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 19914 genes and 13 clinical features across 412 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'.

    • KLHL31 ,  PLVAP ,  TNS3 ,  RPS6KA1 ,  PRR7 ,  ...

  • 30 genes correlated to 'PATHOLOGIC_STAGE'.

    • GATA6 ,  ZBP1 ,  NAT1 ,  ABCG2 ,  ARAP2 ,  ...

  • 30 genes correlated to 'PATHOLOGY_T_STAGE'.

    • NR1D1 ,  THRA ,  GATA6 ,  CXCR2 ,  HSH2D ,  ...

  • 30 genes correlated to 'PATHOLOGY_N_STAGE'.

    • LDHB ,  MRPS28 ,  NCRNA00188 ,  SNORD49A ,  SNORD65 ,  ...

  • 30 genes correlated to 'GENDER'.

    • ALG11__1 ,  UTP14C ,  C2ORF7 ,  CCT7 ,  KIF4B ,  ...

  • 30 genes correlated to 'RADIATION_THERAPY'.

    • PIK3C2B ,  ZNF132 ,  FBXL14 ,  C8ORF44 ,  KIAA1671 ,  ...

  • 30 genes correlated to 'KARNOFSKY_PERFORMANCE_SCORE'.

    • TULP4 ,  KCNH4 ,  ENTPD3 ,  BET3L ,  FAM26D ,  ...

  • 2 genes correlated to 'NUMBER_PACK_YEARS_SMOKED'.

    • HMMR ,  NUDCD2

  • 30 genes correlated to 'NUMBER_OF_LYMPH_NODES'.

    • MIR17 ,  MIR17HG ,  MIR18A ,  MIR19A ,  MIR19B1 ,  ...

  • 30 genes correlated to 'RACE'.

    • SCAMP5 ,  LOC100133161 ,  BTF3 ,  SPRED2 ,  SH3YL1 ,  ...

  • No genes correlated to 'DAYS_TO_DEATH_OR_LAST_FUP', 'PATHOLOGY_M_STAGE', 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=20 younger N=10
PATHOLOGIC_STAGE Kruskal-Wallis test N=30        
PATHOLOGY_T_STAGE Spearman correlation test N=30 higher stage N=20 lower stage N=10
PATHOLOGY_N_STAGE Spearman correlation test N=30 higher stage N=26 lower stage N=4
PATHOLOGY_M_STAGE Wilcoxon test   N=0        
GENDER Wilcoxon test N=30 male N=30 female N=0
RADIATION_THERAPY Wilcoxon test N=30 yes N=30 no N=0
KARNOFSKY_PERFORMANCE_SCORE Spearman correlation test N=30 higher score N=28 lower score N=2
NUMBER_PACK_YEARS_SMOKED Spearman correlation test N=2 higher number_pack_years_smoked N=0 lower number_pack_years_smoked N=2
NUMBER_OF_LYMPH_NODES Spearman correlation test N=30 higher number_of_lymph_nodes N=21 lower number_of_lymph_nodes N=9
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) 0.1-166 (median=17.2)
  censored N = 232
  death N = 179
     
  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) 68.08 (11)
  Significant markers N = 30
  pos. correlated 20
  neg. correlated 10
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
KLHL31 0.2544 1.708e-07 0.00172
PLVAP -0.2521 2.23e-07 0.00172
TNS3 -0.246 4.437e-07 0.00172
RPS6KA1 0.2451 4.905e-07 0.00172
PRR7 0.2431 6.114e-07 0.00172
KIAA1143 0.2422 6.745e-07 0.00172
KIF15 0.2422 6.745e-07 0.00172
CCDC21 0.2419 6.922e-07 0.00172
KLHL8 0.2402 8.354e-07 0.00185
F2RL1 0.2383 1.019e-06 0.00203
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 2
  STAGE II 131
  STAGE III 141
  STAGE IV 136
     
  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
GATA6 4.966e-12 9.89e-08
ZBP1 1.078e-11 1.07e-07
NAT1 2.56e-11 1.7e-07
ABCG2 4.289e-11 1.76e-07
ARAP2 4.427e-11 1.76e-07
NR1D1 7.27e-11 1.85e-07
THRA 7.27e-11 1.85e-07
FGF1 7.443e-11 1.85e-07
ENPP3__2 3.467e-10 7.67e-07
NTN3 5.639e-10 9.95e-07
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) 2.82 (0.7)
  N
  T0 1
  T1 3
  T2 120
  T3 196
  T4 59
     
