Table 1 Selected features from the Human miRNA disease database (HMDD) included in present study .................................................................................... 2
Table 2 List of the miRNA SNPs included in multiplex primer design using the MassARRAY® Assay Design Suite v1.0 software (SEQUENOM Inc., San
Diego, CA, USA) ............................................................................................................................................................................................................................. 3
Table 3 Primer sequences for the miRNA SNPs included in genotyping study using multiplex PCR reaction and MALDI-TOF MS ......................................... 4
Table 4 Allele and genotype frequencies for miRNA SNPs in mir-210 obtained from the GRC-BC population ........................................................................... 5
Table 5 Allele and genotype frequencies for miRNA SNPs in mir-221 and mir-222 from the GRC-BC Population..................................................................... 5
Table 6 Allele and genotype frequencies for miRNA SNPs in mir-21 obtained from the GRC-BC population............................................................................. 6
Table 7 Allele and genotype frequencies for miRNA SNPs in let-7a-1 obtained from the GRC-BC cohort .................................................................................. 6
Table 8 Allele and genotype frequencies for miRNA SNPs in let-7a-2 obtained from the GRC-BC population ........................................................................... 7
Table 9 Allele and genotype frequencies for rs55945735 located in mir-145 obtained from the GRC-BC population .................................................................. 7
Table 10 Allele and genotype frequencies for SNP rs353291 located in mir-145 obtained from the GRC-BC and GU-CCQ BB cohorts ................................... 8
Table 1 Selected features from the Human miRNA disease database (HMDD) included in present study
Selected diseases and/or pathological features related to breast cancer
miRNA-disease
association
dataset
Human miRNA genes in
dataset
503
Human Diseases in dataset
396
Human miRNA genes in
dataset
503
miRNA
function
dataset
Biological and cellular
functions in dataset
43
1. Adenocarcinoma
2. Breast Neoplasms
3. Carcinoma
4. Ductal breast carcinoma
5. Squamous Cell Carcinoma
6. Neoplasms
7. Germ cell and embryonal neoplasm
8. Squamous cell neoplasms
Selected biological and/or cellular function in relation to Cancer
1. Activation of caspases cascade
13. Cell proliferation
2. Akt pathway
14. Chemosensitivity of tumour cells
3. Angiogenesis
15. Chemotaxis
