Aims 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.
Methods 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.
Results 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).
Conclusion 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.
- embryonic and fetal development
Data availability statement
Data are available on reasonable request.
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Handling editor Tahir S Pillay.
YY and GS contributed equally.
Contributors YY, LL and XX designed the study. YY, GS, ZR and YaL performed experiments and analyzed the data. HL, PL, XW, ZD, QZ, PZ and JH performed experiments. XZ, YC, JT, JL, YaL, XZ and YiL collected and analysed clinical data. YY, GS, ZR, LL and XX wrote the manuscript. XX is responsible for the overall content as guarantor. All authors read and approved the final manuscript.
Funding This study was supported by National Natural Science Foundation of China (Grant No. 31871265) and Shanghai Municipal Science and Technology Major Project (Grant No. 2017SHZDZX01).
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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