SUPPLEMENTARY TABLES AND FIGURES
Integrative Analysis of Congenital Muscular Torticollis: from Gene
Expression to Clinical Significance
Shin-Young Yim, MD, PhD1,†, Dukyong Yoon, MD, MS2,†, Myong Chul Park, MD, PhD3, Il Jae
Lee, MD, PhD3, Jang-Hee Kim, MD, MS4, Myung Ae Lee,PhD5, Kyu-Sung Kwack, MD, PhD6,
Jan-Dee Lee, MD, PhD7, Jeong-Hun Lee, MD8, Euy-Young Soh, MD, PhD8, Young-In Na, MS9,
Rae Woong Park, MD, PhD2, KiYoung Lee, PhD2,* and Jae-Bum Jun, MD, PhD9,*
1
The Center for Torticollis, Department of Physical Medicine and Rehabilitation, Ajou University School of
Medicine, Suwon, Republic of Korea; 2Department of Biomedical Informatics, Ajou University School of
Medicine, Suwon, Republic of Korea; 3Department of Plastic and Reconstructive Surgery, Ajou University
School of Medicine, Suwon, Republic of Korea; 4Department of Pathology, Ajou University School of
Medicine, Suwon, Republic of Korea;5Brain Disease Research Center, Ajou University School of Medicine,
Suwon, Republic of Korea; 6Department of Radiology, Ajou University School of Medicine, Suwon, Republic
of Korea; 7Department of Surgery, Yonsei University College of Medicine, Seoul, Republic of
Korea;8Department of Surgery, Ajou University School of Medicine, Suwon, Republic of Korea; 9Department
of Rheumatology, The Hospital for Rheumatic Diseases, Hanyang University College of Medicine, Seoul,
Republic of Korea
1
Table S1 The genes and their primers used in the quantitative real-time PCR analyses.
Gene symbol
Full name
Gene ID
THBS4
Thrombospondin 4
7060
FMOD
Fibromodulin
2331
COL14A1
Collagen, type XIV, alpha 1
7373
CTSK
Cathepsin K
1513
EDIL3
EGF-like repeats and
discoidin I-like domains 3
10085
LOX
Lysyl oxidase
153455
SFRP4
Secreted frizzled-related
protein 4
6424
S100 calcium binding protein
A4
Glyceraldehyde-3-phosphate
GAPDH
dehydrogenase
Fw: forward, Rv: reverse
S100A4
6275
2597
2
Primer sequence
Fw : gacaatgatggtatcccagacc
Rv : ctcacagatgtctcccactcc
Fw : aggtggattccgttcacaat
Rv : cagctcttccaagaagtcctgt
Fw : gcacttaattcttcccatttcaaa
Rv : ggctaactctcctccaaca
Fw : caaccagctgcagaagatcc
Rv : gcagaagctgctgatggag
Fw : aggtcatctgggggttcct
Rv : tgaactctggctgatctgga
Fw : aactgatggaagatcacaagatga
Rv : cacaccaattgcaaaaatgc
Fw : tctctgtggccattgatgc
Rv : gcagctttcatcataatacacacc
Fw : aacgaagccagacaacag
Rv : tccagtatctcctcagggta
Fw : ttcttggctgtgaactgtcg
Rv : ccatgttgagcgttctgaag
Fw : gggattcactgtcagcacaa
Rv: ctcacaggaatagttagcaacaaga
Fw : tctctttgaaatctcataggatagtc
Rv : aactctcagtgccaggaa
Fw : cctggttcctgaatctgac
Rv : ttgccttctaatacggtgaa
Fw : ttagttggctaatgctcaagtatt
Rv : gtcaaattattctcagggtggat
Fw : gtcccgctcattacaaattctt
Rv : agcaattttcaagaagcatcatc
Fw : agggtgacaagttcaagctca
Rv : aagcagcttcatctgtcctttt
Fw : tcgacagtcagccgcatcttcttt
Rv : gcccaatacgaccaaatccgttga
Table S2. Specifications of antibodies used for immunohistochemistry.
