Hemimethylated CpG sites for both normal and tumor cells are identified using the Wilcoxon tests. Table 1 describes the proportions of hemimethylation sites that are in clusters depending on the p-value (p < 0.05) and three mean difference cutoff values. The CpG sites that are not in clusters are called singletons. There are similar numbers of hemimethylation sites in tumor and normal samples, but the proportion in clusters is slightly higher in normal samples. For the rest of this paper, our analysis will focus on the hemimethylation sites identified based on the p-value of 0.05 and the absolute mean difference greater than or equal to 0.4.
Table 1
Number of hemimethylated CpG sites and percentage of sites in clusters. Each row is for a mean difference level. The two panels (3 columns each) are for normal and tumor samples respectively.
|Mean difference| | Normal | Tumor |
Total | Sites in clusters | Percentage | Total | Sites in clusters | Percentage |
≥ 0.4 | 7351 | 1510 | 20.54% | 7330 | 1336 | 18.23% |
≥ 0.6 | 2588 | 348 | 13.45% | 2743 | 282 | 10.28% |
≥ 0.8 | 723 | 53 | 7.33% | 823 | 49 | 5.95% |
Tumor and normal samples’ hemimethylation CpG sites are compared in Table 2. The first row of this table, i.e., the T.MU row, indicates the total number of MU hemimethylation CpG sites in tumor (T) cells. Among these sites, 1697 of them are also hemimethylated in normal cells (N.MU), 1688 of them are not significantly hemimethylated in normal (N.NS), and 217 of them have no data in normal cells (N.NA). The first column of Table 2, i.e., the N.MU column, shows the total number of MU hemimethylation CpG sites in normal (N) cells. Among these sites, 1697 of them are also hemimethylated in tumor cells (T.MU), 1728 of them are not significantly hemimethylated in tumor (T.NS), and 268 of them have no data in tumor cells (T.NA).
Table 2
Comparison of normal and tumorous hemimethylation site patterns. Each row is for the tumor (T) sample and each column is for the normal (N) sample with various hemimethylation types. T.MU refers to CpG sites that are methylated (M) on the forward strand and unmethylated (U) on the reverse strand in tumor (T) samples. N.MU refers to CpG sites with the MU hemimethylation in normal (N) samples.
| N.MU | N.UM | N.NS | N.NA |
T.MU | 1697 | 0 | 1688 | 217 |
T.UM | 0 | 1597 | 1892 | 239 |
T.NS | 1728 | 1789 | 1895429 | 101322 |
T.NA | 268 | 272 | 98209 | 27295013 |
Tumor and normal samples’ hemimethylation clusters are compared in Table 3. This table shows that most clusters only have two or three CpG sites and cluster frequency decreases with increased cluster length, meaning large congregations of hemimethylation are infrequent.
Table 3
Normal and tumor hemimethylation cluster patterns. The first column is the cluster pattern, separating forward and reverse strands by “-”. The second and third columns are the counts of such patterns in normal and tumor samples respectively.
Cluster Pattern | Normal | Tumor |
MMMMMMMMMMMM-UUUUUUUUUUUU | 1 | 1 |
MMMMMMMMMM-UUUUUUUUUU | 1 | 1 |
MMMMMMMM-UUUUUUUU | 2 | 2 |
MMMMMMM-UUUUUUU | 2 | 2 |
MMMMMM-UUUUUU | 5 | 3 |
MMMMM-UUUUU | 6 | 7 |
MMMM-UUUU | 18 | 13 |
MMM-UUU | 55 | 32 |
MM-UU | 168 | 153 |
MMU-UUM | 0 | 1 |
MU-UM | 28 | 32 |
UMM-MUU | 1 | 0 |
UM-MU | 7 | 4 |
UUM-MMU | 1 | 0 |
UU-MM | 195 | 172 |
UUU-MMM | 52 | 44 |
UUUU-MMMM | 22 | 22 |
UUUUU-MMMMM | 9 | 14 |
UUUUUU-MMMMMM | 3 | 4 |
UUUUUUM-MMMMMMU | 0 | 1 |
UUUUUUU-MMMMMMM | 4 | 3 |
UUUUUUUM-MMMMMMMU | 1 | 0 |
UUUUUUUU-MMMMMMMM | 2 | 2 |
Total | 583 | 513 |
The length of a cluster is defined as the total number of base pairs between the first and the last CpG sites in the cluster. Figure 2 shows 4 histograms of cluster lengths. These histograms display the length distributions of polarity patterns in tumor, polarity patterns in normal, regular patterns in tumor, and regular patterns in normal samples. Regular and polarity patterns are analyzed separately because polarity clusters tend to be much shorter. In fact, many of the polarity clusters are less than 40 base pairs long and a majority of them are less than 10 base pairs long (see peaks in the top panels of Fig. 2). Many of the regular clusters are relatively short, i.e., less than 60 base pairs long, but a small amount of them are longer than that with a maximum length of around 100 to 120 base pairs.
