Correlation between gene methylation status and clinical features
Overview
Introduction

This pipeline uses various statistical tests to identify genes whose promoter methylation levels correlated to selected clinical features.

Summary

Testing the association between 19705 genes and 17 clinical features across 497 samples, statistically thresholded by P value < 0.05 and Q value < 0.3, 14 clinical features related to at least one genes.

  • 25 genes correlated to 'Time to Death'.

    • FAM118B__1 ,  SYNGR3 ,  KLHL29 ,  ANKRD53 ,  SYN2__1 ,  ...

  • 511 genes correlated to 'AGE'.

    • MGA ,  INA ,  NHLRC1 ,  SYNGR3 ,  C1ORF59 ,  ...

  • 391 genes correlated to 'NEOPLASM.DISEASESTAGE'.

    • MGA ,  INA ,  TBKBP1 ,  RANBP17 ,  IFT140 ,  ...

  • 288 genes correlated to 'PATHOLOGY.T.STAGE'.

    • TBKBP1 ,  GJD3 ,  IFT140 ,  TMEM204 ,  PDGFB ,  ...

  • 1723 genes correlated to 'PATHOLOGY.N.STAGE'.

    • BMP1 ,  DAGLA ,  ST6GALNAC5 ,  CPNE1__1 ,  RBM12__1 ,  ...

  • 3 genes correlated to 'PATHOLOGY.M.STAGE'.

    • IQCD ,  TPCN1 ,  SLC25A37

  • 33 genes correlated to 'GENDER'.

    • ALG11__2 ,  UTP14C__1 ,  ETF1 ,  KIF4B ,  FAM35A ,  ...

  • 3138 genes correlated to 'HISTOLOGICAL.TYPE'.

    • LEPR ,  LEPROT ,  PON2 ,  GPR115 ,  XDH ,  ...

  • 9 genes correlated to 'RADIATIONS.RADIATION.REGIMENINDICATION'.

    • KLHDC1 ,  C1ORF77 ,  S100A13__1 ,  ZNF782 ,  PPOX ,  ...

  • 389 genes correlated to 'EXTRATHYROIDAL.EXTENSION'.

    • NRCAM ,  TBKBP1 ,  PDGFB ,  IFT140 ,  TMEM204 ,  ...

  • 2 genes correlated to 'COMPLETENESS.OF.RESECTION'.

    • SLC25A37 ,  NECAB2

  • 1299 genes correlated to 'NUMBER.OF.LYMPH.NODES'.

    • FUT2 ,  MET ,  TMEM173 ,  MACF1 ,  TAGLN2 ,  ...

  • 1 gene correlated to 'TUMOR.SIZE'.

    • TGFBR1

  • 49 genes correlated to 'RACE'.

    • SCAMP5 ,  CDKN2AIP ,  CLTC ,  PTGES ,  RAD21L1 ,  ...

  • No genes correlated to 'RADIATIONEXPOSURE', 'MULTIFOCALITY', and 'ETHNICITY'.

Results
Overview of the results

Complete statistical result table is provided in Supplement Table 1

Table 1.  Get Full Table This table shows the clinical features, statistical methods used, and the number of genes that are significantly associated with each clinical feature at P value < 0.05 and Q value < 0.3.

