Stage 4S neuroblastoma tumors show a characteristic DNA methylation portrait

Figures & data

Figure 1. MBD sequencing analysis of MYCN non-amplified stage 4S, stage 4, and stage 1/2 tumors identifies stage-specific promoter methylation portraits. A. Diagram of the promoters identified as hypermethylated in the respective sample groups. For example, 393 and 1,150 promoters are specifically hyper- and hypomethylated in stage 4S, respectively. B. Principal component (PC) analysis using the normalized read counts of the promoters included in the diagram. Shown are the data for the first 2 PCs. PC1 explains 29.9% of the variability of the data and PC2 10.1%.

Figure 2. Stage 4S differentially methylated promoters are not randomly distributed across the genome. A. Percentage of stage 4S hypermethylated promoters per chromosome, based on promoter density corrected numbers. Significantly under- and overrepresented chromosomes are indicated with a dark dot. The vertical line represents the percentage if the stage 4S hypermethylated promoters would be randomly distributed across the genome (null hypothesis). B. Percentage of stage 4S hypomethylated promoters per chromosome, based on promoter density corrected numbers. Significantly under- and overrepresented chromosomes are indicated with a dark dot. The vertical line represents the percentage if the stage 4S hypomethylated promoters would be randomly distributed across the genome (null hypothesis). C. Significantly enriched cytogenetic locations of stage 4S hyper- and hypomethylated promoters. A list of genes located in the enriched regions can be found in Supplemental Table 2.

Figure 3. Stage 4S hypermethylated, and stage 4 and 1/2 hypomethylated promoters are frequently located on subtelomeres. Depicted are the percentages of the stage-specific differentially methylated promoters located in the 1 Mb (A) and 2 Mb (B) subtelomeres. Stage 4S hypermethylated and stage 4 hypomethylated promoters have a significantly larger proportion of 1 Mb and 2 Mb subtelomeric promoters, compared to stage 1/2 and stage 4 hypermethylated and stage 4S hypomethylated promoters. Stage 1/2 hypomethylated promoters also comprise a larger proportion of 2 Mb subtelomeric promoters, compared to stage 1/2 and stage 4 hypermethylated and stage 4S hypomethylated promoters.

Table 1. One third of subtelomeres are enriched in stage 4S hypermethylated promoters.

		Enriched 1Mb subtelomeres				Enriched 2 Mb subtelomeres
Comparison		Subtelomere	NES	Nom P-value	FDR q-value	Subtelomere	NES	Nom P-value	FDR q-value
Stage 4S vs. stage 4	Hypermethylated stage 4S	chr4p	1.58	0.037	0.040	chr1p	2.21	<0.001	<0.001
		chr5p	2.29	<0.001	<0.001	chr4p	1.92	<0.001	0.002
		chr10q	1.87	<0.001	0.002	chr4q	1.47	0.041	0.045
		chr13q	2.12	<0.001	<0.001	chr5p	2.60	<0.001	<0.001
		chr16p	2.32	<0.001	<0.001	chr5q	1.49	0.011	0.042
		chr17q	1.68	0.006	0.022	chr8q	1.35	0.022	0.083
		chr19p	2.28	<0.001	<0.001	chr9q	1.58	<0.001	0.029
						chr10q	1.78	<0.001	0.003
						chr13q	1.97	<0.001	0.001
						chr16p	2.50	<0.001	<0.001
						chr16q	2.17	<0.001	<0.001
						chr17q	1.65	<0.001	0.020
						chr19p	2.80	<0.001	<0.001
						chr20q	1.83	<0.001	0.004
	Hypermethylated stage 4			None				None
Stage 4S vs. stage 1/2	Hypermethylated stage 4S	chr4q	2.04	<0.001	<0.001	chr4q	2.12	<0.001	<0.001
		chr5p	1.59	0.022	0.030	chr5p	1.81	<0.001	0.006
		chr9q	2.30	<0.001	<0.001	chr5q	1.96	<0.001	<0.001
		chr12q	1.60	0.027	0.031	chr8q	1.79	<0.001	0.006
		chr13q	1.72	<0.001	0.013	chr10q	1.73	<0.001	0.008
		chr17p	1.81	0.008	0.008	chr13q	1.75	<0.001	0.009
		chr17q	2.22	<0.001	<0.001	chr20q	3.56	<0.001	<0.001
		chr20q	2.19	<0.001	<0.001	chr21q	1.64	<0.001	0.018
		chr22q	1.53	<0.001	0.044	chr22q	1.28	0.047	0.109
	Hypermethylated stage 1/2	chr1p	1.38	0.035	0.141			None
Stage 1/2 vs. stage 4	Hypermethylated stage 1/2	chr5p	2.29	<0.001	<0.001	chr4p	1.49	0.023	0.054
						chr5p	1.92	<0.001	<0.001
						chr10p	1.49	0.035	0.059
	Hypermethylated stage 4			None				None