Table 4
Regular clusters with corresponding percentages. Bigger clusters (see the fourth row) are the ones with 3 or more hemimethylated CpG sites.
Regular Clusters | Normal | Tumor |
MM-UU | 168 | 30.66% | 153 | 32.075% |
UU-MM | 195 | 35.58% | 172 | 36.059% |
Bigger cluster | 185 | 33.76% | 152 | 31.866% |
Total | 548 | 100% | 477 | 100% |
Table 5
Polarity clusters with corresponding percentages.
Polarity Clusters | Normal | Tumor |
MU-UM | 28 | 80% | 32 | 88.89% |
UM-MU | 7 | 20% | 4 | 11.11% |
Total | 35 | 100% | 36 | 100% |
For the two main hemimethylation cluster patterns, regular cluster and polarity cluster, we summarize them in detail in Table 4 and Table 5. Table 4 describes the proportions of different regular clusters in normal and tumor DNA. Table 5 describes the proportions of different polarity patterns in normal and tumor DNA. Polarity clusters appear less frequently than regular patterns, as seen by the difference in the number of sites between Tables 4 and 5. For example, tumor samples have a total of 477 regular clusters and only 36 polar clusters.
One way to detect which clusters may be related to cancer is to compare the cluster locations between tumor DNA and normal DNA. Some clusters may appear in the same sites in both tumor and normal samples, but others may be found only in tumor or only in normal. We compare the 513 tumor clusters with the 583 normal clusters and summarize the results in Table 6. This table shows that multiple kinds of overlaps can be found between tumor and normal. Hemimethylation clusters that occur only in tumor or normal samples are shown in Column B. 695 (313 tumor only and 382 normal only) clusters fall into these categories, and these are the clusters or regions that may be associated with cancer. Column C counts the number of clusters that are exactly the same for normal and tumor. Column D indicates the situations in which a tumor cluster begins and ends within a normal cluster (i.e., tumor cluster contained within the bounds of a normal cluster), and vice versa as shown in Column E. For example, a tumor cluster’s start and end positions on a chromosome are 150 and 170 base pairs. It is located within a normal cluster that has the start and end positions of 120 and 190 base pairs. Column D, which represents tumor clusters that are embedded in normal clusters, shows different counts for normal and tumor samples because there are two instances of multiple normal clusters located in one tumor cluster. Similarly, Column E, which represents normal clusters that are embedded in tumor clusters, shows different counts because there are three tumor clusters that are located in one normal cluster. Column F represents all other kinds of overlap. For example, there are two normal clusters that have some overlap with the same tumor cluster.
The second row of Table 6 shows that among the 513 tumor clusters, 313 of them belong to tumor only; 140 clusters also show up in normal samples; 25 tumor clusters are short ones and they are located within long normal clusters; 23 tumor clusters are long ones in which short normal clusters are located; and 12 tumor clusters are partially overlapped with normal clusters. The third row of Table 6 shows that among the 583 normal clusters, 382 of them belong to normal only; 140 clusters also show up in tumor samples; 23 normal clusters are long ones and they cover short tumor clusters; 25 normal clusters are short ones and they are located within long tumor clusters; and 13 normal clusters are partially overlapped with tumor clusters.
