1. The document analyzes the correlation between methylation and gene expression of various MAGE genes. 2. Graphs show the relationships between methylation levels and gene expression levels for different MAGE genes. 3. Methylation is found to inhibit the expression of some MAGE genes, which could increase patient survival times.

1. Correlation between methylation and
gene expression
(For organizing data)

2. Search/collect data
• 1, correlation of methylation and
gene expression. MAGEA1
y = 0.011x - 0.353
6 MAGEB10
• 2, correlation of MAGE genes
R² = 0.050 y=-
4
1.5 0.008x
2
MAGEA1
+
0 1 0.201
expression and HLA alelles -2 0 50 100 0.5 R² =
Axis Title
0.130
HLA-DMB 0
-0.5 0 100
expression ( ).
which indicate HLA may present the MAGE CTA -1
-1.5
HLA-DOB
• 3, K-M analysis (survival curve)
• 4, Prediction and stringency of antigenic epitopes of
MAGE genes after targeted MAGE gene determined.
• 5, Palindrome 2-D structure of targeted MAGE genes
• 6, MAGE gene expression distribution in Estonia cohort

3. MAGEA2B
MAGEA2B 0.5
0.5 0.4
Gene expression (2nd )
0.4 0.3 y = -0.121x + 0.083
y = -0.335x + 0.272 R² = 0.021
0.3 0.2
Gene expression
R² = 0.070
0.2 0.1
0.1 0
0 -0.1 0 0.2 0.4 0.6 0.8
-0.1 0 0.2 0.4 0.6 0.8 1
-0.2
-0.2 -0.3
-0.3 Methylation
Methylation
4
3.5 MAGEB2
3 MAGED2
2.5 1.5
2
Gene expression
y = 0.882x - 0.136
y = -1.704x + 1.308 1
R² = 0.039
1.5 R² = 0.039
Gene expression
0.5
1
0.5 0
0 0 0.1 0.2 0.3 0.4
-0.5
-0.5 0 0.2 0.4 0.6 0.8 1
-1
-1
Methylation
-1.5
Methylation
Methylation inhibited the MAGEB2
expression, increase life span

4. MAGEB2
4
3.5
3
2.5 y = -1.096x + 0.786
2 R² = 0.055
Axis Title
1.5
1
0.5
0
-0.5 0 0.2 0.4 0.6 0.8 1
-1
Axis Title

5. MAGEA11 MAGEA11
2
2
1.5
1.5
Gene expression (2nd)
Gene Expression
1
y = 0.135x - 0.031 1
y = 0.628x - 0.167
0.5 R² = 0.001
0.5 R² = 0.021
0
0
0 0.2 0.4 0.6 0.8 1
-0.5 0 0.1 0.2 0.3 0.4 0.5 0.6
-0.5
-1
Methylation -1
Methylation
MAGEH1
1.2
MAGEH1
1 1.2
0.8 1
y = 0.736x - 0.102
0.8
Gene expression (2nd)
Gene expression
0.6 R² = 0.049
0.4 0.6
y = 0.331x - 0.086
0.2 0.4 R² = 0.022
0 0.2
-0.2 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0
-0.4 -0.2 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
-0.6 -0.4
-0.8 -0.6
Methylation -0.8
Methylation

6. MAGED1 MAGED1
2.5
2.5
2
2
y = -0.309x + 0.2 1.5
1.5 R² = 0.000
1
Gene expression
Gene expression
1
0.5
0.5 y = -0.567x + 0.235
0
0 R² = 0.005
-0.5 0 0.1 0.2 0.3 0.4
-0.5 0 0.05 0.1 0.15 0.2 0.25
-1
-1
-1.5
-1.5
-2
-2 Methylation
Methylation
MAGEA12
MAGEA12
3 3
2.5 2.5
y = -0.276x + 0.239
2 R² = 0.001 2 y = -0.435x + 0.320
Gene expression
R² = 0.002
Gene expression
1.5 1.5
1 1
0.5 0.5
0 0
-0.5 0 0.2 0.4 0.6 0.8 1
-0.5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
-1
Methylation
-1
Methylation

7. MAGEC1
3.5
3
2.5
P=0,1274
Gene expression
2
y = -3,7562x + 3,1755
1.5 R² = 0,4691
1
0.5
0
-0.5 0 0.2 0.4 0.6 0.8 1
-1
Methylation
Methylation inhibits the MAGEC1 expression, increased survivals
MAGEC1
3.5
3
2.5
Gene expression
2
1.5
y = -2.115x + 1.684
1
R² = 0.193
0.5
0
-0.5 0 0.2 0.4 0.6 0.8 1 1.2
-1
Methylation

