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 19984 genes and 14 clinical features across 417 samples, statistically thresholded by P value < 0.05 and Q value < 0.3, 10 clinical features related to at least one genes.

  • 24 genes correlated to 'AGE'.

    • KIAA1143 ,  KIF15 ,  BMP8A ,  NHLRC1 ,  CLEC5A ,  ...

  • 2 genes correlated to 'NEOPLASM.DISEASESTAGE'.

    • NOLC1 ,  ENDOD1

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

    • ACBD5 ,  TACC2 ,  SLC41A3 ,  PRDM15 ,  GLT1D1 ,  ...

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

    • POLR2J3__1 ,  GPRIN2 ,  MRPS17 ,  LZIC ,  NMNAT1 ,  ...

  • 191 genes correlated to 'GENDER'.

    • KIF4B ,  EIF4A1 ,  SNORA48 ,  ATP5J ,  GABPA ,  ...

  • 23 genes correlated to 'HISTOLOGICAL.TYPE'.

    • LOC150622 ,  IQGAP1 ,  TRIM8 ,  RILP ,  ZMIZ1 ,  ...

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

    • DNAJC5 ,  MRPS23 ,  C18ORF54 ,  EIF5A ,  SCYL2__1

  • 4 genes correlated to 'NUMBERPACKYEARSSMOKED'.

    • FAM128A__1 ,  LOC150776__1 ,  BHMT2 ,  DMGDH

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

    • APBB3 ,  SLC35A4 ,  CDC23

  • 13 genes correlated to 'RACE'.

    • GPBAR1 ,  LOC100133161 ,  LOC253039 ,  PSMD5 ,  CS ,  ...

  • No genes correlated to 'Time to Death', 'PATHOLOGY.M.STAGE', 'KARNOFSKY.PERFORMANCE.SCORE', 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=0        
AGE Spearman correlation test N=24 older N=13 younger N=11
NEOPLASM DISEASESTAGE Kruskal-Wallis test N=2        
PATHOLOGY T STAGE Spearman correlation test N=52 higher stage N=19 lower stage N=33
PATHOLOGY N STAGE Spearman correlation test N=9 higher stage N=2 lower stage N=7
PATHOLOGY M STAGE Kruskal-Wallis test   N=0        
GENDER Wilcoxon test N=191 male N=191 female N=0
KARNOFSKY PERFORMANCE SCORE Spearman correlation test   N=0        
HISTOLOGICAL TYPE Kruskal-Wallis test N=23        
RADIATIONS RADIATION REGIMENINDICATION Wilcoxon test N=5 yes N=5 no N=0
NUMBERPACKYEARSSMOKED Spearman correlation test N=4 higher numberpackyearssmoked N=0 lower numberpackyearssmoked N=4
COMPLETENESS OF RESECTION Kruskal-Wallis test N=3        
RACE Kruskal-Wallis test N=13        
ETHNICITY Wilcoxon test   N=0        
Clinical variable #1: 'Time to Death'

No gene related to 'Time to Death'.

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

Time to Death Duration (Years) 1-2696 (median=426.5)
  censored N = 292
  death N = 72
     
  Significant markers N = 0
Clinical variable #2: 'AGE'

24 genes related to 'AGE'.

Table S2.  Basic characteristics of clinical feature: 'AGE'

AGE Mean (SD) 65.31 (9.9)
  Significant markers N = 24
  pos. correlated 13
  neg. correlated 11
List of top 10 genes differentially expressed by 'AGE'

Table S3.  Get Full Table List of top 10 genes significantly correlated to 'AGE' by Spearman correlation test

SpearmanCorr corrP Q
KIAA1143 0.2745 3.903e-08 0.00078
KIF15 0.2745 3.903e-08 0.00078
BMP8A 0.2683 8.023e-08 0.0016
NHLRC1 0.2514 5.236e-07 0.0105
CLEC5A 0.2505 5.77e-07 0.0115
B4GALNT4 0.2476 7.849e-07 0.0157
LOC148696 -0.2416 1.465e-06 0.0293
CYP3A4 0.2344 3.046e-06 0.0609
UBE2L6 -0.2342 3.125e-06 0.0624
ELOVL2 0.2337 3.268e-06 0.0653
Clinical variable #3: 'NEOPLASM.DISEASESTAGE'

2 genes related to 'NEOPLASM.DISEASESTAGE'.

