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 19924 genes and 15 clinical features across 457 samples, statistically thresholded by P value < 0.05 and Q value < 0.3, 10 clinical features related to at least one genes.

  • 30 genes correlated to 'YEARS_TO_BIRTH'.

    • MRPS33 ,  C14ORF169 ,  HEATR4__2 ,  RPS2__1 ,  SNHG9__1 ,  ...

  • 30 genes correlated to 'PATHOLOGIC_STAGE'.

    • ENDOD1 ,  SEC11C ,  NOLC1 ,  PLCB3 ,  ZFP112 ,  ...

  • 30 genes correlated to 'PATHOLOGY_T_STAGE'.

    • ACBD5 ,  IRF4 ,  SLC41A3 ,  NHEJ1 ,  DDX21 ,  ...

  • 30 genes correlated to 'PATHOLOGY_N_STAGE'.

    • GPRIN2 ,  C1ORF159 ,  DNAJC5 ,  PLAGL2 ,  EGLN2 ,  ...

  • 30 genes correlated to 'PATHOLOGY_M_STAGE'.

    • C6ORF136 ,  SLC38A3 ,  APLP1 ,  FAM64A ,  NOVA1 ,  ...

  • 30 genes correlated to 'GENDER'.

    • KIF4B ,  EIF4A1 ,  SNORA48 ,  YARS2 ,  FRG1B ,  ...

  • 30 genes correlated to 'HISTOLOGICAL_TYPE'.

    • LOC150622 ,  CANT1 ,  IQGAP1 ,  TRIM8 ,  ZMIZ1 ,  ...

  • 30 genes correlated to 'NUMBER_PACK_YEARS_SMOKED'.

    • FAM128A__1 ,  LOC150776__1 ,  BHMT2 ,  DMGDH ,  PET112L ,  ...

  • 30 genes correlated to 'YEAR_OF_TOBACCO_SMOKING_ONSET'.

    • MOGAT2 ,  C14ORF169 ,  HEATR4__2 ,  S100A16 ,  MRPS33 ,  ...

  • 30 genes correlated to 'RACE'.

    • ZMYM1 ,  C14ORF167 ,  DHRS4 ,  LOC100133161 ,  ATP6V1F ,  ...

  • No genes correlated to 'DAYS_TO_DEATH_OR_LAST_FUP', 'RADIATION_THERAPY', 'KARNOFSKY_PERFORMANCE_SCORE', 'RESIDUAL_TUMOR', 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
DAYS_TO_DEATH_OR_LAST_FUP Cox regression test   N=0        
YEARS_TO_BIRTH Spearman correlation test N=30 older N=27 younger N=3
PATHOLOGIC_STAGE Kruskal-Wallis test N=30        
PATHOLOGY_T_STAGE Spearman correlation test N=30 higher stage N=6 lower stage N=24
PATHOLOGY_N_STAGE Spearman correlation test N=30 higher stage N=20 lower stage N=10
PATHOLOGY_M_STAGE Wilcoxon test N=30 class1 N=30 class0 N=0
GENDER Wilcoxon test N=30 male N=30 female N=0
RADIATION_THERAPY Wilcoxon test   N=0        
KARNOFSKY_PERFORMANCE_SCORE Spearman correlation test   N=0        
HISTOLOGICAL_TYPE Kruskal-Wallis test N=30        
NUMBER_PACK_YEARS_SMOKED Spearman correlation test N=30 higher number_pack_years_smoked N=2 lower number_pack_years_smoked N=28
YEAR_OF_TOBACCO_SMOKING_ONSET Spearman correlation test N=30 higher year_of_tobacco_smoking_onset N=27 lower year_of_tobacco_smoking_onset N=3
RESIDUAL_TUMOR Kruskal-Wallis test   N=0        
RACE Kruskal-Wallis test N=30        
ETHNICITY Wilcoxon test   N=0        
Clinical variable #1: 'DAYS_TO_DEATH_OR_LAST_FUP'

No gene related to 'DAYS_TO_DEATH_OR_LAST_FUP'.

