What Can We Do Together To Understand DBA?

CULTURE OF DBA CELLS

Ficoll gradient

Magnetic

labeling of CD34+ cells

Selection of CD34+ cells

~15,000

Day 0-7 Day 8-14 SCF, TPO, IL-3, SCF, TPO, IL-3, IL-6, EPO (0.5U) IL-6, EPO (3U)

CULTURE OF DBA CELLS

Kelly O’Brien

DBA CELLS PROLIFERATE SLOWLY

D

Control

DBA

0

1,000,000

2,000,000

3,000,000

4,000,000

5,000,000

6,000,000

7,000,000

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Num

ber o

f Cel

ls

cells051415_control.fcsEvent Count: 44982

0 102 103 104 105

<FITC-A>: CD235

0

102

103

104

105

<PE

-A>:

CD

41

23.3 13.7

17.245.9

cells051415_0649.fcsEvent Count: 42750

0 102 103 104 105

<FITC-A>: CD235

0

102

103

104

105

<PE

-A>:

CD

41

22.8 18.9

30.727.6

cells051415_0721.fcsEvent Count: 40972

0 102 103 104 105

<FITC-A>: CD235

0

102

103

104

105

<PE

-A>:

CD

41

25.4 26.9

28.519.2

cells051415_0649.fcsEvent Count: 42750

0 102 103 104 105

<FITC-A>: CD235

0

102

103

104

105

<Per

CP

-Cy5

-5-A

>: C

D44

32.2 53.1

6.198.55

cells051415_control.fcsEvent Count: 44982

0 102 103 104 105

<FITC-A>: CD235

0

102

103

104

105<P

erC

P-C

y5-5

-A>:

CD

4433.7 62.9

1.631.78

cells051415_0721.fcsEvent Count: 40972

0 102 103 104 105

<FITC-A>: CD235

0

102

103

104

105

<Per

CP

-Cy5

-5-A

>: C

D44

30.1 60.3

7.891.73

CD235

CD

44

CONTROL DBA

CD235

CD

44

STEROID RESPONSIVNESS IN A DBA PATIENT

EPIGENETIC REGULATION OF GENE EXPRESSION Chromatin Accessibility

closed open

DNA Methylation

Methylation site Methylation site

cells051415_control.fcsEvent Count: 44982

0 102 103 104 105

<FITC-A>: CD235

0

102

103

104

105

<PE

-A>:

CD

41

23.3 13.7

17.245.9

cells051415_0649.fcsEvent Count: 42750

0 102 103 104 105

<FITC-A>: CD235

0

102

103

104

105

<PE

-A>:

CD

41

22.8 18.9

30.727.6

cells051415_0721.fcsEvent Count: 40972

0 102 103 104 105

<FITC-A>: CD235

0

102

103

104

105

<PE

-A>:

CD

41

25.4 26.9

28.519.2

cells051415_0649.fcsEvent Count: 42750

0 102 103 104 105

<FITC-A>: CD235

0

102

103

104

105

<Per

CP

-Cy5

-5-A

>: C

D44

32.2 53.1

6.198.55

cells051415_control.fcsEvent Count: 44982

0 102 103 104 105

<FITC-A>: CD235

0

102

103

104

105<P

erC

P-C

y5-5

-A>:

CD

4433.7 62.9

1.631.78

cells051415_0721.fcsEvent Count: 40972

0 102 103 104 105

<FITC-A>: CD235

0

102

103

104

105

<Per

CP

-Cy5

-5-A

>: C

D44

30.1 60.3

7.891.73

CD235

CD

44

CONTROL DBA

CD235

CD

44

RNA DNA methylation Chromatin Accessibility

RNA DNA methylation Chromatin Accessibility

RNA DNA methylation Chromatin Accessibility

RNA DNA methylation Chromatin Accessibility

EPIGENETIC ANALYSIS OF DBA PATIENT CELLS

Next Steps: Establish a baseline of transfusion dependent DBA patients and controls. Analyze steroid responsive DBA patients. Analyze DBA patient in remission. Correlate epigenetic changes with changes in transcriptional profiles.

Goal: To identify genes or pathways that are altered in steroid responsive or remission DBA patient cells