4. Anti-cell proliferation
16. Chromatin remodelling
5. Apoptosis
17. DNA Repair
6. Cell cycle related
18. Epithelial-mesenchymal transition
7. Cell death
19. Human embryonic stem cell (hESC) regulation
8. Cell differentiation
20 Immune response
9. Cell division
21. Immune system
10. Cell fate determination
22. Inflammation
11. Cell motility
23 miRNA tumour suppressors
12. Cell proliferation
24. Onco-miRNAs
Table 2 List of the miRNA SNPs included in multiplex primer design using the MassARRAY® Assay Design Suite v1.0 software (SEQUENOM Inc., San Diego, CA, USA)
SNPs near 3'
end
MAF
Location
rs1062099
0.1877
567,627
rs77585961
rs2829801
0.0247
0.4707
9,211,468
26,944,305
45,605,585 - 45,605,694
rs7050391
0.0133
45,605,273
Xp11.3
45,606,421 - 45,606,530
rs2858061
0.4352
45,606,132
MIR21
17q23.1
57,918,627 - 57,918,698
MIRLET7A1
9q22.32
96,938,239 - 96,938,318
rs112394324
rs3851812
rs10761322
rs10739971
0.0032
0.0371
0.4588
0.2601
57,918,251
57,918,370
96,937,478
96,937,680
MIRLET7A2
11q24.1
122,017,230 - 122,017,301
rs629367
0.1451
MIR145
5q32
148,810,209 - 148,810,296
rs55945735
rs73798217
0.2010
0.0545
miRNA
Locus
Location
MIR210
11p15.5
568,089 - 568,198
MIR34A
MIR155
1p36.22
21q21.3
9,211,727 - 9,211,836
26,946,292 - 26,946,356
MIR221
Xp11.3
MIR222
SNPs in
gene region
rs35301225
MAF
NA
Location
9,211,802
SNPs near 5'
end
rs7395206
rs10902173
MAF
Location
0.5000
0.4835
568,211
570,072
rs1547354
rs2858061
rs2858060
rs2858059
rs2858060
rs2858059
rs1292037
rs13137
0.0810
0.4352
0.4991
0.4786
0.4991
0.4786
0.2440
0.2440
26,946,709
45,606,132
45,607,008
45,607,190
45,607,008
45,607,190
57918908
57919031
122,017,014
rs1143770
rs562052
rs693120
0.4867
0.3265
0.3265
122,017,598
122,018,550
122,019,011
148,809,158
148,809,918
rs353291
0.3324
148,810,746
Table 3 Primer sequences for the miRNA SNPs included in genotyping study using multiplex PCR reaction and MALDI-TOF MS
SNP
rs2829801
rs7395206
rs73798217
rs353291
rs55945735
rs10902173
rs112394324
rs1292037
rs562052
rs2858059
rs1062099
rs10739971
rs2858060
rs2858061
rs35301225
rs1547354
rs13137
rs10761322
rs693120
rs1143770
rs77585961
rs7050391
rs3851812
rs629367
Forward primer sequence
ACGTTGGATGTTCCCACTCAGGCATATAGC
ACGTTGGATGTCGGACGCCCAAGTTGGAG
ACGTTGGATGACTGGAGGTTATCAGAGAGG
ACGTTGGATGGTAGAGATGCCACAAGAGGG
ACGTTGGATGGGTCTCAAACTCCTGACTTC
ACGTTGGATGTCACAGGCACCTTTTCTCAG
ACGTTGGATGACTGGAGAGAGAAATTACCC
ACGTTGGATGTACAGCTAGAAAAGTCCCTG
ACGTTGGATGCTCCAGGCTAGTGGAATAAC
ACGTTGGATGCAGTAAGTATTTCTGGGGTG
ACGTTGGATGGACCCGGTCCTGATTTTAAC
ACGTTGGATGCCTAATAAGACCACTTAGTGT
ACGTTGGATGACTGTATTATCCTCAGTTC
ACGTTGGATGGCTTTCAATACTACAAGGG
ACGTTGGATGGGCAGTATACTTGCTGATTG
ACGTTGGATGTTGCAGGTTTTGGCTTGTTC
ACGTTGGATGAGGTGAAAGAGATGAACCAC
ACGTTGGATGGCTTTTGGTTACTAAATCAC
