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

McGowan, Eunike C. LU orcid ; Wu, Ping Chun LU orcid ; Hellberg, Åsa LU ; Lopez, Genghis H. ; Hyland, Catherine A. and Olsson, Martin L. LU orcid (2024) In Transfusion Medicine and Hemotherapy
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.... (More)

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 R2 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.

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author
; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
epub
subject
keywords
Bioinformatics, Blood group, Del, GATA1, Regulation, Weak D
in
Transfusion Medicine and Hemotherapy
publisher
Karger
external identifiers
  • scopus:85193917905
ISSN
1660-3796
DOI
10.1159/000538469
language
English
LU publication?
yes
id
9b651121-d0d9-47e2-8bd4-80a50c3799b9
date added to LUP
2024-06-14 12:29:52
date last changed
2024-06-14 12:29:59
@article{9b651121-d0d9-47e2-8bd4-80a50c3799b9,
  abstract     = {{<p>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 R2 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 RHD 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. Conclusion: The bioinformatic predictions enabled the identification of a novel DEL allele, RHD 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 RHD regulatory investigations.</p>}},
  author       = {{McGowan, Eunike C. and Wu, Ping Chun and Hellberg, Åsa and Lopez, Genghis H. and Hyland, Catherine A. and Olsson, Martin L.}},
  issn         = {{1660-3796}},
  keywords     = {{Bioinformatics; Blood group; Del; GATA1; Regulation; Weak D}},
  language     = {{eng}},
  publisher    = {{Karger}},
  series       = {{Transfusion Medicine and Hemotherapy}},
  title        = {{A Bioinformatically Initiated Approach to Evaluate GATA1 Regulatory Regions in Samples with Weak D, Del, or D- Phenotypes Despite Normal RHD Exons}},
  url          = {{http://dx.doi.org/10.1159/000538469}},
  doi          = {{10.1159/000538469}},
  year         = {{2024}},
}