Table S4.  Basic characteristics of clinical feature: 'NEOPLASM.DISEASESTAGE'

NEOPLASM.DISEASESTAGE Labels N
  STAGE I 4
  STAGE IA 101
  STAGE IB 118
  STAGE IIA 46
  STAGE IIB 59
  STAGE IIIA 60
  STAGE IIIB 9
  STAGE IV 19
     
  Significant markers N = 2
List of 2 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

Table S5.  Get Full Table List of 2 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

ANOVA_P Q
NOLC1 4.964e-06 0.0992
ENDOD1 1.438e-05 0.287
Clinical variable #4: 'PATHOLOGY.T.STAGE'

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

Table S6.  Basic characteristics of clinical feature: 'PATHOLOGY.T.STAGE'

PATHOLOGY.T.STAGE Mean (SD) 1.84 (0.72)
  N
  1 131
  2 232
  3 36
  4 15
     
  Significant markers N = 52
  pos. correlated 19
  neg. correlated 33
List of top 10 genes differentially expressed by 'PATHOLOGY.T.STAGE'

Table S7.  Get Full Table List of top 10 genes significantly correlated to 'PATHOLOGY.T.STAGE' by Spearman correlation test

SpearmanCorr corrP Q
ACBD5 -0.2783 8.412e-09 0.000168
TACC2 -0.2624 6.044e-08 0.00121
SLC41A3 -0.262 6.316e-08 0.00126
PRDM15 0.2536 1.686e-07 0.00337
GLT1D1 0.2535 1.707e-07 0.00341
DDX21 -0.2513 2.212e-07 0.00442
LSS -0.2464 3.833e-07 0.00766
MCM3APAS -0.2464 3.833e-07 0.00766
NME1-NME2 -0.244 5.03e-07 0.01
NME2 -0.244 5.03e-07 0.01
Clinical variable #5: 'PATHOLOGY.N.STAGE'

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

Table S8.  Basic characteristics of clinical feature: 'PATHOLOGY.N.STAGE'

PATHOLOGY.N.STAGE Mean (SD) 0.51 (0.76)
  N
  0 266
  1 78
  2 63
  3 1
     
  Significant markers N = 9
  pos. correlated 2
  neg. correlated 7
List of 9 genes differentially expressed by 'PATHOLOGY.N.STAGE'

Table S9.  Get Full Table List of 9 genes significantly correlated to 'PATHOLOGY.N.STAGE' by Spearman correlation test

SpearmanCorr corrP Q
POLR2J3__1 -0.2319 2.2e-06 0.044
GPRIN2 0.2311 2.38e-06 0.0476
MRPS17 -0.228 3.261e-06 0.0652
LZIC -0.221 6.605e-06 0.132
NMNAT1 -0.221 6.605e-06 0.132
VAMP1 0.2179 8.909e-06 0.178
POP7 -0.2149 1.194e-05 0.239
CHPF -0.2141 1.287e-05 0.257
TMEM198 -0.2141 1.287e-05 0.257
Clinical variable #6: 'PATHOLOGY.M.STAGE'

No gene related to 'PATHOLOGY.M.STAGE'.

Table S10.  Basic characteristics of clinical feature: 'PATHOLOGY.M.STAGE'

PATHOLOGY.M.STAGE Labels N
  M0 272
  M1 12
  M1A 1
  M1B 4
  MX 124
     
  Significant markers N = 0
Clinical variable #7: 'GENDER'

191 genes related to 'GENDER'.

Table S11.  Basic characteristics of clinical feature: 'GENDER'

GENDER Labels N
  FEMALE 223
  MALE 194
     
  Significant markers N = 191
  Higher in MALE 191
  Higher in FEMALE 0
List of top 10 genes differentially expressed by 'GENDER'

Table S12.  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
KIF4B 6656 3.171e-34 6.34e-30 0.8461
EIF4A1 9340 1.351e-23 2.7e-19 0.7841
SNORA48 9340 1.351e-23 2.7e-19 0.7841
ATP5J 31554 6.321e-16 1.26e-11 0.7294
GABPA 31554 6.321e-16 1.26e-11 0.7294
RWDD2B 11880 1.976e-15 3.95e-11 0.7254
RNASEH2C 31281 3.824e-15 7.64e-11 0.7231
COX7C 12006 4.498e-15 8.99e-11 0.7225
FUBP3 12170 1.292e-14 2.58e-10 0.7187
LOC100272217 12170 1.292e-14 2.58e-10 0.7187
Clinical variable #8: 'KARNOFSKY.PERFORMANCE.SCORE'

No gene related to 'KARNOFSKY.PERFORMANCE.SCORE'.

Table S13.  Basic characteristics of clinical feature: 'KARNOFSKY.PERFORMANCE.SCORE'

KARNOFSKY.PERFORMANCE.SCORE Mean (SD) 84.21 (22)
  Significant markers N = 0
Clinical variable #9: 'HISTOLOGICAL.TYPE'

23 genes related to 'HISTOLOGICAL.TYPE'.

