with Weak D, Del, or D- Phenotypes Despite Normal RHD Exons
Transfusion Medicine and Hemotherapy, Vol.51(4), pp.252-264
2024
Abstract
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 RHD gene for samples presenting with weak or apparently negative RhD antigen expression but showing normal RHD exons. Methods: Publicly available open chromatin region data were overlayed with GATA1 motif candidates in RHD. Genomic DNA from weak D, Del or D- samples with normal RHD exons (n = 13) was used to confirm RHD zygosity by quantitative PCR. Then, RHD 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 R-2 haplotype variant (rs675072G>A) was present. GATA1 motifs were intact in 12 of 13 samples. For one sample with a Del phenotype, a novel RHD 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 DEL allele, RHD 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 RHD regulatory investigations.
Details
- Title
- A Bioinformatically Initiated Approach to Evaluate GATA1 Regulatory Regions in Samples with Weak D, Del, or D- Phenotypes Despite Normal RHD Exons
- Authors
- Eunike C. Mcgowan - Lund UniversityPing Chun Wu - Lund UniversityGenghis H. Lopez - University of the Sunshine Coast, Queensland, School of Health and Behavioural Sciences - LegacyCatherine A. Hyland - Australian Red Cross Blood ServiceMartin L. Olsson (Corresponding Author) - Lund University
- Publication details
- Transfusion Medicine and Hemotherapy, Vol.51(4), pp.252-264
- Publisher
- S. Karger AG
- Date published
- 2024
- DOI
- 10.1159/000538469
- ISSN
- 1660-3818; 1660-3796
- Copyright note
- This article is licensed under the Creative Commons AttributionNonCommercial 4.0 International License (CC BY-NC) (http://www.karger.com/Services/OpenAccessLicense). Usage and distribution for commercial purposes requires written permission.
- Data Availability
- All data generated or analyzed during this study are included in this article and its supplementary material files. Further inquiries can be directed to the corresponding author.
- Grant note
- 2019-01683 / Swedish Research Council; 2023-152651 / Royal Physiographic Society of Lund, Sweden; 2020.0234 / Knut and Alice Wallenberg Foundation; Knut & Alice Wallenberg Foundation; ALFSKANE-446521 / Governmental ALF grants
- Organisation Unit
- School of Health; School of Health and Behavioural Sciences - Legacy
- Language
- English
- Record Identifier
- 991034797802621
- Output Type
- Journal article
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- Hematology
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