Introduction: β-Thalassemia/hemoglobin E represents one-half of all the clinically severe β-thalassemias worldwide. Despite similar genetic backgrounds, patients show clinical heterogeneity ranging from nearly asymptomatic to transfusion-dependent thalassemia. The underlying disease modifying factors remain largely obscure. Methods: To elucidate the correlation between ineffective erythropoiesis and β0-thalassemia/hemoglobin E (HbE) disease severity, in vitro culture of erythroid cells derived from patients with different clinical symptoms was established. Cell proliferation, viability, and differentiation were investigated. To identify potential molecular mechanisms leading to the arrested erythroid maturation, the expression levels of erythropoiesis modifying factors were measured. Results: The β0-thalassemia/HbE cells exhibited enhanced proliferation, limited differentiation, and impaired erythroid terminal maturation but did not show accelerated erythroblast differentiation and increased cell death. Erythroblasts derived from mild patients showed the highest proliferation rate with a faster cell division time, while erythroblasts derived from severe patients displayed extremely delayed erythroid maturation. Downregulation of growth differentiation factor 11 and FOXO3a was observed in mild β0-thalassemia/HbE erythroblasts, while upregulation of heat shock protein 70 and activin receptor 2A was revealed in severe erythroblasts. Discussion/Conclusion: The degree of erythroid expansion and maturation arrest contributes to the severity of β0-thalassemia/HbE patients, accounting for the disease heterogeneity. The findings suggest a restoration of erythroid maturation as a promising targeted therapy for severe patients.

1.
Fucharoen
S
,
Winichagoon
P
.
Hemoglobinopathies in Southeast Asia: molecular biology and clinical medicine
.
Hemoglobin
.
1997
;
21
(
4
):
299
319
. .
2.
Fucharoen
S
,
Weatherall
DJ
.
The hemoglobin E thalassemias
.
Cold Spring Harb Perspect Med
.
2012
;
2
(
8
):
a011734
. .
3.
Yuan
J
,
Angelucci
E
,
Lucarelli
G
,
Aljurf
M
,
Snyder
LM
,
Kiefer
CR
,
Accelerated programmed cell death (apoptosis) in erythroid precursors of patients with severe beta-thalassemia (Cooley’s anemia)
.
Blood
.
1993
;
82
(
2
):
374
7
. .
4.
Mathias
LA
,
Fisher
TC
,
Zeng
L
,
Meiselman
HJ
,
Weinberg
KI
,
Hiti
AL
,
Ineffective erythropoiesis in beta-thalassemia major is due to apoptosis at the polychromatophilic normoblast stage
.
Exp Hematol
.
2000
;
28
(
12
):
1343
53
. .
5.
Centis
F
,
Tabellini
L
,
Lucarelli
G
,
Buffi
O
,
Tonucci
P
,
Persini
B
,
The importance of erythroid expansion in determining the extent of apoptosis in erythroid precursors in patients with beta-thalassemia major
.
Blood
.
2000
;
96
(
10
):
3624
9
. .
6.
Pootrakul
P
,
Sirankapracha
P
,
Hemsorach
S
,
Moungsub
W
,
Kumbunlue
R
,
Piangitjagum
A
,
A correlation of erythrokinetics, ineffective erythropoiesis, and erythroid precursor apoptosis in Thai patients with thalassemia
.
Blood
.
2000
;
96
(
7
):
2606
12
. .
7.
Libani
IV
,
Guy
EC
,
Melchiori
L
,
Schiro
R
,
Ramos
P
,
Breda
L
,
Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in beta-thalassemia
.
Blood
.
2008
;
112
(
3
):
875
85
. .
8.
Kittikalayawong
Y
,
Sila-asna
M
,
Bunyaratvej
A
.
Enhanced maturation and proliferation of beta-thalassemia/Hb E erythroid precursor cells in culture
.
Southeast Asian J Trop Med Public Health
.
2005
;
36
(
5
):
1298
303
..
9.
Leecharoenkiat
A
,
Wannatung
T
,
Lithanatudom
P
,
Svasti
S
,
Fucharoen
S
,
Chokchaichamnankit
D
,
Increased oxidative metabolism is associated with erythroid precursor expansion in β0-thalassaemia/Hb E disease
.
Blood Cells Mol Dis
.
2011
;
47
(
3
):
143
57
. .
10.
Leecharoenkiat
A
,
Wannatung
T
,
Smith
DR
.
Erythroblast cell expansion as a marker for disease severity in β0-thalassemia/Hb E disease
.
African J Biotech
.
2012
;
11
(
2
):
504
9
.
11.
Lithanatudom
P
,
Wannatung
T
,
Leecharoenkiat
A
,
Svasti
S
,
Fucharoen
S
,
Smith
DR
.
Enhanced activation of autophagy in β-thalassemia/Hb E erythroblasts during erythropoiesis
.
Ann Hematol
.
2011
;
90
(
7
):
747
58
. .