8. MAGEA8
4
3.5
3
2.5 y = -1.697x + 1.276
Gene expression
R² = 0.110
2
1.5
1
0.5
0
-0.5 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
-1
Methylation
MAGEA10 MAGEA10
5 5
4 4 y = -3.594x + 3.176
R² = 0.143
3 y = -4.002x + 3.595 3
Gene expression
Gene expression
R² = 0.138
2 2
1 1
0 0
0 0.2 0.4 0.6 0.8 1 1.2 0 0.2 0.4 0.6 0.8 1 1.2
-1 -1
-2 -2
Methylation Methylation

9. MAGEE1
0.4
y = 0.184x - 0.044
0.3
R² = 0.023
0.2
0.1
Axis Title
0
-0.1 0 0.1 0.2 0.3 0.4 0.5
-0.2
-0.3
-0.4
Axis Title
MAGEE1
0.4
y = 0.115x - 0.020
0.3 R² = 0.022
0.2
0.1
Axis Title
0
-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6
-0.2
-0.3
-0.4
Axis Title

10. MAGEA5
0.4
0.3
0.2 y = -0.038x + 0.022
R² = 0.001
0.1
Axis Title
0
-0.1 0 0.2 0.4 0.6 0.8 1
-0.2
-0.3
-0.4
Axis Title
MAGEA5
0.4
y = -0.028x + 0.023
0.3 R² = 0.000
0.2
0.1
Axis Title
0
-0.1 0 0.2 0.4 0.6 0.8 1
-0.2
-0.3
-0.4
Axis Title

11. MAGEB3
0.4
0.3 y = 0.140x - 0.075
0.2 R² = 0.019
0.1
Axis Title
0
-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
-0.2
-0.3
-0.4
Axis Title
MAGEB3
0.4
0.3 y = 0.195x - 0.071
0.2 R² = 0.026
0.1
Axis Title
0
-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
-0.2
-0.3
-0.4
Axis Title

12. MAGEA6
y = -0.365x + 0.248
0.4 R² = 0.130
0.3
0.2
0.1
Axis Title
0
0 0.2 0.4 0.6 0.8 1
-0.1
-0.2
-0.3
-0.4
Axis Title
MAGEA6
0.4
0.3 y = -0.371x + 0.260
R² = 0.144
0.2
0.1
Axis Title
0
-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
-0.2
-0.3
-0.4
Axis Title

13. MAGEB4
0.7
0.6
0.5 y = 0.223x - 0.114
0.4 R² = 0.067
0.3
Axis Title
0.2
0.1
0
-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
-0.2
-0.3
-0.4
Axis Title
MAGEB4
0.7
0.6
0.5
0.4 y = 0.246x - 0.157
0.3 R² = 0.104
Axis Title
0.2
0.1
0
-0.1 0 0.2 0.4 0.6 0.8 1
-0.2
-0.3
-0.4
Axis Title

14. Axis Title
MAGEE2
0.5
0.4 y = -0.128x + 0.025
MAGEA9B
0.3 R² = 0.016 5
0.2 4
0.1
3 y = -3.405x + 2.928
0
R² = 0.037
Axis Title
-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 2
-0.2 1
-0.3
0
-0.4
0 0.2 0.4 0.6 0.8 1
Axis Title -1
-2
Axis Title
MAGEB18
0.25
0.2 y = 0.027x - 0.034
0.15 R² = 0.001
0.1
0.05
Axis Title
0
-0.05 0 0.2 0.4 0.6 0.8 1
-0.1
-0.15
-0.2
-0.25
-0.3
Axis Title

15. MAGEC3 MAGEC3
0.4
0.4
0.3
0.3
0.2
0.2
0.1
0.1
Axis Title
y = -0.003x + 0.001
Axis Title
y = -0.000x + 0.000 0
0 R² = 2E-05
R² = 1E-06 -0.1 0 0.2 0.4 0.6 0.8 1
-0.1 0 0.2 0.4 0.6 0.8 1
-0.2
-0.2
-0.3
-0.3
-0.4
-0.4
-0.5
-0.5 Axis Title
Axis Title

16. 1.5
MAGED1 1 MAGED2
2.5
2 0.5
y = 0.882x - 0.136
Axis Title
1.5 R² = 0.039
0
1 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Axis Title
0.5 -0.5
y = -0.309x + 0.2
0 R² = 0.000 -1
-0.5 0 0.05 0.1 0.15 0.2 0.25
-1 -1.5
Axis Title
-1.5
-2
Axis Title