*
Antibody
Cat. no
Source
Elastin (ELN)
ab9519
Abcam,Cambridge, MA
Antibody
dilution
1:50
Asporin (ASPN)
ab58741
Abcam,Cambridge, MA
1:50
Chromodomain helicase
DNA-binding protein 3
(CHD3).
ab109195
Abcam,Cambridge, MA
1:100
HIER in
citrate buffer
1 hour at RT
Tenascin (TNC)
ab82449
Abcam,Cambridge, MA
1:100
HIER in
citrate buffer
24 hours at 4℃
Thrombospondin 4
(THBS4)
sc-20646
Santa Cruz
Biotechnology, Santa
Cruz, CA
1:50
HIER* in
citrate buffer
24 hours at 4℃
EGF-like repeats and
discoidin I-like domains 3
(EDIL3)
12580-1-AP
PTGlab, Chicago, IL
1:100
HIER in
citrate buffer
1 hour at RT**
HIER - Heat Induced Epitope Retrieval
RT- Room temperature
**
3
Antigen
retrieval
Proteinase K
HIER in
citrate buffer
Antibody
incubation
24 hours at 4℃
24 hours at 4℃
Table S3. The demographic characteristics of the subjects.
Age at the
time of
Gestational
Location
Group
No Gender
operation for
period
of CMT
CMT
(weeks)
(months old)
M
Left
26
40
1
2
M
Right
6
40
3
M
Left
7
40
Microarray
4
M
Left
31
39
5
M
Right
476
40
6
M
Right
17
39
7
M
Right
5
37
1
F
Left
48
41
2
M
Right
30
40
3
M
Right
11
39
4
F
Right
101
38
5
M
Left
78
40
6
F
Left
27
38
QRT-PCR
7
M
Right
58
41
8
M
Right
120
40
9
M
Left
31
38
10
M
Right
20
40
11
M
Right
17
40
1
M
Right
11
38
2
M
Right
11
40
3
F
Left
11
38
IHC
4
M
Right
15
40
5
M
Right
12
40
*
Vaginal delivery: Cesarean section
4
Birth
weight
(gram)
Method
of child
birth
(V:C)*
Previous
physical
therapy
3600
4290
3900
3000
3000
3700
3140
3780
3200
3200
2900
3750
3150
4500
3200
3000
3200
4080
3060
3110
3900
3120
3610
V
V
V
V
V
V
C
V
C
V
C
V
V
V
C
V
V
V
C
V
V
V
V
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Table S4 All 269 differentially expressed genes in microarray study.
Name
EDIL3
ASPN
THBS4
TNMD
NOV
SFRP2
SFRP4
MXRA5
FMOD
CTSK
COL14A1
LOX
FAM38B
BGN
GLT8D4
FIBIN
STEAP2
LUM
DPT
THY1
LOC493869
VCAN
COL12A1
CTGF
FNDC1
RAB31
AEBP1
SLC41A2
ACTC1
ENPP1
OGN
IGFN1
XG
ABI3BP
DCLK1
FAP
THBS1
SERTAD4
S100A11
C18ORF30
KDELR3
FRMD4B
MYO1D
DPP4
TMEM45A
NOX4
VCAM1
C7
CRABP2
FN1
C18ORF58
AHNAK2
COL6A3
GALNT5
Fold
change
9.85
8.24
8.20
7.58
5.58
4.56
4.53
4.51
4.25
4.10
4.02
4.01
4.00
3.76
3.60
3.55
3.47
3.44
3.40
3.39
3.38
3.31
3.30
3.21
3.19
3.09
3.07
3.06
3.05
3.05
3.02
3.00
3.00
2.93
2.86
2.86
2.83
2.82
2.80
2.79
2.79
2.78
2.74
2.74
2.72
2.70
2.70
2.68
2.64
2.64
2.63
2.62
2.57
2.57
Name
COMP
CCDC3
CDH11
TIMP1
TUBA1A
MLLT11
ANTXR1
MGP
ALDH1L2
SERPINE2
GOLM1
CD44
OMD
MAP1A
TRIM38
ASAM
BOC
ADIPOQ
SPON1
CCDC80
THBS2
HMCN1
PTPN13
MMP2
TNC
CCND2
COL1A1
LIMA1
ANGPTL2
UNQ1940
F13A1
RAI14
COL1A2
PDGFRL