For each of the 3 sample group comparisons, the subtelomeres significantly enriched for promoters hypermethylated in each of the sample groups are indicated, as well as their corresponding normalized enrichment score (NES), nominal P-value (nom P-value < 0.05), and false discovery rate q-value (FDR q-value < 0.25).

Table 2. Specific transcription factor targets are enriched in stage 4S differentially methylated promoters.

Hypermethylated in stage 4S
		Enrichment analysis						Differential expression analysis
Transcription factor target	Transcription factor	C	E	O	Genes	R	q	Stage 4S vs. stage 4	Stage 4S vs. stage 1/2
								q	q
CCGNMNNTNACG		75	0.28	3	HMG20B, HSPD1, FOSB	10.58	0.035	0.198	0.777
NTNGCGTGNNN	AHR	209	0.79	6	TSSK6, SLC9A5, FHOD1, FOSB, CIRBP, LPHN1	7.60	0.012	0.051	0.292
TTCNRGNNNNTTC		150	0.57	4	MKNK2, HSPD1, FOSB, CCT4	7.06	0.034	0.225	0.944
TAAWWATAG	MEF2A	163	0.62	4	EIF5A, SLC9A5, ALDOA, JSRP1	6.49	0.036	< 0.001	< 0.001
SGCGSSAAA	E2F1 TFDP1 RB1	167	0.63	4	MCM2, MCM7, SLC9A5, FHOD1	6.34	0.038	< 0.001	0.001
TGAMCTTTGMMCYT	HNF4A	260	0.98	6	PRKRA, NDUFA3, PRKCSH, RTN2, C4A, ALDOA	6.11	0.017	0.033	0.147
TTTSGCGS	E2F1	228	0.86	5	GPN3, MCM2, MCM7, SLC9A5, FHOD1	5.80	0.034	< 0.001	0.001
NCSCGCSAAAN	E2F1 TFDP1	234	0.88	5	EIF5A, TNPO2, GPN3, MCM2, MCM7	5.65	0.034	0.053	0.044
GCCATNTTN	YY1	238	0.90	5	NCOR1, PIGL, PRKCSH, PTBP1, LPHN1	5.56	0.034	0.121	0.003
NKCGCGCSAAAN	E2F1 TFDP1	239	0.90	5	EIF5A, TNPO2, MCM2, MCM7, PRKCSH	5.54	0.034	0.053	0.044
Hypomethylated in stage 4S
		Enrichment analysis						Differential expression analysis
Transcription factor target	Transcription factor	C	E	O	Genes	R	q	Stage 4S vs. stage 4	Stage 4S vs. stage 1/2
								q	q
NNTGTTACTAAAAATAGAAMNN		25	0.32	3	SLCO5A1, PPARGC1A, MGST3	9.27	0.011	0.045	0.213
NTGCGTGGGCGK	EGR3	86	1.11	7	CELF4, GPR176, ATP6V1C1, EPHB1, KCNQ5, UBE2W, CLTC	6.29	< 0.001	< 0.001	< 0.001
CARAACTAGGNCAAAGGTCA	PPARA	37	0.48	3	SS18, MARCH10, PPARGC1A	6.27	0.026	0.046	0.379
KMCATNNWGGA		87	1.13	7	CELF4, PDE4D, ADAMTSL1, RUNX1T1, OSBPL6, ZFAT, ARNTL	6.22	< 0.001	0.530	< 0.001
TGATTTRY	GFI1	291	3.77	23	RUNX1T1, FOXN3, WDPCP, AGR2, ALPK2, MYOT, SND1, CREB5, CDK14, IGF2BP3, FOXP2, NRXN3, MIR137HG, PTEN, C12orf42, ALDH1A2, JAZF1, ZNF521, MYBPC1, ZBTB20, PPARGC1A, MAPK10, RIMS2	6.11	< 0.001	< 0.001	< 0.001
NNGAATATKCANNNN	POU2F1	211	2.73	16	ADAMTSL1, NPAS3, RUNX1T1, PHLPP1, NRXN3, FOXP2, MARCH10, PPFIA2, ZNF521, ZBTB20, LPL, PDE4D, CACNA1D, PPARGC1A, RGS8, ARPP21	5.86	< 0.001	< 0.001	< 0.001
CTBATTTCARAAW	CEBPG	252	3.26	19	BRAF, RUNX1T1, NR6A1, FOXN3, PTK2, CREB5, WDR64, KCNQ5, CXCL9, FOXP2, SPAG9, PPFIA2, ALDH1A2, ST18, ZBTB20, C8orf46, SLIRP, PPARGC1A, PABPC1	5.83	< 0.001	< 0.001	0.007
NNNNNNKCTAWAAATAGMNNNN		226	2.92	17	LARS, SLC8A3, CELF4, ALPK2, CKMT2, KCNQ5, CACNA2D3, ARHGEF38, ARNTL, FBXO40, TSC22D1, TNNT2, PPARGC1A, C8orf46, ADRA1A, MLLT3, ARPP21	5.81	< 0.001	0.040	0.067
CNGTAWNTG	MSX1	173	2.24	13	FOXP2, BCAR3, KCNAB1, ADAMTSL1, ELMO1, C12orf42, RUNX1T1, NR6A1, ARNTL, CBLN2, SND1, INHBA, IGF2BP3	5.81	< 0.001	0.340	0.010
AGATAAGATAA	EVI1	55	0.71	4	CREB5, CACNA1D, PPARGC1A, RUNX1T1	5.62	0.014	< 0.001	0.324