Clinical feature Statistical test Significant genes Associated with                 Associated with
Time to Death Cox regression test N=25 shorter survival N=18 longer survival N=7
AGE Spearman correlation test N=511 older N=505 younger N=6
NEOPLASM DISEASESTAGE Kruskal-Wallis test N=391        
PATHOLOGY T STAGE Spearman correlation test N=288 higher stage N=115 lower stage N=173
PATHOLOGY N STAGE Wilcoxon test N=1723 class1 N=1723 class0 N=0
PATHOLOGY M STAGE Kruskal-Wallis test N=3        
GENDER Wilcoxon test N=33 male N=33 female N=0
HISTOLOGICAL TYPE Kruskal-Wallis test N=3138        
RADIATIONS RADIATION REGIMENINDICATION Wilcoxon test N=9 yes N=9 no N=0
RADIATIONEXPOSURE Wilcoxon test   N=0        
EXTRATHYROIDAL EXTENSION Kruskal-Wallis test N=389        
COMPLETENESS OF RESECTION Kruskal-Wallis test N=2        
NUMBER OF LYMPH NODES Spearman correlation test N=1299 higher number.of.lymph.nodes N=39 lower number.of.lymph.nodes N=1260
MULTIFOCALITY Wilcoxon test   N=0        
TUMOR SIZE Spearman correlation test N=1 higher tumor.size N=1 lower tumor.size N=0
RACE Kruskal-Wallis test N=49        
ETHNICITY Wilcoxon test   N=0        
Clinical variable #1: 'Time to Death'

25 genes related to 'Time to Death'.

Table S1.  Basic characteristics of clinical feature: 'Time to Death'

Time to Death Duration (Months) 0-158.8 (median=16.6)
  censored N = 479
  death N = 14
     
  Significant markers N = 25
  associated with shorter survival 18
  associated with longer survival 7
List of top 10 genes differentially expressed by 'Time to Death'

Table S2.  Get Full Table List of top 10 genes significantly associated with 'Time to Death' by Cox regression test

HazardRatio Wald_P Q C_index
FAM118B__1 0 1.92e-07 0.0038 0.226
SYNGR3 211 2.675e-07 0.0053 0.75
KLHL29 0 3.491e-07 0.0069 0.223
ANKRD53 431 4.079e-07 0.008 0.649
SYN2__1 361 9.515e-07 0.019 0.854
RAB6C 34001 9.687e-07 0.019 0.788
LOC150786 10001 1.305e-06 0.026 0.877
SLC6A17 840001 1.701e-06 0.034 0.837
ARSJ 301 2.879e-06 0.057 0.77
GSG1L 0 2.928e-06 0.058 0.216
Clinical variable #2: 'AGE'

511 genes related to 'AGE'.

Table S3.  Basic characteristics of clinical feature: 'AGE'

AGE Mean (SD) 47.13 (16)
  Significant markers N = 511
  pos. correlated 505
  neg. correlated 6
List of top 10 genes differentially expressed by 'AGE'

Table S4.  Get Full Table List of top 10 genes significantly correlated to 'AGE' by Spearman correlation test

SpearmanCorr corrP Q
MGA 0.5323 1.027e-37 2.02e-33
INA 0.4867 6.305e-31 1.24e-26
NHLRC1 0.4847 1.205e-30 2.37e-26
SYNGR3 0.4685 1.769e-28 3.48e-24
C1ORF59 0.4679 2.115e-28 4.17e-24
RANBP17 0.4658 3.91e-28 7.7e-24
ACN9 0.4528 1.697e-26 3.34e-22
OTUD7A 0.4424 3.096e-25 6.1e-21
ZNF518B 0.4301 8.446e-24 1.66e-19
NTNG2 0.4296 9.733e-24 1.92e-19
Clinical variable #3: 'NEOPLASM.DISEASESTAGE'

391 genes related to 'NEOPLASM.DISEASESTAGE'.

Table S5.  Basic characteristics of clinical feature: 'NEOPLASM.DISEASESTAGE'

NEOPLASM.DISEASESTAGE Labels N
  STAGE I 281
  STAGE II 52
  STAGE III 109
  STAGE IV 2
  STAGE IVA 45
  STAGE IVC 6
     
  Significant markers N = 391
List of top 10 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

Table S6.  Get Full Table List of top 10 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

ANOVA_P Q
MGA 1.373e-13 2.71e-09
INA 6.216e-13 1.22e-08
TBKBP1 8.023e-13 1.58e-08
RANBP17 6.083e-12 1.2e-07
IFT140 4.309e-11 8.49e-07
TMEM204 4.309e-11 8.49e-07
ZNF518B 1.007e-10 1.98e-06
SPRY4 2.52e-10 4.96e-06
PDGFB 3.2e-10 6.3e-06
STK40 3.335e-10 6.57e-06
Clinical variable #4: 'PATHOLOGY.T.STAGE'

288 genes related to 'PATHOLOGY.T.STAGE'.

