Transcript The effect of Steroids in DBA

Investigation of blood cell
production and the effect of
steroids in Diamond-Blackfan
Anaemia
Deena Iskander, Clinical Research Fellow
Centre for Haematology, Faculty of Medicine, Hammersmith Hospital, Imperial College London
Paediatric Haematology and Bone Marrow Transplantation Unit, St Mary’s Hospital, Imperial
College Healthcare NHS Trust, London, UK
Imperial Molecular Pathology Laboratory, Hammersmith Hospital, Imperial College Healthcare
Trust
DBA Family Weekend, May 2016
Plan
•
•
•
•
•
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Background to project
Aims
Experimental design
Results
Conclusions
Further work
Diamond-Blackfan Anaemia
Inherited bone marrow
failure disease
5-10 per million babies born
Low haemoglobin:
ANAEMIA requiring blood
transfusions
Other blood count
abnormalities e.g. low white
blood cell count
Physical changes arising at
birth e.g. heart problems,
kidney problems, joint
problems
Problems developing
throughout life e.g. slow
growth, infections
Vlachos A et al. Expert Rev Hematol, 2014
Narla A & Ebert B, Blood 2010
DBA is a caused by a change in the genetic
code -a ‘mutation’- that leads to half the
normal amount of one ribosomal protein
Small
ribosomal
subunit
Large
ribosomal
subunit
Slavov et al Cell Reports 2015
Mutation inherited from one parent in 35% patients and arises
‘out of the blue’ in 65%
The same mutation can cause different
effects in different people
dad
11y son
Preterm birth with poor
growth and low birth weight
Diagnosed at birth (mother
known to have DBA)
RPL11 mutation
Transfusions needed for first
few years
Steroids aged 5y– initial
response but loss of response
by 10y
Second steroid trial no
response
Resistant to steroids
mum
30y old mum
Diagnosed aged 7y
RPL11 mutation
Blood transfusions needed in pregnancy
Treated with steroids
Responsive to steroids- no further blood
transfusions needed
Some of family details changed
DBA is characterised by absent or
reduced red blood cell precursors in
the bone marrow
Non-DBA child BM
DBA BM
How red blood cells develop in the
bone marrow
Red blood cell
‘parents’
Red blood cell
precursor
Red blood cells
Stem cells
What is the identity of the
‘parents’/progenitors of red cells in
humans?
‘Invisible’
Bone marrow
Blood
Different blood cell defects are
implicated in DBA
• Absent progenitors in 7 relapsed patients with
DBA (Nathan D JCI 1978)
• Normal progenitor numbers with impaired
development of progenitors to red blood cells
(Lipton J et al Blood 1989)
What is the origin of red blood cell failure in DBA?
Management of DBA
Diagnosis
Steroid responsive
80%
Dose weaned/stopped
Steroid resistant
20%
Steroid resistant
40%
Prednisolone
steroid tablets
- high dose
Blood transfusions
/Bone marrow
transplantation
Ultimately only 40% patients can use steroids in the long-term
How do steroids work in DBA?
• Act directly on DNA
• Increase haemoglobin
• Increase the number of times that red blood cell progenitors
divide, leading to more and more red blood cells
How do steroids affect red blood cell progenitors in DBA?
Aims
1
a) Define the identities of red blood cell progenitors in human
bone marrow
b) Describe early blood cell development in DBA
2
Understand how steroids improve anaemia in DBA by studying the
cells and genes that are affected by steroids in human red blood
cell progenitors
Experimental design
Bone marrow from patients
with DBA and healthy children
after consent
Isolation of cells of
interest
Looing at the cells directly isolated
from bone marrow
Growing the cells in a ‘soup’ that
promotes red blood cell development
Samples labelled anonymously
once they arrive in the lab
Clinical information
accompanying each sample
stored confidentially
Investigation of
- the markers on the outside of
red blood cell progenitor
populations
- How the cells grow
- What genes the cells express
Patients with DBA compared with
healthy children
Non-DBA (18 people)
DBA (15 people)
Average age (range)
6y7m (1y6m-43y)
6y (10m-17y)
Reason for bone marrow
Donating their bone
marrow for a bone
marrow transplant
13 for follow-up, 1 for low
blood counts, 1 for diagnosis of
DBA
Treatment
NA
10 on regular blood
transfusions (2 steroids not yet
tried, 8 resistant to steroid)
5 steroid responsive
Diagnosis of DBA by DNA sequencing
Not detected
RPS19
RPL5
RPS26
RPS24
RPL11
RPL35a
RPS7
RPL15
RPS29
RPS10
RPS17
RPS15
RPL36
Under investigation
A ribosomal protein mutation likely to be causing DBA has been identified by
sequencing in 69% (68/99) patients tested.
