■系统中已识别出超过360种血型抗原,有抗原,比如RhD,这表明由于基因非编码区的核苷酸变体导致异常剪接或调节机制而导致抗原表达的定量降低。这项研究旨在评估RHD基因中生物信息学预测的GATA1结合调节基序,这些基序表现出RhD抗原表达弱或明显阴性但显示正常RHD外显子的样品。
■公开可用的开放染色质区域数据与RHD中的GATA1基序候选物重叠。来自弱D的基因组DNA,具有正常RHD外显子的Del或D-样品(n=13)用于通过定量PCR确认RHD接合性。然后,RHD启动子,扩增内含子1和内含子2区域用于Sanger测序,以检测GATA1基序候选物中的潜在破坏.进行电泳迁移率变化测定(EMSA)以评估GATA1结合。荧光素酶测定用于评估转录活性。
■生物信息学分析确定了启动子中六个GATA1基序候选物中的五个,内含子1和内含子2在样品中的调查。荧光素酶分析显示,仅在存在R2单倍型变体(rs675072G>A)时,内含子1和内含子2中GATA1基序的转录才增强。在13个样品中的12个中,GATA1基序是完整的。对于一个具有Del表型的样本,一种新的RHDc.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超移位和荧光素酶测定中73%转录活性的支持。两个样品是D+/D-嵌合体。
■生物信息学预测使得能够鉴定新的DEL等位基因,RHDc.1-110A>C,破坏了近端启动子中的GATA1基序。尽管这里调查的大多数样本都无法解释,我们提供GATA1目标,这些目标可能有利于未来的RHD监管调查。
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.