Table S14.  Basic characteristics of clinical feature: 'HISTOLOGICAL.TYPE'

HISTOLOGICAL.TYPE Labels N
  LUNG ACINAR ADENOCARCINOMA 15
  LUNG ADENOCARCINOMA MIXED SUBTYPE 79
  LUNG ADENOCARCINOMA- NOT OTHERWISE SPECIFIED (NOS) 262
  LUNG BRONCHIOLOALVEOLAR CARCINOMA MUCINOUS 5
  LUNG BRONCHIOLOALVEOLAR CARCINOMA NONMUCINOUS 19
  LUNG CLEAR CELL ADENOCARCINOMA 1
  LUNG MICROPAPILLARY ADENOCARCINOMA 3
  LUNG MUCINOUS ADENOCARCINOMA 2
  LUNG PAPILLARY ADENOCARCINOMA 18
  LUNG SIGNET RING ADENOCARCINOMA 1
  LUNG SOLID PATTERN PREDOMINANT ADENOCARCINOMA 5
  MUCINOUS (COLLOID) CARCINOMA 7
     
  Significant markers N = 23
List of top 10 genes differentially expressed by 'HISTOLOGICAL.TYPE'

Table S15.  Get Full Table List of top 10 genes differentially expressed by 'HISTOLOGICAL.TYPE'

ANOVA_P Q
LOC150622 1.263e-08 0.000252
IQGAP1 1.073e-06 0.0214
TRIM8 2.025e-06 0.0405
RILP 2.379e-06 0.0475
ZMIZ1 3.243e-06 0.0648
FAM46B 4.792e-06 0.0957
SHISA4 4.803e-06 0.096
DGKI 5.144e-06 0.103
RICH2 6.027e-06 0.12
AVPI1 8.242e-06 0.165
Clinical variable #10: 'RADIATIONS.RADIATION.REGIMENINDICATION'

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

Table S16.  Basic characteristics of clinical feature: 'RADIATIONS.RADIATION.REGIMENINDICATION'

RADIATIONS.RADIATION.REGIMENINDICATION Labels N
  NO 18
  YES 399
     
  Significant markers N = 5
  Higher in YES 5
  Higher in NO 0
List of 5 genes differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

Table S17.  Get Full Table List of 5 genes differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

W(pos if higher in 'YES') wilcoxontestP Q AUC
DNAJC5 1328 6.084e-06 0.122 0.8151
MRPS23 5849 6.378e-06 0.127 0.8144
C18ORF54 5824 7.041e-06 0.141 0.813
EIF5A 5790 1.105e-05 0.221 0.8062
SCYL2__1 5742 1.504e-05 0.3 0.8015
Clinical variable #11: 'NUMBERPACKYEARSSMOKED'

4 genes related to 'NUMBERPACKYEARSSMOKED'.

Table S18.  Basic characteristics of clinical feature: 'NUMBERPACKYEARSSMOKED'

NUMBERPACKYEARSSMOKED Mean (SD) 41.14 (27)
  Significant markers N = 4
  pos. correlated 0
  neg. correlated 4
List of 4 genes differentially expressed by 'NUMBERPACKYEARSSMOKED'

Table S19.  Get Full Table List of 4 genes significantly correlated to 'NUMBERPACKYEARSSMOKED' by Spearman correlation test

SpearmanCorr corrP Q
FAM128A__1 -0.2923 5.109e-07 0.0102
LOC150776__1 -0.2923 5.109e-07 0.0102
BHMT2 -0.2627 6.98e-06 0.139
DMGDH -0.2627 6.98e-06 0.139
Clinical variable #12: 'COMPLETENESS.OF.RESECTION'

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

Table S20.  Basic characteristics of clinical feature: 'COMPLETENESS.OF.RESECTION'

COMPLETENESS.OF.RESECTION Labels N
  R0 276
  R1 10
  R2 1
  RX 18
     
  Significant markers N = 3
List of 3 genes differentially expressed by 'COMPLETENESS.OF.RESECTION'

Table S21.  Get Full Table List of 3 genes differentially expressed by 'COMPLETENESS.OF.RESECTION'

ANOVA_P Q
APBB3 8.534e-07 0.0171
SLC35A4 8.534e-07 0.0171
CDC23 7.493e-06 0.15
Clinical variable #13: 'RACE'

13 genes related to 'RACE'.

Table S22.  Basic characteristics of clinical feature: 'RACE'

RACE Labels N
  ASIAN 5
  BLACK OR AFRICAN AMERICAN 31
  WHITE 331
     
  Significant markers N = 13
List of top 10 genes differentially expressed by 'RACE'

Table S23.  Get Full Table List of top 10 genes differentially expressed by 'RACE'

ANOVA_P Q
GPBAR1 7.839e-08 0.00157
LOC100133161 1.858e-07 0.00371
LOC253039 1.865e-07 0.00373
PSMD5 1.865e-07 0.00373
CS 3.296e-07 0.00659
WBSCR27 5.301e-07 0.0106
RGPD5 1.181e-06 0.0236
RGPD8 1.181e-06 0.0236
C14ORF167__1 1.377e-06 0.0275
DHRS4__1 1.377e-06 0.0275
Clinical variable #14: 'ETHNICITY'

No gene related to 'ETHNICITY'.

Table S24.  Basic characteristics of clinical feature: 'ETHNICITY'

ETHNICITY Labels N
  HISPANIC OR LATINO 7
  NOT HISPANIC OR LATINO 303
     
  Significant markers N = 0
Methods & Data
Input

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

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

  • Number of patients = 417

  • Number of genes = 19984

  • Number of clinical features = 14

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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