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 20112 genes and 7 clinical features across 39 samples, statistically thresholded by P value < 0.05 and Q value < 0.3, 1 clinical feature related to at least one genes.

  • 6 genes correlated to 'Time to Death'.

    • C16ORF45 ,  KRT14 ,  CMAH ,  TADA2A ,  SEMA7A ,  ...

  • No genes correlated to 'AGE', 'NEOPLASM.DISEASESTAGE', 'PATHOLOGY.T.STAGE', 'PATHOLOGY.N.STAGE', 'PATHOLOGY.M.STAGE', and 'GENDER'.

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=6 shorter survival N=1 longer survival N=5
AGE Spearman correlation test   N=0        
NEOPLASM DISEASESTAGE Kruskal-Wallis test   N=0        
PATHOLOGY T STAGE Spearman correlation test   N=0        
PATHOLOGY N STAGE Spearman correlation test   N=0        
PATHOLOGY M STAGE Kruskal-Wallis test   N=0        
GENDER Wilcoxon test   N=0        
Clinical variable #1: 'Time to Death'

6 genes related to 'Time to Death'.

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

Time to Death Duration (Months) 0.2-91.7 (median=13.3)
  censored N = 10
  death N = 29
     
  Significant markers N = 6
  associated with shorter survival 1
  associated with longer survival 5
List of 6 genes differentially expressed by 'Time to Death'

Table S2.  Get Full Table List of 6 genes significantly associated with 'Time to Death' by Cox regression test

HazardRatio Wald_P Q C_index
C16ORF45 0 1.001e-06 0.02 0.291
KRT14 0 1.881e-06 0.038 0.224
CMAH 491 6.039e-06 0.12 0.689
TADA2A 0 6.242e-06 0.13 0.249
SEMA7A 0 7.81e-06 0.16 0.274
BRPF1 0 9.02e-06 0.18 0.245
Clinical variable #2: 'AGE'

No gene related to 'AGE'.

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

AGE Mean (SD) 64.79 (8.4)
  Significant markers N = 0
Clinical variable #3: 'NEOPLASM.DISEASESTAGE'

No gene related to 'NEOPLASM.DISEASESTAGE'.

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

NEOPLASM.DISEASESTAGE Labels N
  STAGE I 3
  STAGE IA 1
  STAGE IB 1
  STAGE II 11
  STAGE III 19
  STAGE IV 4
     
  Significant markers N = 0
Clinical variable #4: 'PATHOLOGY.T.STAGE'

No gene related to 'PATHOLOGY.T.STAGE'.

Table S5.  Basic characteristics of clinical feature: 'PATHOLOGY.T.STAGE'

PATHOLOGY.T.STAGE Mean (SD) 2.26 (0.82)
  N
  1 7
  2 17
  3 13
  4 2
     
  Significant markers N = 0
Clinical variable #5: 'PATHOLOGY.N.STAGE'

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

Table S6.  Basic characteristics of clinical feature: 'PATHOLOGY.N.STAGE'

PATHOLOGY.N.STAGE Mean (SD) 0.44 (0.73)
  N
  0 25
  1 6
  2 5
     
  Significant markers N = 0
Clinical variable #6: 'PATHOLOGY.M.STAGE'

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

Table S7.  Basic characteristics of clinical feature: 'PATHOLOGY.M.STAGE'

PATHOLOGY.M.STAGE Labels N
  M0 31
  M1 2
  MX 6
     
  Significant markers N = 0
Clinical variable #7: 'GENDER'

No gene related to 'GENDER'.

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

GENDER Labels N
  FEMALE 10
  MALE 29
     
  Significant markers N = 0
Methods & Data
Input

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

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

  • Number of patients = 39

  • Number of genes = 20112

  • Number of clinical features = 7

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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