Supplementary Materials
(4 Tables and 5 Figures)
Table S1 Primers for validating expression levels of six genes by quantitative PCR
Table S2 Information of the four specific modules
Table S3 54 leading genes identified in gene set enrichment analysis (GSEA) of differentially
expressed genes mapped by expression probes in Module 1 (in males)
Table S4 97 leading genes identified in gene set enrichment analysis (GSEA) of differentially
expressed genes mapped by expression probes in Module 2 (in males)
Fig. S1 Assessment of the reproducibility of the Illumina HumanHT-12 v4 Expression
BeadChip assay and validation of the BeadChip expression data of six genes using
quantitative PCR.
Fig. S2 Genome-wide gene expression patterns (at the probe level) in postmortem prefrontal
cortex (PFC) of 46 subjects.
Fig. S3 Genome-wide co-expression analysis of all 23 pairs of subjects (23 AUD cases vs. 23
matched controls).
Fig. S4 Genome-wide co-expression analysis of the seven pairs of subjects (7 female AUD
cases vs. 7 matched female controls).
Fig. S5 Visualization of the protein-protein interaction (PPI) network in the fatty acid
metabolism pathway.
Table S1 Primers for validating expression levels of six genes by quantitative PCR
Gene (GenBank)
Primers
Sequences (5'-3')
PCR product size (bp)
AGT
forward
AGACTGGCTGCTCCCTGAT
185
(NM_000029)
reverse
ACTCCAGTGCTGGAAGGTG
ALDH1L1
(NM_012190)
forward
reverse
AGATTGCAGTGATTGGACAGAG
CCAAAGCCTGGTATTTTGCCA
225
GABRA1
(NM_001127647)
forward
reverse
AGCCGTCATTACAAGATGAACTT
TGGTCTCAGGCGATTGTCATAA
95
GRIN2C
(NM_000835)
forward
reverse
GAGTGGTCAAATTCTCCTACGAC
TGTAGTACACCTCCCCAATCAT
102
PAX6
(NM_001604)
forward
reverse
TGGGCAGGTATTACGAGACTG
ACTCCCGCTTATACTGGGCTA
111
SLC1A3
(NM_004172)
forward
reverse
GGCCAAGAAGAAAGTGCAGA
GCATCTGTAACATCCTCATCAGA
200
Rn18s (reference)
forward
CAGCCACCCGAGATTGAGCA
253
(NM10098)
reverse
TAGTAGCGACGGGCGGTGTG
SYBR green-based real-time quantitative PCRs were performed for detecting the relative expression
levels of six genes that showed significant expression differences (by expression array-based assays)
between 23 AUD cases and 23 matched controls by paired t-tests. About 2 μg of total RNAs were
converted to cDNAs under the standard protocol of the High Capacity RNA-to-cDNA Kit (Applied
Biosystems, Foster City, CA). 20 µL of PCR reaction was prepared in a mixture containing 10µL of 2×
SYBR Green PCR Master Mix, 1 µL of each forward/reverse primer, 2µL of cDNA template (~5.0
ng/μL), and 6 µL of RNase-free ddH2O. Each reaction was performed in triplicate. The cycling conditions
were 95°C 10 min plus 45 three-step cycles: 95°C 30 sec, 57°C 30 sec, and 72°C 30 sec. To normalize
relative expression levels, a calibrator was set up using a cDNA mix that was pooled from cDNAs of the
46 subjects. After real-time PCRs, the Cq (threshold cycle) of the target gene (Cq T) or the reference gene
(Cq E) was analyzed using programs SDS version 2.3 and RQ manager version 1.3 (Applied Biosystems).
Reaction wells with Cq ≥ 40 or differences between Cq and mean Cq greater than 0.3 was excluded for
further analysis. The relative expression level ΔCq (Cq T - Cq E) of the target gene was normalized to the
reference gene Rn18s. Average ΔCq was used to represent the relative expression level of the target
genes that were normalized to the reference gene; the larger ΔCq, the lower relative expression level.