TES
SRPX2
GLIPR1
HTRA1
SULF1
C1QTNF3
CHAD
SLIT2
CLIC1
PRELP
FER1L3
CMKLR1
COL5A2
OLFML1
PDGFD
HGF
C9ORF19
LTBP2
GPNMB
CAMK1D
Fold
change
2.57
2.56
2.54
2.53
2.53
2.52
2.51
2.51
2.47
2.46
2.46
2.45
2.45
2.44
2.43
2.43
2.42
2.41
2.41
2.41
2.40
2.39
2.38
2.38
2.38
2.37
2.34
2.34
2.34
2.34
2.33
2.31
2.30
2.30
2.30
2.30
2.30
2.28
2.28
2.27
2.27
2.26
2.26
2.25
2.25
2.25
2.23
2.22
2.22
2.22
2.21
2.21
2.20
2.20
Name
FKBP10
GPC3
ANXA1
CDO1
DKFZP586H2123
FSTL1
SNX7
MAP1B
CRISPLD1
CYR61
PTGFRN
CHODL
COBLL1
F2R
PYGL
CHD3
CTSO
KIAA1598
DAB2
S100A4
PLEKHA5
MYH10
ANXA2
CCL2
CHRDL1
ANXA2P2
TSHZ2
LOC652811
EMP1
PARVA
RAB23
LEPREL1
FABP4
SPARC
GLI3
ABHD2
KIAA1377
ELN
CD109
CERCAM
GEM
SERPINH1
MFAP5
ARHGAP1
TNFAIP6
LHFPL2
MAMDC2
PLOD2
PDE1A
LRRC17
GJA1
NFATC2
EMP3
SFRP1
5
Fold
change
2.19
2.18
2.18
2.17
2.17
2.15
2.14
2.14
2.13
2.13
2.13
2.13
2.12
2.12
2.12
2.12
2.12
2.11
2.11
2.10
2.10
2.10
2.09
2.08
2.08
2.08
2.08
2.07
2.07
2.07
2.06
2.06
2.06
2.05
2.05
2.05
2.05
2.05
2.05
2.04
2.04
2.04
2.04
2.04
2.03
2.03
2.02
2.02
2.02
2.01
2.01
2.01
2.01
2.00
Name
ZNF521
PLXDC2
TIMP2
GOT2
TMEM16E
ACADSB
DLK1
ADSSL1
UQCRC1
FBXO32
BRP44L
LOC729830
TYRP1
LRRTM4
OSBPL11
CD38
SLC7A2
TMEM159
NT5C1A
DLAT
DDIT4L
GADL1
LPIN1
LMOD1
COX10
FASTKD1
TUBA8
LRRC2
COQ10A
ASB12
APOO
ENO3
SLC47A1
PLCL2
KLHL30
FNDC5
NNT
C1ORF170
PYGM
ACO2
AGBL1
ACADM
PHYH
SLC19A2
PGAM2
KIAA0408
CA2
ESRRG
ACSS2
FABP3
UCP3
TMEM143
PPIF
NIPSNAP3B
Fold
change
2.00
2.00
2.00
-2.00
-2.01
-2.01
-2.01
-2.03
-2.03
-2.03
-2.03
-2.03
-2.04
-2.05
-2.05
-2.05
-2.06
-2.06
-2.07
-2.07
-2.07
-2.08
-2.08
-2.08
-2.09
-2.09
-2.09
-2.10
-2.10
-2.10
-2.11
-2.11
-2.12
-2.12
-2.13
-2.13
-2.14
-2.14
-2.15
-2.15
-2.15
-2.16
-2.16
-2.17
-2.17
-2.17
-2.19
-2.19
-2.19
-2.20
-2.20
-2.21
-2.22
-2.23
Name
PPP1R1A
SLC2A4
RPL3L
ADHFE1
ECH1
FIT1
HRASLS
IDH2
RORC
ALDH1L1
HADHB
ASB2
ZADH1
MAP2K6
MPP7
CMBL
LPL
HSDL2
ETFDH
ITGB6
ASB15
ASB4
PPP1R3A
C21ORF82
ASB14
OCC-1
LOC121952
PHKA1
SLC38A4
CABC1
MAOB
SLC36A2
GPD1L
PDE11A
C3ORF43
PFKFB1
ALPK2
SLC38A3
CLCN1
PAIP2B
C8ORFK36
ASB11
MYLK2
ART3
PEBP4
GPT2
SLC25A30
DHRS7C
MYLK3
MLF1
C8ORF22
NEK10
AQP4
Fold
change
-2.24
-2.26
-2.27
-2.27
-2.27
-2.28
-2.29
-2.33
-2.33
-2.33
-2.34
-2.34
-2.34
-2.36
-2.41
-2.42
-2.44
-2.44
-2.44
-2.45
-2.46
-2.49
-2.51
-2.51
-2.52
-2.55
-2.59
-2.66
-2.67
-2.67
-2.69
-2.69
-2.79
-2.79
-2.88
-2.90
-2.92
-2.93
-3.03
-3.03
-3.03
-3.08
-3.13
-3.15
-3.33
-3.41
-3.44
-3.47
-3.79
-4.53
-5.41
-6.60
-9.81
Table S5 Semi-quantitation of immunohistochemical study according to proteins encoded by the differentially
expressed genes of CMT.