17. MAGED4 (1)
3
2.5
2
1.5 MAGED4 (2)
1
Axis Title
3
0.5
2.5
0
2
-0.5 0 0.1 0.2 0.3 y = -1.886x + 0.367 0.5
0.4 0.6
R² = 0.066 1.5
-1
1
Axis Title
-1.5
0.5
-2
Axis Title 0
-0.5 0 0.1 0.2 0.3 0.4 y = -1.651x + 0.416
0.5 0.6 0.7
-1 R² = 0.121
-1.5
-2
Axis Title

18. P=0,0703
Km5

19. Km2

20. Km7

21. Km1

22. Km3

23. Km4
P=0,0734

24. Km6
P=0.0026

25. Correlation of methylation and gene
expression

27. (A) Correlation between ID4
expression and ID4 promoter
methylation in human breast
cancer
For this purpose, a part of the same breast cancer
cohort (n = 46) used previously for methylation
analysis was re-assessed (Figure 2A ).
We found an increased risk for tumour recurrence in
breast cancer patients with ID4 promoter methylation
(P = 0.036) compared to patients with lack of ID4
methylation (Figure 2B ).

28. Methylation headmap of MAGE genes?

29. MAGE antigen presented by HLA
alelles?
MAGEA1
5 MAGEA3
y = 0.011x - 0.353 4
R² = 0.050
4
3
3
2
Axis Title
2
MAGEA1
1
y = 0.009x - 0.178
R² = 0.034
1
0
0 20 40 60 80 100
0
-1
0 20 40 60 80 100
-1
-2
Axis Title
-2
HLA-DMB

30. MAGEA5
MAGED4
0.4
3
0.3 2.5
0.2 2
0.1 1.5
Axis Title
y = 0.000x - 0.015 1
Axis Title
0 R² = 0.017 y = 0.004x + 0.103
0.5
R² = 0.016
-0.1 0 20 40 60 80 100
0
-0.2 -0.5 0 20 40 60 80 100
-0.3 -1
-1.5
-0.4
HLA-DMB -2
HLA-DMB
MAGEH1
1.2
MAGEE1
1 0.4
0.8 0.3 y = -0.001x + 0.042
0.6 R² = 0.055
0.2
0.4
Axis Title
Axis Title 0.1
0.2 y = 0.002x - 0.118
0 R² = 0.051 0
-0.2 0 20 40 60 80 100 -0.1 0 20 40 60 80 100
-0.4 -0.2
-0.6
-0.3
-0.8
HLA-DMB -0.4
HLA-DMB

31. HLA-DOB
MAGEE2 MAGEB18
0.25 y = -0.000x + 0.029
0.5
0.2 R² = 0.054
0.15
Axis Title
y = 0.000x - 0.018 0.1
0 R² = 0.013 0.05
Axis Title
0 20 40 60 80 100
0
-0.5 -0.05 0 20 40 60 80 100
HLA-DOB -0.1
-0.15
-0.2
MAGEA1 -0.25
-0.3
6 HLA-DOB
4
Axis Title
2
y = 0.011x - 0.353
0 R² = 0.050
-2
0 20 40 60 80 100 MAGED4
HLA-DOB
3
2.5
2
1.5
MAGEB10 + 0.201 1
Axis Title
y = -0.008x
y = 0.004x + 0.103
2 R² = 0.130 0.5
R² = 0.016
Axis Title
0
0 -0.5 0 20 40 60 80 10
0 20 40 60 80 100 -1
-2 -1.5
Axis Title
-2
HLA-DOB

32. HLA-DRA
MAGEA1 MAGEA3
4
6
3
4 2
Axis Title
Axis Title
2 1 y = 0.009x - 0.178
y = 0.011x - 0.353 0 R² = 0.034
0 R² = 0.050
-1 0 20 40 60 80 100
0 20 40 60 80 100
-2 -2
HLA-DRA HLA-DRA MAGEE1
0.5 y = -0.001x + 0.042
R² = 0.055
Axis Title
0
0 20 40 60 80 100
MAGEA9B MAGEB3
-0.5
Axis Title
5 0.4
4 0.3 y = -0.000x + 0.037
R² = 0.025
0.2
3
0.1
Axis Title
Axis Title
2
0
1 y = 0.013x - 0.344
R² = 0.04 -0.1 0 20 40 60 80 100
0
-0.2
0 20 40 60 80 100
-1 -0.3
-2 -0.4
HLA-DRA Axis Title

