1. Genome editing in K562 cells suggests a functional role for the XmnI Gg polymorphism: a widely used genetic marker in β-thalassemia and sickle cell disease patients.
- Author
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Ahmadifard A, Maroofi N, Maleki Tehrani M, Dabestani T, Sadat Mousavi Maleki M, Bayrami S, and Banan M
- Subjects
- Humans, K562 Cells, Genetic Markers genetics, Deoxyribonucleases, Type II Site-Specific metabolism, Deoxyribonucleases, Type II Site-Specific genetics, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Fetal Hemoglobin genetics, Fetal Hemoglobin metabolism, Base Sequence, beta-Thalassemia genetics, Gene Editing methods, Anemia, Sickle Cell genetics, CRISPR-Cas Systems genetics, Polymorphism, Single Nucleotide genetics, Alleles
- 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.
- Published
- 2024
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