Table 6
Tumor and normal cluster comparison results. Columns are for different overlap (or non-overlap) patterns. The two rows are for tumor and normal, respectively.
A | B | C | D | E | F |
Tumor Total 513 | Tumor Only 313 | Exact Overlap 140 | Tumor in Normal 25 | Normal in Tumor 23 | Other Overlap 12 |
Normal Total 583 | Normal Only 382 | Exact Overlap 140 | Tumor in Normal 23 | Normal in Tumor 25 | Other Overlap 13 |
After identifying hemimethylated CpG sites, we may also map them back to genes. That is, we provide the annotation for each CpG site by providing the gene name in whose gene body or promoter region a hemimethylation site is located. We call this analysis gene annotation and summarizing such will provide the frequency on how many hemimethylated CpG sites a gene has. This annotation analysis is important because highly hemimethylated genes may play an important role. Table 7 shows the frequency of hemimethylated CpG sites in gene bodies. Each column shows how many genes have n hemimethylated CpG sites in their gene bodies, where n is given in the first row. The second row describes the distribution for tumor genes and the third row describes the distribution for normal genes. Similarly, Table 8 describes the frequency of hemimethylated CpG sites in promoter regions. Table 7 displays that the large majority of gene bodies have at most 3 hemimethylated CpG sites in both tumor and normal samples, but a few have more than 10. When looking at promoter regions, Table 8 shows none have 10 or more and the large majority of genes have 1 or 2 hemimethylated CpG sites.
Table 7
Hemimethylation frequency measured in gene bodies for both tumor and normal samples.
No. of Hemimethylation sites per gene body | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | >=10 |
Tumor | 1133 | 250 | 79 | 37 | 17 | 4 | 7 | 2 | 0 | 4 |
Normal | 1118 | 229 | 73 | 32 | 11 | 4 | 3 | 1 | 1 | 5 |
Table 8
Hemimethylation frequency measured in promoter regions for both tumor and normal samples.
No. of Hemimethylation sites per prom region | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
Tumor | 223 | 23 | 5 | 6 | 0 | 2 | 0 | 1 |
Normal | 256 | 36 | 13 | 3 | 2 | 1 | 1 | 0 |
With the gene annotation analysis, we can identify genes that have relatively more hemimethylation sites. In particular, we select the genes that have at least 5 hemimethylation sites in tumor only, in normal only, and in both normal and tumor samples. These genes are summarized in Tables 9, 10, and 11 respectively. In each of these tables, the first column is the gene name, the second column is the number of hemimethylation sites belonging to this gene, and the third column is the description of this gene. Terms in the tables that are followed by * are gene families, e.g., transcription factor and oncogene families. Otherwise they are general gene descriptions. The description and gene family of each gene are summarized based on the Molecular Signature Database [16] and the GeneCards database [17] .
There are 41 genes with the most hemimethylation in tumor DNA, see Table 9. Among these genes, TP73 [18–20], GNAS [21–25], and NOTCH1 [26, 27] are notable ones with known relations to cancer. Table 9 shows that among these 41 genes, 1 is a tumor suppressor (WT1), 3 are oncogenes (GNAS, NOTCh1, and PRDM16), and of those 3, 2 are translocated cancer genes (NOTCH1 and PRDM16). There are also 8 transcription factors in this table (HDAC4, IRX2, NFATC1, PRDM16, RUNX3, SIX3, TP73, and WT1). Table 10 shows 35 genes with the most hemimethylation in normal DNA. Among these genes, 4 are oncogenes (CBFA2T3, GNAS, PDGFB and PRDM16). Of the oncogenes, 3 are translocated cancer genes (CBFA2T3, PDGFB and PRDM16). There are also 7 transcription factors in this table (CBFA2T3, HOXA3, IRX2, MEIS1, NFIC, PRDM16, and ZFPM1). Note that no tumor suppressor genes are hemimethylated in the normal cells. Table 11 shows 36 genes with the most hemimethylation in both normal and tumor DNA. Among these genes, 2 are oncogenes and also translocated cancer genes (CBFA2T3 and PRDM16). There are also 6 transcription factors in this table (KLF5, HOXA2, CBFA2T3, HOXA3, ISL2, and PRDM16). All three gene tables have some transcription factor genes, which may affect the gene expression of other cancer-related genes that are not found to be hemimethylated.