Shown are the top 10 significantly (q < 0.05) enriched transcription factor targets (motifs) in stage 4S hyper- and hypomethylated promoters. For each enriched motif, matching transcription factor annotation is shown (if available), as well as the number of genes in the category (C), the expected number in the category (E), the number of genes hyper- or hypomethylated in stage 4S in the category (O), the annotation for the genes in O, the ratio of enrichment (R), and the q-value. Global test q-values of the differential expression analyses are also indicated.

Table 3. Oncogenic signatures are enriched in stage 4S hypomethylated promoters.

	Enrichment analysis						Differential expression analysis
	Stage 4S vs. stage 4			Stage 4S vs. stage 1/2			Stage 4S vs. stage 4	Stage 4S vs. stage 1/2
Gene set	NES	Nom P-value	FDR q-value	NES	Nom P-value	FDR q-value	q	q	Description of gene set
BCAT_BILD_ET_AL_DN	1.38	0.033	0.053	1.47	0.002	0.026	0.002	0.205	Genes downregulated in primary epithelial breast cancer cell culture overexpressing activated CTNNB1 (PMID: 16273092)
BMI1_DN.V1_DN	1.30	0.025	0.088	1.30	0.002	0.102	0.002	< 0.001	Genes downregulated in DAOY medulloblastoma cells upon knockdown of BMI1 by RNAi (PMID: 17452456)
CAHOY_NEURONAL	1.49	0.001	0.042	1.27	0.021	0.131	< 0.001	< 0.001	Genes upregulated in neurons (PMID: 18171944)
CRX_NRL_DN.V1_DN	1.38	0.006	0.056	1.23	0.030	0.182	< 0.001	< 0.001	Genes downregulated in retina cells from CRX and NRL double knockout mice (PMID: 17653270)
EGFR_UP.V1_DN	1.40	0.001	0.056	1.26	0.007	0.131	< 0.001	< 0.001	Genes downregulated in MCF7 breast cancer cells positive for ESR1 and engineered to express ligand-activated EGFR (PMID: 11888208)
EIF4E_DN	1.31	0.035	0.076	1.42	< 0.001	0.036	< 0.001	< 0.001	Genes downregulated in HMEC primary mammary epithelium cells upon overexpression of EIF4E (PMID: 17638893)
ERB2_UP.V1_DN	1.25	0.041	0.145	1.41	< 0.001	0.038	< 0.001	0.005	Genes downregulated in MCF7 breast cancer cells positive for ESR1 and engineered to express ligand-activated ERBB2 (PMID: 11888208)
KRAS.AMP.LUNG_UP.V1_UP	1.37	0.006	0.063	1.30	0.006	0.113	0.003	< 0.001	Genes upregulated in epithelial lung cancer cell lines overexpressing KRAS (PMID: 19847166)
KRAS.KIDNEY_UP.V1_UP	1.66	< 0.001	0.004	1.50	< 0.001	0.021	< 0.001	< 0.001	Genes upregulated in epithelial kidney cancer cell lines overexpressing an oncogenic form of KRAS