33. y = -0.001x + 0.042 y = 0.013x - 0.344
R² = 0.055 MAGEE1 R² = 0.04 MAGEA9B
0.5
HLA-C 6
Axis Title
4
Axis Title
0 2
0 50 100 0
-0.5
Axis Title -2 0 20 40 60 80 100
MAGEA1 Axis Title
5
MAGEB18 + 0.029
y = -0.000x
R² = 0.054 4
0.3
0.2 3
0.1
Axis Title
Axis Title
2
0
-0.1 0 20 40 60 80 100 1 y = 0.011x - 0.353
-0.2 R² = 0.050
0
-0.3
HLA-C 0 20 40 60 80 100
-1
MAGEA1= 0.011x - 0.353
y
R² = 0.050 -2
6 HLA-C
4 MAGEA12
MAGEH1
Axis Title
y = 0.002x - 0.118
2 R² = 0.051 3
1.2
0
1 2
-2 0 50 100
Axis Title
0.8
HLA-C 1
0.6 y = 0.007x - 0.184
0.4
Axis Title
0 R² = 0.037
0.2
0 20 40 60 80 100
0 -1
-0.2 0 HLA-C
20 40 60 80 100
-0.4
-0.6
-0.8
HLA-C

34. y = -0.000x + 0.029
R² = 0.054
0.3
MAGEB18 HLA-B
0.2
0.1
Axis Title
0
-0.1 0 50 100
-0.2
-0.3
HLA-B

35. MAGEE1
The sequence you input has 13375 nucleotides in length. There are 2 possible CpG island(s) found.
Homo sapiens melanoma antigen family E, 1 (MAGEE1), RefSeqGene on chromosome X.
CDS 5279..8152
MAGEE2
There are NO possible CpG islands found.
Homo sapiens melanoma antigen family E, 2 (MAGEE2), RefSeqGene on chromosome X.

36. MAGEC1
Homo sapiens melanoma antigen family C, 1 (MAGEC1), RefSeqGene on chromosome X.
Homo sapiens melanoma antigen family A, 2B (MAGEA2B), mRNA

37. Homo sapiens melanoma antigen family A, 11
(MAGEA11), RefSeqGene on chromosome X

38. Homo sapiens melanoma antigen family A, 10 (MAGEA10), transcript
variant 1, mRNA
NCBI Reference Sequence: NM_001011543.2
Homo sapiens melanoma antigen family B, 18 (MAGEB18), RefSeqGene on
chromosome X
NCBI Reference Sequence: NG_016370.1

39. Homo sapiens melanoma antigen family C, 3 Homo sapiens melanoma antigen family D, 1
(MAGEC3), RefSeqGene on chromosome X (MAGED1), RefSeqGene on chromosome X
NCBI Reference Sequence: NG_012559.1
NCBI Reference Sequence: NG_013272.1

40. Homo sapiens melanoma antigen
Homo sapiens melanoma antigen family
family H, 1 (MAGEH1), RefSeqGene
D, 2 (MAGED2), RefSeqGene on
on chromosome X chromosome X
NCBI Reference Sequence: NCBI Reference Sequence: NG_012844.1
NG_013248.1

41. Homo sapiens melanoma antigen family B, 4
(MAGEB4), RefSeqGene on chromosome X
NCBI Reference Sequence: NG_015870.1

42. Homo sapiens melanoma antigen family B, 1 (MAGEB1), RefSeqGene
on chromosome X
NCBI Reference Sequence: NG_016336.1

43. Homo sapiens melanoma antigen family A, 3 (MAGEA3), RefSeqGene on
chromosome X
NCBI Reference Sequence: NG_013244.1
There are 1 possible CpG island(s) found.
Homo sapiens melanoma antigen family A, 6 (MAGEA6), transcript variant 2, mRNA
NCBI Reference Sequence: NM_175868.1

44. Homo sapiens melanoma antigen family A, 5 (MAGEA5), RefSeqGene
on chromosome X
NCBI Reference Sequence: NG_016417.1

45. Homo sapiens MAGED4 protein (MAGED4) mRNA, complete cds
GenBank: AF329733.1
There are 2 possible CpG island(s) found.
http://www.urogene.org/methprimer

46. Homo sapiens melanoma antigen family B, 10 (MAGEB10), RefSeqGene on
chromosome X
NCBI Reference Sequence: NG_017094.1
There are 1 possible CpG island(s) found.

47. Homo sapiens melanoma antigen family C, 2 (MAGEC2), RefSeqGene
on chromosome X
NCBI Reference Sequence: NG_015872.1

48. Homo sapiens melanoma antigen family B, 2 (MAGEB2), RefSeqGene
on chromosome X
NCBI Reference Sequence: NG_013246.1
PREDICTED: Homo sapiens melanoma antigen family B, 5 (MAGEB5), mRNA
NCBI Reference Sequence: XM_293407.7

49. Homo sapiens melanoma antigen family A, 12
(MAGEA12), RefSeqGene on chromosome X
NCBI Reference Sequence: NG_015852.1
Homo sapiens melanoma antigen family A, 9 (MAGEA9), mRNA
NCBI Reference Sequence: NM_005365.4