Subjects with CMT
Subjects with normal SCM
Proteins
P value
1
2
3
4
5
1
2
3
4
5
Elastin (ELN)
2
2
3
2
2
1
1
1
1
1
0.008
Asporin (ASPN)
3
3
3
3
2
1
1
2
1
1
0.008
Chromodomain helicase DNAbinding protein 3 (CHD3).
Tenascin (TNC)
Thrombospondin 4
(THBS4)
EGF-like repeats and discoidin
I-like domains 3 (EDIL3)
*
2
2
1
1
1
0
0
0
0
0
0.008
2
1
1
2
1
1
1
1
1
1
> 0.05
1
2
0
0
0
1
1
0
0
0
> 0.05
0
0
0
0
0
0
0
0
0
0
> 0.05
thrombospondin 4
EGF-like repeats and discoidin I-like domains 3
**
6
Figure S1. The decision tree model for discriminating T-CMT and T-control
We examined the discriminant power of the DEGs using decision tree method implemented in Clementine 12.0
(SPSS Inc., Chicago, IL, USA). 8 samples (4 T-CMT and 4 T-control) from 4 subjects were used for training
data and the others for test. Decision tree models showed a mean sensitivity of 0.70 and a mean specificity of
0.82. That means that the DEGs identified from the microarray study were able to accurately differentiate the TCMTs from the T-controls. This figure showed one example of the decision tree model. The expressed level of
S100A4 in the T-CMT was greater than 225.683 in all 4 T-CMTs, while the expressed levels of S100A4 in the
T-control was 225.683 or less.
7
Figure S2. The Gene Ontology enrichment analysis on molecular function
Each portion represents the Gene Ontology term (p-value) and the number of genes. (a) The significant subordinate concepts
of the ‘developmental process’ were ‘anatomical structure morphogenesis’ (pdevelopment’ (ppment’ (p), etc. (b) The
enriched subordinate GO terms of ‘extracellular region part’ in the cellular components included ‘extracellular matrix’ (p-value=3.44E-25, 39 genes), ‘collagen’ (pvalue=1.79E-7, 8 genes). (c) This diagram shows the subordinate GO terms of ‘binding’ such as collagen binding, pattern
binding and carbohydrate binding.
8
Figure S3. A graph showing the correlation between the quantitative real-time PCR (QRT-PCR) data and the
microarray data for 8 DEGs
The full name of the genes are as follows; thrombospondin 4 (THBS4), fibromodulin (FMOD), collagen, type XIV, alpha 1
(COL14A1), cathepsin K (CTSK), epidermal growth factor (EGF)-like repeats and discoidin I-like domains 3 (EDIL3), lysyl
oxidase (LOX, secreted frizzled-related protein 4 (SFRP4), and S100 calcium binding protein A4 (S100A4). R2 is 0.41.
9
Figure S4. The MRI images of the remaining 8 CMT patients (#4~#11)
We measured the mean color intensity of the SCM with and without CMT on the axial T1 weighted, pre-operational MRI
image using the region of interest (ROI) method on the same axial T1 weighted image using the same ROI method. Red and
green circles indicate the cross sectional areas of the SCM with and without CMT, respectively. The red arrows indicate the
lowest signal areas of CMT.
10