Table S2 Information of the four specific modules
Correlation with
Total
Probes
AUDs
Modules
probes
(P<0.05)
r
P
Module 1
(male:magenta)
561
177
0.42
0.020
Module 2
(male:red)
Module 3
(male:brown)
Module 4
(female:yellow)
GSEA
mapped genes
P
134
0.028
815
326
0.41
0.020
243
0.004
1,446
255
-0.38
0.030
197
0.566
683
62
0.64
0.010
46
0.539
Table S3 54 leading genes identified in gene set enrichment analysis (GSEA) of differentially expressed
genes mapped by expression probes in Module 1 (in males)
PROBE
GENE SYMBOL
1
C1orf54
FAM65B
PLXNB2
ITGB5
MSI2
PITPNC1
CYB5B
AKR1C3
PSPC1
TAP1
ARAP3
BAG3
SEMA3E
NRG1
CLEC2B
ALDH1L1
GNA12
LPP
RYK
AHSA1
IMPA2
DHRS3
GLIS3
CCDC91
SCARA3
MOV10
ALDH7A1
FERMT2
ACAA2
FEZ1
MASP1
PALLD
ASPSCR1
ATP7B
IGDCC4
CIDEA
ABCA1
C10orf54
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
RANK METRIC SCORE
RUNNING ES
CORE ENRICHMENT
60
4.644
0.018
187
3.907
0.029
278
3.682
0.041
403
3.451
0.049
432
3.416
0.064
440
3.411
0.079
478
3.365
0.092
561
3.26
0.103
857
3.01
0.1
1030
2.893
0.103
1069
2.873
0.114
1210
2.81
0.119
1262
2.783
0.129
1264
2.783
0.142
1281
2.777
0.154
1436
2.708
0.157
1493
2.684
0.166
1592
2.646
0.173
1613
2.638
0.184
1752
2.582
0.188
1788
2.572
0.198
1835
2.554
0.207
1873
2.544
0.217
1882
2.542
0.228
1968
2.511
0.235
2005
2.499
0.244
2319
2.402
0.237
2512
2.346
0.237
2686
2.297
0.238
2955
2.223
0.233
2977
2.219
0.242
2981
2.218
0.252
3111
2.192
0.254
3438
2.125
0.245
3521
2.108
0.25
3629
2.085
0.254
3652
2.081
0.262
3727
2.068
0.268
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
TIMP3
GALM
WFS1
PTTG1IP
HSDL2
TGFBR3
CPT2
PXN
DTNA
IFITM2
SLC7A2
PARP9
FEM1C
PRMT2
LAMC1
LYN
3743
2.066
0.276
3757
2.063
0.285
4153
1.987
0.271
4237
1.979
0.276
4517
1.932
0.269
4776
1.883
0.263
4818
1.876
0.269
4895
1.866
0.273
4918
1.863
0.28
4928
1.86
0.289
4945
1.857
0.296
4981
1.851
0.303
4984
1.851
0.311
5070
1.839
0.315
5165
1.821
0.318
5175
1.821
0.326
Table S4 97 leading genes identified in gene set enrichment analysis (GSEA) of differentially expressed
genes mapped by expression probes in Module 2 (in males)
PROBE
GENE SYMBOL
RUNNING ES
CORE ENRICHMENT
DARS
RANK METRIC
SCORE
44
1
4.77
0.0096
2
RYR1
147
4.027
0.014
3
DDAH1
155
3.991
0.0238
4
CPE
161
3.971
0.0336
5
NFIB
208
3.83
0.0407
6
ITPR2
271
3.699
0.0465
7
AUTS2
281
3.674
0.0554
8
SLC39A12
282
3.674
0.0647
9
NTM
305
3.622
0.0727
10
RYR3
306
3.622
0.0819
11
LPAR3
349
3.539
0.0885
12
FYN
470
3.375
0.0902
13
CADM1
502
3.332
0.0969
14
SPON1
545
3.277
0.1028
15
NTRK2
600
3.221
0.1079
16
GPC5
620
3.201
0.115
17
PAX6
652
3.172
0.1213
18
LRRC16A
660
3.164
0.1289
19
GPR98
823
3.034
0.1273
20
BCL2
897
2.987
0.1307
21
AHCTF1
1003
2.907
0.132
22
NAV2
1020
2.9
0.1385
23
SPRED1
1036
2.889
0.145
24
EYA2
1114
2.854
0.1478
25
PCDHGC3
1170
2.827
0.1519
26
KCNN3
1213
2.807
0.1566
27
CGNL1
1233
2.799
0.1626
28
PAMR1
1396
2.726
0.1602
29
RGL1
1518
2.674
0.1601
30
PAPLN
1736
2.588
0.1541
31
MYO5A
1740
2.587
0.1605
32
NEBL
1827
2.556
0.1621
33
CYBRD1
1870
2.544
0.1662
34
GRAMD3
1904
2.535
0.1707
35
PRDM16
1925
2.526
0.176
36
HADH
1938
2.52
0.1817
37
ACACB
2088
2.472
0.1794
38
MICAL2
2116
2.465
0.1841
39
ACSL6
2299
2.41
0.1798
40
ABLIM1
2301
2.41
0.1859
41
RAB31
2312
2.405
0.1914
42
CACHD1
2390
2.381
0.193
43
PPAP2B
2518
2.344
0.1917
44
NT5C2
2526
2.342
0.1972
45
WWC1
2613
2.315
0.1982
46
SMO
2646
2.307
0.2022
47
EPHX2
2647
2.307
0.2081
48
CABLES1
2648
2.307
0.214
49
SLC14A1
2650
2.306
0.2198
50
RERG
2679
2.298
0.224
51
CNTFR
2684
2.297
0.2297
52
GCNT2
2731
2.285
0.2328
53
PRODH
2777
2.272
0.236
54
SLC22A5
2814
2.261
0.2397
55
CYP2U1
2867
2.248
0.2424
56
APCDD1
2937
2.228
0.2441
57
RHPN2
2948
2.225
0.2492
58
SLC1A2
3030
2.208
0.2502
59
MAP3K5
3071
2.201
0.2535
60
RBM43
3095
2.194
0.2577
61
FGGY
3125
2.189
0.2616
62
PHYHD1
3140
2.185
0.2664
63
ZIC2
3196
2.176
0.2688
64
ATP1B2
3219
2.171
0.273
65
EYA1
3337
2.146
0.2717
66
OAF
3398
2.133
0.2737
67
BBS2
3399
2.132
0.2791
68
CHPT1
3471
2.115
0.2804
69
LRIG1
3542
2.103
0.2818
70
WIF1
3593
2.092
0.2842
71
PLCG1
3608
2.09
0.2887
72
