The composition of the α-like and β-like subunits of haemoglobin changes over the course of development to cater for varying demands of oxygen in the developing body. This tightly-regulated process is known as globin switching, which involves two switches in the composition of β-globin, first from embryonic-to-foetal globin during gestation, and secondly from foetal-to-adult after birth. β-haemoglobinopathies such as β-thalassemia and sickle-cell anaemia arise due to mutations in the genes encoding the β-globin subunits of haemoglobin.
Heterogeneity in the clinical manifestations of these blood disorders are partly attributed to variations in foetal-haemoglobin (HbF) expression. Persistence of HbF expression into adulthood is known to reduce morbidity and mortality from these blood disorders. The level of HbF varies between individuals, and it has been hypothesised that multiple loci influence γ-globin expression. GWAS studies have identified single nucleotide polymorphisms that are associated with elevated HbF within the β-globin locus, BCL11A, and the intergenic region of HBS1L-MYB.
The C>T single-base substitution at position -158 of the Gγ-globin gene (XmnI-Gγ) has long been proposed as a genetic marker of reduced β-haemoglobinopathy disease severity. The presence of XmnI-Gγ is correlated with increased HbF, however, a question remains concerning whether XmnI-Gγ is causative, or whether the site is in linkage disequilibrium with another polymorphism that is directly responsible for modulating γ-globin expression.
Here we report on red blood cell progenitor (HUDEP-2) cells engineered by CRISPR-Cas9 to contain the -158 C>T base substitution. In these cells, levels of HbF at the mRNA (RT-PCR) and the protein level (HPLC) are significantly increased, suggesting that the XmnI-Gγ site may indeed be causative. Furthermore, differential binding by electrophoretic mobility shift assays suggests that a DNA-binding protein is directly involved with the XmnI- Gγ region, further implicating the site in having a direct impact of γ-globin expression.