  Significant markers N = 30
  pos. correlated 20
  neg. correlated 10
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
NR1D1 -0.3275 6.32e-11 6.29e-07
THRA -0.3275 6.32e-11 6.29e-07
GATA6 -0.319 2.059e-10 1.37e-06
CXCR2 0.3155 3.306e-10 1.65e-06
HSH2D 0.3079 9.145e-10 3.64e-06
PRPF6 0.3036 1.605e-09 4.57e-06
SAMD10 0.3036 1.605e-09 4.57e-06
C21ORF49 0.2998 2.606e-09 5.11e-06
C21ORF66 0.2998 2.606e-09 5.11e-06
TBX4 -0.2992 2.828e-09 5.11e-06
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) 0.61 (0.89)
  N
  N0 238
  N1 47
  N2 76
  N3 9
     
  Significant markers N = 30
  pos. correlated 26
  neg. correlated 4
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
LDHB 0.2629 2.891e-07 0.0039
MRPS28 0.2532 8.022e-07 0.0039
NCRNA00188 0.2513 9.798e-07 0.0039
SNORD49A 0.2513 9.798e-07 0.0039
SNORD65 0.2513 9.798e-07 0.0039
MST1R 0.2446 1.93e-06 0.00459
CIITA 0.2385 3.495e-06 0.00459
TADA2B__1 0.2384 3.523e-06 0.00459
TULP4 0.2383 3.582e-06 0.00459
MIR17 0.2379 3.69e-06 0.00459
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
  class0 196
  class1 11
     
  Significant markers N = 0
Clinical variable #7: 'GENDER'

30 genes related to 'GENDER'.

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

GENDER Labels N
  FEMALE 108
  MALE 304
     
  Significant markers N = 30
  Higher in MALE 30
  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
ALG11__1 31600 2.768e-46 2.76e-42 0.9625
UTP14C 31600 2.768e-46 2.76e-42 0.9625
C2ORF7 26882 7.186e-23 3.58e-19 0.8188
CCT7 26882 7.186e-23 3.58e-19 0.8188
KIF4B 6912 3.882e-19 1.55e-15 0.7895
C6ORF174 10535 3.166e-08 0.000105 0.6791
FASTKD2__1 11056 4.61e-07 0.00115 0.6633
MDH1B__1 11056 4.61e-07 0.00115 0.6633
APOC1 11201 9.321e-07 0.00206 0.6588
C10ORF57 11310 1.564e-06 0.00283 0.6555
Clinical variable #8: 'RADIATION_THERAPY'

30 genes related to 'RADIATION_THERAPY'.

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

RADIATION_THERAPY Labels N
  NO 366
  YES 19
     
  Significant markers N = 30
  Higher in YES 30
  Higher in NO 0
List of top 10 genes differentially expressed by 'RADIATION_THERAPY'

Table S14.  Get Full Table List of top 10 genes differentially expressed by 'RADIATION_THERAPY'

W(pos if higher in 'YES') wilcoxontestP Q AUC
PIK3C2B 1441 1.679e-05 0.0927 0.7928
ZNF132 1447 1.778e-05 0.0927 0.7919
FBXL14 1454 1.9e-05 0.0927 0.7909
C8ORF44 1491 2.692e-05 0.0927 0.7856
KIAA1671 1499 2.9e-05 0.0927 0.7844
PLA2G2A 1537 4.117e-05 0.0927 0.779
RHOT2 1538 4.155e-05 0.0927 0.7788
WDR90__1 1538 4.155e-05 0.0927 0.7788
RND1 1556 4.894e-05 0.0927 0.7762
CCDC81 5394 5.074e-05 0.0927 0.7757
Clinical variable #9: 'KARNOFSKY_PERFORMANCE_SCORE'

30 genes related to 'KARNOFSKY_PERFORMANCE_SCORE'.

