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 19880 genes and 12 clinical features across 364 samples, statistically thresholded by Q value < 0.05, 11 clinical features related to at least one genes.

13 genes correlated to 'AGE'.

GDNF , HPSE2 , ZNF334 , EBF4 , PENK , ...

1973 genes correlated to 'PRIMARY.SITE.OF.DISEASE'.

CCDC85A , TEF , DCTN5__1 , PALB2__1 , EIF2AK3 , ...

422 genes correlated to 'NEOPLASM.DISEASESTAGE'.

JOSD2 , LYSMD2 , TMOD2 , FAM86B2 , KCNK4 , ...

2 genes correlated to 'PATHOLOGY.T.STAGE'.

SYN2 , TIMP4

19 genes correlated to 'PATHOLOGY.N.STAGE'.

UBE2L6 , CASP1__1 , SP140L , CASP5 , IL12RB1 , ...

290 genes correlated to 'PATHOLOGY.M.STAGE'.

JOSD2 , LYSMD2 , TMOD2 , FAM86B2 , KCNK4 , ...

10 genes correlated to 'GENDER'.

GPX1 , KIF4B , POLDIP3 , RNU12 , MIR220B , ...

875 genes correlated to 'HISTOLOGICAL.TYPE'.

ZNF646 , ZNF668 , METTL3 , HIST1H2AL , C20ORF43__1 , ...

275 genes correlated to 'RADIATIONS.RADIATION.REGIMENINDICATION'.

INHA__1 , OBSL1__1 , ZNF599 , ZNF665 , FAM69C , ...

65 genes correlated to 'COMPLETENESS.OF.RESECTION'.

FUCA1 , ERO1L , CAPNS2 , LPCAT2 , ZNF140 , ...

13 genes correlated to 'NUMBER.OF.LYMPH.NODES'.

UBE2L6 , CASP1__1 , IL12RB1 , MAST3 , CASP5 , ...

No genes correlated to 'Time to Death'

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 Q value < 0.05.

Clinical feature	Statistical test	Significant genes	Associated with		Associated with
Time to Death	Cox regression test	N=0
AGE	Spearman correlation test	N=13	older	N=13	younger	N=0
PRIMARY SITE OF DISEASE	t test	N=1973	rectum	N=193	colon	N=1780
NEOPLASM DISEASESTAGE	ANOVA test	N=422
PATHOLOGY T STAGE	Spearman correlation test	N=2	higher stage	N=2	lower stage	N=0
PATHOLOGY N STAGE	Spearman correlation test	N=19	higher stage	N=17	lower stage	N=2
PATHOLOGY M STAGE	ANOVA test	N=290
GENDER	t test	N=10	male	N=2	female	N=8
HISTOLOGICAL TYPE	ANOVA test	N=875
RADIATIONS RADIATION REGIMENINDICATION	t test	N=275	yes	N=267	no	N=8
COMPLETENESS OF RESECTION	ANOVA test	N=65
NUMBER OF LYMPH NODES	Spearman correlation test	N=13	higher number.of.lymph.nodes	N=13	lower number.of.lymph.nodes	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 (Months)	0.1-140.4 (median=13)
	censored	N = 289
	death	N = 59

	Significant markers	N = 0

Clinical variable #2: 'AGE'

13 genes related to 'AGE'.

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


AGE	Mean (SD)	64.64 (13)
	Significant markers	N = 13
	pos. correlated	13
	neg. correlated	0

List of top 10 genes significantly correlated to 'AGE' by Spearman correlation test

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

	SpearmanCorr	corrP	Q
GDNF	0.2944	1.086e-08	0.000216
HPSE2	0.2642	3.283e-07	0.00653
ZNF334	0.2635	3.525e-07	0.00701
EBF4	0.262	4.132e-07	0.00821
PENK	0.2603	4.917e-07	0.00977
NKX2-2	0.2586	5.897e-07	0.0117
KIAA1755	0.2585	5.966e-07	0.0119
VIM	0.2588	5.975e-07	0.0119
ZNF75A	0.2578	6.361e-07	0.0126
OLFM2	0.2555	8.117e-07	0.0161

Figure S1. Get High-res Image As an example, this figure shows the association of GDNF to 'AGE'. P value = 1.09e-08 with Spearman correlation analysis. The straight line presents the best linear regression.

Clinical variable #3: 'PRIMARY.SITE.OF.DISEASE'

1973 genes related to 'PRIMARY.SITE.OF.DISEASE'.

