***
4
DNA M ethylation [% ] ) ) ) ) 0 20 40 60 80 100 ** ** *** * Efcab4a ** * *** *** * Drd4 ** * *** *** ** * *** *** Adam11 ** * ** ** Fosb ** * *** *** * ** * Chr1:107998952- 108000925 Cdh15 ** * *** *** ** * *** ** Stc2 Chr8:125563887-125565053 ** * *** Additional data file 4: Validation of screening results by quantitative high-resolution DNA methylation assessment DNA M ethylation [% ] ) ) ) ) 0 20 40 60 80 100 DNA M ethylation [% ] ) ) ) ) 0 20 40 60 80 100 DNA M ethylation [% ] ) ) ) ) 0 20 40 60 80 100 DNA M ethylation [% ] ) ) ) ) 0 20 40 60 80 100 Hic1 DNA M ethylation [% ] ) ) ) ) 0 20 40 60 80 100 ** * *** *** Egr3 *** ** * Il4i1 Zfp775 * * * DNA M ethylation [% ] ) ) ) ) 0 20 40 60 80 100 DNA M ethylation [% ] ) ) ) ) 0 20 40 60 80 100 DNA M ethylation [% ] ) ) ) ) 0 20 40 60 80 100 DNA M ethylation [% ] ) ) ) ) 0 20 40 60 80 100 DNA M ethylation [% ] ) ) ) ) 0 20 40 60 80 100 DNA M ethylation [% ] ) ) ) ) 0 20 40 60 80 100 Wildt ype (n=19) Prel eukem ic (n=7) Early leuke mic ( n=5) Late l eukem ic (n =7) Wildtype (n=19) Preleu kem i c (n=7) Early leukemic ( n=5) Late l eukem ic (n =6) Wildtype (n=19) Preleu kemic (n=7 ) Early leuke mic ( n=5) Late l eukem ic (n =7) Wildtype (n=19) Prel eukem ic (n=7) Early leuke mic ( n=5) Late l eukem ic (n=7) Wildtype (n=19) Preleu kemic (n=7 ) Early leuke mic ( n=5) Late l eukem ic (n =7) Wildtype (n=19) Preleu kemic (n=7 ) Early leuke mic ( n=5) Late l eukem ic (n =6) Wildtype (n=19) Preleu kem i c (n=7) Early leuke mic ( n=5) Late l eukem ic (n =7) Wildt ype (n=19) Prel eukem ic (n=7) Early leuke mic ( n=5) Late l eukem ic (n =7) Wildt ype (n=18) Prel eukem ic (n=7) Earl y leu kemic (n=5) Late leukem ic (n =7) Wildtype (n=19) Preleu kem i c (n=7) Early leuke mic ( n=5) Late l eukem ic (n =7) Wildtype (n=18) Preleu kemic (n=7 ) Early leuke mic ( n=5) Late l eukem ic (n =7) Wildtype (n=18) Preleu kemic (n=7 ) Early leuke mic ( n=5) Late l eukem ic (n =7)
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Additional data file 4: Validation of screening results by quantitative high-resolution DNA methylation assessment. Il4i1. Hic1. Efcab4a. Drd4. *. *. *. **. ***. ***. ***. ***. ***. **. ***. ***. ***. **. Wildtype (n=19). Wildtype (n=19). Wildtype (n=18). - PowerPoint PPT Presentation
Transcript of ***
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=6
)0
20
40
60
80
100
**
**
***
*Efcab4a
***
***
***
* Drd4
***
***
***
***
***
***
Adam11
***
**
**
Fosb
***
***
***
*
**
*
Chr1:107998952-108000925
Cdh15
***
***
***
***
***
**
Stc2 Chr8:125563887-125565053
***
***
Additional data file 4: Validation of screening results by quantitative high-resolution DNA methylation assessment
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
Hic1
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=6
)0
20
40
60
80
100
***
***
***
Egr3
***
**
*Il4i1
Zfp775
*
*
*
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=18
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100D
NA
Met
hyl
atio
n [
%]
Wild
type
(n=18
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=18
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=6
)
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=6
)
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)W
ildtyp
e (n
=19)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=18
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=18
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)W
ildtyp
e (n
=18)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
***
**
***
12000 Mab21l1
***
*
**
Chr6:030888000-030889519
***
***
**
**
*Chr5:067705737-067707409
***
***
***
*****
***
**
**Chr10:012631455-012632051
Gabbr
***
**
***
****
***
*
*Gm16792410137
****
***
*
**
***
*
Myh14
***
***Il17re
***
******
**Mapk11 Mdga
***
*****
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=18
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100D
NA
Met
hyl
atio
n [
%]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=18
)
Prele
ukem
ic (n
=6)
Transi
tional
(n=5)
Final
(n=6
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=18
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=5
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=18
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100D
NA
Met
hyl
atio
n [
%]
Wild
type
(n=18
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=18
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=18
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=5
)
Wild
type
(n=18
)
Preleu
kem
ic (n
=6)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=6
)
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)W
ildtyp
e (n
=19)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=18
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=18
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)W
ildtyp
e (n
=19)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=18
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=4)
Final
(n=7
)0
20
40
60
80
100
Ltbp3
***
**
**
Map3k6
***
*****
Nfix
*****
***
*
Pde4d
**
***
Bsn
***
***
***Spen
***
***
***
Lmna***
**
*
Ptrf
Cxcl14
*
*
Zfp574 Robo3
***
*****
**
*
***
**
***
***
*Prdm16
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=17
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=14
)
Prele
ukem
ic (n
=5)
Transi
tional
(n=3)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100D
NA
Met
hyl
atio
n [
%]
Wild
type
(n=18
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=16
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=3)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=18
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100D
NA
Met
hyl
atio
n [
%]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=17
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=14
)
Preleu
kem
ic (n
=5)
Early
leuke
mic
(n=3
)
Late
leuk
emic
(n=7
)
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=16
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=3
)
Late
leuk
emic
(n=7
)
Wild
type
(n=18
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)W
ildtyp
e (n
=19)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=18
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)W
ildtyp
e (n
=18)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=4
)
Late
leuk
emic
(n=7
)
Il17rc
***
****
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=19
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
DN
A M
eth
ylat
ion
[%
]
Wild
type
(n=18
)
Prele
ukem
ic (n
=7)
Transi
tional
(n=5)
Final
(n=7
)0
20
40
60
80
100
Tcp11
***
****
Wild
type
(n=19
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Wild
type
(n=18
)
Preleu
kem
ic (n
=7)
Early
leuke
mic
(n=5
)
Late
leuk
emic
(n=7
)
Validation of array-based screening results by quantitative DNA methylation analysis (MassARRAY). 40 preleukemic hypermethylated genes were randomly selected for MassARRAY analysis (see also Fig. 2 for Fzd5 and Fzd8). Dotplots depict average methylation per amplicon and sample, the median of a sample group is represented by a black bar. Mann-Whitney U test was used to test for differences between wild type and the different disease stages and also between the disease stages (* p < 0.05, ** p < 0.01, *** p ≤ 0.001).