Table S1.  Basic characteristics of clinical feature: 'DAYS_TO_DEATH_OR_LAST_FUP'

DAYS_TO_DEATH_OR_LAST_FUP Duration (Months) 0.1-238.3 (median=19.5)
  censored N = 307
  death N = 149
     
  Significant markers N = 0
Clinical variable #2: 'YEARS_TO_BIRTH'

30 genes related to 'YEARS_TO_BIRTH'.

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

YEARS_TO_BIRTH Mean (SD) 65.03 (10)
  Significant markers N = 30
  pos. correlated 27
  neg. correlated 3
List of top 10 genes differentially expressed by 'YEARS_TO_BIRTH'

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

SpearmanCorr corrP Q
MRPS33 0.438 1.732e-21 3.45e-17
C14ORF169 0.3713 1.934e-15 1.28e-11
HEATR4__2 0.3713 1.934e-15 1.28e-11
RPS2__1 0.3353 1.051e-12 3.49e-09
SNHG9__1 0.3353 1.051e-12 3.49e-09
SNORA78__1 0.3353 1.051e-12 3.49e-09
KIAA1143 0.3231 7.47e-12 1.86e-08
KIF15 0.3231 7.47e-12 1.86e-08
FOXD2 0.3002 2.294e-10 5.08e-07
ACN9 0.2926 7.078e-10 1.41e-06
Clinical variable #3: 'PATHOLOGIC_STAGE'

30 genes related to 'PATHOLOGIC_STAGE'.

Table S4.  Basic characteristics of clinical feature: 'PATHOLOGIC_STAGE'

PATHOLOGIC_STAGE Labels N
  STAGE I 5
  STAGE IA 120
  STAGE IB 124
  STAGE II 1
  STAGE IIA 49
  STAGE IIB 62
  STAGE IIIA 64
  STAGE IIIB 9
  STAGE IV 21
     
  Significant markers N = 30
List of top 10 genes differentially expressed by 'PATHOLOGIC_STAGE'

Table S5.  Get Full Table List of top 10 genes differentially expressed by 'PATHOLOGIC_STAGE'

kruskal_wallis_P Q
ENDOD1 1.668e-06 0.0332
SEC11C 3.618e-06 0.036
NOLC1 5.709e-06 0.0379
PLCB3 1.308e-05 0.0421
ZFP112 1.352e-05 0.0421
ST14 1.664e-05 0.0421
PADI1 1.68e-05 0.0421
C11ORF41 1.689e-05 0.0421
MOCOS 2.471e-05 0.0547
EXT1 3.838e-05 0.0695
Clinical variable #4: 'PATHOLOGY_T_STAGE'

30 genes related to 'PATHOLOGY_T_STAGE'.

Table S6.  Basic characteristics of clinical feature: 'PATHOLOGY_T_STAGE'

PATHOLOGY_T_STAGE Mean (SD) 1.82 (0.73)
  N
  T1 154
  T2 243
  T3 41
  T4 16
     
  Significant markers N = 30
  pos. correlated 6
  neg. correlated 24
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 -0.2899 3.06e-10 6.1e-06
IRF4 0.27 5.219e-09 4.25e-05
SLC41A3 -0.2682 6.397e-09 4.25e-05
NHEJ1 -0.262 1.513e-08 7.54e-05
DDX21 -0.2546 3.765e-08 0.00015
NME1-NME2 -0.2519 5.281e-08 0.00015
NME2 -0.2519 5.281e-08 0.00015
NR1D2 -0.2479 8.686e-08 0.000204
GLT1D1 0.2466 1.021e-07 0.000204
PDE1C 0.2453 1.201e-07 0.000204
Clinical variable #5: 'PATHOLOGY_N_STAGE'

30 genes related to 'PATHOLOGY_N_STAGE'.