Table S4. Basic characteristics of clinical feature: 'PRIMARY.SITE.OF.DISEASE'


PRIMARY.SITE.OF.DISEASE	Labels	N
	COLON	269
	RECTUM	93

	Significant markers	N = 1973
	Higher in RECTUM	193
	Higher in COLON	1780

List of top 10 genes differentially expressed by 'PRIMARY.SITE.OF.DISEASE'

Table S5. Get Full Table List of top 10 genes differentially expressed by 'PRIMARY.SITE.OF.DISEASE'

	T(pos if higher in 'RECTUM')	ttestP	Q	AUC
CCDC85A	-10.67	1.553e-22	3.09e-18	0.8364
TEF	-10.66	1.875e-22	3.73e-18	0.8494
DCTN5__1	-11.4	1.15e-21	2.29e-17	0.8375
PALB2__1	-11.4	1.15e-21	2.29e-17	0.8375
EIF2AK3	-10.25	2.087e-21	4.15e-17	0.8218
ATP5C1__1	-10.24	7.212e-21	1.43e-16	0.8345
KIN	-10.24	7.212e-21	1.43e-16	0.8345
AIDA	-10.03	9.33e-21	1.85e-16	0.8015
C1ORF58	-10.03	9.33e-21	1.85e-16	0.8015
PPCS__1	-9.85	3.44e-20	6.84e-16	0.8052

Figure S2. Get High-res Image As an example, this figure shows the association of CCDC85A to 'PRIMARY.SITE.OF.DISEASE'. P value = 1.55e-22 with T-test analysis.

Clinical variable #4: 'NEOPLASM.DISEASESTAGE'

422 genes related to 'NEOPLASM.DISEASESTAGE'.

Table S6. Basic characteristics of clinical feature: 'NEOPLASM.DISEASESTAGE'


NEOPLASM.DISEASESTAGE	Labels	N
	STAGE I	51
	STAGE IA	1
	STAGE II	25
	STAGE IIA	98
	STAGE IIB	7
	STAGE IIC	3
	STAGE III	12
	STAGE IIIA	19
	STAGE IIIB	53
	STAGE IIIC	31
	STAGE IV	26
	STAGE IVA	24
	STAGE IVB	1

	Significant markers	N = 422

List of top 10 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

Table S7. Get Full Table List of top 10 genes differentially expressed by 'NEOPLASM.DISEASESTAGE'

	ANOVA_P	Q
JOSD2	5.518e-208	1.1e-203
LYSMD2	7.73e-119	1.54e-114
TMOD2	7.73e-119	1.54e-114
FAM86B2	6.6e-113	1.31e-108
KCNK4	1.988e-85	3.95e-81
FZD3	1.982e-63	3.94e-59
GBP5	3.264e-55	6.49e-51
ZNF136	1.868e-51	3.71e-47
MTUS2	1.99e-50	3.95e-46
PRELID1	2.659e-46	5.28e-42

Figure S3. Get High-res Image As an example, this figure shows the association of JOSD2 to 'NEOPLASM.DISEASESTAGE'. P value = 5.52e-208 with ANOVA analysis.

Clinical variable #5: 'PATHOLOGY.T.STAGE'

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

Table S8. Basic characteristics of clinical feature: 'PATHOLOGY.T.STAGE'


PATHOLOGY.T.STAGE	Mean (SD)	2.91 (0.63)
		N
	0	1
	1	10
	2	54
	3	253
	4	44

	Significant markers	N = 2
	pos. correlated	2
	neg. correlated	0

List of 2 genes significantly correlated to 'PATHOLOGY.T.STAGE' by Spearman correlation test

Table S9. Get Full Table List of 2 genes significantly correlated to 'PATHOLOGY.T.STAGE' by Spearman correlation test

	SpearmanCorr	corrP	Q
SYN2	0.2502	1.436e-06	0.0285
TIMP4	0.2502	1.436e-06	0.0285

Figure S4. Get High-res Image As an example, this figure shows the association of SYN2 to 'PATHOLOGY.T.STAGE'. P value = 1.44e-06 with Spearman correlation analysis.