ACGTTGGATGGTAGATGGCACATATAGAAA
ACGTTGGATGCTGAACAATTTAATGCCTTC
ACGTTGGATGGTTTCCTTCTCTGCAAGACG
ACGTTGGATGGTAAGGCAGTATGATTAGGC
ACGTTGGATGTTTTCCTCCCAAGCAAAAC
ACGTTGGATGTATGCAGCATTTTTGTGAC
Reverse primer sequence
ACGTTGGATGTGGAGTCTATGTCCACTTCC
ACGTTGGATGTCACACGCACAGTGGGTCT
ACGTTGGATGATGTGTATTCCCCAGTCTCC
ACGTTGGATGAAACCTTAAGTCTTCGTTCG
ACGTTGGATGCTGCATCTGAGCACTTCAAG
ACGTTGGATGGAAGCCTGGGTATTAGGATG
ACGTTGGATGGTTGAAACCAGAGTACATGC
ACGTTGGATGGGAGGGAGGATTTTATGGAG
ACGTTGGATGCTACTGCACTGATCGTGTTC
ACGTTGGATGCTCCCATGATACAATGAAGG
ACGTTGGATGTGTGTTTCTGCCGCTTCAGT
ACGTTGGATGATGCACTAACATACAACGAG
ACGTTGGATGACTTGGGTAATCTAGCAATG
ACGTTGGATGAATGATACCTTTCATAGGGG
ACGTTGGATGGCTGTGAGTGTTTCTTTGGC
ACGTTGGATGGGAGGTTAGTAGTCCTTCTA
ACGTTGGATGAAAGCATTCCCAAAATGCTC
ACGTTGGATGCTTCATATTTAGGAGGTAGC
ACGTTGGATGCATCCCTTAACTGTAAGTTC
ACGTTGGATGTTCAGTTTTACCAGAGGAAC
ACGTTGGATGCACTTACTATGCAGGAAGGC
ACGTTGGATGGCCTCAACTGTCAAAGATTG
ACGTTGGATGTTCTTGCCGTTCTGTAAGTG
ACGTTGGATGATTCTGTTTCCTCGGGTTAG
iPLEX® (Extension) primer sequence
TGGGCTAAGCAACCA
GGTGGGCGGGCGGAG
ACCATTCAGTTCCTAGC
GGTTGTTCTCTGGCTGC
TGATGCCTGGCGGGGCG
ACCTTTTCTCAGCATCTG
CAGATACGACAGAGTGTG
AACCTTTTCAAAACCCACA
CAGATTCAAATGCCATAGCA
TGGGGTGGATAAATGAATAG
TTTAACAGTAGACTTGAGAAG
CCACCTACTCATTTATCCCATG
GTATTATCCTCAGTTCGTAACA
GTAAAACAAAAACAGGTAAGAG
GGATTATTGCTCACAACAACCAG
TTTGATTCAACTGTTAGAAATGTG
TCCCAAAATGCTCTATTTTAGATAG
GGCATATTTAGGAGGTAGCTACTAC
GATTGTCAAATGAAAAGAAGAATAT
CCCCCATGCCTTCTGATATCTGTTGA
CCTACATGATGTAATACACTTACAATA
TGTTCATAATTATTATCAGAAGGCATA
GGTGGAAGTGTTTTATTCTTAGTGTGA
GGGGTCAGCATTTTTGTGACAATGGACA
Table 4 Allele and genotype frequencies for miRNA SNPs in MIR210 obtained from the GRC-BC population
rs1062099
Control
Cases
Hapmap
(%)
G
(%)
300
(83.3)
263
(80.7)
81.7
Allele
C
p-value
(%)
60
(16.7)
0.36
63
(19.3)
18.3
GG
(%)
126
(70.0)
106
(65.0)
66.8
rs10902173
Genotype
CG
CC
(%)
(%)
48
6
(26.7) (3.3)
51
6
(31.3) (3.7)
29.8
p-value
0.61
3.4
C
(%)
203
(59.0)
216
(59.3)
60.4
Allele
T
p-value
(%)
141
(41.0)
0.93
148
(40.7)
39.6
CC
(%)
63
(36.6)
63
(34.6)
34.8
Genotype
TC
TT
(%)
(%)
90
29
(49.5) (15.9)
77
32
(44.8) (18.6)
51.2
p-value
0.64
14.0
Table 5 Allele and genotype frequencies for miRNA SNPs in MIR221 and MIR222 from the GRC-BC Population
rs2858061
Control
Cases
Hapmap
(%)
G
(%)
323
(87.8)
295
(85.8)
86.2
Allele
C
p-value
(%)
45
(12.2)
0.43
49
(14.2)
13.8
GG
(%)
142
(77.2)
130
(75.6)
80.2
rs2858060
Genotype
CG
CC
(%)
(%)
39
3
(21.2) (1.6)
35
7
(20.3) (4.1)
12.4
7.4
p-value
0.38
C
(%)
219
(69.3)
218
(68.1)
72.8
Allele
G
p-value
(%)
97
(30.7)
0.75
102
(31.9)
27.2
CC
(%)
81
(51.3)
74
(46.3)
62.3
Genotype
GC
GG
(%)
(%)
57
20
(36.1) (12.7)
70
16
(43.8) (10.0)
20.6
17.2
p-value
0.35
Table 6 Allele and genotype frequencies for miRNA SNPs in MIR21 obtained from the GRC-BC population