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

KARNOFSKY_PERFORMANCE_SCORE Mean (SD) 83.09 (14)
  Significant markers N = 30
  pos. correlated 28
  neg. correlated 2
List of top 10 genes differentially expressed by 'KARNOFSKY_PERFORMANCE_SCORE'

Table S16.  Get Full Table List of top 10 genes significantly correlated to 'KARNOFSKY_PERFORMANCE_SCORE' by Spearman correlation test

SpearmanCorr corrP Q
TULP4 0.4665 1.037e-08 0.000104
KCNH4 0.4665 1.043e-08 0.000104
ENTPD3 0.4472 4.801e-08 0.000224
BET3L 0.4443 6.015e-08 0.000224
FAM26D 0.4443 6.015e-08 0.000224
MFSD2A 0.4428 6.735e-08 0.000224
STXBP3 0.4356 1.158e-07 0.000329
S1PR5 0.4317 1.543e-07 0.000342
ACBD4 0.4302 1.719e-07 0.000342
PLCD3 0.4302 1.719e-07 0.000342
Clinical variable #10: 'NUMBER_PACK_YEARS_SMOKED'

2 genes related to 'NUMBER_PACK_YEARS_SMOKED'.

Table S17.  Basic characteristics of clinical feature: 'NUMBER_PACK_YEARS_SMOKED'

NUMBER_PACK_YEARS_SMOKED Mean (SD) 39.04 (53)
  Significant markers N = 2
  pos. correlated 0
  neg. correlated 2
List of 2 genes differentially expressed by 'NUMBER_PACK_YEARS_SMOKED'

Table S18.  Get Full Table List of 2 genes significantly correlated to 'NUMBER_PACK_YEARS_SMOKED' by Spearman correlation test

SpearmanCorr corrP Q
HMMR -0.3339 3.092e-07 0.00308
NUDCD2 -0.3339 3.092e-07 0.00308
Clinical variable #11: 'NUMBER_OF_LYMPH_NODES'

30 genes related to 'NUMBER_OF_LYMPH_NODES'.

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

NUMBER_OF_LYMPH_NODES Mean (SD) 2.09 (7)
  Significant markers N = 30
  pos. correlated 21
  neg. correlated 9
List of top 10 genes differentially expressed by 'NUMBER_OF_LYMPH_NODES'

Table S20.  Get Full Table List of top 10 genes significantly correlated to 'NUMBER_OF_LYMPH_NODES' by Spearman correlation test

SpearmanCorr corrP Q
MIR17 0.2597 5.781e-06 0.0164
MIR17HG 0.2597 5.781e-06 0.0164
MIR18A 0.2597 5.781e-06 0.0164
MIR19A 0.2597 5.781e-06 0.0164
MIR19B1 0.2597 5.781e-06 0.0164
MIR20A 0.2597 5.781e-06 0.0164
MIR92A1 0.2597 5.781e-06 0.0164
IGF2R 0.2532 1.003e-05 0.025
MRPS28 0.2459 1.821e-05 0.0403
KCNG1 -0.2425 2.388e-05 0.0475
Clinical variable #12: 'RACE'

30 genes related to 'RACE'.

Table S21.  Basic characteristics of clinical feature: 'RACE'

RACE Labels N
  ASIAN 44
  BLACK OR AFRICAN AMERICAN 23
  WHITE 327
     
  Significant markers N = 30
List of top 10 genes differentially expressed by 'RACE'

Table S22.  Get Full Table List of top 10 genes differentially expressed by 'RACE'

kruskal_wallis_P Q
SCAMP5 7.396e-15 1.47e-10
LOC100133161 6.77e-12 4.56e-08
BTF3 6.871e-12 4.56e-08
SPRED2 1.031e-11 5.13e-08
SH3YL1 1.514e-11 5.24e-08
SETMAR 1.661e-11 5.24e-08
C10ORF58 1.844e-11 5.24e-08
AGPHD1 2.405e-11 5.99e-08
SLC44A3 4.697e-11 8.35e-08
BIN3 4.842e-11 8.35e-08
Clinical variable #13: 'ETHNICITY'

No gene related to 'ETHNICITY'.

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

ETHNICITY Labels N
  HISPANIC OR LATINO 9
  NOT HISPANIC OR LATINO 371
     
  Significant markers N = 0
Methods & Data
Input
  • Expresson data file = BLCA-TP.meth.by_min_clin_corr.data.txt

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

  • Number of patients = 412

  • Number of genes = 19914

  • Number of clinical features = 13

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)