Clinical variable #6: 'PATHOLOGY.N.STAGE'

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

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


PATHOLOGY.N.STAGE	Mean (SD)	0.62 (0.76)
		N
	0	199
	1	99
	2	62

	Significant markers	N = 19
	pos. correlated	17
	neg. correlated	2

List of top 10 genes significantly correlated to 'PATHOLOGY.N.STAGE' by Spearman correlation test

Table S11. Get Full Table List of top 10 genes significantly correlated to 'PATHOLOGY.N.STAGE' by Spearman correlation test

	SpearmanCorr	corrP	Q
UBE2L6	0.3572	2.857e-12	5.68e-08
CASP1__1	0.3357	6.249e-11	1.24e-06
SP140L	0.305	3.446e-09	6.85e-05
CASP5	0.3001	6.299e-09	0.000125
IL12RB1	0.2979	8.223e-09	0.000163
APOL1	0.2968	9.402e-09	0.000187
MARCH8	0.2873	2.85e-08	0.000566
C8ORF80	0.2778	8.37e-08	0.00166
ASPHD2	0.2709	1.783e-07	0.00354
UBA7	0.2696	2.058e-07	0.00409

Figure S5. Get High-res Image As an example, this figure shows the association of UBE2L6 to 'PATHOLOGY.N.STAGE'. P value = 2.86e-12 with Spearman correlation analysis.

Clinical variable #7: 'PATHOLOGY.M.STAGE'

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

Table S12. Basic characteristics of clinical feature: 'PATHOLOGY.M.STAGE'


PATHOLOGY.M.STAGE	Labels	N
	M0	255
	M1	38
	M1A	8
	M1B	1
	MX	55

	Significant markers	N = 290

List of top 10 genes differentially expressed by 'PATHOLOGY.M.STAGE'

Table S13. Get Full Table List of top 10 genes differentially expressed by 'PATHOLOGY.M.STAGE'

	ANOVA_P	Q
JOSD2	4.257e-219	8.46e-215
LYSMD2	4.203e-130	8.36e-126
TMOD2	4.203e-130	8.36e-126
FAM86B2	1.84e-122	3.66e-118
KCNK4	6.228e-93	1.24e-88
FZD3	4.54e-71	9.02e-67
ZNF136	2.521e-57	5.01e-53
GBP5	7.494e-54	1.49e-49
PRELID1	1.097e-53	2.18e-49
RAB24	1.097e-53	2.18e-49

Figure S6. Get High-res Image As an example, this figure shows the association of JOSD2 to 'PATHOLOGY.M.STAGE'. P value = 4.26e-219 with ANOVA analysis.

Clinical variable #8: 'GENDER'

10 genes related to 'GENDER'.

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


GENDER	Labels	N
	FEMALE	163
	MALE	201

	Significant markers	N = 10
	Higher in MALE	2
	Higher in FEMALE	8

List of 10 genes differentially expressed by 'GENDER'

Table S15. Get Full Table List of 10 genes differentially expressed by 'GENDER'

	T(pos if higher in 'MALE')	ttestP	Q	AUC
GPX1	-11.97	6.296e-28	1.25e-23	0.8216
KIF4B	-11.3	2.521e-25	5.01e-21	0.8015
POLDIP3	-10.06	7.981e-21	1.59e-16	0.7974
RNU12	-10.06	7.981e-21	1.59e-16	0.7974
MIR220B	7.79	7.253e-14	1.44e-09	0.7594
TUBB4	7.79	7.253e-14	1.44e-09	0.7594
UBAP2	-7.35	1.359e-12	2.7e-08	0.7059
PAFAH1B2	-6.99	1.429e-11	2.84e-07	0.6991
YARS2	-5.59	4.894e-08	0.000973	0.6661
ZNF839	-5.54	5.793e-08	0.00115	0.6674

Figure S7. Get High-res Image As an example, this figure shows the association of GPX1 to 'GENDER'. P value = 6.3e-28 with T-test analysis.

Clinical variable #9: 'HISTOLOGICAL.TYPE'

875 genes related to 'HISTOLOGICAL.TYPE'.

Table S16. Basic characteristics of clinical feature: 'HISTOLOGICAL.TYPE'


HISTOLOGICAL.TYPE	Labels	N
	COLON ADENOCARCINOMA	236
	COLON MUCINOUS ADENOCARCINOMA	33
	RECTAL ADENOCARCINOMA	87
	RECTAL MUCINOUS ADENOCARCINOMA	6

	Significant markers	N = 875

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'

	ANOVA_P	Q
ZNF646	6.375e-44	1.27e-39
ZNF668	6.375e-44	1.27e-39
METTL3	4.193e-38	8.33e-34
HIST1H2AL	1.073e-30	2.13e-26
C20ORF43__1	3.197e-30	6.35e-26
DCTN5__1	1.012e-29	2.01e-25
PALB2__1	1.012e-29	2.01e-25
PILRB__1	5.456e-27	1.08e-22
POU2F1	1.704e-26	3.39e-22
HYI	2.515e-24	5e-20

Figure S8. Get High-res Image As an example, this figure shows the association of ZNF646 to 'HISTOLOGICAL.TYPE'. P value = 6.38e-44 with ANOVA analysis.

