Quantification of human embryonic ζ-globin chains in Southeast Asian deletion (--^SEA) carriers

Reactivation of embryonic ζ-globin is a promising strategy for genetic treatment of α-thalassaemia. However, quantification of ζ-globin as a quantitative trait in α-thalassaemia carriers and patients remains incompletely understood. In this study, we aimed to set up a reliable approach for the quantification of ζ-globin in α-thalassaemia carriers, followed by a population study to investigate its expression patterns.

ζ-globin was purified as monomers from cord blood haemolysate of a Hb Bart's fetus, followed by absolute protein quantification, which was then tested by in-house ELISA system and introduced as protein standard. It was then used for large-scale quantification in peripheral blood samples from 6179 individuals. Finally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) introduced as an independent validating approach by measuring ζ-globin expression in a second cohort of 141^-SEA/αα carriers.

The ELISA system was proved sensitive in distinguishing individuals with varied extent of ζ-globin. Large scale quantitative study of this --^SEA/αα carrier cohort indicated the high diversity of ζ-globin expression ranging from 0.00155 g/L to 1.48778 g/L. Significant positive correlation between ELISA and LC-MS/MS (R=0.400, p<0.001) was observed and it is more sensitive in distinguishing the samples with extreme expression of ζ-globin (R=0.650, p<0.001).

Our study has reported reliable approaches for the quantification of ζ-globin and presented the expression patterns of ζ-globin among the --^SEA/αα carrier population, which might lay a foundation on subsequent genotype-phenotype studies on mechanisms of delayed haemoglobin switch in α-thalassaemia.