Table S8.  Basic characteristics of clinical feature: 'PATHOLOGY_N_STAGE'

PATHOLOGY_N_STAGE Mean (SD) 0.48 (0.75)
  N
  N0 298
  N1 83
  N2 65
  N3 1
     
  Significant markers N = 30
  pos. correlated 20
  neg. correlated 10
List of top 10 genes differentially expressed by 'PATHOLOGY_N_STAGE'

Table S9.  Get Full Table List of top 10 genes significantly correlated to 'PATHOLOGY_N_STAGE' by Spearman correlation test

SpearmanCorr corrP Q
GPRIN2 0.2254 1.475e-06 0.0294
C1ORF159 0.2103 7.312e-06 0.065
DNAJC5 0.2074 9.786e-06 0.065
PLAGL2 0.1978 2.536e-05 0.0874
EGLN2 0.1938 3.698e-05 0.0874
SEC11C 0.1912 4.717e-05 0.0874
MYADM -0.1903 5.151e-05 0.0874
NPEPPS -0.1897 5.415e-05 0.0874
VAMP1 0.189 5.812e-05 0.0874
JOSD1 -0.1883 6.167e-05 0.0874
Clinical variable #6: 'PATHOLOGY_M_STAGE'

30 genes related to 'PATHOLOGY_M_STAGE'.

Table S10.  Basic characteristics of clinical feature: 'PATHOLOGY_M_STAGE'

PATHOLOGY_M_STAGE Labels N
  class0 295
  class1 19
     
  Significant markers N = 30
  Higher in class1 30
  Higher in class0 0
List of top 10 genes differentially expressed by 'PATHOLOGY_M_STAGE'

Table S11.  Get Full Table List of top 10 genes differentially expressed by 'PATHOLOGY_M_STAGE'

W(pos if higher in 'class1') wilcoxontestP Q AUC
C6ORF136 1099 9.005e-06 0.179 0.8039
SLC38A3 1236 4.453e-05 0.208 0.7795
APLP1 1248 5.093e-05 0.208 0.7773
FAM64A 1254 5.444e-05 0.208 0.7763
NOVA1 1287 7.826e-05 0.208 0.7704
RALBP1 1306 9.614e-05 0.208 0.767
SEC14L5 1316 0.000107 0.208 0.7652
MRPL11 1325 0.0001178 0.208 0.7636
C15ORF24 1335 0.000131 0.208 0.7618
PGBD4 1335 0.000131 0.208 0.7618
Clinical variable #7: 'GENDER'

30 genes related to 'GENDER'.

Table S12.  Basic characteristics of clinical feature: 'GENDER'

GENDER Labels N
  FEMALE 243
  MALE 214
     
  Significant markers N = 30
  Higher in MALE 30
  Higher in FEMALE 0
List of top 10 genes differentially expressed by 'GENDER'

Table S13.  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 7852 5.673e-38 1.13e-33 0.849
EIF4A1 11475 6.318e-25 4.2e-21 0.7793
SNORA48 11475 6.318e-25 4.2e-21 0.7793
YARS2 11691 3.079e-24 1.53e-20 0.7752
FRG1B 12042.5 3.856e-23 1.54e-19 0.7684
COX7C 13614 1.467e-18 4.87e-15 0.7382
RWDD2B 14794 1.797e-15 5.12e-12 0.7155
RNASEH2C 37153 2.462e-15 6.13e-12 0.7145
TBCA 15522 1.023e-13 2.27e-10 0.7015
FUBP3 15667 2.219e-13 4.02e-10 0.6987
Clinical variable #8: 'RADIATION_THERAPY'

No gene related to 'RADIATION_THERAPY'.

Table S14.  Basic characteristics of clinical feature: 'RADIATION_THERAPY'

RADIATION_THERAPY Labels N
  NO 328
  YES 49
     
  Significant markers N = 0
Clinical variable #9: 'KARNOFSKY_PERFORMANCE_SCORE'

No gene related to 'KARNOFSKY_PERFORMANCE_SCORE'.