Table 9
Genes with ≥ 5 hemimethylation sites in tumor samples. The “*” beside certain genes indicates a specific gene family (e.g., transcription factor or oncogene family) that a gene belongs to.
Gene name | Count | Gene Description |
RGS14 | 17 | regulator of G protein signaling 14 |
MEX3A | 16 | mex-3 RNA binding family member A |
WT1* | 11 | tumor suppressor, transcription factor*, WT1 transcription factor |
PRDM16* | 10 | oncogene, translocated cancer gene, transcription factor*, PR/SET domain 16 |
ZDHHC9 | 10 | zinc finger DHHC-type containing 9 |
AGAP2 | 8 | ArfGAP with GTPase domain, ankyrin repeat and PH domain 2 |
GNAS* | 8 | oncogene*, GNAS complex locus |
EXOC3L2 | 8 | exocyst complex component 3 like 2 |
PTPRN2 | 7 | protein tyrosine phosphatase receptor type N2 |
FANK1 | 7 | fibronectin type III and ankyrin repeat domains 1 |
UNC93B1 | 7 | unc-93 homolog B1, TLR signaling regulator |
IGSF9B | 7 | immunoglobulin superfamily member 9B |
GNAS-AS1 | 7 | GNAS antisense RNA 1 |
MAD1L1 | 7 | mitotic arrest deficient 1 like 1 |
TSPAN9 | 7 | tetraspanin 9 |
PTPRM | 7 | protein tyrosine phosphatase receptor type M |
TP73* | 6 | transcription factor*, tumor protein p73 |
IFT140 | 6 | intraflagellar transport 140 |
NFATC1* | 6 | transcription factor*, nuclear factor of activated T cells 1 |
DGKA | 6 | diacylglycerol kinase alpha |
FMNL1 | 6 | formin like 1 |
CACNA1I | 6 | calcium voltage-gated channel subunit alpha1 I |
LOC101927636 | 6 | RNA Gene affiliated with the lncRNA class |
HDAC4* | 5 | transcription factor*, histone deacetylase 4 |
IRX2* | 5 | homeodomain protein, transcription factor*, iroquois homeobox 2 |
ANKRD33B | 5 | ankyrin repeat domain 33B |
LINC00537 | 5 | Long Intergenic Non-Protein Coding RNA 537 |
NOTCH1* | 5 | oncogene, translocated cancer gene*, notch receptor 1 |
ANO2 | 5 | anoctamin 2 |
CACNA1H | 5 | calcium voltage-gated channel subunit alpha1 H |
RUNX3* | 5 | transcription factor*, runt related transcription factor 3 |
SIX3* | 5 | homeodomain protein, transcription factor*, SIX homeobox 3 |
FZD7 | 5 | frizzled class receptor 7 |
ADGRA2 | 5 | adhesion G protein-coupled receptor A2 |
IFFO1 | 5 | intermediate filament family orphan 1 |
CHTF18 | 5 | chromosome transmission fidelity factor 18 |
TMEM204 | 5 | transmembrane protein 204 |
RECQL5 | 5 | RecQ like helicase 5 |
SMIM5 | 5 | small integral membrane protein 5 |
MAPK1* | 5 | protein kinase*, mitogen-activated protein kinase 1 |
SYN1 | 5 | synapsin I |
Table 10
Genes with ≥ 5 hemimethylation sites in normal samples. The “*” beside certain genes indicates a specific gene family (e.g., the transcription factor family) that a gene belongs to.