Table S7.  Basic characteristics of clinical feature: 'PATHOLOGY.T.STAGE'

PATHOLOGY.T.STAGE Mean (SD) 2.13 (0.88)
  N
  1 141
  2 167
  3 165
  4 21
     
  Significant markers N = 288
  pos. correlated 115
  neg. correlated 173
List of top 10 genes differentially expressed by 'PATHOLOGY.T.STAGE'

Table S8.  Get Full Table List of top 10 genes significantly correlated to 'PATHOLOGY.T.STAGE' by Spearman correlation test

SpearmanCorr corrP Q
TBKBP1 0.3115 1.412e-12 2.78e-08
GJD3 0.2967 1.701e-11 3.35e-07
IFT140 0.2957 1.995e-11 3.93e-07
TMEM204 0.2957 1.995e-11 3.93e-07
PDGFB 0.2854 1.04e-10 2.05e-06
GGT7 0.2759 4.406e-10 8.68e-06
PSD3 -0.2689 1.258e-09 2.48e-05
BTBD3 0.2671 1.636e-09 3.22e-05
FLJ42875 0.2661 1.885e-09 3.71e-05
CKMT2 0.2648 2.266e-09 4.46e-05
Clinical variable #5: 'PATHOLOGY.N.STAGE'

1723 genes related to 'PATHOLOGY.N.STAGE'.

Table S9.  Basic characteristics of clinical feature: 'PATHOLOGY.N.STAGE'

PATHOLOGY.N.STAGE Labels N
  class0 225
  class1 222
     
  Significant markers N = 1723
  Higher in class1 1723
  Higher in class0 0
List of top 10 genes differentially expressed by 'PATHOLOGY.N.STAGE'

Table S10.  Get Full Table List of top 10 genes differentially expressed by 'PATHOLOGY.N.STAGE'

W(pos if higher in 'class1') wilcoxontestP Q AUC
BMP1 13046 2.434e-18 4.8e-14 0.7388
DAGLA 13061 2.682e-18 5.28e-14 0.7385
ST6GALNAC5 13073 2.898e-18 5.71e-14 0.7383
CPNE1__1 13184 5.92e-18 1.17e-13 0.7361
RBM12__1 13184 5.92e-18 1.17e-13 0.7361
SLC34A2 13232 8.046e-18 1.59e-13 0.7351
MET 13236 8.254e-18 1.63e-13 0.735
PON2 13335 1.548e-17 3.05e-13 0.733
MACF1 13458 3.357e-17 6.61e-13 0.7306
CAPN2 13516 4.822e-17 9.5e-13 0.7294
Clinical variable #6: 'PATHOLOGY.M.STAGE'

3 genes related to 'PATHOLOGY.M.STAGE'.

Table S11.  Basic characteristics of clinical feature: 'PATHOLOGY.M.STAGE'

PATHOLOGY.M.STAGE Labels N
  M0 274
  M1 9
  MX 213
     
  Significant markers N = 3
List of 3 genes differentially expressed by 'PATHOLOGY.M.STAGE'

Table S12.  Get Full Table List of 3 genes differentially expressed by 'PATHOLOGY.M.STAGE'

ANOVA_P Q
IQCD 1.034e-05 0.204
TPCN1 1.034e-05 0.204
SLC25A37 1.348e-05 0.266
Clinical variable #7: 'GENDER'

33 genes related to 'GENDER'.