Table S15.  Basic characteristics of clinical feature: 'KARNOFSKY_PERFORMANCE_SCORE'

KARNOFSKY_PERFORMANCE_SCORE Mean (SD) 79.83 (28)
  Significant markers N = 0
Clinical variable #10: 'HISTOLOGICAL_TYPE'

30 genes related to 'HISTOLOGICAL_TYPE'.

Table S16.  Basic characteristics of clinical feature: 'HISTOLOGICAL_TYPE'

HISTOLOGICAL_TYPE Labels N
  LUNG ACINAR ADENOCARCINOMA 19
  LUNG ADENOCARCINOMA MIXED SUBTYPE 91
  LUNG ADENOCARCINOMA- NOT OTHERWISE SPECIFIED (NOS) 280
  LUNG BRONCHIOLOALVEOLAR CARCINOMA MUCINOUS 5
  LUNG BRONCHIOLOALVEOLAR CARCINOMA NONMUCINOUS 19
  LUNG CLEAR CELL ADENOCARCINOMA 1
  LUNG MICROPAPILLARY ADENOCARCINOMA 2
  LUNG MUCINOUS ADENOCARCINOMA 2
  LUNG PAPILLARY ADENOCARCINOMA 22
  LUNG SIGNET RING ADENOCARCINOMA 1
  LUNG SOLID PATTERN PREDOMINANT ADENOCARCINOMA 5
  MUCINOUS (COLLOID) CARCINOMA 10
     
  Significant markers N = 30
List of top 10 genes differentially expressed by 'HISTOLOGICAL_TYPE'

Table S17.  Get Full Table List of top 10 genes differentially expressed by 'HISTOLOGICAL_TYPE'

kruskal_wallis_P Q
LOC150622 1.54e-10 3.07e-06
CANT1 3.487e-07 0.00347
IQGAP1 8.641e-07 0.00476
TRIM8 9.55e-07 0.00476
ZMIZ1 1.986e-06 0.00792
SEPW1 3.075e-06 0.00888
LIMD1 3.529e-06 0.00888
RILP 4.738e-06 0.00888
SCMH1 5.106e-06 0.00888
KRT13 5.179e-06 0.00888
Clinical variable #11: 'NUMBER_PACK_YEARS_SMOKED'

30 genes related to 'NUMBER_PACK_YEARS_SMOKED'.

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

NUMBER_PACK_YEARS_SMOKED Mean (SD) 40.68 (27)
  Significant markers N = 30
  pos. correlated 2
  neg. correlated 28
List of top 10 genes differentially expressed by 'NUMBER_PACK_YEARS_SMOKED'

Table S19.  Get Full Table List of top 10 genes significantly correlated to 'NUMBER_PACK_YEARS_SMOKED' by Spearman correlation test

SpearmanCorr corrP Q
FAM128A__1 -0.2957 9.812e-08 0.000977
LOC150776__1 -0.2957 9.812e-08 0.000977
BHMT2 -0.2482 8.843e-06 0.0388
DMGDH -0.2482 8.843e-06 0.0388
PET112L -0.2471 9.726e-06 0.0388
NLE1 -0.2385 2.007e-05 0.0666
LOC284798 -0.2326 3.253e-05 0.0926
GSTK1 -0.23 3.995e-05 0.0995
LILRB1 -0.2282 4.597e-05 0.102
EME1__1 -0.2215 7.763e-05 0.122
Clinical variable #12: 'YEAR_OF_TOBACCO_SMOKING_ONSET'

30 genes related to 'YEAR_OF_TOBACCO_SMOKING_ONSET'.