12.
Ponnikorn
S
,
Panichakul
T
,
Sresanga
K
,
Wongborisuth
C
,
Roytrakul
S
,
Hongeng
S
,
Phosphoproteomic analysis of apoptotic hematopoietic stem cells from hemoglobin E/β-thalassemia
.
J Transl Med
.
2011
;
9
:
96
. .
13.
Kheansaard
W
,
Panichob
P
,
Fucharoen
S
,
Tanyong
DI
.
Cytokine-induced apoptosis of beta-thalassemia/hemoglobin E erythroid progenitor cells via nitric oxide-mediated process in vitro
.
Acta Haematol
.
2011
;
126
(
4
):
224
30
. .
14.
Watanapokasin
R
,
Sanmund
D
,
Winichagoon
P
,
Muta
K
,
Fucharoen
S
.
Hydroxyurea responses and fetal hemoglobin induction in beta-thalassemia/HbE patients’ peripheral blood erythroid cell culture
.
Ann Hematol
.
2006
;
85
:
164
9
. .
15.
Sripichai
O
,
Makarasara
W
,
Munkongdee
T
,
Kumkhaek
C
,
Nuchprayoon
I
,
Chuansumrit
A
,
A scoring system for the classification of beta-thalassemia/Hb E disease severity
.
Am J Hematol
.
2008
;
83
(
6
):
482
4
. .
16.
McGrath
KE
,
Bushnell
TP
,
Palis
J
.
Multispectral imaging of hematopoietic cells: where flow meets morphology
.
J Immunol Methods
.
2008
;
336
(
2
):
91
7
. .
17.
Vermes
I
,
Haanen
C
,
Steffens-Nakken
H
,
Reutelingsperger
C
.
A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V
.
J Immunol Methods
.
1995
;
184
(
1
):
39
51
. .
18.
Nuinoon
M
,
Makarasara
W
,
Mushiroda
T
,
Setianingsih
I
,
Wahidiyat
PA
,
Sripichai
O
,
A genome-wide association identified the common genetic variants influence disease severity in beta 0-thalassemia/hemoglobin E
.
Hum Genet
.
2010
;
127
(
3
):
303
14
. .
19.
Sripichai
O
,
Fucharoen
S
.
Fetal hemoglobin regulation in beta-thalassemia: heterogeneity, modifiers and therapeutic approaches
.
Expert Rev Hematol
.
2016
;
9
(
12
):
1129
37
.
20.
Dzierzak
E
,
Philipsen
S
.
Erythropoiesis: development and differentiation
.
Cold Spring Harb Perspect Med
.
2013
;
3
(
4
):
a011601
. .
21.
Olivieri
NF
.
The beta-thalassemias
.
N Engl J Med
.
1999
;
341
(
2
):
99
109
. .
22.
Chaisiripoomkere
W
,
Jootar
S
,
Chanjarunee
S
,
Ungkanont
A
.
Serum erythropoietin levels in thalassemia major and intermedia
.
Southeast Asian J Trop Med Public Health
.
1999
;
30
(
4
):
786
8
..
23.
Forster
L
,
Cornwall
S
,
Finlayson
J
,
Ghassemifar
R
.
Cell cycle, proliferation and apoptosis in erythroblasts cultured from patients with β-thalassaemia major
.
Br J Haematol
.
2016
;
175
(
3
):
539
42
. .
24.
Grosso
R
,
Fader
CM
,
Colombo
MI
.
Autophagy: a necessary event during erythropoiesis
.
Blood Rev
.
2017
;
31
(
5
):
300
5
. .
25.
Zhang
J
,
Wu
K
,
Xiao
X
,
Liao
J
,
Hu
Q
,
Chen
H
,
Autophagy as a regulatory component of erythropoiesis
.
Int J Mol Sci
.
2015
;
16
(
2
):
4083
94
. .
26.
Rivella
S
.
Ineffective erythropoiesis and thalassemias
.
Curr Opin Hematol
.
2009
;
16
(
3
):
187
94
. .
27.
Ribeil
JA
,
Arlet
JB
,
Dussiot
M
,
Moura
IC
,
Courtois
G
,
Hermine
O
.
Ineffective erythropoiesis in β -thalassemia
.
ScientificWorldJournal
.
2013
;
2013
:
394295
. .
28.
Morikawa
M
,
Derynck
R
,
Miyazono
K
.
TGF-beta and the TGF-beta Family: context-dependent roles in cell and tissue physiology
.
Cold Spring Harb Perspect Biol
.
2016
;
8
(
5
):
a021873
.
29.
Zermati
Y
,
Fichelson
S
,
Valensi
F
,
Freyssinier
JM
,
Rouyer-Fessard
P
,
Cramer
E
,
Transforming growth factor inhibits erythropoiesis by blocking proliferation and accelerating differentiation of erythroid progenitors
.
Exp Hematol
.
2000
;
28
(
8
):
885
94
. .
30.