1
Gerrard G, Valgañón M, En Foong H et al. British Journal Haematol. 2013
Results
Aim 1a: What is the identity of the
‘parents’/progenitors of red cells?
Characterisation of red blood cell
progenitors using protein labels on their
surface
Early red cell
progenitors
Late red cell
progenitors
x10
x10
A
B
Hypotheses:
A cells are early red blood cell progenitors
B cells are late red blood cell progenitors
Iskander D et al, Blood, 2015
Indeed we found that these newly
defined progenitors look different…..
Sorted
A
EEP
Sorted
B
LEP
50mm
…..and express different genes
GATA-1
GATA-2
Gene expression
relative to GAPDH
100
80
60
40
20
5
4
3
2
1
0
2.0
1.5
1.0
0.5
G
M
P
EB
P
B
LE
P
A
EE
EP
M
G
M
P
EB
P
B
LE
P
A
EE
EP
0.0
M
Gene expression
relative to GAPDH
P
G
M
EB
P
B
LE
P
A
EE
EP
M
Gene expression
relative to GAPDH
CD36
500
400
300
200
100
20
15
10
5
0
N=3
Iskander D et al, Blood, 2015
Conclusions I: Normal blood cell
production
We have identified the early and late red blood cell progenitor
populations in the bone marrow of healthy children.
The same populations were also found in adult bone marrow.
The above findings have since been corroborated by other
groups. (Mori Y, PNAS, 2015)
This is important because it means we can capture these
cells from human bone marrow samples and study them in
health and diseases, which should lead to understanding
these diseases better.
Results II
Aim 1b: Describe early blood cell development in DBA
Aim 2: Understand how steroids improve anaemia
in DBA
DBA progenitors do not grow normally
Non-DBA
x10
No. colonies/100
cells
60
*
50
40
30
BFU-E
CFU-E
CFU-G/M/GM
Erythroid clusters
Erythroid
clusters
DBA
20
10
x20
0
CON DBA CON DBA CON DBA
CMP
MEP
GMP
N=4 CON
N=5 DBA
Iskander D et al, Blood, 2015
Transfusion-dependent (TD) versus
steroid-responsive (SR) patients
DBA TD
CON
NON-DBA
CD71
10 5
10
4
10
3
10
2
DBA SR
5
10
78.8%
90.7%
9.48%
104
3
10
102
0
0
0
10
3
10
4
CD41a
CD105
10
10
5
5
10
4
10
3
0
10
LEP
0.9%
EEP
2.8%
10 2
0
0
10
3
10
4
5
104
105
5
10 4
10
3
10
2
LEP
1.7%
LEP
0.0%
EEP
6.9%
EEP
0.7%
0
10
103
0
10
3
10
4
10
5
CD36
Iskander D et al, Blood, 2015
Conclusions II: DBA blood cell
production
• In transfusion-dependent DBA
– There are fewer red blood cell progenitors and
they don’t work normally
• Red blood cell progenitor abnormalities are
restored in steroid-responsive DBA
Summary of progress and future work
We have identified the early and late red blood cell progenitor
populations in the bone marrow of healthy children.
We have used these definitions to identify the places in red cell
production where things are going wrong in DBA.
We will continue to study these cells with the aim of identifying why
things go wrong in DBA and how steroids can help.
The overall aim of this work is to find new approaches and gene
targets that we can use to treat DBA.
Acknowledgements
Tassos Karadimitris
Irene Roberts
Josu de la Fuente
Beth Psaila, Valentina Caputo, Aris Chaidos,
Katerina Goudevenou, Andi Roy
Qais Al-Oqaily, Neha Bhatnagar, Joana Costa,
Maialen Lasa, Kalliopi Makarona, David O’Connor,
Kyriake Petivi, Kanagaraju Ponnusamy, Antonella
Rotolo, Nikolaos Trasanidis
Mauritius Kleijnen, David Pitcher
Holger Auner, Sandra Loaiza, Katarzyna Parzych
Niklas Feldhahn, Bryant Boulianne
Paediatric Haematology team, St Mary’s Hospital
Yvonne Harrington, SpRs
Mark Layton, Hammersmith Hospital
The patients and their familiesAll of you!
NIHR BRC Imaging and FACS Facility, Imperial
College
John Goldman Centre for Cellular Therapy
Imperial Molecular Pathology lab
Letizia Foroni, Jamshid Khorashad, Gareth Gerrard,
Hui en Foong , Sandra Hing