Table S20.  Basic characteristics of clinical feature: 'YEAR_OF_TOBACCO_SMOKING_ONSET'

YEAR_OF_TOBACCO_SMOKING_ONSET Mean (SD) 1965.27 (12)
  Significant markers N = 30
  pos. correlated 27
  neg. correlated 3
List of top 10 genes differentially expressed by 'YEAR_OF_TOBACCO_SMOKING_ONSET'

Table S21.  Get Full Table List of top 10 genes significantly correlated to 'YEAR_OF_TOBACCO_SMOKING_ONSET' by Spearman correlation test

SpearmanCorr corrP Q
MOGAT2 0.314 3.785e-07 0.00592
C14ORF169 -0.3043 8.911e-07 0.00592
HEATR4__2 -0.3043 8.911e-07 0.00592
S100A16 0.2854 4.345e-06 0.0216
MRPS33 -0.2811 6.091e-06 0.0243
CALML4 0.2779 7.877e-06 0.0262
BTBD17 0.2732 1.129e-05 0.0321
ZNF362 0.268 1.677e-05 0.0378
AATK 0.2678 1.706e-05 0.0378
RNF39 0.2641 2.244e-05 0.0447
Clinical variable #13: 'RESIDUAL_TUMOR'

No gene related to 'RESIDUAL_TUMOR'.

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

RESIDUAL_TUMOR Labels N
  R0 293
  R1 10
  R2 1
  RX 23
     
  Significant markers N = 0
Clinical variable #14: 'RACE'

30 genes related to 'RACE'.

Table S23.  Basic characteristics of clinical feature: 'RACE'

RACE Labels N
  ASIAN 6
  BLACK OR AFRICAN AMERICAN 49
  WHITE 352
     
  Significant markers N = 30
List of top 10 genes differentially expressed by 'RACE'

Table S24.  Get Full Table List of top 10 genes differentially expressed by 'RACE'

kruskal_wallis_P Q
ZMYM1 2.804e-12 5.59e-08
C14ORF167 7.719e-11 5.13e-07
DHRS4 7.719e-11 5.13e-07
LOC100133161 1.246e-10 6.21e-07
ATP6V1F 2.109e-10 8.27e-07
LOC253039 2.904e-10 8.27e-07
PSMD5 2.904e-10 8.27e-07
GPBAR1 8.831e-10 2.2e-06
LOC147804 1.703e-09 3.39e-06
ZNF761 1.703e-09 3.39e-06
Clinical variable #15: 'ETHNICITY'

No gene related to 'ETHNICITY'.

Table S25.  Basic characteristics of clinical feature: 'ETHNICITY'

ETHNICITY Labels N
  HISPANIC OR LATINO 7
  NOT HISPANIC OR LATINO 341
     
  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 = 457

  • Number of genes = 19924

  • Number of clinical features = 15

Selected clinical features

  • Further details on clinical features selected for this analysis, please find a documentation on selected CDEs (Clinical Data Elements). The first column of the file is a formula to convert values and the second column is a clinical parameter name.

  • Survival time data

    • Survival time data is a combined value of days_to_death and days_to_last_followup. For each patient, it creates a combined value 'days_to_death_or_last_fup' using conversion process below.

      • if 'vital_status'==1(dead), 'days_to_last_followup' is always NA. Thus, uses 'days_to_death' value for 'days_to_death_or_fup'

      • if 'vital_status'==0(alive),

        • if 'days_to_death'==NA & 'days_to_last_followup'!=NA, uses 'days_to_last_followup' value for 'days_to_death_or_fup'

        • if 'days_to_death'!=NA, excludes this case in survival analysis and report the case.

      • if 'vital_status'==NA,excludes this case in survival analysis and report the case.

    • cf. In certain diesase types such as SKCM, days_to_death parameter is replaced with time_from_specimen_dx or time_from_specimen_procurement_to_death .

  • This analysis excluded clinical variables that has only NA values.

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

Wilcoxon rank sum test (Mann-Whitney U test)

For two groups (mutant or wild-type) of continuous type of clinical data, wilcoxon rank sum test (Mann and Whitney, 1947) was applied to compare their mean difference using 'wilcox.test(continuous.clinical ~ as.factor(group), exact=FALSE)' function in R. This test is equivalent to the Mann-Whitney test.

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] Mann and Whitney, On a Test of Whether one of Two Random Variables is Stochastically Larger than the Other, Annals of Mathematical Statistics 18 (1), 50-60 (1947)
[4] 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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