Table S13.  Basic characteristics of clinical feature: 'GENDER'

GENDER Labels N
  FEMALE 365
  MALE 132
     
  Significant markers N = 33
  Higher in MALE 33
  Higher in FEMALE 0
List of top 10 genes differentially expressed by 'GENDER'

Table S14.  Get Full Table List of top 10 genes differentially expressed by 'GENDER'. 0 significant gene(s) located in sex chromosomes is(are) filtered out.

W(pos if higher in 'MALE') wilcoxontestP Q AUC
ALG11__2 47962 6.111e-64 1.2e-59 0.9955
UTP14C__1 47962 6.111e-64 1.2e-59 0.9955
ETF1 47544 8.751e-62 1.72e-57 0.9868
KIF4B 4554 2.055e-43 4.05e-39 0.9055
FAM35A 7530 1.121e-31 2.21e-27 0.8437
GLUD1__1 7530 1.121e-31 2.21e-27 0.8437
MTFR1 40283 2.315e-30 4.56e-26 0.8361
WBP11P1 38801 2.396e-25 4.72e-21 0.8053
ANKRD20A4 35762 1.531e-16 3.01e-12 0.7423
DACH1 12597 4.382e-16 8.63e-12 0.7385
Clinical variable #8: 'HISTOLOGICAL.TYPE'

3138 genes related to 'HISTOLOGICAL.TYPE'.

Table S15.  Basic characteristics of clinical feature: 'HISTOLOGICAL.TYPE'

HISTOLOGICAL.TYPE Labels N
  OTHER SPECIFY 9
  THYROID PAPILLARY CARCINOMA - CLASSICAL/USUAL 353
  THYROID PAPILLARY CARCINOMA - FOLLICULAR (>= 99% FOLLICULAR PATTERNED) 100
  THYROID PAPILLARY CARCINOMA - TALL CELL (>= 50% TALL CELL FEATURES) 35
     
  Significant markers N = 3138
List of top 10 genes differentially expressed by 'HISTOLOGICAL.TYPE'

Table S16.  Get Full Table List of top 10 genes differentially expressed by 'HISTOLOGICAL.TYPE'

ANOVA_P Q
LEPR 5.525e-30 1.09e-25
LEPROT 5.525e-30 1.09e-25
PON2 8.991e-30 1.77e-25
GPR115 1.081e-28 2.13e-24
XDH 4.686e-28 9.23e-24
SLC34A2 8.607e-28 1.7e-23
CAPN2 2.08e-27 4.1e-23
MICAL2 2.962e-27 5.83e-23
SEC14L2 3.828e-27 7.54e-23
C3ORF26__1 4.642e-27 9.14e-23
Clinical variable #9: 'RADIATIONS.RADIATION.REGIMENINDICATION'

9 genes related to 'RADIATIONS.RADIATION.REGIMENINDICATION'.

Table S17.  Basic characteristics of clinical feature: 'RADIATIONS.RADIATION.REGIMENINDICATION'

RADIATIONS.RADIATION.REGIMENINDICATION Labels N
  NO 14
  YES 483
     
  Significant markers N = 9
  Higher in YES 9
  Higher in NO 0
List of 9 genes differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

Table S18.  Get Full Table List of 9 genes differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

W(pos if higher in 'YES') wilcoxontestP Q AUC
KLHDC1 906 2.995e-06 0.059 0.866
C1ORF77 981 5.91e-06 0.116 0.8549
S100A13__1 981 5.91e-06 0.116 0.8549
ZNF782 988 6.291e-06 0.124 0.8539
PPOX 991 6.585e-06 0.13 0.8531
COPS8 1013 7.852e-06 0.155 0.8502
C13ORF23 1050 1.086e-05 0.214 0.8447
NHLRC3 1050 1.086e-05 0.214 0.8447
WBP4 1080 1.407e-05 0.277 0.8403
Clinical variable #10: 'RADIATIONEXPOSURE'

No gene related to 'RADIATIONEXPOSURE'.