FABP3
3609
2.089
0.294
73
HEATR2
3622
2.086
0.2987
74
JARID2
3646
2.082
0.3026
75
DOCK7
3651
2.081
0.3077
76
TPD52L1
3733
2.067
0.3083
77
OGFOD1
3865
2.042
0.306
78
MYBPC1
4074
2.004
0.2991
79
GLUD1
4160
1.987
0.2992
80
GLI3
4184
1.983
0.3029
81
C14orf159
4255
1.975
0.3039
82
ATP13A4
4315
1.963
0.3055
83
TPP1
4467
1.939
0.3018
84
SLC1A3
4508
1.932
0.3044
85
CALML4
4522
1.932
0.3085
86
LARS
4579
1.921
0.3102
87
DYNLL2
4591
1.921
0.3145
88
EGFR
4638
1.91
0.3167
89
SERPINE2
4865
1.87
0.3084
90
RANBP3L
4943
1.857
0.3087
91
APPL2
4948
1.857
0.3132
92
PPP1R1B
4954
1.857
0.3176
93
IL17RB
5053
1.839
0.3167
94
PLOD2
5154
1.824
0.3155
95
RERE
5220
1.812
0.3164
96
INHBB
5335
1.799
0.3144
97
UNG
5353
1.799
0.318
Fig. S1 Assessment of the reproducibility of the Illumina HumanHT-12 v4 Expression BeadChip assay
and validation of the BeadChip expression data of six genes using quantitative PCR.
(a) Correlation of genome-wide gene expression levels of Sample #38 measured by Chip 1 and Chip 2. (b)
Correlation of genome-wide gene expression levels of Sample #38 measured by Chip 1 and Chip 3. (b)
Correlation of genome-wide gene expression levels of Sample #38 measured by Chip 2 and Chip 3. (d)
Correlation of expression levels of six genes (AGT, ALDH1L1, GABRA1, GRIN2C, PAX6, and SLC1A3)
measured by the Illumina HumanHT-12 v4 Expression BeadChip assay and quantitative PCR (indicated
by delta Ct values normalized to the reference gene Rn18s). Expression levels of six genes measured by
the expression array-based approach were validated using quantitative PCR in all 46 postmortem
prefrontal cortex (PFC) RNA samples. Correlation of the mean expression levels of the six genes
measured by the two methods was analyzed and the correlation coefficient was -0.869.
Fig. S2 Genome-wide gene expression patterns (at the probe level) in postmortem prefrontal
cortex (PFC) of 46 subjects.
X axis: RNA sample IDs (#1 - #23: healthy subjects; #24 - #46: subjects with alcohol use
disorders or AUDs). Y axis: normalized gene expression levels. From top to bottom, statistics
such as maximum, 3rd quantile, median, 1st quantile, and minimum values are shown in the box
plot.
Fig. S3 Genome-wide co-expression analysis of all 23 pairs of subjects (23 AUD cases vs. 23
matched controls).
Weighted gene co-expression network analysis (WGCNA) was performed to assess the intercorrelation of the intensities of 21,521 expression probes in all 23 pairs of subjects. (a)
Dendrogram of 11 modules identified by WGCNA under default setting. (b) Association of 11
modules with clinical traits [alcohol use disorders (AUDs), age, postmortem internal (PMI), or
alcohol daily use in grams (AlcGram)].
Fig. S4 Genome-wide co-expression analysis of the seven pairs of subjects (7 female AUD
cases vs. 7 matched female controls).
Weighted gene co-expression network analysis (WGCNA) was performed to assess the intercorrelation of the intensities of genome-wide expression probes (n = 21,521) in the seven pairs of
subjects. (a) Dendrogram of 15 modules identified by WGCNA under default setting. (b)
Association of 15 modules with clinical traits [alcohol use disorders (AUDs), age, postmortem
internal (PMI), or alcohol daily use in grams (AlcGram)].
Fig. S5 Visualization of the protein-protein interaction (PPI) network in the fatty acid
metabolism pathway.
The protein-protein interaction (PPI) network was obtained from the STRING 9.0 database built
in program EnrichNet for 327 unique genes from two AUDs-associated modules (Modules 1 and
2) identified in the 16 pairs of male subjects (16 male AUD cases vs. 16 male controls). The
visualization consisted of a force-directed graphic layout of the sub-network containing 327
uploaded genes (the blue nodes), 32 genes (the red nodes) in the fatty acid metabolism pathway,
and nine overlapped genes (the green nodes) in a human protein-protein interaction (PPI)
network (and three of these genes, including ALDH9A1, ALDH7A1, and ALDH2, are involved in
aldehyde detoxification).