MEK_UP.V1_DN	1.47	< 0.001	0.044	1.37	< 0.001	0.054	< 0.001	< 0.001	Genes downregulated in MCF7 breast cancer cells positive for ESR1; MCF7 cells stably overexpressing constitutively active MAP2K1 (PMID: 16585219)
MEL18_DN.V1_DN	1.36	0.004	0.062	1.20	0.040	0.244	< 0.001	0.006	Genes downregulated in DAOY medulloblastoma cells upon knockdown of PCGF2 gene by RNAi (PMID: 17452456)
PIGF_UP.V1_UP	1.31	0.009	0.076	1.38	< 0.001	0.053	< 0.001	0.005	Genes upregulated in HUVEC endothelium cells by treatment with PIGF (PMID: 15516835)
PRC2_EDD_UP.V1_DN	1.30	0.014	0.088	1.32	< 0.001	0.106	< 0.001	< 0.001	Genes downregulated in TIG3 fibroblast cells upon knockdown of EED (PMID: 16618801)
PRC2_EZH2_UP.V1_DN	1.33	0.003	0.071	1.21	0.034	0.209	0.021	< 0.001	Genes downregulated in TIG3 fibroblast cells upon knockdown of EZH2 (PMID: 16618801)
RAF_UP.V1_DN	1.39	< 0.001	0.056	1.30	< 0.001	0.105	0.002	< 0.001	Genes downregulated in MCF7 breast cancer cells positive for ESR1; MCF7 cells stably overexpressing constitutively active RAF1 (PMID: 16585219)
RAF_UP.V1_UP	1.33	0.007	0.068	1.25	0.009	0.139	0.002	< 0.001	Genes upregulated in MCF7 breast cancer cells positive for ESR1; MCF7 cells stably overexpressing constitutively active RAF1 (PMID: 16585219)
STK33_NOMO_UP	1.21	0.040	0.204	1.27	< 0.001	0.127	0.007	< 0.001	Genes upregulated in NOMO1 acute myeloid leukemia cells after knockdown of STK33 by RNAi (PMID: 19490892)
STK33_SKM_UP	1.27	0.007	0.115	1.28	< 0.001	0.120	0.002	< 0.001	Genes upregulated in SKM1 acute myeloid leukemia cells after knockdown of STK33 by RNAi (PMID: 19490892)
STK33_UP	1.33	< 0.001	0.070	1.31	< 0.001	0.109	0.004	0.004	Genes upregulated in NOMO1 and SKM1 acute myeloid leukemia cells after knockdown of STK33 by RNAi (PMID: 19490892)

For each gene set significantly enriched in promoters hypomethylated in stage 4S compared to stage 4 and stage 1/2, the corresponding normalized enrichment score (NES), nominal P-value (nom P-value < 0.05) and false discovery rate q-value (FDR q-value < 0.25) are shown. Global test q-values of the differential expression analyses are also indicated, as well as a description of the gene set (with PubMed ID of the source publication, as described in the Molecular Signatures Database).¹⁸

Table 4. Multiple stage 4S differentially methylated genes are also differentially expressed.