Table S19.  Basic characteristics of clinical feature: 'RADIATIONEXPOSURE'

RADIATIONEXPOSURE Labels N
  NO 418
  YES 17
     
  Significant markers N = 0
Clinical variable #11: 'EXTRATHYROIDAL.EXTENSION'

389 genes related to 'EXTRATHYROIDAL.EXTENSION'.

Table S20.  Basic characteristics of clinical feature: 'EXTRATHYROIDAL.EXTENSION'

EXTRATHYROIDAL.EXTENSION Labels N
  MINIMAL (T3) 130
  MODERATE/ADVANCED (T4A) 16
  NONE 330
  VERY ADVANCED (T4B) 1
     
  Significant markers N = 389
List of top 10 genes differentially expressed by 'EXTRATHYROIDAL.EXTENSION'

Table S21.  Get Full Table List of top 10 genes differentially expressed by 'EXTRATHYROIDAL.EXTENSION'

ANOVA_P Q
NRCAM 1.164e-12 2.29e-08
TBKBP1 3.518e-12 6.93e-08
PDGFB 1.099e-11 2.17e-07
IFT140 2.863e-11 5.64e-07
TMEM204 2.863e-11 5.64e-07
SLC34A2 3.163e-11 6.23e-07
COL1A1 4.502e-11 8.87e-07
BBC3 4.71e-11 9.28e-07
GPR115 6.271e-11 1.24e-06
MICAL2 1.04e-10 2.05e-06
Clinical variable #12: 'COMPLETENESS.OF.RESECTION'

2 genes related to 'COMPLETENESS.OF.RESECTION'.

Table S22.  Basic characteristics of clinical feature: 'COMPLETENESS.OF.RESECTION'

COMPLETENESS.OF.RESECTION Labels N
  R0 380
  R1 51
  R2 4
  RX 30
     
  Significant markers N = 2
List of 2 genes differentially expressed by 'COMPLETENESS.OF.RESECTION'

Table S23.  Get Full Table List of 2 genes differentially expressed by 'COMPLETENESS.OF.RESECTION'

ANOVA_P Q
SLC25A37 3.952e-06 0.0779
NECAB2 1.209e-05 0.238
Clinical variable #13: 'NUMBER.OF.LYMPH.NODES'

1299 genes related to 'NUMBER.OF.LYMPH.NODES'.

Table S24.  Basic characteristics of clinical feature: 'NUMBER.OF.LYMPH.NODES'

NUMBER.OF.LYMPH.NODES Mean (SD) 3.56 (6.2)
  Significant markers N = 1299
  pos. correlated 39
  neg. correlated 1260
List of top 10 genes differentially expressed by 'NUMBER.OF.LYMPH.NODES'

Table S25.  Get Full Table List of top 10 genes significantly correlated to 'NUMBER.OF.LYMPH.NODES' by Spearman correlation test

SpearmanCorr corrP Q
FUT2 -0.4216 2.508e-18 4.94e-14
MET -0.4203 3.252e-18 6.41e-14
TMEM173 -0.4127 1.476e-17 2.91e-13
MACF1 -0.4048 6.87e-17 1.35e-12
TAGLN2 -0.4 1.721e-16 3.39e-12
CAPN2 -0.3988 2.126e-16 4.19e-12
CPNE1__1 -0.3976 2.684e-16 5.29e-12
RBM12__1 -0.3976 2.684e-16 5.29e-12
SMURF1 -0.3963 3.417e-16 6.73e-12
DUSP6 -0.3933 5.936e-16 1.17e-11
Clinical variable #14: 'MULTIFOCALITY'

No gene related to 'MULTIFOCALITY'.

Table S26.  Basic characteristics of clinical feature: 'MULTIFOCALITY'

MULTIFOCALITY Labels N
  MULTIFOCAL 225
  UNIFOCAL 262
     
  Significant markers N = 0
Clinical variable #15: 'TUMOR.SIZE'

One gene related to 'TUMOR.SIZE'.