Gene name | Count | Gene Description |
ZFPM1* | 14 | transcription factor*, zinc finger protein, FOG family member 1 |
GNAS* | 13 | oncogene*, GNAS complex locus |
RGPD2 | 12 | RANBP2 like and GRIP domain containing 2 |
SHANK3 | 11 | SH3 and multiple ankyrin repeat domains 3 |
IRX2* | 10 | homeodomain protein, transcription factor*, iroquois homeobox 2 |
LTB4R | 9 | leukotriene B4 receptor |
CPEB3 | 8 | cytoplasmic polyadenylation element binding protein 3 |
PTPRN2 | 7 | protein tyrosine phosphatase receptor type N2 |
MIR1268A | 7 | microRNA 1268a |
GNAS-AS1 | 7 | GNAS antisense RNA 1 |
CYP26C1 | 7 | cytochrome P450 family 26 subfamily C member 1 |
TBL1XR1 | 6 | transducin beta like 1 X-linked receptor 1 |
HOXA3* | 6 | homeodomain protein, transcription factor*, homeobox A3 |
CACNA1H | 6 | calcium voltage-gated channel subunit alpha1 H |
NPEPPS | 6 | aminopeptidase puromycin sensitive |
SEMA6B* | 6 | cytokine or growth factor*, semaphorin 6B |
HOMER3 | 6 | homer scaffold protein 3 |
PINLYP | 6 | phospholipase A2 inhibitor and LY6/PLAUR domain containing |
GDI1 | 6 | GDP dissociation inhibitor 1 |
HS3ST2 | 6 | heparan sulfate-glucosamine 3-sulfotransferase 2 |
PRDM16 | 5 | transcription factor, oncogene, translocated cancer gene*, PR/SET domain 16 |
PLK3* | 5 | protein kinase*, polo like kinase 3 |
GREM2* | 5 | cytokine or growth factor*, gremlin 2, DAN family BMP antagonist |
MEIS1* | 5 | homeodomain protein, transcription factor*, Meis homeobox 1 |
MEIS1-AS2 | 5 | MEIS1 antisense RNA 2 |
POLH | 5 | DNA polymerase eta |
HOXA-AS2 | 5 | HOXA cluster antisense RNA 2 |
EBF3 | 5 | EBF transcription factor 3 |
CBFA2T3* | 5 | transcription factor, oncogene, translocated cancer gene*, CBFA2/RUNX1 translocation partner 3 |
RPL13 | 5 | ribosomal protein L13 |
NFIC* | 5 | transcription factor*, nuclear factor I C |
CDH4 | 5 | cadherin 4 |
PDGFB* | 5 | cytokine or growth factor, oncogene, translocated cancer gene*, platelet derived growth factor subunit B |
CCNT1 | 5 | cyclin T1 |
SNORD68 | 5 | small nucleolar RNA, C/D box 68 |
Table 11
Genes with ≥ 5 hemimethylation sites identical in both tumor and normal samples. The “*” beside certain genes indicates a specific gene family (e.g., transcription factor or oncogene family) that a gene belongs to.
Gene name | Count | Gene Description |
RGPD5 | 16 | RANBP2 like and GRIP domain containing 5 |
RGPD8 | 16 | RANBP2 like and GRIP domain containing 8 |
ROCK1P1 | 13 | Rho associated coiled-coil containing protein kinase 1 pseudogene 1 |
THAP4 | 8 | THAP domain containing 4 |
SGTA | 8 | small glutamine rich tetratricopeptide repeat containing alpha |
PTPRN2 | 7 | protein tyrosine phosphatase receptor type N2 |
CNTNAP3 | 7 | contactin associated protein like 3 |
NUTM2A-AS1 | 7 | NUTM2A antisense RNA 1 |
RBFOX3 | 7 | RNA binding fox-1 homolog 3 |
ESPNP | 6 | espin pseudogene |
FOXK1 | 6 | forkhead box K1 |
HOXA3* | 6 | homeodomain protein, transcription factor*, homeobox A3 |
LMF1 | 6 | lipase maturation factor 1 |
USP45 | 6 | ubiquitin specific peptidase 45 |
LOC101928782 | 6 | RNA Gene affiliated with the lncRNA class |
PRDM16* | 5 | oncogene, translocated cancer gene, transcription factor*, PR/SET domain 16 |
RGPD4 | 5 | RANBP2 like and GRIP domain containing 4 |
MERTK* | 5 | protein kinase*, MER proto-oncogene, tyrosine kinase |
FAM160A1 | 5 | family with sequence similarity 160 member A1 |
PRKAR1B | 5 | protein kinase cAMP-dependent type I regulatory subunit beta |
MAD1L1 | 5 | mitotic arrest deficient 1 like 1 |
HOXA2* | 5 | homeodomain protein, transcription factor*, homeobox A2 |
DPP6 | 5 | dipeptidyl peptidase like 6 |
DIP2C | 5 | disco interacting protein 2 homolog C |
FANK1 | 5 | fibronectin type III and ankyrin repeat domains 1 |