Clinical variable #10: 'RADIATIONS.RADIATION.REGIMENINDICATION'

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

Table S18. Basic characteristics of clinical feature: 'RADIATIONS.RADIATION.REGIMENINDICATION'


RADIATIONS.RADIATION.REGIMENINDICATION	Labels	N
	NO	8
	YES	356

	Significant markers	N = 275
	Higher in YES	267
	Higher in NO	8

List of top 10 genes differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

Table S19. Get Full Table List of top 10 genes differentially expressed by 'RADIATIONS.RADIATION.REGIMENINDICATION'

	T(pos if higher in 'YES')	ttestP	Q	AUC
INHA__1	16.55	7.809e-41	1.55e-36	0.7925
OBSL1__1	16.55	7.809e-41	1.55e-36	0.7925
ZNF599	15.01	1.102e-38	2.19e-34	0.7858
ZNF665	13.98	1.934e-35	3.85e-31	0.7093
FAM69C	12.66	1.783e-30	3.54e-26	0.7433
LRGUK	12.53	4.028e-30	8.01e-26	0.8292
LOC285548	13.37	6.948e-29	1.38e-24	0.8023
SEMA5B	12.58	3.327e-28	6.61e-24	0.8395
ST3GAL1	12.19	7.315e-28	1.45e-23	0.7114
ZNF586	11.97	7.418e-28	1.47e-23	0.7609

Figure S9. Get High-res Image As an example, this figure shows the association of INHA__1 to 'RADIATIONS.RADIATION.REGIMENINDICATION'. P value = 7.81e-41 with T-test analysis.

Clinical variable #11: 'COMPLETENESS.OF.RESECTION'

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

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


COMPLETENESS.OF.RESECTION	Labels	N
	R0	232
	R1	4
	R2	6
	RX	27

	Significant markers	N = 65

List of top 10 genes differentially expressed by 'COMPLETENESS.OF.RESECTION'

Table S21. Get Full Table List of top 10 genes differentially expressed by 'COMPLETENESS.OF.RESECTION'

	ANOVA_P	Q
FUCA1	7.097e-28	1.41e-23
ERO1L	4.044e-11	8.04e-07
CAPNS2	9.142e-11	1.82e-06
LPCAT2	9.142e-11	1.82e-06
ZNF140	2.109e-10	4.19e-06
ZFP14	2.838e-10	5.64e-06
C17ORF71	8.826e-10	1.75e-05
ASB7__1	1.275e-09	2.53e-05
LOC440354	1.554e-09	3.09e-05
BMS1P1	1.613e-09	3.21e-05

Figure S10. Get High-res Image As an example, this figure shows the association of FUCA1 to 'COMPLETENESS.OF.RESECTION'. P value = 7.1e-28 with ANOVA analysis.

Clinical variable #12: 'NUMBER.OF.LYMPH.NODES'

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

Table S22. Basic characteristics of clinical feature: 'NUMBER.OF.LYMPH.NODES'


NUMBER.OF.LYMPH.NODES	Mean (SD)	2.23 (4.8)
	Significant markers	N = 13
	pos. correlated	13
	neg. correlated	0

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

Table S23. Get Full Table List of top 10 genes significantly correlated to 'NUMBER.OF.LYMPH.NODES' by Spearman correlation test

	SpearmanCorr	corrP	Q
UBE2L6	0.3465	9.715e-11	1.93e-06
CASP1__1	0.3429	1.549e-10	3.08e-06
IL12RB1	0.3023	2.114e-08	0.00042
MAST3	0.2748	3.941e-07	0.00783
CASP5	0.2735	4.529e-07	0.009
APOL1	0.2716	5.455e-07	0.0108
MARCH8	0.2708	5.928e-07	0.0118
SP140L	0.2689	7.096e-07	0.0141
C8ORF80	0.264	1.151e-06	0.0229
ACTA2__1	0.2575	2.145e-06	0.0426

Figure S11. Get High-res Image As an example, this figure shows the association of UBE2L6 to 'NUMBER.OF.LYMPH.NODES'. P value = 9.71e-11 with Spearman correlation analysis. The straight line presents the best linear regression.

Methods & Data

Input

Expresson data file = COADREAD-TP.meth.by_min_clin_corr.data.txt
Clinical data file = COADREAD-TP.merged_data.txt
Number of patients = 364
Number of genes = 19880
Number of clinical features = 12

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

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

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

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] Lehmann and Romano, Testing Statistical Hypotheses (3E ed.), New York: Springer. ISBN 0387988645 (2005)

[4] Howell, D, Statistical Methods for Psychology. (5th ed.), Duxbury Press:324-5 (2002)

[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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