Table S27.  Basic characteristics of clinical feature: 'TUMOR.SIZE'

TUMOR.SIZE Mean (SD) 2.97 (1.6)
  Significant markers N = 1
  pos. correlated 1
  neg. correlated 0
List of one gene differentially expressed by 'TUMOR.SIZE'

Table S28.  Get Full Table List of one gene significantly correlated to 'TUMOR.SIZE' by Spearman correlation test

SpearmanCorr corrP Q
TGFBR1 0.2298 3.628e-06 0.0715
Clinical variable #16: 'RACE'

49 genes related to 'RACE'.

Table S29.  Basic characteristics of clinical feature: 'RACE'

RACE Labels N
  AMERICAN INDIAN OR ALASKA NATIVE 1
  ASIAN 49
  BLACK OR AFRICAN AMERICAN 26
  WHITE 322
     
  Significant markers N = 49
List of top 10 genes differentially expressed by 'RACE'

Table S30.  Get Full Table List of top 10 genes differentially expressed by 'RACE'

ANOVA_P Q
SCAMP5 1.888e-15 3.72e-11
CDKN2AIP 5.284e-11 1.04e-06
CLTC 6.565e-09 0.000129
PTGES 9.557e-09 0.000188
RAD21L1 3.123e-08 0.000615
SLC25A1 3.284e-08 0.000647
RFWD3 5.38e-08 0.00106
LOC253039 7.272e-08 0.00143
PSMD5 7.272e-08 0.00143
NLRP1 7.36e-08 0.00145
Clinical variable #17: 'ETHNICITY'

No gene related to 'ETHNICITY'.

Table S31.  Basic characteristics of clinical feature: 'ETHNICITY'

ETHNICITY Labels N
  HISPANIC OR LATINO 38
  NOT HISPANIC OR LATINO 351
     
  Significant markers N = 0
Methods & Data
Input

  • Expresson data file = THCA-TP.meth.by_min_clin_corr.data.txt

  • Clinical data file = THCA-TP.merged_data.txt

  • Number of patients = 497

  • Number of genes = 19705

  • Number of clinical features = 17

Survival analysis

For survival clinical features, Wald's test in univariate Cox regression analysis with proportional hazards model (Andersen and Gill 1982) was used to estimate the P values using the 'coxph' function in R. Kaplan-Meier survival curves were plot using the four quartile subgroups of patients based on expression levels

Correlation analysis

For continuous numerical clinical features, Spearman's rank correlation coefficients (Spearman 1904) and two-tailed P values were estimated using 'cor.test' function in R

ANOVA analysis

For multi-class clinical features (ordinal or nominal), one-way analysis of variance (Howell 2002) was applied to compare the log2-expression levels between different clinical classes using 'anova' function in R

Student's t-test analysis

For two-class clinical features, two-tailed Student's t test with unequal variance (Lehmann and Romano 2005) was applied to compare the log2-expression levels between the two clinical classes using 't.test' function in R

Q value calculation

For multiple hypothesis correction, Q value is the False Discovery Rate (FDR) analogue of the P value (Benjamini and Hochberg 1995), defined as the minimum FDR at which the test may be called significant. We used the 'Benjamini and Hochberg' method of 'p.adjust' function in R to convert P values into Q values.

References
[1] Andersen and Gill, Cox's regression model for counting processes, a large sample study, Annals of Statistics 10(4):1100-1120 (1982)
[2] Spearman, C, The proof and measurement of association between two things, Amer. J. Psychol 15:72-101 (1904)
[3] Howell, D, Statistical Methods for Psychology. (5th ed.), Duxbury Press:324-5 (2002)
[4] Lehmann and Romano, Testing Statistical Hypotheses (3E ed.), New York: Springer. ISBN 0387988645 (2005)
[5] Benjamini and Hochberg, Controlling the false discovery rate: a practical and powerful approach to multiple testing, Journal of the Royal Statistical Society Series B 59:289-300 (1995)
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