Malik
J
,
Kim
AR
,
Tyre
KA
,
Cherukuri
AR
,
Palis
J
.
Erythropoietin critically regulates the terminal maturation of murine and human primitive erythroblasts
.
Haematologica
.
2013
;
98
(
11
):
1778
87
. .
31.
Suragani
RN
,
Cawley
SM
,
Li
R
,
Wallner
S
,
Alexander
MJ
,
Mulivor
AW
,
Modified activin receptor IIB ligand trap mitigates ineffective erythropoiesis and disease complications in murine β-thalassemia
.
Blood
.
2014
;
123
(
25
):
3864
72
. .
32.
Gupta
R
,
Musallam
KM
,
Taher
AT
,
Rivella
S
.
Ineffective erythropoiesis: anemia and iron overload
.
Hematol Oncol Clin North Am
.
2018
;
32
(
2
):
213
21
. .
33.
Dussiot
M
,
Maciel
TT
,
Fricot
A
,
Chartier
C
,
Negre
O
,
Veiga
J
,
An activin receptor IIA ligand trap corrects ineffective erythropoiesis in β-thalassemia
.
Nat Med
.
2014
;
20
(
4
):
398
407
. .
34.
Verma
A
,
Suragani
RN
,
Aluri
S
,
Shah
N
,
Bhagat
TD
,
Alexander
MJ
,
Biological basis for efficacy of activin receptor ligand traps in myelodysplastic syndromes
.
J Clin Invest
.
2020
;
130
(
2
):
582
9
. .
35.
Suragani
RN
,
Cadena
SM
,
Cawley
SM
,
Sako
D
,
Mitchell
D
,
Li
R
,
Transforming growth factor-β superfamily ligand trap ACE-536 corrects anemia by promoting late-stage erythropoiesis
.
Nat Med
.
2014
;
20
(
4
):
408
14
. .
36.
Zhou
L
,
Nguyen
AN
,
Sohal
D
,
Ying Ma
J
,
Pahanish
P
,
Gundabolu
K
,
Inhibition of the TGF-beta receptor I kinase promotes hematopoiesis in MDS
.
Blood
.
2008
;
112
(
8
):
3434
43
. .
37.
Mayer
MP
,
Bukau
B
.
Hsp70 chaperones: cellular functions and molecular mechanism
.
Cell Mol Life Sci
.
2005
;
62
(
6
):
670
84
. .
38.
Gurbuxani
S
,
Schmitt
E
,
Cande
C
,
Parcellier
A
,
Hammann
A
,
Daugas
E
,
Heat shock protein 70 binding inhibits the nuclear import of apoptosis-inducing factor
.
Oncogene
.
2003
;
22
(
43
):
6669
78
. .
39.
Rosenzweig
R
,
Nillegoda
NB
,
Mayer
MP
,
Bukau
B
.
The Hsp70 chaperone network
.
Nat Rev Mol Cell Biol
.
2019
;
20
(
11
):
665
80
. .
40.
Ribeil
JA
,
Zermati
Y
,
Vandekerckhove
J
,
Cathelin
S
,
Kersual
J
,
Dussiot
M
,
Hsp70 regulates erythropoiesis by preventing caspase-3-mediated cleavage of GATA-1
.
Nature
.
2007
;
445
(
7123
):
102
5
. .
41.
Arlet
JB
,
Ribeil
JA
,
Guillem
F
,
Negre
O
,
Hazoume
A
,
Marcion
G
,
HSP70 sequestration by free α-globin promotes ineffective erythropoiesis in β-thalassaemia
.
Nature
.
2014
;
514
(
7521
):
242
6
. .
42.
Marinkovic
D
,
Zhang
X
,
Yalcin
S
,
Luciano
JP
,
Brugnara
C
,
Huber
T
,
Foxo3 is required for the regulation of oxidative stress in erythropoiesis
.
J Clin Invest
.
2007
;
117
(
8
):
2133
44
. .
43.
Liang
R
,
Campreciós
G
,
Kou
Y
,
McGrath
K
,
Nowak
R
,
Catherman
S
,
A systems approach identifies essential FOXO3 functions at key steps of terminal erythropoiesis
.
PLoS Genet
.
2015
;
11
(
10
):
e1005526
. .
44.
Wang
H
,
Li
Y
,
Wang
S
,
Zhang
Q
,
Zheng
J
,
Yang
Y
,
Knockdown of transcription factor forkhead box O3 (FOXO3) suppresses erythroid differentiation in human cells and zebrafish
.
Biochem Biophys Res Commun
.
2015
;
460
(
4
):
923
30
. .
45.
Franco
SS
,
De Falco
L
,
Ghaffari
S
,
Brugnara
C
,
Sinclair
DA
,
Matte
A
,
Resveratrol accelerates erythroid maturation by activation of FoxO3 and ameliorates anemia in beta-thalassemic mice
.
Haematologica
.
2014
;
99
(
2
):
267
75
. .
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