GAL3ST3 | 5 | galactose-3-O-sulfotransferase 3 |
FLJ12825 | 5 | RNA Gene affiliated with the lncRNA class |
KLF5* | 5 | transcription factor*, Kruppel like factor 5 |
ISL2* | 5 | homeodomain protein, transcription factor*, ISL LIM homeobox 2 |
CBFA2T3* | 5 | oncogene, translocated cancer gene, transcription factor*, CBFA2/RUNX1 translocation partner 3 |
SBNO2 | 5 | strawberry notch homolog 2 |
GIPR | 5 | gastric inhibitory polypeptide receptor |
SCAF1 | 5 | SR-related CTD associated factor 1 |
COL6A1 | 5 | collagen type VI alpha 1 chain |
NEXMIF | 5 | neurite extension and migration factor |
GK5 | 5 | glycerol kinase 5 |
In order to understand the functions and relationships of these genes, we further analyze their biological interactions using the ConsensusPath Database (CPDB) software package [28–30], see Figs. 3, 4, 5, and 6. Figure 3 describes the different types of biological relationships between genes based on the CPDB software. A gene with a black label is known to be hemimethylated (i.e., identified by our analysis) and a gene with a purple label is a gene that is not provided in our hemimethylation gene list but it interacts with one of the known genes. Figure 3 is the legend for Figs. 4, 5, and 6. This legend figure summarizes the relationships for gene lists in Tables 9, 10, and 11 as shown in Figs. 4, 5, and 6, respectively. These figures show the extent to which these highly hemimethylated genes interact and possibly affect the cell function of related genes.
Figure 4 shows genetic interactions between genes with the most hemimethylation in tumor samples, and these genes are recorded in Table 9. The gene network in Fig. 4 contains a number of hub genes with complex interactions. These hub genes include GNAS, NFATC1, NOTCH1, MAPK1, HOAC4, TP73, and EGR1. We can see that if a hub gene like MAPK1 is hemimethylated, it may interact with dozens of other genes. Some of these genes, e.g., EGR1 [31–34] and UNC5B [35–38], are known to be associated with cancer.
Figure 5 shows genetic interactions between genes with the most hemimethylation in normal DNA, and these genes are recorded in Table 10. In this figure, we can see that GNAS is a hub gene interacting with many other genes that may not be hemimethylated themselves. GNAS is observed in both tumor and normal samples, as well as in the hemimethylation study for breast cancer cell lines [8]. MEIS1 is also a hub gene that interacts with genes like KMT2A [39] and TK1 [40]. While these genes are not hemimethylated in our samples, they are known to be associated with cancer. MEIS1 plays a crucial role in normal development [17] and it is also reported as an important gene related to leukemia [41–43]. Therefore, it is possible that the hemimethylation of hub genes like MEIS1 affects protein, biochemical, or regulatory functions of genes that are associated with cancer.
Figure 6 shows genetic interactions between genes with the most hemimethylation on identical locations in tumor and normal samples. These genes are recorded in Table 11. This means that the hemimethylation of CpG sites in this network are unchanged or unaffected by the formation of cancer. The HNRNPL gene is a major hub in this gene network. While we do not detect any hemimethylation in this gene, it directly interacts with 10 genes that we know to be hemimethylated. Some of these genes, like PTPRN2 and MAD1L1, can also be found in the tumor gene network, see Fig. 4. There appears to be no common genes between Fig. 5 (hemimethylated genes in normal samples) and Fig. 6 (hemimethylated genes in both tumor and normal samples). Therefore, genes that have a large number of hemimethylated CpG sites found only in normal DNA seem to have very few CpG sites that remain the same when cancer forms.