Hypermethylated in stage 4S
		Stage 4S vs. stage 4			Stage 4S vs. stage 1/2
Enriched gene set	Gene	q	logFC	Overexpression in	q	logFC	Overexpression in
E2F1, E2F1_TFDP1_RB1 and MEF2A	SLC9A5	<0.001	0.833	stage 4	0.002	0.500	stage 1/2
Hypomethylated in stage 4S
		Stage 4S vs. stage 4			Stage 4S vs. stage 1/2
Enriched gene set	Gene	q	logFC	Overexpression in	q	logFC	Overexpression in
CAHOY_NEURONAL	DYNC1I1	0.010	1.035	stage 4	< 0.001	1.517	stage 1/2
	HS3ST2	0.021	0.680	stage 4	0.031	0.589	stage 1/2
	NSF	0.006	0.470	stage 4	< 0.001	0.538	stage 1/2
	GPR22	< 0.001	1.272	stage 4S	0.016	0.847	stage 4S
	CALB1	< 0.001	1.718	stage 4S	0.048	0.850	stage 4S
CRX_NRL_DN.V1_DN	GPR124	0.011	0.471	stage 4S	0.050	0.344	stage 4S
	PVRL3	< 0.001	1.153	stage 4S	0.009	0.719	stage 4S
EGFR_UP.V1_DN	C9orf3	< 0.001	0.833	stage 4S	0.011	0.574	stage 4S
	DERA	< 0.001	0.897	stage 4S	0.023	0.451	stage 4S
EGR3	ATP6V1C1	0.002	0.380	stage 4	< 0.001	0.444	stage 1/2
	CLTC	0.008	0.391	stage 4	0.002	0.419	stage 1/2
	GPR176	0.031	0.184	stage 4	0.018	0.185	stage 1/2
	UBE2W	0.048	0.177	stage 4	0.031	0.177	stage 1/2
EIF4E_DN	CLTC	0.008	0.391	stage 4	0.002	0.419	stage 1/2
	NPEPPS	0.002	0.348	stage 4	0.004	0.305	stage 1/2
	TP53BP1	0.027	0.287	stage 4	0.010	0.304	stage 1/2
	LMO7	0.001	0.490	stage 4S	0.028	0.323	stage 4S
	SH3YL1	0.004	0.428	stage 4S	0.049	0.282	stage 4S
ERB2_UP.V1_DN	NPEPPS	0.002	0.348	stage 4	0.004	0.305	stage 1/2
	C9orf3	< 0.001	0.833	stage 4S	0.011	0.574	stage 4S
	LAMB1	< 0.001	0.690	stage 4S	0.022	0.354	stage 4S
KRAS.AMP.LUNG_UP.V1_UP	DYNC1I1	0.010	1.035	stage 4	< 0.001	1.517	stage 1/2
	C8B	0.038	0.340	stage 4S	0.006	0.408	stage 4S
	GCKR	0.027	0.240	stage 4S	0.032	0.215	stage 4S
KRAS.KIDNEY_UP.V1_UP	DYNC1I1	0.010	1.035	stage 4	< 0.001	1.517	stage 1/2
	ESRRG	0.021	0.475	stage 4	0.018	0.449	stage 1/2
	CALB1	< 0.001	1.718	stage 4S	0.048	0.850	stage 4S
MEK_UP.V1_DN	NPEPPS	0.002	0.348	stage 4	0.004	0.305	stage 1/2
	C9orf3	< 0.001	0.833	stage 4S	0.011	0.574	stage 4S
	TCF12	0.001	0.370	stage 4S	0.015	0.261	stage 4S
MEL18_DN.V1_DN	DIO2	0.048	0.460	stage 4	0.019	0.497	stage 1/2
PIGF_UP.V1_UP	LRRC17	< 0.001	0.568	stage 4S	0.031	0.283	stage 4S
PRC2_EDD_UP.V1_DN	CBX8	< 0.001	0.815	stage 4	0.002	0.588	stage 1/2
RAF_UP.V1_DN	C9orf3	< 0.001	0.833	stage 4S	0.011	0.574	stage 4S
	PALLD	0.042	0.330	stage 4S	0.019	0.347	stage 4S
	TCF12	0.001	0.370	stage 4S	0.015	0.261	stage 4S
RAF_UP.V1_UP	CABYR	0.003	0.462	stage 4	< 0.001	0.589	stage 1/2
	GATA2	0.032	0.711	stage 4S	0.012	0.760	stage 4S
STK33_NOMO_UP	KIAA1467	< 0.001	0.560	stage 4	0.002	0.447	stage 1/2
	NPEPPS	0.002	0.348	stage 4	0.004	0.305	stage 1/2
	UBE2W	0.048	0.177	stage 4	0.031	0.177	stage 1/2
STK33_SKM_UP	UBE2W	0.048	0.177	stage 4	0.031	0.177	stage 1/2
	PALLD	0.042	0.330	stage 4S	0.019	0.347	stage 4S
STK33_UP	UBE2W	0.048	0.177	stage 4	0.031	0.177	stage 1/2

For each gene set differentially expressed in stage 4S compared to stage 4 and stage 1/2 (Table 2 and Table 3), differentially expressed individual genes are shown. Q-values are indicated, as well as the log₂ fold change (logFC) and the group in which the gene is overexpressed.