McGowan Eunike C, Wu Ping Chun, Hellberg Åsa, Lopez Genghis H, Hyland Catherine A, Olsson Martin L
Division of Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University, Lund, Sweden.
Department of Clinical Immunology and Transfusion Medicine, Office for Medical Services, Region Skåne, Lund, Sweden.
Transfus Med Hemother. 2024 May 10;51(4):252-264. doi: 10.1159/000538469. eCollection 2024 Aug.
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.
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.
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.
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.
在已识别的血型系统中有超过360种血型抗原，存在一些抗原，如RhD，由于基因非编码区的核苷酸变异导致异常剪接或调控机制，其抗原表达呈现定量减少。本研究旨在通过生物信息学方法评估RhD基因中预测的GATA1结合调控基序，该基因样本表现为RhD抗原表达弱或明显阴性，但外显子正常。
将公开可用的开放染色质区域数据与RhD基因中的GATA1基序候选序列进行比对。使用来自外显子正常的弱D、Del或D-样本（n = 13）的基因组DNA通过定量PCR来确认纯合性。然后，扩增RhD启动子、内含子1和内含子2区域用于桑格测序，以检测GATA1基序候选序列中的潜在破坏。进行电泳迁移率变动分析（EMSA）以评估GATA1结合。使用荧光素酶测定来评估转录活性。
生物信息学分析在启动子、内含子1和内含子2中识别出六个GATA1基序候选序列中的五个，用于在样本中进行研究。荧光素酶测定显示，仅当RhD单倍型变体（rs675072G>A）存在时，内含子1和内含子2中的GATA1基序转录增强。13个样本中有12个样本的GATA1基序完整。对于一个具有Del表型的样本，一个新的RhD c.1-110A>C变体破坏了启动子中的GATA1基序，这得到了EMSA中缺乏GATA1超迁移以及荧光素酶测定中73%转录活性的支持。两个样本是D+/D-嵌合体。
生物信息学预测能够识别一个新的RhD等位基因，即c.1-110A>C，其破坏了近端启动子中的GATA1基序。尽管此处研究的大多数样本仍无法解释，但我们提供了GATA1靶点，这可能有益于未来的RhD调控研究。