rs1292037
Control
Cases
Hapmap
(%)
A
(%)
300
(80.6)
274
(80.1)
81.0
Allele
G
p-value
(%)
72
(19.4)
0.86
68
(19.9)
19.0
rs13137
Genotype
GA
GG
(%)
(%)
56
8
(30.1) (4.3)
54
7
(31.6) (4.1)
AA
(%)
122
(65.6%)
110
(64.3)
65.4
31.1
p-value
0.95
3.4
T
(%)
299
(80.8)
276
(80.2)
81.0
Allele
A
p-value
(%)
71
(19.2)
0.85
68
(19.8)
TT
(%)
122
(65.9)
111
(64.5)
19.0
65.4
Genotype
AT
AA
(%)
(%)
55
8
(29.7) (4.3)
54
7
(31.4) (4.1)
31.1
p-value
0.94
3.4
Table 7 Allele and genotype frequencies for miRNA SNPs in MIRLET7A1 obtained from the GRC-BC cohort
rs10761322
Control
Cases
Hapmap
(%)
T
(%)
225
(61.8)
207
(60.5)
61.9
Allele
C
p-value
(%)
139
(38.2)
0.73
135
(39.5)
38.1
TT
(%)
76
(41.8)
59
(34.5)
40.9
rs10739971
Genotype
TC
CC
(%)
(%)
73
33
(40.1)
(18.1)
89
23
(52.0)
(13.5)
42.0
17.2
p-value
0.08
G
(%)
243
(67.5)
223
(64.5)
68.1
Allele
A
p-value
(%)
117
(32.5)
0.39
123
(35.5)
31.9
GG
(%)
89
(49.4)
74
(42.8)
46.4
Genotype
AG
AA
(%)
(%)
65
26
(36.1) (14.4)
75
24
(43.4) (13.9)
43.3
10.3
p-value
0.36
Table 8 Allele and genotype frequencies for miRNA SNPs in MIRLET7A2 obtained from the GRC-BC population
rs629367
Control
Cases
Hapmap
(%)
Allele
A
C
(%)
(%)
302
64
(82.5) (17.5)
289
57
(83.5) (16.5)
84.6
pvalue
0.72
AA
(%)
125
(68.3)
122
(70.5)
15.4
75.4
rs562052
Genotype
CA
CC
(%)
(%)
52
6
(28.4) (3.3)
45
6
(26.0) (3.5)
18.5
Allele
pvalue
G (%)
A (%)
pvalue
0.88
242
(65.4)
230
(66.5)
128
(34.6)
116
(33.5)
0.76
68.1
31.9
6.2
GG
(%)
81
(43.8)
74
(42.8)
46.2
rs693120
Genotype
AG
AA
(%)
(%)
80
24
(43.2) (13.0)
82
17
(47.4) (9.8)
43.8
10.0
Table 9 Allele and genotype frequencies for rs55945735 located in MIR145 obtained from the GRC-BC population
rs55945735
Control
Cases
Hapmap
(%)
A
(%)
193
(59.9)
182
(64.1)
62.8
Allele
G
p-value
(%)
129
(40.1)
0.29
102
(35.9)
37.2
AA
(%)
61
(37.9)
60
(42.3)
38.3
Genotype
GA
GG
(%)
(%)
71
29
(44.1) (18.0)
62
20
(43.7) (14.1)
49.1
12.7
p-value
0.58
pvalue
0.57
Allele
G
A
(%)
(%)
230
112
(67.3) (32.7)
225
115
(66.2) (33.8)
68.1
31.9
pvalue
0.77
GG
(%)
82
(48.0)
72
(42.4)
46.2
Genotype
AG
AA
(%)
(%)
66
23
(38.6) (13.5)
81
17
(47.6) (10.0)
43.8
10.0
pvalue
0.21
Table 10 Allele and genotype frequencies for SNP rs353291 located in MIR145 obtained from the GRC-BC and GU-CCQ BB cohorts
GRC-BC Population
Control
Cases
Hapmap
(%)
A
(%)
211
(58.6)
171
(50.9)
62.7
Allele
G
p-value
(%)
149
(41.4)
0.041
165
(49.1)
37.3
AA
(%)
61
(33.9)
40
(23.8)
40.6
GU-CCQ BB Population
Genotype
GA
GG
(%)
(%)
89
30
(49.4) (16.7)
91
37
(54.2) (22.0)
44.1
15.3
p-value
0.09
A
(%)
386
(62.7)
769
(57.2)
62.7
Allele
G
p-value
(%)
230
(37.3)
0.023
575
(42.8)
37.3
AA
(%)
128
(41.6)
229
(34.1)
40.6
Genotype
GA
GG
(%)
(%)
130
50
(42.2) (16.2)
311
132
(46.3) (19.6)
44.1
15.3
p-value
0.07