Compensated variability in the expression of globin-related genes in erythroblasts generated ex vivo from different donors.
BACKGROUND
Ex vivo generated erythroblasts are being evaluated for transfusion. Expression of balanced levels of globin mRNA is essential for normal red blood cell function and survival but it is unknown whether the expression of the globin genes in ex vivo expanded cells is balanced.


METHODS
Immature erythroblasts (IEs) were expanded in human erythroid massive amplification cultures from blood mononuclear cells of 19 normal donors and four beta(0)-thalassemia patients (for comparison) and induced to mature for 4 days in the presence of erythropoietin. mRNA was prepared from IEs and mature erythroblasts to evaluate the expression of alpha-, beta-, and gamma-globin genes and of adult hemoglobin-stabilizing protein (AHSP) and BCL11A, two proteins directly controlling globin function and/or production. Results were analyzed using Pearson's correlation coefficient, the Wilcoxon signed rank, and the Mann-Whitney rank sum tests.


RESULTS
The absolute levels of globin, AHSP, and BCL11A mRNA expressed by erythroblasts generated ex vivo from normal donors were distributed along a 2-log range. With maturation, the levels of gamma-globin and BCL11A mRNA did not decrease while those of alpha-globin, gamma + beta-globins, and AHSP mRNA greatly increased. In normal cells, the modest imbalance (two- to fourfold) observed between alpha- and gamma + beta-globin mRNA was fully compensated by AHSP expression. Thus, the levels of alpha-globin mRNA were correlated with those of gamma + beta-globin (R(2) = 0.93, p < 0.0001) and AHSP (R(2) = 0.86, p < 0.0001).


CONCLUSIONS
Ex vivo expanded erythroblasts from normal donors express modestly imbalanced levels of alpha-globin and gamma + beta-globin fully compensated by AHSP expression, likely ensuring normal function and survival.
Varricchio
Lilian
L
Istituto Superiore Sanità, Rome, Italy.
Fabucci
Maria Elena
ME
Alfani
Elena
E
Godbold
James
J
Migliaccio
Anna Rita
AR
D000328:Adult; D001798:Blood Proteins / Q000096:biosynthesis; D002352:Carrier Proteins / Q000096:biosynthesis; D002470:Cell Survival; D002478:Cells, Cultured; D004900:Erythroblasts / Q000166:cytology / Q000378:metabolism*; D004921:Erythropoietin / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation; D005914:Globins / Q000096:biosynthesis*; D006801:Humans; D008297:Male; D018832:Molecular Chaperones / Q000096:biosynthesis; D009687:Nuclear Proteins / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D012097:Repressor Proteins; D017086:beta-Thalassemia / Q000378:metabolism / Q000473:pathology
C468768:AHSP protein, human; C486563:BCL11A protein, human; D001798:Blood Proteins; D002352:Carrier Proteins; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D004921:Erythropoietin; D005914:Globins