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

Aims Reactivation of embryonic zeta-globin is a promising strategy for genetic treatment of alpha-thalassaemia. However, quantification of zeta-globin as a quantitative trait in alpha-thalassaemia carriers and patients remains incompletely understood. In this study, we aimed to set up a reliable approach for the quantification of zeta-globin in alpha-thalassaemia carriers, followed by a population study to investigate its expression patterns. Methods zeta-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 zeta-globin expression in a second cohort of 141(-SEA)/alpha alpha carriers. Results The ELISA system was proved sensitive in distinguishing individuals with varied extent of zeta-globin. Large scale quantitative study of this --(SEA)/alpha alpha carrier cohort indicated the high diversity of zeta-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 zeta-globin (R=0.650, p<0.001). Conclusion Our study has reported reliable approaches for the quantification of zeta-globin and presented the expression patterns of zeta-globin among the --(SEA)/alpha alpha carrier population, which might lay a foundation on subsequent genotype-phenotype studies on mechanisms of delayed haemoglobin switch in alpha-thalassaemia.