Abstract：
The XmnI Gg -158 C/T polymorphism has been widely associated with fetal hemoglobin (HbF) levels, the severity of disease, and the response to the drug hydroxyurea (HU) in both β-thalassemia (β-thal) and sickle cell disease (SCD) patients. However, the functional significance of this single nucleotide polymorphism (SNP) remains unclear. To gain insight, green fluorescence protein (GFP) cassettes harboring the XmnI C or T alleles in their left homology arms (i.e. Gg promoters) were knocked into the Gg gene(s) of K562 cells via CRISPR/Cas9. Subsequently, the GFP fluorescence levels were compared in the ensuing cell populations and isolated clones. In both instances, median fluorescence intensities (MFI) of the knockin cells having the inserted XmnI T allele were higher than those having the XmnI C allele. Our results suggest that the XmnI T allele can increase Gg expression in K562 cells. The possible functional significance of the XmnI Gg -158 C/T polymorphism provides a rationale for the aforementioned associations. Furthermore, the XmnI polymorphism as a functional SNP substantiates its importance as a prognostic marker.
摘要：
XmnIGg-158C/T多态性已广泛与胎儿血红蛋白(HbF)水平相关,疾病的严重程度，以及β-地中海贫血（β-thal）和镰状细胞病（SCD）患者对药物羟基脲（HU）的反应。然而,这种单核苷酸多态性(SNP)的功能意义尚不清楚.为了获得洞察力，通过CRISPR/Cas9将在其左侧同源臂(即Gg启动子)中含有XmnIC或T等位基因的绿色荧光蛋白(GFP)盒敲入K562细胞的Gg基因。随后，在随后的细胞群体和分离的克隆中比较GFP荧光水平。在这两种情况下，具有插入的XmnIT等位基因的敲入细胞的中值荧光强度（MFI）高于具有XmnIC等位基因的敲入细胞。我们的结果表明，XmnIT等位基因可以增加K562细胞中Gg的表达。XmnIGg-158C/T多态性的可能功能意义为上述关联提供了理论基础。此外，作为功能性SNP的XmnI多态性证实了其作为预后标志物的重要性.
